Also proteolysis, occurring naturally or induced, limited or massive, is a way through which proteins' activities are naturally or artificially switched on/off, or anyhow tuned. Thus, oligomerization can also affect proteolysis: in fact, a domain that in a native monomer is exposed to the action of a proteolytic agent can be partially or totally hidden by dimerization or oligomerization, and proteolysis can be slowed down or even blocked; otherwise, a region which in the native monomer is well structured and therefore protected, or even hidden, can be destabilized, destructured and exposed after oligomerization, becoming susceptible to proteolysis. This is what happens with RNase A, whose ND is definitely more susceptible to

Another interesting case is represented by the pore forming toxin (PFT) families, which are most often produced as soluble monomers, proteolitically cleaved by host proteases leading to their oligomerization and pore formation. This occurs, for example, with aerolysin [145], with protective antigen (PA) from anthrax [146], and with thiol-activated cytolysin (TACY)

Finally, it has to be underlined that all factors able to stabilize/destabilize protein oligomers, i.e., pH, temperature, ionic strength, protein concentration, molecular crowding, mutations, are not independent of each other. This makes the scenario more complicated than expected and clearly indicates that an 'absolute stability' of a protein can not be easily defined. This is true, for example, for RNase A domain-swapped oligomers (see Figure 9). Their high number of conformers and the possible interconversions between oligomers make the picture quite complicated [74], and only some data concerning dimers' stabilities have been reported to date [89, 148, 149]. With an apparent contradiction, RNase A-ND was sometimes reported to be more stable than CD while in other environmental conditions the situation is the opposite. These data clearly indicate that different combinations of all the environmental conditions reported above

Natural oligomerization of proteins has been settled by evolution in order to obtain a control of their biological features, for instance of enzyme activity. In some cases, proteins are inactive unless they dimerize or oligomerize because of the high hydrophobic surface the monomer exposes to the solvent, as reported above for PLP-enzymes. Oligomerization can be constitu‐ tive, such as for PLP-enzymes, or induced by signal molecules, like for membrane proteins. Self-or hetero-association of proteins can also be artificial, but can in any case lead to new activities or block activities that in certain situations can become unwanted, for example in the

Dimerization and oligomerization can also increase or lower pre-existing activities. In this case the phenomenon can be considered as a sharp controller and tuner of important

are crucial to stabilize or destabilize different dimeric or oligomeric structures.

**5. Oligomeric proteins: functional vs aberrant interactions**

**5.1. Gain or loss of function(s) after protein oligomerization**

feedback control of some enzymes.

proteolysis than the CD conformer [144].

260 Oligomerization of Chemical and Biological Compounds

pore forming family [147].

An interesting case in which protein oligomerization induces a gain of function concerns RNase A. The native monomeric enzyme only degrades single-stranded (ss) RNA and is not cytotoxic, while its artificial dimers and oligomers, either forming through non-covalent 3D-DS or covalent bonds become also active against double-stranded (ds) RNA [29, 57, 73, 152]. Moreover, they can become selectively cytotoxic towards cancer cells both *in vitro* and *in vivo* [153-156], although more recent results indicate that cytotoxicity can be dependent also on the type of cell line studied [32]. This acquired cytotoxic power can be considered benign, given the selectivity towards malignant cells, and can be mainly ascribable to the possibility of oligomeric RNases to evade the ribonuclease inhibitor (RI), which is designed for tightly trap monomeric RNases [157]. This is also the main reason why only MxM BS-RNase (see Figure 8) is cytotoxic: in fact, the cytosolic reducing environment allows only the MxM domainswapped isoform to survive as a non-covalent dimer (NCD) and to evade RI, while M=M becomes a monomeric, RI-susceptible, derivative [158]. Another example of cytotoxic protein oligomers is represented by the ones formed by p13suc1 [159]. The native cell-cycle regulatory protein is a monomer/DS-dimer mixture [160] that can form large native-like cytotoxic aggregates through 3D-DS, and whose structural determinants governing the swapping mechanism have been extensively studied as reported above [62, 68]. Thus, the artificial induction, quench, or control of the oligomerization event(s) can be useful to avoid, induce, or tune several biological properties of proteins, such as for example enzymatic activity, or the incoming of cytotoxicity.

#### **5.2. Protein oligomerization towards fibrillization and/or amyloidosis**

Protein self- or cross-association can be naturally or artificially controlled to the degree of oligomers or multimers, but can sometimes undergo uncontrolled massive aggregation, often resulting in fibers, as it has already been reported in the domain swapping section. These supramolecular structures can be benign, such as in muscle tissues (actin [150]), or very often harmful. The latter case is true for sickle cell HbS insoluble fibrous malignant polymers, or for amyloid or amyloid-like fibrils, which are often, although not always [2, 15], associated with deleterious neurodegenerative diseases [37]. Proteins can become prone to fibrillization because of ageing, or after changes in the environmental conditions, such as crowding, pH or temperature shocks. In addition, also point mutations can induce and often speed up the fibrillization phenomena, as for TTR V30M or L55P [161, 162], or for the human prion protein [163], or also for the homo-tetrameric p53 tumor suppressor protein [164, 165]. Several lethal amyloidoses, for example linked to TTR or β2-m, or to other toxic fibrillogenic proteins, like prions, display a premature incoming when associated to familial pathogenic mutations or overpass the species transmission barriers [166]. Then, also cancer has been recently considered a possible prio-like disease, due to the fibrillogenic behavior of some p53 mutants [167].

ments they can undergo (Figure 12) to follow a harmful fibrillogenic pathway or a non

Protein Oligomerization http://dx.doi.org/10.5772/57489 263

**Figure 12.** Evolution of partially or totally unfolded protein monomers towards ordered fibrils through oligomeric in‐

Besides artificial chemistry products, industry has often used natural or artificially modified bio-products to produce bio-materials or bio-fibers. In this context, nano-particles and nano-

Peptides and proteins can be driven towards controlled oligomerization, polymerization and/ or fibrillization to form products displaying useful physico-chemical and mechanical features to be adopted for new or renewed industrial applications. Incidentally, several efforts have been performed in the engineering of short peptides and small biomolecules [168-170], while definitely less is known on the industrial application of protein oligomers, multimers and/or fibrils. The most common materials formed from peptides and proteins are hydrogels, which can be applied in tissue engineering and in drug delivery [169]. These materials are typically formed by hydrated cross-linked fibers that somehow resemble the agarose or polyacrylamide

The 'intima' of these hydrogel structures and their morphology define size, biocompatibility, and mechanical, elastic or rigid properties. Anyway, only some hints concerning the interest‐

For example, the 3D-DS mechanism has been reported as to be applicable in material design [94]. Indeed, domain-swapped oligomeric peptides have been produced to obtain hydrogels [171], while also proteins can be artificially designed to undergo 3D-DS to form oligomers which could be useful to produce biomaterials [172]. Beyond small oligomers, the ability of some proteins to become fibrillogenic through propagative or runaway domain swapping [80, 94] could, or should, be exploited in order to engineer variants that could form harmless 3D-

fibrillogenic harmless destiny [7] can be found in that review [2].

termediates displaying different sizes and conformations. (Modified from [2] and [88]).

materials certainly represent a new fascinating frontier to be developed.

ing 'industrial' applications of protein oligomers will be given here.

**6. Oligomeric proteins and industry**

gels extensively used in biochemical laboratories.

In this complex scenario, it is known [163] and is more and more accepted that the first oligomeric/multimeric species preceding the formation of protofibrils and fibrils are the toxic agents responsible for the incoming of the associated pathology(ies) [2]. Thus, a lot of work is presently devoted to discover the structural and functional properties of these 'toxic oligom‐ ers', which is a very difficult endeavor indeed, because these species show a great tendency to fibrillize quickly.

In the last five years, the evidence became clear that oligomers produced by the same protein can be toxic or non toxic depending on the way they are produced [2, 7, 15]. This was found, for example, with the *Escherichia Coli* hyperforin-N (HypF-N) spherical oligomers [15] and also with other amyloidogenic proteins. Several different supramolecular large structures can be detected with new techniques, such as solid-state NMR, Cryo-TEM, High-Resolution Atomic-Force Spectroscopy, Molecular Modeling. For example, TTR has been discovered to fibrillize through the formation of annular oligomers deriving from the monomer which had been in turn detached from the native tetramer [10].

Several other amyloidogenic proteins have been extensively studied. Among them, the number of those that follow the 3D-DS mechanism is continuously increasing [80], such as for hPrP, hCC, β2-m, the properties of which have already been discussed. In the last years several proteins initially not considered to follow 3D-DS were discovered instead to undergo this mechanism. Nevertheless, the mechanism by which an oligomer/fiber may form essentially depends on the symmetry of the interfacial association and 3D-DS can be not mandatory to reach this requisite. Thus, the formation of the cross-β-spine fibrils intima could follow an end-to-end stacking mechanism, the same followed by non-amyloidogen‐ ic proteins, such as hemoglobin-S [164], or by non-harmful proteins, such as actin [170] or tubulin [171]. Anyway, considering that amyloid fibers are not oligomers, several oligomer‐ ic precursors of amyloidogenic proteins are continuously studied, despite their transient nature.  Further detailed studies, which are beyond the topic of this chapter and focused on the nature of amyloidogenic oligomers and their differences from the non-amyloidogenic ones have been recently reviewed [2]. Several interesting news concerning the oligomers' polymorphism and the structural rearrange‐ ments they can undergo (Figure 12) to follow a harmful fibrillogenic pathway or a non fibrillogenic harmless destiny [7] can be found in that review [2].

**Figure 12.** Evolution of partially or totally unfolded protein monomers towards ordered fibrils through oligomeric in‐ termediates displaying different sizes and conformations. (Modified from [2] and [88]).

#### **6. Oligomeric proteins and industry**

because of ageing, or after changes in the environmental conditions, such as crowding, pH or temperature shocks. In addition, also point mutations can induce and often speed up the fibrillization phenomena, as for TTR V30M or L55P [161, 162], or for the human prion protein [163], or also for the homo-tetrameric p53 tumor suppressor protein [164, 165]. Several lethal amyloidoses, for example linked to TTR or β2-m, or to other toxic fibrillogenic proteins, like prions, display a premature incoming when associated to familial pathogenic mutations or overpass the species transmission barriers [166]. Then, also cancer has been recently considered a possible prio-like disease, due to the fibrillogenic behavior of some p53 mutants [167].

In this complex scenario, it is known [163] and is more and more accepted that the first oligomeric/multimeric species preceding the formation of protofibrils and fibrils are the toxic agents responsible for the incoming of the associated pathology(ies) [2]. Thus, a lot of work is presently devoted to discover the structural and functional properties of these 'toxic oligom‐ ers', which is a very difficult endeavor indeed, because these species show a great tendency to

In the last five years, the evidence became clear that oligomers produced by the same protein can be toxic or non toxic depending on the way they are produced [2, 7, 15]. This was found, for example, with the *Escherichia Coli* hyperforin-N (HypF-N) spherical oligomers [15] and also with other amyloidogenic proteins. Several different supramolecular large structures can be detected with new techniques, such as solid-state NMR, Cryo-TEM, High-Resolution Atomic-Force Spectroscopy, Molecular Modeling. For example, TTR has been discovered to fibrillize through the formation of annular oligomers deriving from the monomer which had been in

Several other amyloidogenic proteins have been extensively studied. Among them, the number of those that follow the 3D-DS mechanism is continuously increasing [80], such as for hPrP, hCC, β2-m, the properties of which have already been discussed. In the last years several proteins initially not considered to follow 3D-DS were discovered instead to undergo this mechanism. Nevertheless, the mechanism by which an oligomer/fiber may form essentially depends on the symmetry of the interfacial association and 3D-DS can be not mandatory to reach this requisite. Thus, the formation of the cross-β-spine fibrils intima could follow an end-to-end stacking mechanism, the same followed by non-amyloidogen‐ ic proteins, such as hemoglobin-S [164], or by non-harmful proteins, such as actin [170] or tubulin [171]. Anyway, considering that amyloid fibers are not oligomers, several oligomer‐ ic precursors of amyloidogenic proteins are continuously studied, despite their transient nature.  Further detailed studies, which are beyond the topic of this chapter and focused on the nature of amyloidogenic oligomers and their differences from the non-amyloidogenic ones have been recently reviewed [2]. Several interesting news concerning the oligomers' polymorphism and the structural rearrange‐

fibrillize quickly.

turn detached from the native tetramer [10].

262 Oligomerization of Chemical and Biological Compounds

Besides artificial chemistry products, industry has often used natural or artificially modified bio-products to produce bio-materials or bio-fibers. In this context, nano-particles and nanomaterials certainly represent a new fascinating frontier to be developed.

Peptides and proteins can be driven towards controlled oligomerization, polymerization and/ or fibrillization to form products displaying useful physico-chemical and mechanical features to be adopted for new or renewed industrial applications. Incidentally, several efforts have been performed in the engineering of short peptides and small biomolecules [168-170], while definitely less is known on the industrial application of protein oligomers, multimers and/or fibrils. The most common materials formed from peptides and proteins are hydrogels, which can be applied in tissue engineering and in drug delivery [169]. These materials are typically formed by hydrated cross-linked fibers that somehow resemble the agarose or polyacrylamide gels extensively used in biochemical laboratories.

The 'intima' of these hydrogel structures and their morphology define size, biocompatibility, and mechanical, elastic or rigid properties. Anyway, only some hints concerning the interest‐ ing 'industrial' applications of protein oligomers will be given here.

For example, the 3D-DS mechanism has been reported as to be applicable in material design [94]. Indeed, domain-swapped oligomeric peptides have been produced to obtain hydrogels [171], while also proteins can be artificially designed to undergo 3D-DS to form oligomers which could be useful to produce biomaterials [172]. Beyond small oligomers, the ability of some proteins to become fibrillogenic through propagative or runaway domain swapping [80, 94] could, or should, be exploited in order to engineer variants that could form harmless 3D- DS fibers with special morphological and mechanical properties. These supra-molecular structures could also become materials devoid of direct biological applications, but with industrial and ecologic relevance. Indeed, proteins that for example combine 3D-DS with a covalent stabilization of their fibrillar products through the formation of novel disulfides (like β2-m and recombinant PrP) [110, 112], should be useful to obtain reversible products that could be easily unstructured and recycled under reducing conditions, and, thus, without negative ecologic consequences.

**Author details**

Giovanni Gotte\*

Verona, Verona, Italy

FEBS J. 2010;277:1348-58.

**References**

and Massimo Libonati

\*Address all correspondence to<EMAIL_ADDRESS>
Department of Life and Reproduction Sciences, Biological Chemistry Section, University of

Protein Oligomerization http://dx.doi.org/10.5772/57489 265

[1] Levy ED, Teichmann S. Structural, evolutionary, and assembly principles of protein

[2] Bemporad F, Chiti F. Protein misfolded oligomers: experimental approaches, mecha‐ nism of formation, and structure-toxicity relationships. Chem Biol. 2012;19:315-27.

[3] Haass C, Selkoe DJ. Soluble protein oligomers in neurodegeneration: lessons from the Alzheimer's amyloid beta-peptide. Nat Rev Mol Cell Biol. 2007;8:101-12.

[4] Sakono M, Zako T. Amyloid oligomers: formation and toxicity of Abeta oligomers.

[5] Libonati M, Gotte G. Oligomerization of bovine ribonuclease A: structural and func‐

[6] Cozza G, Moro S, Gotte G. Elucidation of the ribonuclease a aggregation process mediated by 3D domain swapping: A computational approach reveals possible new

[7] Glabe CG. Structural classification of toxic amyloid oligomers. The Journal of Biolog‐

[8] Fandrich M. Oligomeric intermediates in amyloid formation: structure determination and mechanisms of toxicity. Journal of Molecular Biology. 2012;421:427-40.

[9] Barghorn S, Nimmrich V, Striebinger A, Krantz C, Keller P, Janson B, et al. Globular amyloid beta-peptide oligomer - a homogenous and stable neuropathological protein

[10] Pires RH, Karsai A, Saraiva MJ, Damas AM, Kellermayer MS. Distinct annular oligomers captured along the assembly and disassembly pathways of transthyretin

[11] Lashuel HA, Hartley D, Petre BM, Walz T, Lansbury PT, Jr. Neurodegenerative dis‐

ease: amyloid pores from pathogenic mutations. Nature. 2002;418:291.

tional features of its multimers. The Biochemical Journal. 2004;380:311-27.

oligomerization. Prog Mol Biol Transl Sci. 2013;117:25-51.

multimeric structures. Biopolymers. 2008;89:26-39.

in Alzheimer's disease. J Neurochem. 2005;95:834-47.

amyloid protofibrils. PLoS One. 2012;7:e44992.

ical Chemistry. 2008;283:29639-43.

Many examples of protein 'benign' (non-amyloidogenic) fibers could become precious for industry applications. An example can be represented by the trimeric hexon protein. display‐ ing a novel triple β-spiral fibrous fold with implications for the design of a new class of artificial, silk-like fibrous materials [173].

Anyway, all the potential industrial applications of protein oligomers, multimers, and fibrils are certainly far to be completely explored, and what we have here reported about 3D-DS applications confirms that further deep investigations deserve to be performed.

#### **7. Conclusions**

All the notions reported in this chapter indicate that the complexity of the protein oligomeri‐ zation topic augments every day. In particular, the increasing number of studies focused on natural protein oligomerization and the improvement of the quality of the investigation techniques have greatly enlarged the complexity of the analysis of the structural and functional features of protein oligomers. Furthermore, the sharpening of the strategies used by chemistry to obtain cross-linked artificial oligomers allowed industry and laboratories to obtain less heterogeneous products, which were, in addition, scarcely modified with respect to the native monomeric protein(s).

Again, the discovery that some proteins can naturally oligomerize by combining covalent linkings with weak interactions made the scenario even more complicated. This is the case, for example, of proteins that form domain-swapped fibrils additionally stabilized by newly forming disulfides. The same is true also for membrane proteins that associate and sometimes covalently stabilize their interaction as a response to effectors which can play the role of activators or quench cell signals.

Thus, the aim of this chapter is to focus the attention of the reader on the principal features of protein oligomerization. We kept separated, when possible, the covalent linking oligomers from the non-covalent protein self-association products, as well as the natural, constitutive or induced, events from the artificial ones. When we were not able to separate these aspects well, we tried to give a picture as clear as possible. On the other hand, this underlines how much increasing interest has and how many further studies deserves the topic of protein oligome‐ rization.

#### **Author details**

DS fibers with special morphological and mechanical properties. These supra-molecular structures could also become materials devoid of direct biological applications, but with industrial and ecologic relevance. Indeed, proteins that for example combine 3D-DS with a covalent stabilization of their fibrillar products through the formation of novel disulfides (like β2-m and recombinant PrP) [110, 112], should be useful to obtain reversible products that could be easily unstructured and recycled under reducing conditions, and, thus, without negative

Many examples of protein 'benign' (non-amyloidogenic) fibers could become precious for industry applications. An example can be represented by the trimeric hexon protein. display‐ ing a novel triple β-spiral fibrous fold with implications for the design of a new class of artificial,

Anyway, all the potential industrial applications of protein oligomers, multimers, and fibrils are certainly far to be completely explored, and what we have here reported about 3D-DS

All the notions reported in this chapter indicate that the complexity of the protein oligomeri‐ zation topic augments every day. In particular, the increasing number of studies focused on natural protein oligomerization and the improvement of the quality of the investigation techniques have greatly enlarged the complexity of the analysis of the structural and functional features of protein oligomers. Furthermore, the sharpening of the strategies used by chemistry to obtain cross-linked artificial oligomers allowed industry and laboratories to obtain less heterogeneous products, which were, in addition, scarcely modified with respect to the native

Again, the discovery that some proteins can naturally oligomerize by combining covalent linkings with weak interactions made the scenario even more complicated. This is the case, for example, of proteins that form domain-swapped fibrils additionally stabilized by newly forming disulfides. The same is true also for membrane proteins that associate and sometimes covalently stabilize their interaction as a response to effectors which can play the role of

Thus, the aim of this chapter is to focus the attention of the reader on the principal features of protein oligomerization. We kept separated, when possible, the covalent linking oligomers from the non-covalent protein self-association products, as well as the natural, constitutive or induced, events from the artificial ones. When we were not able to separate these aspects well, we tried to give a picture as clear as possible. On the other hand, this underlines how much increasing interest has and how many further studies deserves the topic of protein oligome‐

applications confirms that further deep investigations deserve to be performed.

ecologic consequences.

**7. Conclusions**

monomeric protein(s).

rization.

activators or quench cell signals.

silk-like fibrous materials [173].

264 Oligomerization of Chemical and Biological Compounds

Giovanni Gotte\* and Massimo Libonati

\*Address all correspondence to<EMAIL_ADDRESS>
Department of Life and Reproduction Sciences, Biological Chemistry Section, University of Verona, Verona, Italy

#### **References**

[12] Fandrich M. On the structural definition of amyloid fibrils and other polypeptide ag‐ gregates. Cell Mol Life Sci. 2007;64:2066-78.

[27] Nenci A, Gotte G, Bertoldi M, Libonati M. Structural properties of trimers and tet‐ ramers of ribonuclease A. Protein Science: a publication of the Protein Society.

Protein Oligomerization http://dx.doi.org/10.5772/57489 267

[28] Gotte G, Libonati M. Oligomerization of ribonuclease A: two novel three-dimension‐ al domain-swapped tetramers. The Journal of Biological Chemistry. 2004;279:36670-9.

[29] Gotte G, Laurents DV, Libonati M. Three-dimensional domain-swapped oligomers of ribonuclease A: identification of a fifth tetramer, pentamers and hexamers, and de‐ tection of trace heptameric, octameric and nonameric species. Biochimica et Biophy‐

[30] Simons BL, King MC, Cyr T, Hefford MA, Kaplan H. Covalent cross-linking of pro‐ teins without chemical reagents. Protein Science: a publication of the Protein Society.

[31] Simons BL, Kaplan H, Fournier SM, Cyr T, Hefford MA. A novel cross-linked RNase

[32] Vottariello F, Costanzo C, Gotte G, Libonati M. "Zero-length" dimers of ribonuclease A: further characterization and no evidence of cytotoxicity. Bioconjug Chem.

[33] Fancy DA, Kodadek T. Chemistry for the analysis of protein-protein interactions: rapid and efficient cross-linking triggered by long wavelength light. Proceedings of the National Academy of Sciences of the United States of America. 1999;96:6020-4. [34] Lumb MJ, Danpure CJ. Functional synergism between the most common polymor‐ phism in human alanine:glyoxylate aminotransferase and four of the most common disease-causing mutations. The Journal of Biological Chemistry. 2000;275:36415-22.

[35] Cellini B, Montioli R, Paiardini A, Lorenzetto A, Maset F, Bellini T, et al. Molecular defects of the glycine 41 variants of alanine glyoxylate aminotransferase associated with primary hyperoxaluria type I. Proceedings of the National Academy of Sciences

[36] Levy ED, Boeri Erba E, Robinson CV, Teichmann SA. Assembly reflects evolution of

[37] Eisenberg D, Jucker M. The amyloid state of proteins in human diseases. Cell.

[38] Monaco HL, Rizzi M, Coda A. Structure of a complex of two plasma proteins: trans‐

[39] Blake C, Serpell L. Synchrotron X-ray studies suggest that the core of the transthyre‐ tin amyloid fibril is a continuous beta-sheet helix. Structure. 1996;4:989-98.

thyretin and retinol-binding protein. Science. 1995;268:1039-41.

of the United States of America. 2010;107:2896-901.

protein complexes. Nature. 2008;453:1262-5.

A dimer with enhanced enzymatic properties. Proteins. 2007;66:183-95.

2001;10:2017-27.

2002;11:1558-64.

2010;21:635-45.

2012;148:1188-203.

sica Acta. 2006;1764:44-54.

[27] Nenci A, Gotte G, Bertoldi M, Libonati M. Structural properties of trimers and tet‐ ramers of ribonuclease A. Protein Science: a publication of the Protein Society. 2001;10:2017-27.

[12] Fandrich M. On the structural definition of amyloid fibrils and other polypeptide ag‐

[13] Nelson R, Sawaya MR, Balbirnie M, Madsen AO, Riekel C, Grothe R, et al. Structure

[14] Sipe JD, Cohen AS. Review: history of the amyloid fibril. Journal of Structural Biolo‐

[15] Campioni S, Mannini B, Zampagni M, Pensalfini A, Parrini C, Evangelisti E, et al. A causative link between the structure of aberrant protein oligomers and their toxicity.

[16] D'Alessio G, Parente A, Guida C, Leone E. Dimeric structure of seminal ribonuclease.

[18] Hartman FC, Wold F. Cross-linking of bovine pancreatic ribonuclease A with di‐

[19] Wang D, Wilson G, Moore S. Preparation of cross-linked dimers of pancreatic ribo‐

[20] Green NS, Reisler E, Houk KN. Quantitative evaluation of the lengths of homobi‐ functional protein cross-linking reagents used as molecular rulers. Protein Science: a

[21] Fracasso G, Bellisola G, Castelletti D, Tridente G, Colombatti M. Immunotoxins and other conjugates: preparation and general characteristics. Mini Rev Med Chem.

[22] Ayers NA, Nadeau OW, Read MW, Ray P, Carlson GM. Effector-sensitive cross-link‐ ing of phosphorylase b kinase by the novel cross-linker 4-phenyl-1,2,4-triazoline-3,5-

[23] Sheehan JCH, J.J. The cross-linking of gelatin using a water-soluble carbodiimide.

[24] Lopez-Alonso JP, Diez-Garcia F, Font J, Ribo M, Vilanova M, Scholtz JM, et al. Carbo‐ diimide EDC Induces Cross-Links That Stabilize RNase A C-Dimer against Dissocia‐ tion: EDC Adducts Can Affect Protein Net Charge, Conformation, and Activity.

[25] Ciglic MI, Jackson PJ, Raillard SA, Haugg M, Jermann TM, Opitz JG, et al. Origin of dimeric structure in the ribonuclease superfamily. Biochemistry. 1998;37:4008-22.

[26] Lin SH, Konishi Y, Denton ME, Scheraga HA. Influence of an extrinsic cross-link on the folding pathway of ribonuclease A. Conformational and thermodynamic analysis of cross-linked (lysine7-lysine41)-Ribonuclease A. Biochemistry. 1984;23:5504-12.

of the cross-beta spine of amyloid-like fibrils. Nature. 2005;435:773-8.

[17] Urry DW. What is elastin; what is not. Ultrastruct Pathol. 1983;4:227-51.

methyl adipimidate. Biochemistry. 1967;6:2439-48.

publication of the Protein Society. 2001;10:1293-304.

dione. The Biochemical Journal. 1998;331 (Pt 1):137-41.

Journal of the American Chemical Society. 1957;79.

gregates. Cell Mol Life Sci. 2007;64:2066-78.

Nature Chemical Biology. 2010;6:140-7.

nuclease. Biochemistry. 1976;15:660-5.

FEBS Letters. 1972;27:285-8.

gy. 2000;130:88-98.

266 Oligomerization of Chemical and Biological Compounds

2004;4:545-62.

Bioconjug Chem. 2009.

[40] Montioli R, Cellini B, Bertoldi M, Paiardini A, Voltattorni CB. An engineered folded PLP-bound monomer of Treponema denticola cystalysin reveals the effect of the di‐ meric structure on the catalytic properties of the enzyme. Proteins. 2009;74:304-17.

[54] Crestfield AM, Stein WH, Moore S. On the aggregation of bovine pancreatic ribonu‐

Protein Oligomerization http://dx.doi.org/10.5772/57489 269

[55] Mazzarella L, Capasso S, Demasi D, Di Lorenzo G, Mattia CA, Zagari A. Bovine seminal ribonuclease: structure at 1.9 A resolution. Acta Crystallogr D Biol Crystal‐

[56] Gronenborn AM. Protein acrobatics in pairs--dimerization via domain swapping.

[57] Libonati M. Molecular aggregates of ribonucleases. Some enzymatic properties. Ital J

[58] Libonati M, Bertoldi M, Sorrentino S. The activity on double-stranded RNA of aggre‐ gates of ribonuclease A higher than dimers increases as a function of the size of the

[59] Piccoli R, D'Alessio G. Relationships between nonhyperbolic kinetics and dimeric structure in ribonucleases. The Journal of biological chemistry. 1984;259:693-5.

[60] Piccoli R, Tamburrini M, Piccialli G, Di Donato A, Parente A, D'Alessio G. The dualmode quaternary structure of seminal RNase. Proceedings of the National Academy

[61] Rousseau F, Schymkowitz JW, Itzhaki LS. The unfolding story of three-dimensional

[62] Rousseau F, Schymkowitz JW, Wilkinson HR, Itzhaki LS. Intermediates control do‐ main swapping during folding of p13suc1. The Journal of Biological Chemistry.

[63] Ercole C, Spadaccini R, Alfano C, Tancredi T, Picone D. A new mutant of bovine seminal ribonuclease with a reversed swapping propensity. Biochemistry.

[64] Picone D, Di Fiore A, Ercole C, Franzese M, Sica F, Tomaselli S, et al. The role of the hinge loop in domain swapping. The special case of bovine seminal ribonuclease.

[65] Ercole C, Avitabile F, Del Vecchio P, Crescenzi O, Tancredi T, Picone D. Role of the hinge peptide and the intersubunit interface in the swapping of N-termini in dimeric

[66] Yang S, Levine H, Onuchic JN. Protein oligomerization through domain swapping: role of inter-molecular interactions and protein concentration. Journal of Molecular

[67] Rousseau F, Schymkowitz JW, Wilkinson HR, Itzhaki LS. The structure of the transi‐ tion state for folding of domain-swapped dimeric p13suc1. Structure. 2002;10:649-57.

clease. Archives of Biochemistry and Biophysics. 1962;Suppl 1:217-22.

Current Opinion in Structural Biology. 2009;19:39-49.

aggregates. The Biochemical Journal. 1996;318 ( Pt 1):287-90.

of Sciences of the United States of America. 1992;89:1870-4.

The Journal of Biological Chemistry. 2005;280:13771-8.

bovine seminal RNase. Eur J Biochem. 2003;270:4729-35.

domain swapping. Structure. 2003;11:243-51.

logr. 1993;49:389-402.

Biochem. 1969;18:407-17.

2004;279:8368-77.

2007;46:2227-32.

Biology. 2005;352:202-11.

[54] Crestfield AM, Stein WH, Moore S. On the aggregation of bovine pancreatic ribonu‐ clease. Archives of Biochemistry and Biophysics. 1962;Suppl 1:217-22.

[40] Montioli R, Cellini B, Bertoldi M, Paiardini A, Voltattorni CB. An engineered folded PLP-bound monomer of Treponema denticola cystalysin reveals the effect of the di‐ meric structure on the catalytic properties of the enzyme. Proteins. 2009;74:304-17.

[41] Zhang X, Roe SM, Hou Y, Bartlam M, Rao Z, Pearl LH, et al. Crystal structure of ala‐ nine:glyoxylate aminotransferase and the relationship between genotype and enzy‐ matic phenotype in primary hyperoxaluria type 1. Journal of Molecular Biology.

[42] Burkhard P, Dominici P, Borri-Voltattorni C, Jansonius JN, Malashkevich VN. Struc‐ tural insight into Parkinson's disease treatment from drug-inhibited DOPA decar‐

[43] Flydal MI, Martinez A. Phenylalanine hydroxylase: function, structure, and regula‐

[44] Grueninger D, Treiber N, Ziegler MO, Koetter JW, Schulze MS, Schulz GE. Designed

[45] Marianayagam NJ, Sunde M, Matthews JM. The power of two: protein dimerization

[46] Hebert TE, Bouvier M. Structural and functional aspects of G protein-coupled recep‐

[47] Terrillon S, Bouvier M. Roles of G-protein-coupled receptor dimerization. EMBO

[48] Renatus M, Stennicke HR, Scott FL, Liddington RC, Salvesen GS. Dimer formation drives the activation of the cell death protease caspase 9. Proceedings of the National

[49] Agre P, Kozono D. Aquaporin water channels: molecular mechanisms for human

[50] Bennett MJ, Choe S, Eisenberg D. Domain swapping: entangling alliances between proteins. Proceedings of the National Academy of Sciences of the United States of

[51] Steere B, Eisenberg D. Characterization of high-order diphtheria toxin oligomers. Bi‐

[52] Bennett MJ, Schlunegger MP, Eisenberg D. 3D domain swapping: a mechanism for oligomer assembly. Protein science : a publication of the Protein Society.

[53] Benito A, Laurents DV, Ribo M, Vilanova M. The structural determinants that lead to the formation of particular oligomeric structures in the pancreatic-type ribonuclease

Academy of Sciences of the United States of America. 2001;98:14250-5.

boxylase. Nature Structural Biology. 2001;8:963-7.

protein-protein association. Science. 2008;319:206-9.

tor oligomerization. Biochem Cell Biol. 1998;76:1-11.

in biology. Trends Biochem Sci. 2004;29:618-25.

diseases. FEBS Letters. 2003;555:72-8.

family. Curr Protein Pept Sci. 2008;9:370-93.

America. 1994;91:3127-31.

ochemistry. 2000;39:15901-9.

1995;4:2455-68.

tion. IUBMB Life. 2013;65:341-9.

2003;331:643-52.

268 Oligomerization of Chemical and Biological Compounds

Rep. 2004;5:30-4.

[68] Rousseau F, Schymkowitz JW, Wilkinson HR, Itzhaki LS. Three-dimensional domain swapping in p13suc1 occurs in the unfolded state and is controlled by conserved pro‐ line residues. Proceedings of the National Academy of Sciences of the United States of America. 2001;98:5596-601.

C-swapped tetramers and multimers with increasing biological activities. PLoS One.

Protein Oligomerization http://dx.doi.org/10.5772/57489 271

[82] Koharudin LM, Liu L, Gronenborn AM. Different 3D domain-swapped oligomeric cyanovirin-N structures suggest trapped folding intermediates. Proceedings of the National Academy of Sciences of the United States of America. 2013;110:7702-7.

[83] Chen KE, Richards AA, Ariffin JK, Ross IL, Sweet MJ, Kellie S, et al. The mammalian DUF59 protein Fam96a forms two distinct types of domain-swapped dimer. Acta

[84] Adinolfi S, Piccoli R, Sica F, Mazzarella L. BS-RNase tetramers: an example of do‐

[85] Spadaccini R, Ercole C, Gentile MA, Sanfelice D, Boelens R, Wechselberger R, et al. NMR studies on structure and dynamics of the monomeric derivative of BS-RNase:

[86] Sica F, Pica A, Merlino A, Russo Krauss I, Ercole C, Picone D. The multiple forms of bovine seminal ribonuclease: Structure and stability of a C-terminal swapped dimer.

[87] Lopez-Alonso JP, Bruix M, Font J, Ribo M, Vilanova M, Jimenez MA, et al. NMR spectroscopy reveals that RNase A is chiefly denatured in 40% acetic acid: implica‐ tions for oligomer formation by 3D domain swapping. Journal of the American

[88] Dobson CM. Protein misfolding, evolution and disease. Trends Biochem Sci.

[89] Gotte G, Donadelli M, Laurents DV, Vottariello F, Morbio M, Libonati M. Increase of RNase a N-terminus polarity or C-terminus apolarity changes the two domains' pro‐ pensity to swap and form the two dimeric conformers of the protein. Biochemistry.

[90] Russo N, Antignani A, D'Alessio G. In vitro evolution of a dimeric variant of human

[91] Canals A, Pous J, Guasch A, Benito A, Ribo M, Vilanova M, et al. The structure of an engineered domain-swapped ribonuclease dimer and its implications for the evolu‐

[92] Merlino A, Avella G, Di Gaetano S, Arciello A, Piccoli R, Mazzarella L, et al. Structur‐ al features for the mechanism of antitumor action of a dimeric human pancreatic ri‐ bonuclease variant. Protein Science: a publication of the Protein Society. 2009;18:50-7.

[93] Schlunegger MP, Bennett MJ, Eisenberg D. Oligomer formation by 3D domain swap‐ ping: a model for protein assembly and misassembly. Adv Protein Chem.

pancreatic ribonuclease. Biochemistry. 2000;39:3585-91.

tion of proteins toward oligomerization. Structure. 2001;9:967-76.

Crystallogr D Biol Crystallogr. 2012;68:637-48.

FEBS Letters. 2013;587:3755-62.

Chemical Society. 2010;132:1621-30.

1999;24:329-32.

2006;45:10795-806.

1997;50:61-122.

main-swapped oligomers. FEBS Letters. 1996;398:326-32.

new insights for 3D domain swapping. PLoS One. 2012;7:e29076.

2012;7:e46804.

C-swapped tetramers and multimers with increasing biological activities. PLoS One. 2012;7:e46804.

[82] Koharudin LM, Liu L, Gronenborn AM. Different 3D domain-swapped oligomeric cyanovirin-N structures suggest trapped folding intermediates. Proceedings of the National Academy of Sciences of the United States of America. 2013;110:7702-7.

[68] Rousseau F, Schymkowitz JW, Wilkinson HR, Itzhaki LS. Three-dimensional domain swapping in p13suc1 occurs in the unfolded state and is controlled by conserved pro‐ line residues. Proceedings of the National Academy of Sciences of the United States

[69] Di Donato A, D'Alessio G. Interchain disulfide bridges in ribonuclease BS-1. Biochem

[70] Berisio R, Sica F, De Lorenzo C, Di Fiore A, Piccoli R, Zagari A, et al. Crystal struc‐ ture of the dimeric unswapped form of bovine seminal ribonuclease. FEBS Letters.

[71] Liu Y, Hart PJ, Schlunegger MP, Eisenberg D. The crystal structure of a 3D domainswapped dimer of RNase A at a 2.1-A resolution. Proceedings of the National Acade‐

[72] Liu Y, Gotte G, Libonati M, Eisenberg D. A domain-swapped RNase A dimer with implications for amyloid formation. Nature Structural Biology. 2001;8:211-4.

[73] Gotte G, Bertoldi M, Libonati M. Structural versatility of bovine ribonuclease A. Dis‐ tinct conformers of trimeric and tetrameric aggregates of the enzyme. Eur J Biochem.

[74] Lopez-Alonso JP, Gotte G, Laurents DV. Kinetic analysis provides insight into the mechanism of ribonuclease A oligomer formation. Archives of Biochemistry and Bio‐

[75] Liu Y, Gotte G, Libonati M, Eisenberg D. Structures of the two 3D domain-swapped RNase A trimers. Protein Science: a publication of the Protein Society. 2002;11:371-80.

[76] Liu Y, Eisenberg D. 3D domain swapping: as domains continue to swap. Protein Sci‐

[77] Park C, Raines RT. Dimer formation by a "monomeric" protein. Protein Science: a

[78] Gotte G, Vottariello F, Libonati M. Thermal aggregation of ribonuclease A. A contri‐ bution to the understanding of the role of 3D domain swapping in protein aggrega‐

[79] Geiger R, Gautschi M, Thor F, Hayer A, Helenius A. Folding, quality control, and se‐ cretion of pancreatic ribonuclease in live cells. The Journal of Biological Chemistry.

[80] Bennett MJ, Sawaya MR, Eisenberg D. Deposition diseases and 3D domain swap‐

[81] Gotte G, Mahmoud Helmy A, Ercole C, Spadaccini R, Laurents DV, Donadelli M, et al. Double domain swapping in bovine seminal RNase: formation of distinct N- and

ence: a publication of the Protein Society. 2002;11:1285-99.

tion. The Journal of Biological Chemistry. 2003;278:10763-9.

publication of the Protein Society. 2000;9:2026-33.

my of Sciences of the United States of America. 1998;95:3437-42.

of America. 2001;98:5596-601.

270 Oligomerization of Chemical and Biological Compounds

2003;554:105-10.

1999;265:680-7.

physics. 2009;489:41-7.

2011;286:5813-22.

ping. Structure. 2006;14:811-24.

Biophys Res Commun. 1973;55:919-28.

[94] Nagarkar RP, Hule RA, Pochan DJ, Schneider JP. Domain swapping in materials de‐ sign. Biopolymers. 2010;94:141-55.

[107] Janowski R, Kozak M, Jankowska E, Grzonka Z, Grubb A, Abrahamson M, et al. Hu‐ man cystatin C, an amyloidogenic protein, dimerizes through three-dimensional do‐

Protein Oligomerization http://dx.doi.org/10.5772/57489 273

[108] Luhrs T, Zahn R, Wuthrich K. Amyloid formation by recombinant full-length prion proteins in phospholipid bicelle solutions. Journal of Molecular Biology.

[109] Hafner-Bratkovic I, Bester R, Pristovsek P, Gaedtke L, Veranic P, Gaspersic J, et al. Globular domain of the prion protein needs to be unlocked by domain swapping to support prion protein conversion. The Journal of Biological Chemistry.

[110] Lee S, Eisenberg D. Seeded conversion of recombinant prion protein to a disulfidebonded oligomer by a reduction-oxidation process. Nature Structural Biology.

[111] Wahlbom M, Wang X, Lindstrom V, Carlemalm E, Jaskolski M, Grubb A. Fibrillogen‐ ic oligomers of human cystatin C are formed by propagated domain swapping. The

[112] Liu C, Sawaya MR, Eisenberg D. beta(2)-microglobulin forms three-dimensional do‐ main-swapped amyloid fibrils with disulfide linkages. Nature Structural & Molecu‐

[113] Staniforth RA, Giannini S, Higgins LD, Conroy MJ, Hounslow AM, Jerala R, et al. Three-dimensional domain swapping in the folded and molten-globule states of cys‐ tatins, an amyloid-forming structural superfamily. The EMBO Journal.

[114] Sanders A, Jeremy Craven C, Higgins LD, Giannini S, Conroy MJ, Hounslow AM, et al. Cystatin forms a tetramer through structural rearrangement of domain-swapped dimers prior to amyloidogenesis. Journal of Molecular Biology. 2004;336:165-78. [115] Nilsson M, Wang X, Rodziewicz-Motowidlo S, Janowski R, Lindstrom V, Onnerfjord P, et al. Prevention of domain swapping inhibits dimerization and amyloid fibril for‐ mation of cystatin C: use of engineered disulfide bridges, antibodies, and carboxyme‐ thylpapain to stabilize the monomeric form of cystatin C. The Journal of Biological

[116] Orlikowska M, Jankowska E, Kolodziejczyk R, Jaskolski M, Szymanska A. Hingeloop mutation can be used to control 3D domain swapping and amyloidogenesis of

[117] Olafsson I, Grubb A. Hereditary cystatin C amyloid angiopathy. Amyloid.

human cystatin C. Journal of Structural Biology. 2011;173:406-13.

main swapping. Nature Structural Biology. 2001;8:316-20.

Journal of Biological Chemistry. 2007;282:18318-26.

2006;357:833-41.

2011;286:12149-56.

2003;10:725-30.

2001;20:4774-81.

2000;7:70-9.

lar Biology. 2011;18:49-55.

Chemistry. 2004;279:24236-45.

[107] Janowski R, Kozak M, Jankowska E, Grzonka Z, Grubb A, Abrahamson M, et al. Hu‐ man cystatin C, an amyloidogenic protein, dimerizes through three-dimensional do‐ main swapping. Nature Structural Biology. 2001;8:316-20.

[94] Nagarkar RP, Hule RA, Pochan DJ, Schneider JP. Domain swapping in materials de‐

[95] Barrientos LG, Gronenborn AM. The highly specific carbohydrate-binding protein cyanovirin-N: structure, anti-HIV/Ebola activity and possibilities for therapy. Mini

[96] Barrientos LG, Louis JM, Botos I, Mori T, Han Z, O'Keefe BR, et al. The domainswapped dimer of cyanovirin-N is in a metastable folded state: reconciliation of X-

[97] Margoliash E, Lustgarten J. Interconversion of horse heart cytochrome C monomer

[98] Hirota S, Hattori Y, Nagao S, Taketa M, Komori H, Kamikubo H, et al. Cytochrome c polymerization by successive domain swapping at the C-terminal helix. Proceedings of the National Academy of Sciences of the United States of America.

[99] O'Neill JW, Manion MK, Maguire B, Hockenbery DM. BCL-XL dimerization by three-dimensional domain swapping. Journal of Molecular Biology. 2006;356:367-81.

[100] Denisov AY, Sprules T, Fraser J, Kozlov G, Gehring K. Heat-induced dimerization of

[101] Vendome J, Posy S, Jin X, Bahna F, Ahlsen G, Shapiro L, et al. Molecular design prin‐ ciples underlying beta-strand swapping in the adhesive dimerization of cadherins.

[102] Hadjithomas M, Moudrianakis EN. Experimental evidence for the role of domain swapping in the evolution of the histone fold. Proceedings of the National Academy

[103] Zegers I, Deswarte J, Wyns L. Trimeric domain-swapped barnase. Proceedings of the National Academy of Sciences of the United States of America. 1999;96:818-22. [104] Schiering N, Casale E, Caccia P, Giordano P, Battistini C. Dimer formation through domain swapping in the crystal structure of the Grb2-SH2-Ac-pYVNV complex. Bio‐

[105] Perutz MF, Johnson T, Suzuki M, Finch JT. Glutamine repeats as polar zippers: their possible role in inherited neurodegenerative diseases. Proceedings of the National

[106] Knaus KJ, Morillas M, Swietnicki W, Malone M, Surewicz WK, Yee VC. Crystal structure of the human prion protein reveals a mechanism for oligomerization. Na‐

Academy of Sciences of the United States of America. 1994;91:5355-8.

BCL-xL through alpha-helix swapping. Biochemistry. 2007;46:734-40.

Nature Structural & Molecular Biology. 2011;18:693-700.

of Sciences of the United States of America. 2011;108:13462-7.

and polymers. The Journal of Biological Chemistry. 1962;237:3397-405.

sign. Biopolymers. 2010;94:141-55.

ray and NMR structures. Structure. 2002;10:673-86.

Rev Med Chem. 2005;5:21-31.

272 Oligomerization of Chemical and Biological Compounds

2010;107:12854-9.

chemistry. 2000;39:13376-82.

ture Structural Biology. 2001;8:770-4.

[118] Eakin CM, Attenello FJ, Morgan CJ, Miranker AD. Oligomeric assembly of nativelike precursors precedes amyloid formation by beta-2 microglobulin. Biochemistry. 2004;43:7808-15.

[130] Sambashivan S, Liu Y, Sawaya MR, Gingery M, Eisenberg D. Amyloid-like fibrils of ribonuclease A with three-dimensional domain-swapped and native-like structure.

Protein Oligomerization http://dx.doi.org/10.5772/57489 275

[131] Mizuno H, Fujimoto Z, Koizumi M, Kano H, Atoda H, Morita T. Structure of coagu‐ lation factors IX/X-binding protein, a heterodimer of C-type lectin domains. Nature

[132] Vatzaki EH, Allen SC, Leonidas DD, Trautwein-Fritz K, Stackhouse J, Benner SA, et al. Crystal structure of a hybrid between ribonuclease A and bovine seminal ribonu‐

[133] Sica F, Di Fiore A, Merlino A, Mazzarella L. Structure and stability of the non-cova‐ lent swapped dimer of bovine seminal ribonuclease: an enzyme tailored to evade ri‐ bonuclease protein inhibitor. The Journal of Biological Chemistry. 2004;279:36753-60.

[134] Ercole C, Colamarino RA, Pizzo E, Fogolari F, Spadaccini R, Picone D. Comparison of the structural and functional properties of RNase A and BS-RNase: a stepwise muta‐

[135] Merlino A, Picone D, Ercole C, Balsamo A, Sica F. Chain termini cross-talk in the swapping process of bovine pancreatic ribonuclease. Biochimie. 2012;94:1108-18.

[136] Meersman F, Heremans K. Temperature-induced dissociation of protein aggregates:

[137] Babu CR, Hilser VJ, Wand AJ. Direct access to the cooperative substructure of pro‐ teins and the protein ensemble via cold denaturation. Nature Structural & Molecular

[138] Jaremko M, Jaremko L, Kim HY, Cho MK, Schwieters CD, Giller K, et al. Cold dena‐ turation of a protein dimer monitored at atomic resolution. Nature Chemical Biology.

[139] Merlino A, Vitagliano L, Ceruso MA, Mazzarella L. Dynamic properties of the N-ter‐

[140] Merlino A, Ceruso MA, Vitagliano L, Mazzarella L. Open interface and large quater‐ nary structure movements in 3D domain swapped proteins: insights from molecular dynamics simulations of the C-terminal swapped dimer of ribonuclease A. Biophys J.

[141] Selwood T, Jaffe EK. Dynamic dissociating homo-oligomers and the control of pro‐ tein function. Archives of Biochemistry and Biophysics. 2012;519:131-43.

[142] Zhou HX, Rivas G, Minton AP. Macromolecular crowding and confinement: bio‐ chemical, biophysical, and potential physiological consequences. Annual Review of

minal swapped dimer of ribonuclease A. Biophys J. 2004;86:2383-91.

accessing the denatured state. Biochemistry. 2003;42:14234-41.

clease--the basic surface, at 2.0 A resolution. Eur J Biochem. 1999;260:176-82.

Nature. 2005;437:266-9.

Biology. 2004;11:352-7.

2013;9:264-70.

2005;88:2003-12.

Biophysics. 2008;37:375-97.

Structural Biology. 1997;4:438-41.

genesis approach. Biopolymers. 2009;91:1009-17.

[130] Sambashivan S, Liu Y, Sawaya MR, Gingery M, Eisenberg D. Amyloid-like fibrils of ribonuclease A with three-dimensional domain-swapped and native-like structure. Nature. 2005;437:266-9.

[118] Eakin CM, Attenello FJ, Morgan CJ, Miranker AD. Oligomeric assembly of nativelike precursors precedes amyloid formation by beta-2 microglobulin. Biochemistry.

[119] Byeon IJ, Louis JM, Gronenborn AM. A protein contortionist: core mutations of GB1 that induce dimerization and domain swapping. Journal of Molecular Biology.

[120] Kirsten Frank M, Dyda F, Dobrodumov A, Gronenborn AM. Core mutations switch monomeric protein GB1 into an intertwined tetramer. Nature Structural Biology.

[121] Li J, Hoop CL, Kodali R, Sivanandam VN, van der Wel PC. Amyloid-like fibrils from a domain-swapping protein feature a parallel, in-register conformation without na‐

tive-like interactions. The Journal of Biological Chemistry. 2011;286:28988-95.

[122] Guo Z, Eisenberg D. Runaway domain swapping in amyloid-like fibrils of T7 endo‐ nuclease I. Proceedings of the National Academy of Sciences of the United States of

[123] Bader R, Seeliger MA, Kelly SE, Ilag LL, Meersman F, Limones A, et al. Folding and fibril formation of the cell cycle protein Cks1. The Journal of Biological Chemistry.

[124] Laidman J, Forse GJ, Yeates TO. Conformational change and assembly through edge beta strands in transthyretin and other amyloid proteins. Accounts of Chemical Re‐

[125] Serag AA, Altenbach C, Gingery M, Hubbell WL, Yeates TO. Arrangement of subu‐ nits and ordering of beta-strands in an amyloid sheet. Nature Structural Biology.

[126] Sawaya MR, Sambashivan S, Nelson R, Ivanova MI, Sievers SA, Apostol MI, et al. Atomic structures of amyloid cross-beta spines reveal varied steric zippers. Nature.

[127] Goldschmidt L, Teng PK, Riek R, Eisenberg D. Identifying the amylome, proteins ca‐ pable of forming amyloid-like fibrils. Proceedings of the National Academy of Scien‐

[128] Teng PK, Anderson NJ, Goldschmidt L, Sawaya MR, Sambashivan S, Eisenberg D. Ribonuclease A suggests how proteins self-chaperone against amyloid fiber forma‐

[129] Torrent M, Odorizzi F, Nogues MV, Boix E. Eosinophil cationic protein aggregation: identification of an N-terminus amyloid prone region. Biomacromolecules.

tion. Protein Science: a publication of the Protein Society. 2012;21:26-37.

ces of the United States of America. 2010;107:3487-92.

2004;43:7808-15.

274 Oligomerization of Chemical and Biological Compounds

2003;333:141-52.

2002;9:877-85.

America. 2006;103:8042-7.

2006;281:18816-24.

search. 2006;39:576-83.

2002;9:734-9.

2007;447:453-7.

2010;11:1983-90.

[143] Ercole C, Lopez-Alonso JP, Font J, Ribo M, Vilanova M, Picone D, et al. Crowding agents and osmolytes provide insight into the formation and dissociation of RNase A oligomers. Archives of Biochemistry and Biophysics. 2011;506:123-9.

[156] Cafaro V, Bracale A, Di Maro A, Sorrentino S, D'Alessio G, Di Donato A. New mu‐ teins of RNase A with enhanced antitumor action. FEBS Letters. 1998;437:149-52. [157] Kobe B, Deisenhofer J. Mechanism of ribonuclease inhibition by ribonuclease inhibi‐ tor protein based on the crystal structure of its complex with ribonuclease A. Journal

Protein Oligomerization http://dx.doi.org/10.5772/57489 277

[158] Kim JS, Soucek J, Matousek J, Raines RT. Mechanism of ribonuclease cytotoxicity.

[159] Rousseau F, Wilkinson H, Villanueva J, Serrano L, Schymkowitz JW, Itzhaki LS. Do‐ main swapping in p13suc1 results in formation of native-like, cytotoxic aggregates.

[160] Schymkowitz JW, Rousseau F, Irvine LR, Itzhaki LS. The folding pathway of the cellcycle regulatory protein p13suc1: clues for the mechanism of domain swapping.

[161] Thylen C, Wahlqvist J, Haettner E, Sandgren O, Holmgren G, Lundgren E. Modifica‐ tions of transthyretin in amyloid fibrils: analysis of amyloid from homozygous and heterozygous individuals with the Met30 mutation. The EMBO Journal.

[162] McCutchen SL, Colon W, Kelly JW. Transthyretin mutation Leu-55-Pro significantly alters tetramer stability and increases amyloidogenicity. Biochemistry.

[163] Apostol MI, Sawaya MR, Cascio D, Eisenberg D. Crystallographic studies of prion protein (PrP) segments suggest how structural changes encoded by polymorphism at residue 129 modulate susceptibility to human prion disease. The Journal of Biological

[164] Levy CB, Stumbo AC, Ano Bom AP, Portari EA, Cordeiro Y, Silva JL, et al. Co-locali‐ zation of mutant p53 and amyloid-like protein aggregates in breast tumors. The In‐

[165] Ano Bom AP, Rangel LP, Costa DC, de Oliveira GA, Sanches D, Braga CA, et al. Mu‐ tant p53 aggregates into prion-like amyloid oligomers and fibrils: implications for

[166] Apostol MI, Wiltzius JJ, Sawaya MR, Cascio D, Eisenberg D. Atomic structures sug‐ gest determinants of transmission barriers in mammalian prion disease. Biochemis‐

[167] Silva JL, Rangel LP, Costa DC, Cordeiro Y, De Moura Gallo CV. Expanding the prion concept to cancer biology: dominant-negative effect of aggregates of mutant p53 tu‐

ternational Journal of Biochemistry & Cell Biology. 2011;43:60-4.

cancer. The Journal of Biological Chemistry. 2012;287:28152-62.

mour suppressor. Bioscience Reports. 2013;33.

of Molecular Biology. 1996;264:1028-43.

Structure. 2000;8:89-100.

1993;12:743-8.

1993;32:12119-27.

try. 2011;50:2456-63.

Chemistry. 2010;285:29671-5.

The Journal of Biological Chemistry. 1995;270:31097-102.

Journal of Molecular Biology. 2006;363:496-505.

[156] Cafaro V, Bracale A, Di Maro A, Sorrentino S, D'Alessio G, Di Donato A. New mu‐ teins of RNase A with enhanced antitumor action. FEBS Letters. 1998;437:149-52.

[143] Ercole C, Lopez-Alonso JP, Font J, Ribo M, Vilanova M, Picone D, et al. Crowding agents and osmolytes provide insight into the formation and dissociation of RNase A

[144] Nenci A, Gotte G, Maras B, Libonati M. Different susceptibility of the two dimers of ribonuclease A to subtilisin. Implications for their structure. Biochimica et Biophysica

[145] Abrami L, Fivaz M, van der Goot FG. Adventures of a pore-forming toxin at the tar‐

[146] Milne JC, Furlong D, Hanna PC, Wall JS, Collier RJ. Anthrax protective antigen forms oligomers during intoxication of mammalian cells. The Journal of Biological Chemis‐

[147] Billington SJ, Jost BH, Songer JG. Thiol-activated cytolysins: structure, function and

[148] Bucci E, Vitagliano L, Barone R, Sorrentino S, D'Alessio G, Graziano G. On the ther‐ mal stability of the two dimeric forms of ribonuclease A. Biophysical Chemistry.

[149] Vottariello F, Giacomelli E, Frasson R, Pozzi N, De Filippis V, Gotte G. RNase A oli‐ gomerization through 3D domain swapping is favoured by a residue located far

[150] Rafelski SM, Theriot JA. Crawling toward a unified model of cell mobility: spatial and temporal regulation of actin dynamics. Annual Review of Biochemistry.

[151] Dell'Orco D. A physiological role for the supramolecular organization of rhodopsin

[152] Gotte G, Testolin L, Costanzo C, Sorrentino S, Armato U, Libonati M. Cross-linked trimers of bovine ribonuclease A: activity on double-stranded RNA and antitumor

[153] Bartholeyns J, Baudhuin P. Inhibition of tumor cell proliferation by dimerized ribo‐ nuclease. Proceedings of the National Academy of Sciences of the United States of

[154] Matousek J, Gotte G, Pouckova P, Soucek J, Slavik T, Vottariello F, et al. Antitumor activity and other biological actions of oligomers of ribonuclease A. The Journal of

[155] Merlino A, Russo Krauss I, Perillo M, Mattia CA, Ercole C, Picone D, et al. Toward an antitumor form of bovine pancreatic ribonuclease: the crystal structure of three non‐

and transducin in rod photoreceptors. FEBS letters. 2013;587:2060-6.

role in pathogenesis. FEMS, Microbiology Letters. 2000;182:197-205.

oligomers. Archives of Biochemistry and Biophysics. 2011;506:123-9.

get cell surface. Trends in Microbiology. 2000;8:168-72.

from the swapping domains. Biochimie. 2011;93:1846-57.

Acta. 2001;1545:255-62.

276 Oligomerization of Chemical and Biological Compounds

try. 1994;269:20607-12.

2005;116:89-95.

2004;73:209-39.

action. FEBS Letters. 1997;415:308-12.

Biological Chemistry. 2003;278:23817-22.

covalent dimeric mutants. Biopolymers. 2009;91:1029-37.

America. 1976;73:573-6.

[168] Bucciantini M, Giannoni E, Chiti F, Baroni F, Formigli L, Zurdo J, et al. Inherent tox‐ icity of aggregates implies a common mechanism for protein misfolding diseases. Nature. 2002;416:507-11.

**Chapter 9**

**Oligomerization of Proteins and Neurodegenerative**

Oligomerization of amino acids by binding their peptide bonds (-CONH-) forms proteins (or peptides), which are the major components of our bodies. Although the primary sequence (the linear sequence of amino acids) of the protein mainly determines its characteristics, its secondary structures (the conformations) are also critical determinants of their shapes and functions. The conformation (random coil, α-helix, and ß-sheet) is restricted by the circum‐ stances nearby proteins. The hydrogen bond between the amino acids in the peptide chain forms the α-helix structure. Meanwhile, the ß-sheets (ß-plated sheets) consist of ß-strands

Recent neurochemical evidence indicates that the oligomerization of proteins and the forma‐ tion of ß-sheet structures are linked with several neurodegenerative diseases such as Alz‐ heimer's disease (AD), prion diseases, triplet repeat diseases, dementia with Lewy bodies (DLB). The disease-related proteins, such as ß-amyloid protein (AßP) in AD, prion protein in prion diseases, polyglutamine in triplet repeat disease, α-synuclein in DLB, are identical in each disease (Table 1). However, all of these amyloidogenic proteins share common charac‐ teristics in the formation of amyloid with ß-sheet structures, and in the exhibition of cytotox‐ icity. Therefore, a new concept termed "conformational disease" was proposed, suggesting that protein conformation is an important determinant of its toxicity, and consequently, the

These conformational diseases are included in amyloidosis. At 1853, Virchow found the abnormal accumulates in tissues and named "amyloid", since they exhibited similar charac‐ teristics with *amylum*. At 1968, amyloid was determined to be the oligomers of proteins with ß-sheet structures. The accumulation of amyloid causes various diseases (amyloidosis) including familial amyloid polyneuropathy (FAP), amyloid-light chain amyloidosis, dialysis

> © 2014 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**Diseases**

Dai Mizuno and Masahiro Kawahara

http://dx.doi.org/10.5772/57482

development of the related disease [1].

**1. Introduction**

Additional information is available at the end of the chapter

which are laterally connected peptide bonds with hydrogen bonds.

## **Oligomerization of Proteins and Neurodegenerative Diseases**

[168] Bucciantini M, Giannoni E, Chiti F, Baroni F, Formigli L, Zurdo J, et al. Inherent tox‐ icity of aggregates implies a common mechanism for protein misfolding diseases.

[169] Dykes G, Crepeau RH, Edelstein SJ. Three-dimensional reconstruction of the fibres of

[170] Holmes KC, Popp D, Gebhard W, Kabsch W. Atomic model of the actin filament. Na‐

[171] Downing KH, Nogales E. Crystallographic structure of tubulin: implications for dy‐

[172] Chiti F, Dobson CM. Amyloid formation by globular proteins under native condi‐

[173] Rajagopal K, Schneider JP. Self-assembling peptides and proteins for nanotechnologi‐

[174] Bonzani IC, George JH, Stevens MM. Novel materials for bone and cartilage regener‐

[175] Terzaki K, Kalloudi E, Mossou E, Mitchell EP, Forsyth VT, Rosseeva E, et al. Mineral‐ ized self-assembled peptides on 3D laser-made scaffolds: a new route toward 'scaf‐

[176] Nagarkar RP, Hule RA, Pochan DJ, Schneider JP. De novo design of strand-swapped beta-hairpin hydrogels. Journal of the American Chemical Society. 2008;130:4466-74.

[177] Ogihara NL, Ghirlanda G, Bryson JW, Gingery M, DeGrado WF, Eisenberg D. Design of three-dimensional domain-swapped dimers and fibrous oligomers. Proceedings of the National Academy of Sciences of the United States of America. 2001;98:1404-9.

[178] van Raaij MJ, Mitraki A, Lavigne G, Cusack S. A triple beta-spiral in the adenovirus fibre shaft reveals a new structural motif for a fibrous protein. Nature.

namics and drug binding. Cell Structure and Function. 1999;24:269-75.

cal applications. Current Opinion in Structural Biology. 2004;14:480-6.

fold on scaffold' hard tissue engineering. Biofabrication. 2013;5:045002.

ation. Current Opinion in Chemical Biology. 2006;10:568-75.

Nature. 2002;416:507-11.

278 Oligomerization of Chemical and Biological Compounds

ture. 1990;347:44-9.

1999;401:935-8.

sickle cell haemoglobin. Nature. 1978;272:506-10.

tions. Nature Chemical Biology. 2009;5:15-22.

Dai Mizuno and Masahiro Kawahara

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/57482

#### **1. Introduction**

Oligomerization of amino acids by binding their peptide bonds (-CONH-) forms proteins (or peptides), which are the major components of our bodies. Although the primary sequence (the linear sequence of amino acids) of the protein mainly determines its characteristics, its secondary structures (the conformations) are also critical determinants of their shapes and functions. The conformation (random coil, α-helix, and ß-sheet) is restricted by the circum‐ stances nearby proteins. The hydrogen bond between the amino acids in the peptide chain forms the α-helix structure. Meanwhile, the ß-sheets (ß-plated sheets) consist of ß-strands which are laterally connected peptide bonds with hydrogen bonds.

Recent neurochemical evidence indicates that the oligomerization of proteins and the forma‐ tion of ß-sheet structures are linked with several neurodegenerative diseases such as Alz‐ heimer's disease (AD), prion diseases, triplet repeat diseases, dementia with Lewy bodies (DLB). The disease-related proteins, such as ß-amyloid protein (AßP) in AD, prion protein in prion diseases, polyglutamine in triplet repeat disease, α-synuclein in DLB, are identical in each disease (Table 1). However, all of these amyloidogenic proteins share common charac‐ teristics in the formation of amyloid with ß-sheet structures, and in the exhibition of cytotox‐ icity. Therefore, a new concept termed "conformational disease" was proposed, suggesting that protein conformation is an important determinant of its toxicity, and consequently, the development of the related disease [1].

These conformational diseases are included in amyloidosis. At 1853, Virchow found the abnormal accumulates in tissues and named "amyloid", since they exhibited similar charac‐ teristics with *amylum*. At 1968, amyloid was determined to be the oligomers of proteins with ß-sheet structures. The accumulation of amyloid causes various diseases (amyloidosis) including familial amyloid polyneuropathy (FAP), amyloid-light chain amyloidosis, dialysis

© 2014 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

or histidine (His) or phosphorylated amino acids, and cause cross-linking of the proteins (Fig. 1). Furthermore, all of these amyloidogenic proteins were reported to have the ability to bind metals as shown in Table 1. Our and other numerous studies reported that oligomers cause neurodegeneration by induction of Ca2+ dyshomeostasis through the formation of amyloid channels on neuronal membranes [3,4]. The beneficial characteristics of carnosine (ß alanyl histidine) as a drug for the treatment for these neurodegenerative diseases are also discussed.

Amyloidogenic protein Oligomers with ß-sheet structures

Alzheimer's disease (AD) is a severe type of senile dementia, affecting a large portion of elderly people worldwide. It is characterized by profound memory loss and inability to form new memories. The pathological hallmarks of AD are the presence of numerous extracellular deposits (senile plaques) and intraneuronal neurofibrillary tangles (NFTs). The degeneration of synapses and neurons in the hippocampus or cerebral cortex is also observed. The major components of NFTs are phosphorylated tau proteins, and that of senile plaques are ß-amyloid proteins (AßPs) [5]. Although the precise cause of AD remains elusive, numerous biochemical, cell biological, and genetic studies have supported the idea termed "amyloid cascade hypoth‐ esis" that the AßP accumulation and the consequent neurodegeneration play a central role in AD [6]. Moreover, recent studies on the identified AßP species have indicated that the oligomerization of AßP and the conformational changes are critical in the neurodegeneration

AßP is a small peptide of 39–43 amino acid long. It is derived from the proteolytic cleavage of a large precursor protein (amyloid precursor protein; APP). AßP is secreted by the cleavage of its N-terminal by ß-secretase (BACE), followed by the intra-membrane cleavage of its Cterminal by γ-secretase. Genetic studies of early-onset cases of familial AD indicated that APP mutations and AßP metabolism are associated with AD. It was also revealed that mutations

M

M

M

Oligomerization of Proteins and Neurodegenerative Diseases

http://dx.doi.org/10.5772/57482

281

M

M

Phosphorylated amino acid

M

**Figure 1.** Trace elements acts cross-linkers of amyloidogenic proteins.

**2. Alzheimer's disease and oligomerization of AßP**

His Tyr Arg

**2.1. Amyloid cascade hypothesis**

M stands for metal.

process [7].

\* The sequence of fragment peptide of each amyloidogenic protein (PrP106-126, NAC, polyglutamine) is indicated in italic form.

**Table 1.** Characteristics of amyloidogenic proteins and the related peptides

amyloidosis, *etc* [2]. All of theses diseases share common properties about the deposition of amyloids in various tissues or organs with protease-resistant, insoluble fibril-like structures (amyloid fibrils), and stained by congo-red, ß-sheet specific dye. However, the component of amyloid is different in each disease. For example, the major component of amyloid in FAP patients is transthyretin, and ß2-microglobulin deposits in patients with dialysis amyloidosis. There are no effective treatments for amyloidosis.

In this chapter, we review the implication of protein oligomerization in the pathogenesis of these neurodegenerative diseases. Considering that the amyloidogenic proteins are commonly present in our brain, factors which influence oligomerization play crucial roles in their pathogenesis. As such factors, we focus on trace elements such as Al, Zn, Cu, and Fe. Metals have a property of firmly binding to metal-binding residues of proteins, such as tyrosine (Tyr) or histidine (His) or phosphorylated amino acids, and cause cross-linking of the proteins (Fig. 1). Furthermore, all of these amyloidogenic proteins were reported to have the ability to bind metals as shown in Table 1. Our and other numerous studies reported that oligomers cause neurodegeneration by induction of Ca2+ dyshomeostasis through the formation of amyloid channels on neuronal membranes [3,4]. The beneficial characteristics of carnosine (ß alanyl histidine) as a drug for the treatment for these neurodegenerative diseases are also discussed.

**Figure 1.** Trace elements acts cross-linkers of amyloidogenic proteins.

### **2. Alzheimer's disease and oligomerization of AßP**

#### **2.1. Amyloid cascade hypothesis**

amyloidosis, *etc* [2]. All of theses diseases share common properties about the deposition of amyloids in various tissues or organs with protease-resistant, insoluble fibril-like structures (amyloid fibrils), and stained by congo-red, ß-sheet specific dye. However, the component of amyloid is different in each disease. For example, the major component of amyloid in FAP patients is transthyretin, and ß2-microglobulin deposits in patients with dialysis amyloidosis.

\* The sequence of fragment peptide of each amyloidogenic protein (PrP106-126, NAC, polyglutamine) is indicated in

In this chapter, we review the implication of protein oligomerization in the pathogenesis of these neurodegenerative diseases. Considering that the amyloidogenic proteins are commonly present in our brain, factors which influence oligomerization play crucial roles in their pathogenesis. As such factors, we focus on trace elements such as Al, Zn, Cu, and Fe. Metals have a property of firmly binding to metal-binding residues of proteins, such as tyrosine (Tyr)

There are no effective treatments for amyloidosis.

**Disease The primary sequence of amyloidogenic protein or its fragment peptide**

> *AßP(1-42) and AßP(1-42)truncated C-terminal DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGG*

> MANLGCWMLVLFVATWSDLGLCKKRPKPGGWNTGG SRYPGQGSPGGNRYPPQGGGGWGQPHGGGWGQPH GGGWGQPHGGGWGQPHGGGWGQGGGTHSQWN KPSKP*KTNMKHMAGAAAAGAVVGG*LGGYVLGSAMS RPIIHFGSDYEDRYYRENMHRYPNQVYYRPMDEYSNQ NNFVHDCVNITIKQHTVTTTTKGENFTETDVKMMERVV EQMCITQYERESQAYYQRGSSMVLFSSPPVILLISFLIFL

> α-synuclein; *NAC* ( a fragment of α-synuclein ) MDVFMKGLSKAKEGVVAAAEKTKQGVAEAAGKTKEG VLYVGSKTKEGVVHGVTTVAEKTKEQVSNVGGAVVTG VTAVAHKTVEGAGNFAAATGLVKKDQKNESGFGPEG TMENSENMPVNPNNETYEMPPEEEYQDYDPEA

> MATLEKLMKAFESLKSF*QQQQQQQQQQQQQQQQQ QQQQQQ*PPPPPPPPPPPQLPQPPPQAQPLLPQPQPPPP

KCNTATCATQRLANFLVHSSNNFGAILSSTNVGSNTY

**Table 1.** Characteristics of amyloidogenic proteins and the related peptides

Alzheimer's disease

Dementia with Lewy bodies (DLB)

Triplet- repeat disease

italic form.

*VVIA*

IVG

Polyglutamine

PPPPPPGP

Diabetes mellitusHuman amylin

Prion disease Prion protein: *PrP106-126*

280 Oligomerization of Chemical and Biological Compounds

**Metal ß-sheet**

Al, Zn, Cu, Fe

> Zn, Cu, M, Fe

Cu, Fe, Al

**formation**

**Cytotoxicity**

+ + +

+ + +

+ + +

Fe + + +

Cu, Al + + +

**Poreformation**

> Alzheimer's disease (AD) is a severe type of senile dementia, affecting a large portion of elderly people worldwide. It is characterized by profound memory loss and inability to form new memories. The pathological hallmarks of AD are the presence of numerous extracellular deposits (senile plaques) and intraneuronal neurofibrillary tangles (NFTs). The degeneration of synapses and neurons in the hippocampus or cerebral cortex is also observed. The major components of NFTs are phosphorylated tau proteins, and that of senile plaques are ß-amyloid proteins (AßPs) [5]. Although the precise cause of AD remains elusive, numerous biochemical, cell biological, and genetic studies have supported the idea termed "amyloid cascade hypoth‐ esis" that the AßP accumulation and the consequent neurodegeneration play a central role in AD [6]. Moreover, recent studies on the identified AßP species have indicated that the oligomerization of AßP and the conformational changes are critical in the neurodegeneration process [7].

> AßP is a small peptide of 39–43 amino acid long. It is derived from the proteolytic cleavage of a large precursor protein (amyloid precursor protein; APP). AßP is secreted by the cleavage of its N-terminal by ß-secretase (BACE), followed by the intra-membrane cleavage of its Cterminal by γ-secretase. Genetic studies of early-onset cases of familial AD indicated that APP mutations and AßP metabolism are associated with AD. It was also revealed that mutations

in the presenilin genes account for the majority of cases of early-onset familial AD. Presenilins have been revealed to be one of γ -secretases, and their mutations also influence the production of AßP and its neurotoxicity.

AßP is secreted from its precursor protein, APP by transmembrane cleavage. Sequences of primate AßP(1-42) and ro‐ dent AßP(1-42) are shown. The comparison between the sequence of primate (human or monkey) Aß(1-42) and ro‐ dent (rat or mouse) Aß(1-42) is depicted.

bral administration of the conditioned medium with cultured cells transfected with the human APP gene inhibited long-term potentiation (LTP), which is a form of synaptic information storage well known as a paradigm of memory mechanisms. They also demonstrated that LTP was blocked by SDS-stable low-molecular-weight oligomers (dimers, trimers, or tetramers) but not AßP monomers or larger aggregates. The natural AßP oligomers (derived from the cerebrospinal fluid of AD patients) cause the loss of dendritic spines, synapses, and LTP blockage. Klein and the colleagues reported that AßP-derived diffusible ligands (ADDLs) obtained from sedimentation by clustering are highly toxic to cultured neurons. They also reported that ADDLs inhibited LTP and exhibited adverse effects on synaptic plasticity such as abnormal spine morphology, decreased spine density, and decreased synaptic proteins. Based on these and other numerous findings, it is widely accepted that AßP oligomers are synaptotoxic and neurotoxic, but not monomer or fibrils. These studies further strengthened and modified the amyloid cascade hypothesis, which suggest that AßP oligomers are neuro‐

AßP monomers exhibit random or a-helix structures. However, under aging conditions or in the presence of some ac‐ celeratory factors, Aß self-aggregates and forms several types of oligomers (SDS-soluble oligomers, ADDLS, globulom‐ ers, or protofibrils *etc.*) and finally forms insoluble aggregates termed amyloid fibrils. Oligomeric soluble Aßs are toxic,

*monomer Soluble oligomer Amyloid fibril*

• ß-sheet • soluble • toxic

Toxicity

*protofibril ADDLs*

*globulomer etc.*

Oligomerization of Proteins and Neurodegenerative Diseases

• ß-sheet • insoluble • less or non-toxic

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283

Considering that AßP is secreted from APP into the brain of young people or of normal subjects, factors which influence (accelerate or delay) the oligomerization may become important determinants of the pathogenesis of AD. Various factors, such as the concentration of peptides, the oxidations, mutations, and racemization of AßP, pH, composition of solvents, temperature, and trace elements, can influence the oligomerization processes. A considerable amount of asparagines (Asp) or serine (Ser) residues of AßP accumulated in senile plaques are

toxic and crucial for the pathogeneis of AD [8,9].

• aging (incubation) • metals (Al, Cu, Zn, *etc.*)

Acceleratory factors

• oxidation • Mutation • racemization *etc.*

AßP

• -helix or randam coil • soluble • non-toxic

• rifampicin • curcumine • polyphenol • aspirin

Inhibitory factors

although the monomeric and fibril ones are rather nontoxic.

**Figure 3.** Oligomerization of AßP

• ß-sheet breaker peptide *etc.*

**2.2. Metal-induced oligomerization of AßP**

**Figure 2.** Structure of AßP

Yankner *et al.* reported that the first 40 amino acid residues of AßP (AßP(1–40)) caused the death of cultured rat hippocampal neurons or the neurodegeneration in the brains of experi‐ mental animals. Thereafter, it was agreed upon that the aggregation and the subsequent conformational change of AßP contribute to its neurotoxicity. AßP is a hydrophobic peptide with an intrinsic tendency to self-assemble to form insoluble oligomers with ß-pleated sheet structures. Pike *et al.* revealed that aged AßP(1–40) (aggregated under incubation at 37°C for several days) were considerably more toxic to cultured neurons as compared to freshly prepared AßP(1–40). Simmons *et al*. revealed ß-sheet contents of AßP observed by circular dichroism (CD) spectroscopy correlates with its neurotoxicity.

Furthermore, the longer peptide variant, AßP(1–42), has the characteristics of immediate polymerization compared to AßP(1–40). AßP(1–42) enhances the aggregation of AßP(1–40) and becomes a seed of the amyloid fibrils. AßP (1–42) is more abundant in the brains of AD patients as compared to those of age matched controls. The mutations of APP and those of presenilin genes induce the increased production of AßP (1–42) in the transfected cell lines.

Recent approaches using size-exclusion chromatography, gel electrophoresis, and atomic force microscopy have demonstrated that there are several stable types of soluble oligomers: naturally occurring soluble oligomers (dimers or trimers), ADDLs (AßP-derived diffusible ligands), AßP globulomers, or protofibrils. Hartley separated aggregated AßP(1–40) into low– molecular-weight (mainly monomer), protofibrillar, and fibril fractions by size-exclusion chromatography, and found that the protofibrillar fraction caused marked changes in the electrical activity of cultured neurons and neurotoxicity. Walsh *et al.* found that the intracere‐

AßP monomers exhibit random or a-helix structures. However, under aging conditions or in the presence of some ac‐ celeratory factors, Aß self-aggregates and forms several types of oligomers (SDS-soluble oligomers, ADDLS, globulom‐ ers, or protofibrils *etc.*) and finally forms insoluble aggregates termed amyloid fibrils. Oligomeric soluble Aßs are toxic, although the monomeric and fibril ones are rather nontoxic.

**Figure 3.** Oligomerization of AßP

in the presenilin genes account for the majority of cases of early-onset familial AD. Presenilins have been revealed to be one of γ -secretases, and their mutations also influence the production

membrane

ß-secretase g-secretase

Primate AßP: DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIATVIVITLVMLKKK-

AßP is secreted from its precursor protein, APP by transmembrane cleavage. Sequences of primate AßP(1-42) and ro‐ dent AßP(1-42) are shown. The comparison between the sequence of primate (human or monkey) Aß(1-42) and ro‐

Yankner *et al.* reported that the first 40 amino acid residues of AßP (AßP(1–40)) caused the death of cultured rat hippocampal neurons or the neurodegeneration in the brains of experi‐ mental animals. Thereafter, it was agreed upon that the aggregation and the subsequent conformational change of AßP contribute to its neurotoxicity. AßP is a hydrophobic peptide with an intrinsic tendency to self-assemble to form insoluble oligomers with ß-pleated sheet structures. Pike *et al.* revealed that aged AßP(1–40) (aggregated under incubation at 37°C for several days) were considerably more toxic to cultured neurons as compared to freshly prepared AßP(1–40). Simmons *et al*. revealed ß-sheet contents of AßP observed by circular

Furthermore, the longer peptide variant, AßP(1–42), has the characteristics of immediate polymerization compared to AßP(1–40). AßP(1–42) enhances the aggregation of AßP(1–40) and becomes a seed of the amyloid fibrils. AßP (1–42) is more abundant in the brains of AD patients as compared to those of age matched controls. The mutations of APP and those of presenilin genes induce the increased production of AßP (1–42) in the transfected cell lines.

Recent approaches using size-exclusion chromatography, gel electrophoresis, and atomic force microscopy have demonstrated that there are several stable types of soluble oligomers: naturally occurring soluble oligomers (dimers or trimers), ADDLs (AßP-derived diffusible ligands), AßP globulomers, or protofibrils. Hartley separated aggregated AßP(1–40) into low– molecular-weight (mainly monomer), protofibrillar, and fibril fractions by size-exclusion chromatography, and found that the protofibrillar fraction caused marked changes in the electrical activity of cultured neurons and neurotoxicity. Walsh *et al.* found that the intracere‐

Rodent AßP: DAEF*G*HDSG*F*EV*R*HQKLVFFAEDVGSNKGAIIGLMVGGVVIAT

dichroism (CD) spectroscopy correlates with its neurotoxicity.

of AßP and its neurotoxicity.

282 Oligomerization of Chemical and Biological Compounds

dent (rat or mouse) Aß(1-42) is depicted.

**Figure 2.** Structure of AßP

APP

bral administration of the conditioned medium with cultured cells transfected with the human APP gene inhibited long-term potentiation (LTP), which is a form of synaptic information storage well known as a paradigm of memory mechanisms. They also demonstrated that LTP was blocked by SDS-stable low-molecular-weight oligomers (dimers, trimers, or tetramers) but not AßP monomers or larger aggregates. The natural AßP oligomers (derived from the cerebrospinal fluid of AD patients) cause the loss of dendritic spines, synapses, and LTP blockage. Klein and the colleagues reported that AßP-derived diffusible ligands (ADDLs) obtained from sedimentation by clustering are highly toxic to cultured neurons. They also reported that ADDLs inhibited LTP and exhibited adverse effects on synaptic plasticity such as abnormal spine morphology, decreased spine density, and decreased synaptic proteins. Based on these and other numerous findings, it is widely accepted that AßP oligomers are synaptotoxic and neurotoxic, but not monomer or fibrils. These studies further strengthened and modified the amyloid cascade hypothesis, which suggest that AßP oligomers are neuro‐ toxic and crucial for the pathogeneis of AD [8,9].

#### **2.2. Metal-induced oligomerization of AßP**

Considering that AßP is secreted from APP into the brain of young people or of normal subjects, factors which influence (accelerate or delay) the oligomerization may become important determinants of the pathogenesis of AD. Various factors, such as the concentration of peptides, the oxidations, mutations, and racemization of AßP, pH, composition of solvents, temperature, and trace elements, can influence the oligomerization processes. A considerable amount of asparagines (Asp) or serine (Ser) residues of AßP accumulated in senile plaques are racemized. Tomiyama *et al.* reported that racemized D-Asp23-AßP easily aggregates compared to the L-type. Meanwhile, several substances such as rifampicin, curcumin, and aspirin have been reported to inhibit AßP oligomerization *in vitr*o. Rifampicin, a drug used to treat Hansen's disease, may be an interesting inhibitor of oligomerization since patients with Hansen's disease have a low susceptibility to AD. Aspirin and other NSAIDs (non-steroidal anti-inflammatory drugs) inhibit the AßP oligomerization and simultaneously attenuate inflammation.

rization of AßP and attenuates the accumulation of amyloid in the brains of experimental animals. Clinical trials using its analogue PBT2 are under investigation. DFO, a chelator of Al and Fe, attenuates the decline of daily living skills in AD patients. Silicates, which couple with

There is a considerable interest regarding the mechanisms by which AßP oligomers cause neurotoxicity. Exposure to AßP causes various adverse effects on neuronal survivals such as the production of reactive oxygen species, the induction of cytokines, the induction of endoplasmic reticulum (ER) stresses, and the abnormal increase of intracellular calcium levels ([Ca2+]i), *etc* [18]. Although these effects may be interwoven, the disruption of Ca2+ homeostasis is regarded to be an important determinant considering it occurs upstream of the other effects [19,20]. Ca2+ ions are essential for the normal brain functions. They are involved with key enzymes such as kinases, phosphatases, and proteases. Therefore, its influx is severely

*etc*. Ca2+ is also implicated in the phosphorylation of the tau protein or in APP sequestration. Increasing evidence indicates that presenilins are involved in capacitative Ca2+ entry or endoplasmic reticulum (ER) Ca2+ signaling, and that their mutations affect Ca2+-regulated

There is considerable interest regarding the mechanism by which AßPs interact with neurons and disrupt Ca2+ homeostasis. In 1993, Arispe *et al.* first demonstrated that AßP(1–40) directly incorporates into artificial lipid bilayer membranes and forms cation-selective ion channels [22]. The channels termed "amyloid channels" were revealed to be giant multi-level pores and can allow a large amount of Ca2+ to pass through. Their activity was blocked by Zn2+ ions, which are abundantly present in the brain. Furthermore, soluble AßP oligomers but not amyloid fibrils were reported to increase the membrane permeability. Durell *et al.* proposed a 3-D structural model of the amyloid channels obtained from a computer simulation of the secon‐ dary structure of AßP(1–40) in membranes that showed 5- to 8-mers aggregating to form porelike structures on the membranes. The multimeric (tetramer to hexamer) pore-like structures of AßPs on reconstituted membranes were observed using atomic force microscopy. Jang *et al.* established a model of amyloid channels on the membranes and observed that pentamer AßPs form pores, and their dimensions, shapes, and subunit organizations are in good agreement with AFM studies [23]. These results strongly support the hypothetical idea termed "amyloid channel hypothesis", which suggests that the direct incorporation of AßPs and the subsequent imbalances of Ca2+ and other ions through amyloid channels might be the primary event in AßP neurotoxicity. In this respect, AßP might share the mechanism of toxicity with a similar mechanism underlying the toxicity of various antimicrobial or antifungal peptides that

To determine whether AßPs form channels on neuronal cell membranes as well as artificial lipid bilayers, we employed membrane patches from a neuroblastoma cell line (GT1-7 cells), which exhibit several neuronal characteristics such as the extension of neuritis and the expression of neuron-specific proteins or receptors [24]. After exposing the excised membrane

) are strictly conserved by Ca2+ channels,

Oligomerization of Proteins and Neurodegenerative Diseases

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285

Al and reduce its toxicity, are also candidates for chelation therapy in AD [17].

**2.2. Oligomerization-induced neurotoxicity of AßP**

controlled, and the intracellular Ca2+ levels ([Ca2+]i

also exhibit channel-forming activity and cell toxicity.

functions including AßP production [21].

Among these factors, trace elements such as aluminum (Al), zinc (Zn), copper (Cu), iron (Fe) are of particular interest. The accumulation of AßP is rarely observed in the brains of rodents (rats or mice) as compared to humans or monkeys. As shown in Fig.2, the amino acid sequence of human and rodent AßP are similar, yet they differ by three amino acids. However, rodent AßP exhibits less tendency to oligomerization compared to human AßP [10]. Considering that these three amino acids (Arg5 , Tyr10, and His13) have the ability to bind metals and that trace metals have cross-linking ability, trace elements might play important roles in the accumula‐ tion of AßP in the human brain.

Exley *et al.* first demonstrated that Al induces a conformational change in AßP(1-40) by CD spectroscopy. Furthermore, exposure to Al causes the accumulation of AßP in cultured neurons or in brains of experimental animals or human. Pratico *et al.* found that Al-fed mice transfected with the human APP gene (Tg 2576) exhibited pathological changes similar to those of the AD brain, including a marked increase in the amount of AßP both in the secreted form and the accumulated form: an increased deposition of senile plaques was also observed [11]. The neuropathological case study of the accidental Al-exposure that occurred in 1988 at Camelford (Cornwall, U.K.) indicated that the exposure to Al, even if it is short-term, could cause the accumulation of AßP and exhibit severe amyloid angiopathy [12]. Since there have been studies indicating the link between Al in drinking water and the pathogenesis of AD, Alinduced oligomerization may directly implicated in AD pathogenesis [13].

Bush *et al.* demonstrated that Zn2+ and Cu2+caused the oligomerization of AβP [14]. However, the metal-induced oligomerization of AßP and other amyloidogenic proteins are complex and controversial. The morphology of AßP oligomers treated with Al, Cu, Fe, and Zn were reported to be quite different. Zatta and his colleagues demonstrated that metals including Al, Cu, Fe, Zn differentially alter the oligomerization of AßP and its toxicity. We have shown that Al enhances the polymerization of AßP(1-40) and forms SDS-stable oligomers *in vitro* by immu‐ noblotting and precipitation [15,16]. The oligomerized AßP(1-40) is heat- or SDS-stable but redissolves on adding deferoxamine, a chelator of Al. The oligomerization induced by Al is more marked than that induced by other metals, including Zn, Fe, Cu, and Cd. Furthermore, while Zn-aggregated AßPs are rarely observed on the surface of cultured neurons several days after its exposure, Al-aggregated AßPs bind tightly to the surface of cultured neurons and form fibrillar deposits. These results suggest that Al-induced AßP oligomers have a strong affinity to membrane surfaces and undergo minimal degradation by proteases compared to Zninduced oligomers. Furthermore, AßP coupled with Al was reported to be highly toxic compared to normal AßP.

Considering the implications of metals in AD pathogenesis, chelation therapy for AD treat‐ ment is of great interest. Clioquinol (quinoform), a chelator of Cu2+ or Zn2+, inhibits oligome‐ rization of AßP and attenuates the accumulation of amyloid in the brains of experimental animals. Clinical trials using its analogue PBT2 are under investigation. DFO, a chelator of Al and Fe, attenuates the decline of daily living skills in AD patients. Silicates, which couple with Al and reduce its toxicity, are also candidates for chelation therapy in AD [17].

#### **2.2. Oligomerization-induced neurotoxicity of AßP**

racemized. Tomiyama *et al.* reported that racemized D-Asp23-AßP easily aggregates compared to the L-type. Meanwhile, several substances such as rifampicin, curcumin, and aspirin have been reported to inhibit AßP oligomerization *in vitr*o. Rifampicin, a drug used to treat Hansen's disease, may be an interesting inhibitor of oligomerization since patients with Hansen's disease have a low susceptibility to AD. Aspirin and other NSAIDs (non-steroidal anti-inflammatory

Among these factors, trace elements such as aluminum (Al), zinc (Zn), copper (Cu), iron (Fe) are of particular interest. The accumulation of AßP is rarely observed in the brains of rodents (rats or mice) as compared to humans or monkeys. As shown in Fig.2, the amino acid sequence of human and rodent AßP are similar, yet they differ by three amino acids. However, rodent AßP exhibits less tendency to oligomerization compared to human AßP [10]. Considering that

metals have cross-linking ability, trace elements might play important roles in the accumula‐

Exley *et al.* first demonstrated that Al induces a conformational change in AßP(1-40) by CD spectroscopy. Furthermore, exposure to Al causes the accumulation of AßP in cultured neurons or in brains of experimental animals or human. Pratico *et al.* found that Al-fed mice transfected with the human APP gene (Tg 2576) exhibited pathological changes similar to those of the AD brain, including a marked increase in the amount of AßP both in the secreted form and the accumulated form: an increased deposition of senile plaques was also observed [11]. The neuropathological case study of the accidental Al-exposure that occurred in 1988 at Camelford (Cornwall, U.K.) indicated that the exposure to Al, even if it is short-term, could cause the accumulation of AßP and exhibit severe amyloid angiopathy [12]. Since there have been studies indicating the link between Al in drinking water and the pathogenesis of AD, Al-

Bush *et al.* demonstrated that Zn2+ and Cu2+caused the oligomerization of AβP [14]. However, the metal-induced oligomerization of AßP and other amyloidogenic proteins are complex and controversial. The morphology of AßP oligomers treated with Al, Cu, Fe, and Zn were reported to be quite different. Zatta and his colleagues demonstrated that metals including Al, Cu, Fe, Zn differentially alter the oligomerization of AßP and its toxicity. We have shown that Al enhances the polymerization of AßP(1-40) and forms SDS-stable oligomers *in vitro* by immu‐ noblotting and precipitation [15,16]. The oligomerized AßP(1-40) is heat- or SDS-stable but redissolves on adding deferoxamine, a chelator of Al. The oligomerization induced by Al is more marked than that induced by other metals, including Zn, Fe, Cu, and Cd. Furthermore, while Zn-aggregated AßPs are rarely observed on the surface of cultured neurons several days after its exposure, Al-aggregated AßPs bind tightly to the surface of cultured neurons and form fibrillar deposits. These results suggest that Al-induced AßP oligomers have a strong affinity to membrane surfaces and undergo minimal degradation by proteases compared to Zninduced oligomers. Furthermore, AßP coupled with Al was reported to be highly toxic

Considering the implications of metals in AD pathogenesis, chelation therapy for AD treat‐ ment is of great interest. Clioquinol (quinoform), a chelator of Cu2+ or Zn2+, inhibits oligome‐

induced oligomerization may directly implicated in AD pathogenesis [13].

, Tyr10, and His13) have the ability to bind metals and that trace

drugs) inhibit the AßP oligomerization and simultaneously attenuate inflammation.

these three amino acids (Arg5

284 Oligomerization of Chemical and Biological Compounds

tion of AßP in the human brain.

compared to normal AßP.

There is a considerable interest regarding the mechanisms by which AßP oligomers cause neurotoxicity. Exposure to AßP causes various adverse effects on neuronal survivals such as the production of reactive oxygen species, the induction of cytokines, the induction of endoplasmic reticulum (ER) stresses, and the abnormal increase of intracellular calcium levels ([Ca2+]i), *etc* [18]. Although these effects may be interwoven, the disruption of Ca2+ homeostasis is regarded to be an important determinant considering it occurs upstream of the other effects [19,20]. Ca2+ ions are essential for the normal brain functions. They are involved with key enzymes such as kinases, phosphatases, and proteases. Therefore, its influx is severely controlled, and the intracellular Ca2+ levels ([Ca2+]i ) are strictly conserved by Ca2+ channels, *etc*. Ca2+ is also implicated in the phosphorylation of the tau protein or in APP sequestration. Increasing evidence indicates that presenilins are involved in capacitative Ca2+ entry or endoplasmic reticulum (ER) Ca2+ signaling, and that their mutations affect Ca2+-regulated functions including AßP production [21].

There is considerable interest regarding the mechanism by which AßPs interact with neurons and disrupt Ca2+ homeostasis. In 1993, Arispe *et al.* first demonstrated that AßP(1–40) directly incorporates into artificial lipid bilayer membranes and forms cation-selective ion channels [22]. The channels termed "amyloid channels" were revealed to be giant multi-level pores and can allow a large amount of Ca2+ to pass through. Their activity was blocked by Zn2+ ions, which are abundantly present in the brain. Furthermore, soluble AßP oligomers but not amyloid fibrils were reported to increase the membrane permeability. Durell *et al.* proposed a 3-D structural model of the amyloid channels obtained from a computer simulation of the secon‐ dary structure of AßP(1–40) in membranes that showed 5- to 8-mers aggregating to form porelike structures on the membranes. The multimeric (tetramer to hexamer) pore-like structures of AßPs on reconstituted membranes were observed using atomic force microscopy. Jang *et al.* established a model of amyloid channels on the membranes and observed that pentamer AßPs form pores, and their dimensions, shapes, and subunit organizations are in good agreement with AFM studies [23]. These results strongly support the hypothetical idea termed "amyloid channel hypothesis", which suggests that the direct incorporation of AßPs and the subsequent imbalances of Ca2+ and other ions through amyloid channels might be the primary event in AßP neurotoxicity. In this respect, AßP might share the mechanism of toxicity with a similar mechanism underlying the toxicity of various antimicrobial or antifungal peptides that also exhibit channel-forming activity and cell toxicity.

To determine whether AßPs form channels on neuronal cell membranes as well as artificial lipid bilayers, we employed membrane patches from a neuroblastoma cell line (GT1-7 cells), which exhibit several neuronal characteristics such as the extension of neuritis and the expression of neuron-specific proteins or receptors [24]. After exposing the excised membrane patches of GT1-7 cells in the bath solution to AßP(1–40), the current derived from the amyloid channels appeared. The amyloid channels formed on the GT1-7 cell membranes were cationselective, multilevel, voltage-independent, long-lasting ones; the channel activity was inhib‐ ited by the addition of Zn2+, and recovered by a zinc chelator, *o*-phenanthroline [25]. These features were considerably similar to those observed on artificial lipid bilayers. Meanwhile, AßP(40–1), a peptide bearing the reversed sequence of AßP(1–40), did not form any channels. Thus, we can conclude that AßPs are directly incorporated into neuronal membranes to form calcium-permeable pores. In order to test the amyloid channel hypothesis, we examined whether AßP altered the [Ca2+]i levels in neurons by a high-resolution multi-site video imaging system with fura-2 as the cytosolic free calcium reporter fluorescent probe. This multisite fluorometry system enables the simultaneous long-term observation of temporal changes in [Ca2+]i of more than 50 neurons. We could observe AßP-induced abnormal increase in [Ca2+]i in GT1-7 cells [26-28] as well as in primary cultured rat hippocampal neurons [29]. Shortly after exposure to AßP (1–40), a marked increase in [Ca2+]i occurred among many, but not all neurons. We also observed apoptotic death of cultured neurons after the exposure to AßPs and the consequent rise in the [Ca2+]i levels.

[Ca2+]i

protective function in AD.

 is initiated. However, zinc ions (Zn2+), which are secreted into synaptic clefts in a neuronal activity-dependent manner, inhibit AßP-induced Ca2+ entry, and thus have a

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Once AßP channels are formed on neuronal membranes, homeostasis of Ca2+ and other-ion will be disrupted. Disruption of Ca2+ homeostasis triggers several apoptotic pathways such as the activation of calpain, the induction of caspase, and promote numerous degenerative processes, including the production of reactive oxygen species (ROS) and the phosphorylation of tau, thereby accelerating neuronal death. Mutations of presenilins cause disturbances in the capacitive Ca2+ entry and may influence these pathways. Free radicals also induce membrane disruption, by which unregulated Ca2+ influx is further amplified. The disruption of Ca2+ homeostasis also influences the production and processing of APP. Thus, a vicious cycle of neurodegeneration is initiated. This hypothesis explains the long delay in AD development; AD occurs only in senile subjects despite the fact that Aßs are normally secreted also in younger or in normal subjects. Various environmental factors, such as foods or trace metals, as well as

genetic factors will influence these processes and contribute to AD pathogenesis [31].

The disease-related amyloidogenic proteins exhibit similarities in the formation of ß-pleated sheet structures, abnormal deposition as amyloid fibrils in the tissues, and introduction of apoptotic degeneration. Prion diseases, including human kuru, Creutzfeldt-Jakob disease, and bovine spongiform encephalopathy (BSE), are associated with the conversion of a normal prion protein (PrPC) to an abnormal scrapie isoform (PrPSC) [32]. The ß-sheet region of PrPSC is suggested to play a crucial role in its transmissible degenerative processes. A peptide fragment of PrP corresponding to residues 106–126 (PrP106–126) has been reported to cause death in cultured hippocampal neurons. We investigated the oligomerization of PrP106-126 and its neurotoxicity on primary cultured rat hippocampal neurons [33]. As AßP, PrP106-126 formed amyloid-like fibrils with ß–sheet structures by observation with atomic force microspope and by thioflavin T staining during the aging process. The oligomerization and formation ß-sheet structure enhanced the neurotoxicity of PrP106-126. The co-existence of Zn or Cu inhibited ßsheet formation of PrP106-126 and attenuated its neurotoxicity. Furthermore, the thickness of

Electrophysiological and morphological studies have revealed that PrP106-126 exhibits similarities in the formation of amyloid channels as well as AßP [34]. Lin *et al.* reported that PrP106–126 forms cation-permeable pores in artificial lipid bilayers. The activity of PrP channels was also blocked by Zn2+. Kourie *et al.* investigated the detailed characteristics of channels formed by PrP106–126, concluding that it was directly incorporated into lipid bilayers and formed cation-selective, copper-sensitive ion channels. They also revealed that quinacrine,

The oligomerization and fibrillation of α-synuclein has been implicated in the formation of abnormal inclusions, termed Lewy bodies, and the etiology of dementia with Lewy bodies

a potent therapeutic drug, possibly blocks amyloid channels induced by PrP106-126.

**3. Prion diseases and other amyloidosis**

PrP106-126 fibrils was decreased in the presence of Zn or Cu.

Considering the results of our study together with those of the other studies, we propose the following hypothetical scheme of neurodegeneration induced by oligomerization of AßP (Fig. 4).

AßPs are normally secreted from APP into the cerebrospinal fluid and are usually degraded proteolytically by neprilysin within a short period. However, upregulation of the AßP secretion from APP, or an increased ratio of AßP(1–42) to AßP(1–40) may render AßPs liable to be retained in the brain. It has been demonstrated that APP or presenilin gene mutations promote this process. AßP possesses positive charges at neutral pH. Therefore, the net charge of the outer membrane surface may be a determinant when secreted AßPs bind to cellular membranes (Fig.4 (A)). The distribution of phospholipids on cellular membranes is usually asymmetrical and negatively charged phospholipids such as PS exist on the inner membrane surfaces. Disruption of the assymetrical distribution is the first hallmark of apoptotic cell death [30]. Therefore, the binding of AßP to neuronal membranes seldom occur in normal and young brains. This idea may explain why AD occurs in aged subjects meanwhile AßPs are secreted in the brains of young subjects. After incorporation into the membrane, the conformation of AßPs change and the accumulated AßPs aggregate on the membranes (Fig. 4(B)). The ratio of cholesterol to phospholipids in the membrane may alter membrane fluidity, thereby affecting the process from step (A) to (B). AßP oligomerization *in vitro* will also enhance the channel formation velocity. Considering that natural oligomers (dimers or trimers) are more toxic as compared to monomers or fibrils, it is provable that these oligomers might form tetrameric or hexameric pores and exhibit neurotoxicity. Micro-circumstances on the membranes, such as rafts, are suitable locations that facilitate this process. Finally, aggregated AßP oligomers form ion channels (Fig.4 (C)) leading to the various neurodegenerative processes. The processes required for channel formation (from steps (A) to (C)) may require a long life span and determine the rate of the entire process. Unlike endogenous Ca2+ channels, these AßP channels are not regulated by usual blockers. Thus, once formed on membranes, a continuous flow of [Ca2+]i is initiated. However, zinc ions (Zn2+), which are secreted into synaptic clefts in a neuronal activity-dependent manner, inhibit AßP-induced Ca2+ entry, and thus have a protective function in AD.

Once AßP channels are formed on neuronal membranes, homeostasis of Ca2+ and other-ion will be disrupted. Disruption of Ca2+ homeostasis triggers several apoptotic pathways such as the activation of calpain, the induction of caspase, and promote numerous degenerative processes, including the production of reactive oxygen species (ROS) and the phosphorylation of tau, thereby accelerating neuronal death. Mutations of presenilins cause disturbances in the capacitive Ca2+ entry and may influence these pathways. Free radicals also induce membrane disruption, by which unregulated Ca2+ influx is further amplified. The disruption of Ca2+ homeostasis also influences the production and processing of APP. Thus, a vicious cycle of neurodegeneration is initiated. This hypothesis explains the long delay in AD development; AD occurs only in senile subjects despite the fact that Aßs are normally secreted also in younger or in normal subjects. Various environmental factors, such as foods or trace metals, as well as genetic factors will influence these processes and contribute to AD pathogenesis [31].

#### **3. Prion diseases and other amyloidosis**

patches of GT1-7 cells in the bath solution to AßP(1–40), the current derived from the amyloid channels appeared. The amyloid channels formed on the GT1-7 cell membranes were cationselective, multilevel, voltage-independent, long-lasting ones; the channel activity was inhib‐ ited by the addition of Zn2+, and recovered by a zinc chelator, *o*-phenanthroline [25]. These features were considerably similar to those observed on artificial lipid bilayers. Meanwhile, AßP(40–1), a peptide bearing the reversed sequence of AßP(1–40), did not form any channels. Thus, we can conclude that AßPs are directly incorporated into neuronal membranes to form calcium-permeable pores. In order to test the amyloid channel hypothesis, we examined

system with fura-2 as the cytosolic free calcium reporter fluorescent probe. This multisite fluorometry system enables the simultaneous long-term observation of temporal changes in [Ca2+]i of more than 50 neurons. We could observe AßP-induced abnormal increase in [Ca2+]i in GT1-7 cells [26-28] as well as in primary cultured rat hippocampal neurons [29]. Shortly

neurons. We also observed apoptotic death of cultured neurons after the exposure to AßPs

Considering the results of our study together with those of the other studies, we propose the following hypothetical scheme of neurodegeneration induced by oligomerization of AßP (Fig.

AßPs are normally secreted from APP into the cerebrospinal fluid and are usually degraded proteolytically by neprilysin within a short period. However, upregulation of the AßP secretion from APP, or an increased ratio of AßP(1–42) to AßP(1–40) may render AßPs liable to be retained in the brain. It has been demonstrated that APP or presenilin gene mutations promote this process. AßP possesses positive charges at neutral pH. Therefore, the net charge of the outer membrane surface may be a determinant when secreted AßPs bind to cellular membranes (Fig.4 (A)). The distribution of phospholipids on cellular membranes is usually asymmetrical and negatively charged phospholipids such as PS exist on the inner membrane surfaces. Disruption of the assymetrical distribution is the first hallmark of apoptotic cell death [30]. Therefore, the binding of AßP to neuronal membranes seldom occur in normal and young brains. This idea may explain why AD occurs in aged subjects meanwhile AßPs are secreted in the brains of young subjects. After incorporation into the membrane, the conformation of AßPs change and the accumulated AßPs aggregate on the membranes (Fig. 4(B)). The ratio of cholesterol to phospholipids in the membrane may alter membrane fluidity, thereby affecting the process from step (A) to (B). AßP oligomerization *in vitro* will also enhance the channel formation velocity. Considering that natural oligomers (dimers or trimers) are more toxic as compared to monomers or fibrils, it is provable that these oligomers might form tetrameric or hexameric pores and exhibit neurotoxicity. Micro-circumstances on the membranes, such as rafts, are suitable locations that facilitate this process. Finally, aggregated AßP oligomers form ion channels (Fig.4 (C)) leading to the various neurodegenerative processes. The processes required for channel formation (from steps (A) to (C)) may require a long life span and determine the rate of the entire process. Unlike endogenous Ca2+ channels, these AßP channels are not regulated by usual blockers. Thus, once formed on membranes, a continuous flow of

levels.

levels in neurons by a high-resolution multi-site video imaging

occurred among many, but not all

whether AßP altered the [Ca2+]i

286 Oligomerization of Chemical and Biological Compounds

and the consequent rise in the [Ca2+]i

4).

after exposure to AßP (1–40), a marked increase in [Ca2+]i

The disease-related amyloidogenic proteins exhibit similarities in the formation of ß-pleated sheet structures, abnormal deposition as amyloid fibrils in the tissues, and introduction of apoptotic degeneration. Prion diseases, including human kuru, Creutzfeldt-Jakob disease, and bovine spongiform encephalopathy (BSE), are associated with the conversion of a normal prion protein (PrPC) to an abnormal scrapie isoform (PrPSC) [32]. The ß-sheet region of PrPSC is suggested to play a crucial role in its transmissible degenerative processes. A peptide fragment of PrP corresponding to residues 106–126 (PrP106–126) has been reported to cause death in cultured hippocampal neurons. We investigated the oligomerization of PrP106-126 and its neurotoxicity on primary cultured rat hippocampal neurons [33]. As AßP, PrP106-126 formed amyloid-like fibrils with ß–sheet structures by observation with atomic force microspope and by thioflavin T staining during the aging process. The oligomerization and formation ß-sheet structure enhanced the neurotoxicity of PrP106-126. The co-existence of Zn or Cu inhibited ßsheet formation of PrP106-126 and attenuated its neurotoxicity. Furthermore, the thickness of PrP106-126 fibrils was decreased in the presence of Zn or Cu.

Electrophysiological and morphological studies have revealed that PrP106-126 exhibits similarities in the formation of amyloid channels as well as AßP [34]. Lin *et al.* reported that PrP106–126 forms cation-permeable pores in artificial lipid bilayers. The activity of PrP channels was also blocked by Zn2+. Kourie *et al.* investigated the detailed characteristics of channels formed by PrP106–126, concluding that it was directly incorporated into lipid bilayers and formed cation-selective, copper-sensitive ion channels. They also revealed that quinacrine, a potent therapeutic drug, possibly blocks amyloid channels induced by PrP106-126.

The oligomerization and fibrillation of α-synuclein has been implicated in the formation of abnormal inclusions, termed Lewy bodies, and the etiology of dementia with Lewy bodies (DLB). Non-amyloid component (NAC), a fragment peptide of α-synuclein, accumulates in Alzheimer's senile plaques and causes apoptotic neuronal death. Lashuel *et al.* demonstrated by electron microscope observation that α-synuclein forms annular pore-like structures [35].

The elongation of a polyglutamine-coding CAG triplet repeat in the responsible genes is based on the pathogenesis of triplet-repeat disease such as Huntington's disease or Machado-Joseph disease. Hirakura *et al.* reported that polyglutamine formed ion channels in lipid bilayers.

Lal *et al*. investigated the oligomerization and conformational changes of AßP, synuclein, amylin, and other amyloidogenic proteins using gel electrophoresis and AFM imaging, and demonstrated that these amyloidogenic proteins form annular channel-like structures on bilayer membranes [36]. We have demonstrated that these amyloidogenic peptide also cause the elevations in [Ca2+]<sup>i</sup> as well as AßP [3,31]. Considering these results together as shown in Table 1, it is suggested that the oligomerization of disease-related amyloidogenic proteins and the introduction of apoptotic degeneration by disruption of calcium homeostasis *via* unregu‐ lated amyloid channels may be the molecular basis of neurotoxicity of these diseases.

#### **4. Conclusion**

This hypothesis about the pathogenesis of conformational diseases may help in the develop‐ ment of drugs for these diseases. We focus carnosine (ß-alanyl histidine) as such a protective drug. Carnosine is a naturally occurring dipeptide and is commonly present in vertebrate tissues, particularly within the skeletal muscles and nervous tissues [37]. It is found at high concentrations in the muscles of animals or fish which exhibit high levels of exercise, such as horses, chickens, and whales. Thus, it is believed that carnosine plays important roles in the buffering capacities of muscle tissue and the administration of carnosine has been reported to induce hyperactivity in animals.

Secretion from synapses of AßP, and its direct incorporation into membranes and formation of oligomeric amyloid channels are depicted. Details are discussed in the text.

In the brain, a considerable amount of carnosine is localized in the neurons of the olfactory bulb. It is secreted into synaptic clefts along with the excitatory neurotransmitter glutamate during neuronal excitation. Carnosine reportedly has several beneficial effects including the antioxidant activity, the chelating ability to metal ions, the inhibition of the Maillard reaction. Furthermore, carnosine is reported to have anti-crosslinking properties. Attanasio *et al.* reported that carnosine inhibited the fibrillation of alpha-crystallin. It was also demonstrated that carnosine inhibited the oligomerization and subsequent neurotoxicity of AßP. Corona *et al.* showed that dietary supplementation of carnosine attenuated mitochondrial dysfunction and the accumulation of AßP in Alzheimer's model mice [38]. We also showed that carnosine attenuated the neuronal death induced by prion protein fragment peptide (PrP106-126) by changing its conformation [33]. Carnosine level is significantly reduced in the serum of AD patients. These results suggest possible beneficial effects of carnosine as a treatment for AD and prion diseases. We also demonstrated that carnosine attenuates Zn-induced neuronal

death and becomes a candidate for drugs of vascular dementia [39,40]. All of these functions of carnosine (e.g., antioxidant, anti-glycating, anti-crosslinking, and scavenging toxic alde‐ hydes) are related to the aging processes. The level of carnosine varies during development and is low in the aged animals. Therefore, it is highly possible that carnosine protects against

monomer Oligomer

Carnosine

Metals

Zn2+

APP

synapse

AßP

PC PS cholesterol

**Figure 4.** Amyloid channel hypothesis

Ca2+ Zn2+

Disruption of Ca homeostasis

Degeneration of synapses

Alzheimer' s disease

Neuronal death

Senile plaque

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Membrane composition (fluidity, charge)

Neurosteroids

ApoE

 Free radical formation phosphorylation of tau *etc.*

Foods

Estradiol

**Figure 4.** Amyloid channel hypothesis

(DLB). Non-amyloid component (NAC), a fragment peptide of α-synuclein, accumulates in Alzheimer's senile plaques and causes apoptotic neuronal death. Lashuel *et al.* demonstrated by electron microscope observation that α-synuclein forms annular pore-like structures [35].

The elongation of a polyglutamine-coding CAG triplet repeat in the responsible genes is based on the pathogenesis of triplet-repeat disease such as Huntington's disease or Machado-Joseph disease. Hirakura *et al.* reported that polyglutamine formed ion channels in lipid bilayers.

Lal *et al*. investigated the oligomerization and conformational changes of AßP, synuclein, amylin, and other amyloidogenic proteins using gel electrophoresis and AFM imaging, and demonstrated that these amyloidogenic proteins form annular channel-like structures on bilayer membranes [36]. We have demonstrated that these amyloidogenic peptide also cause the elevations in [Ca2+]<sup>i</sup> as well as AßP [3,31]. Considering these results together as shown in Table 1, it is suggested that the oligomerization of disease-related amyloidogenic proteins and the introduction of apoptotic degeneration by disruption of calcium homeostasis *via* unregu‐

lated amyloid channels may be the molecular basis of neurotoxicity of these diseases.

This hypothesis about the pathogenesis of conformational diseases may help in the develop‐ ment of drugs for these diseases. We focus carnosine (ß-alanyl histidine) as such a protective drug. Carnosine is a naturally occurring dipeptide and is commonly present in vertebrate tissues, particularly within the skeletal muscles and nervous tissues [37]. It is found at high concentrations in the muscles of animals or fish which exhibit high levels of exercise, such as horses, chickens, and whales. Thus, it is believed that carnosine plays important roles in the buffering capacities of muscle tissue and the administration of carnosine has been reported to

Secretion from synapses of AßP, and its direct incorporation into membranes and formation

In the brain, a considerable amount of carnosine is localized in the neurons of the olfactory bulb. It is secreted into synaptic clefts along with the excitatory neurotransmitter glutamate during neuronal excitation. Carnosine reportedly has several beneficial effects including the antioxidant activity, the chelating ability to metal ions, the inhibition of the Maillard reaction. Furthermore, carnosine is reported to have anti-crosslinking properties. Attanasio *et al.* reported that carnosine inhibited the fibrillation of alpha-crystallin. It was also demonstrated that carnosine inhibited the oligomerization and subsequent neurotoxicity of AßP. Corona *et al.* showed that dietary supplementation of carnosine attenuated mitochondrial dysfunction and the accumulation of AßP in Alzheimer's model mice [38]. We also showed that carnosine attenuated the neuronal death induced by prion protein fragment peptide (PrP106-126) by changing its conformation [33]. Carnosine level is significantly reduced in the serum of AD patients. These results suggest possible beneficial effects of carnosine as a treatment for AD and prion diseases. We also demonstrated that carnosine attenuates Zn-induced neuronal

of oligomeric amyloid channels are depicted. Details are discussed in the text.

**4. Conclusion**

induce hyperactivity in animals.

288 Oligomerization of Chemical and Biological Compounds

death and becomes a candidate for drugs of vascular dementia [39,40]. All of these functions of carnosine (e.g., antioxidant, anti-glycating, anti-crosslinking, and scavenging toxic alde‐ hydes) are related to the aging processes. The level of carnosine varies during development and is low in the aged animals. Therefore, it is highly possible that carnosine protects against external toxins and acts as an endogenous protective substance against neuronal injury, senescence, and aging. We have applied patents for carnosine and related compounds as drugs for vascular type of senile dementia (Patent No. 5382633, Patent No. JP5294194).

[7] Kawahara M, Negishi-Kato M, Sadakane Y. Calcium dyshomeostasis and neurotoxic‐

Oligomerization of Proteins and Neurodegenerative Diseases

http://dx.doi.org/10.5772/57482

291

[8] Walsh DM and Selkoe DJ. Aß oligomers - a decade of discovery. *J. Neurochem*. 2007;

[9] Klyubin I *et al.* Alzheimer's disease Aβ assemblies mediating rapid disruption of syn‐ aptic plasticity and memory. *Mol Brain*. 2012; 5: doi: 10.1186/1756-6606-5-25.

[10] Dyrks T *et al.* Amyloidogenicity of rodent and human ß A4 sequences. *FEBS letters*

[11] Pratico D *et al.* Aluminum modulates brain amyloidosis through oxidative stress in

[12] Exley C and Esiri MM. Severe cerebral congophilic angiopathy coincident with in‐ creased brain aluminium in a resident of Camelford, Cornwall, UK. *J Neurol Neuro‐*

[13] Kawahara M and Kato-Negishi M. Link between aluminum and the pathogenesis of Alzheimer's disease: the integration of the aluminum and amyloid cascade hypothe‐

[14] Bush AI. The metal theory of Alzheimer's disease. *J Alzheimers Dis.* 2013; 33 Suppl 1:

[15] Kawahara M *et al.* Aluminum promotes the aggregation of Alzheimer's β-amyloid

[16] Kawahara M *et al.* Effects of aluminum on the neurotoxicity of primary cultured neu‐ rons and on the aggregation of β-amyloid protein. *Brain Res Bull* 2001; 55: 211-217.

[17] Faux NG *et al.* PBT2 rapidly improves cognition in Alzheimer's Disease: additional

[18] Small DH, Mok SS and Bornstein JC. Alzheimer's disease and Aß toxicity: from top

[19] Brorson JR *et al.* The Ca2+ influx induced by beta-amyloid peptide 25-35 in cultured hippocampal neurons results from network excitation. *J Neurobiol*. 1995; 26:325-38.

[20] Camandola S and Mattson MP. Aberrant subcellular neuronal calcium regulation in

[21] Green KN and LaFerla FM. Linking calcium to Aß and Alzheimer's disease. *Neuron*

[22] Arispe N, Pollard HB, and Rojas E. Alzheimer disease amyloid ß protein forms calci‐ um channels in bilayer membranes: Blockade by tromethamine and aluminum. *Proc*

aging and Alzheimer's disease. *Biochim Biophys Acta*. 2011;1813:965-73.

ity of Alzheimer's ß-amyloid protein. *Expert Rev Neurother*. 2009; 9: 681-93.

101: 1172-1184.

1993; 324: 231-236.

S277-81.

2008; 59: 190-4.

*surg Psychiatry* 2006; 77: 877-9.

APP transgenic mice. *FASEB J* 2002; 16: 1138-40.

ses, *Int. J. Alzheimer Dis* 2011; doi: 10.4061/2011/276393.

phase II analyses. *J Alzheimers Dis*. 2010;20(2):509-16.

to bottom. *Nat Rev Neurosci.* 2001; 2: 595-8.

*Natl Acad Sci USA*, 1993; 90: 567-571.

protein *in vitro*. *Biochem Biophys Res Commun* 1994; 198: 531-535.

In conclusion, further research into the role of protein oligomerization and Ca homeostasis via amyloid channels might lead to the development of new treatments for neurodegenerative diseases.

#### **Acknowledgements**

The authors would like to thank Mr. M. Yanagita, Ms. A. Komuro, and Ms. N. Kato for their technical assistance. This work was partially supported by a Grant-in Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan and by a Grant from Cooperation for Innovative Technology and Advanced Research in Evolutional Area (CITY AREA) from the Miyazaki Prefectural Industrial Support Foundation.

#### **Author details**

Dai Mizuno\* and Masahiro Kawahara

Department of Bio-Analytical Chemistry, Musashino University, Research Institute of Phar‐ maceutical Sciences, Musashino University, Nishitokyo-shi, Tokyo, Japan

#### **References**

[7] Kawahara M, Negishi-Kato M, Sadakane Y. Calcium dyshomeostasis and neurotoxic‐ ity of Alzheimer's ß-amyloid protein. *Expert Rev Neurother*. 2009; 9: 681-93.

external toxins and acts as an endogenous protective substance against neuronal injury, senescence, and aging. We have applied patents for carnosine and related compounds as drugs

In conclusion, further research into the role of protein oligomerization and Ca homeostasis via amyloid channels might lead to the development of new treatments for neurodegenerative

The authors would like to thank Mr. M. Yanagita, Ms. A. Komuro, and Ms. N. Kato for their technical assistance. This work was partially supported by a Grant-in Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan and by a Grant from Cooperation for Innovative Technology and Advanced Research in Evolutional Area (CITY AREA) from the Miyazaki Prefectural Industrial Support Foundation.

Department of Bio-Analytical Chemistry, Musashino University, Research Institute of Phar‐

[1] Carrell, R.W. & Lomas, D.A. Conformational disease. *The Lancet*, 1997; 350: 134-138.

[2] Loo D *et al.* Proteomics in molecular diagnosis: typing of amyloidosis. *J Biomed Bio‐*

[3] Kawahara M. Role of calcium dyshomeostasis via amyloid channels in the pathogen‐ esis of Alzheimer's disease. *Current Pharmaceutical Design* 2010; 16: 2779-2789.

[4] Demuro A *et al.* Calcium dysregulation and membrane disruption as a ubiquitous neurotoxic mechanism of soluble amyloid oligomers. *J Biol Chem* 2005; 280:

[5] Selkoe, D.J. The molecular pathology of Alzheimer's disease. *Neuron* 1991; 6: 487-498.

[6] Hardy J and Selkoe DJ. The amyloid hypothesis of Alzheimer's disease: progress and

problems on the road to therapeutics. *Science* 2002; 297: 353-6.

maceutical Sciences, Musashino University, Nishitokyo-shi, Tokyo, Japan

*technol.* 2011; 754109. doi: 10.1155/2011/754109.

for vascular type of senile dementia (Patent No. 5382633, Patent No. JP5294194).

diseases.

**Acknowledgements**

290 Oligomerization of Chemical and Biological Compounds