regulatory atpases is to open the gate in the 20s that blocks the entry of substrates into the degradation chamber. the mechanism by which the proteasomal atpase open this gate has been recently elucidated. 20s gate opening, and thus substrate degradation, requires the c-termini of the proteasomal atpases, which contains a specific motif (i.e., hbyx motif). the atpases c-termini bind into pockets in the top of the 20s, and tether the atpase complex to the 20s proteolytic complex, thus joining the substrate unfolding equipment with the 20s degradation machinery. binding of these c-termini into these 20s pockets by themselves stimulates opening of the gate in the 20s in much the same way that a "key-in-a-lock" opens a door. the precise mechanism by which this "key-in-a-lock" mechanism functions has been structurally elucidated in the context of human 26s proteasome at near-atomic resolution, suggesting that the insertion of five c-termini of atpase subunits rpt1/2/3/5/6 into the 20s surface pockets are required to fully open the 20s gate. other regulatory particles 20s proteasomes can also associate with a second type of regulatory particle, the 11s regulatory particle, a heptameric structure that does not contain any atpases and can promote the degradation of short peptides but not of complete proteins. it is presumed that this is because the complex cannot unfold larger substrates. this structure is also known as pa28, reg, or pa26. the mechanisms by which it binds to the core particle through the c-terminal tails of its subunits and induces α-ring conformational changes to open the 20s gate suggest a similar mechanism for the 19s particle. the expression of the 11s particle is induced by interferon gamma and is responsible, in conjunction with the immunoproteasome β subunits, for the generation of peptides that bind to the major histocompatibility complex. yet another type of non-atpase regulatory particle is the blm10 (yeast) or pa200/psme4 (human). it opens only one α subunit in the 20s gate and itself folds into a dome with a very small pore over it. assembly the assembly of the proteasome is a complex process due to the number of subunits that must associate to form an active complex. the β subunits are synthesized with n-terminal "propeptides" that are post-translationally modified during the assembly of the 20s particle to expose the proteolytic active site. the 20s particle is assembled from two half-proteasomes, each of which consists of a seven-membered pro-β ring attached to a seven-membered α ring. the association of the β rings of the two half-proteasomes triggers threonine-dependent autolysis of the propeptides to expose the active site. these β interactions are mediated mainly by salt bridges and hydrophobic interactions between conserved alpha helices whose disruption by mutation damages the proteasome's ability to assemble. the assembly of the half-proteasomes, in turn, is initiated by the assembly of the α subunits into their heptameric ring, forming a template for the association of the corresponding pro-β ring. the assembly of α subunits has not been characterized. only recently, the assembly process of the 19s regulatory particle has been elucidated to considerable extent. the 19s regulatory particle assembles as two distinct subcomponents, the base and the lid. assembly of the base complex is facilitated by four assembly chaperones, hsm3/s5b, nas2/p27, rpn14/paaf1, and nas6/gankyrin (names for yeast/mammals). these assembly chaperones bind to the aaa-atpase subunits and their main function seems to be to ensure proper assembly of the heterohexameric aaa-atpase ring. to date it is still under debate whether the base complex assembles separately, whether the assembly is templated by the 20s core particle, or whether alternative assembly pathways exist. in addition to the four assembly chaperones, the deubiquitinating enzyme ubp6/usp14 also promotes base assembly, but it is not essential. the lid assembles separately in a specific order and does not require assembly chaperones. protein degradation process ribbon diagram of ubiquitin, the highly conserved protein that serves as a molecular tag targeting proteins for degradation by the proteasome ubiquitination and targeting proteins are targeted for degradation by the proteasome with covalent modification of a lysine residue that requires the coordinated reactions of three enzymes. in the first step, a ubiquitin-activating enzyme (known as e1) hydrolyzes atp and adenylylates a ubiquitin molecule. this is then transferred to e1's active-site cysteine residue in concert with the adenylylation of a second ubiquitin. this adenylylated ubiquitin is then transferred to a cysteine of a second enzyme, ubiquitin-conjugating enzyme (e2). in the last step, a member of a highly diverse class of enzymes known as ubiquitin ligases (e3) recognizes the specific protein to be ubiquitinated and catalyzes the transfer of ubiquitin from e2 to this target protein. a target protein must be labeled with at least four ubiquitin monomers (in the form of a polyubiquitin chain) before it is recognized by the proteasome lid. it is therefore the e3 that confers substrate specificity to this system. the number of e1, e2, and e3 proteins expressed depends on the organism and cell type, but there are many different e3 enzymes present in humans, indicating that there is a huge number of targets for the ubiquitin proteasome system. the mechanism by which a polyubiquitinated protein is targeted to the proteasome is not fully understood. a few high-resolution snapshots of the proteasome bound to a polyubiquitinated protein suggest that ubiquitin receptors might be coordinated with deubiquitinase rpn11 for initial substrate targeting and engagement. ubiquitin-receptor proteins have an n-terminal ubiquitin-like (ubl) domain and one or more ubiquitin-associated (uba) domains. the ubl domains are recognized by the 19s proteasome caps and the uba domains bind ubiquitin via three-helix bundles. these receptor proteins may escort polyubiquitinated proteins to the proteasome, though the specifics of this interaction and its regulation are unclear. the ubiquitin protein itself is 76 amino acids long and was named due to its ubiquitous nature, as it has a highly conserved sequence and is found in all known eukaryotic organisms. the genes encoding ubiquitin in eukaryotes are arranged in tandem repeats, possibly due to the heavy transcription demands on these genes to produce enough ubiquitin for the cell. it has been proposed that ubiquitin is the slowest-evolving protein identified to date. ubiquitin contains seven lysine residues to which another ubiquitin can be ligated, resulting in different types of polyubiquitin chains. chains in which each additional ubiquitin is linked to lysine 48 of the previous ubiquitin have a role in proteasome targeting, while other types of chains may be involved in other processes. the ubiquitination pathway deubiquitylation ubiquitin chains conjugated to a protein targeted for proteasomal degradation are normally removed by any one of the three proteasome-associated deubiquitylating enzymes (dubs), which are rpn11, ubp6/usp14 and uch37. this process recycles ubiquitin and is essential to maintain the ubiquitin reservoir in cells. rpn11 is an intrinsic, stoichiometric subunit of the 19s regulatory particle and is essential for the function of 26s proteasome. the dub activity of rpn11 is enhanced in the proteasome as compared to its monomeric form. how rpn11 removes a ubiquitin chain en bloc from a protein substrate was captured by an atomic structure of the substrate-engaged human proteasome in a conformation named eb. interestingly, this structure also shows how the dub activity is coupled to the substrate recognition by the proteasomal aaa-atpase. in contrast to rpn11, usp14 and uch37 are the dubs that do not always associated with the proteasome. in cells, about 10-40% of the proteasomes were found to have usp14 associated. both ubp6/usp14 and uch37 are largely activated by the proteasome and exhibit a very low dub activity alone. once activated, usp14 was found to suppress proteasome function by its dub activity and by inducing parallel pathways of proteasome conformational transitions, one of which turned out to directly prohibit substrate insertion into the aaa-atpase, as intuitively observed by time-resolved cryogenic electron microscopy. it appears that usp14 regulates proteasome function at multiple checkpoints by both catalytically competing with rpn11 and allosterically reprogramming the aaa-atpase states, which is rather unexpected for a dub. these observations imply that the proteasome regulation may depend on its dynamic transitions of conformational states. unfolding and translocation after a protein has been ubiquitinated, it is recognized by the 19s regulatory particle in an atp-dependent binding step. the substrate protein must then enter the interior of the 20s subunit to come in contact with the proteolytic active sites. because the 20s particle's central channel is narrow and gated by the n-terminal tails of the α ring subunits, the substrates must be at least partially unfolded before they enter the core. the passage of the unfolded substrate into the core is called translocation and necessarily occurs after deubiquitination. however, the order in which substrates are deubiquitinated and unfolded is not yet clear. which of these processes is the rate-limiting step in the overall proteolysis reaction depends on the specific substrate; for some proteins, the unfolding process is rate-limiting, while deubiquitination is the slowest step for other proteins. the extent to which substrates must be unfolded before translocation is suggested to be around 20 amino acid residues by the atomic structure of the substrate-engaged 26s proteasome in the deubiquitylation-compatible state, but substantial tertiary structure, and in particular nonlocal interactions such as disulfide bonds, are sufficient to inhibit degradation. the presence of intrinsically disordered protein segments of sufficient size, either at the protein terminus or internally, has also been proposed to facilitate efficient initiation of degradation. the gate formed by the α subunits prevents peptides longer than about four residues from entering the interior of the 20s particle. the atp molecules bound before the initial recognition step are hydrolyzed before translocation. while energy is needed for substrate unfolding, it is not required for translocation. the assembled 26s proteasome can degrade unfolded proteins in the presence of a non-hydrolyzable atp analog, but cannot degrade folded proteins, indicating that energy from atp hydrolysis is used for substrate unfolding. passage of the unfolded substrate through the opened gate occurs via facilitated diffusion if the 19s cap is in the atp-bound state. the mechanism for unfolding of globular proteins is necessarily general, but somewhat dependent on the amino acid sequence. long sequences of alternating glycine and alanine have been shown to inhibit substrate unfolding, decreasing the efficiency of proteasomal degradation; this results in the release of partially degraded byproducts, possibly due to the decoupling of the atp hydrolysis and unfolding steps. such glycine-alanine repeats are also found in nature, for example in silk fibroin; in particular, certain epstein–barr virus gene products bearing this sequence can stall the proteasome, helping the virus propagate by preventing antigen presentation on the major histocompatibility complex. a cutaway view of the proteasome 20s core particle illustrating the locations of the active sites. the α subunits are represented as green spheres and the β subunits as protein backbones colored by individual polypeptide chain. the small pink spheres represent the location of the active-site threonine residue in each subunit. light blue chemical structures are the inhibitor bortezomib bound to the active sites. proteolysis the proteasome functions as an endoprotease. the mechanism of proteolysis by the β subunits of the 20s core particle is through a threonine-dependent nucleophilic attack. this mechanism may depend on an associated water molecule for deprotonation of the reactive threonine hydroxyl. degradation occurs within the central chamber formed by the association of the two β rings and normally does not release partially degraded products, instead reducing the substrate to short polypeptides typically 7–9 residues long, though they can range from 4 to 25 residues, depending on the organism and substrate. the biochemical mechanism that determines product length is not fully characterized. although the three catalytic β subunits have a common mechanism, they have slightly different substrate specificities, which are considered chymotrypsin-like, trypsin-like, and peptidyl-glutamyl peptide-hydrolyzing (phgh)-like. these variations in specificity are the result of interatomic contacts with local residues near the active sites of each subunit. each catalytic β subunit also possesses a conserved lysine residue required for proteolysis. although the proteasome normally produces very short peptide fragments, in some cases these products are themselves biologically active and functional molecules. certain transcription factors regulating the expression of specific genes, including one component of the mammalian complex nf-κb, are synthesized as inactive precursors whose ubiquitination and subsequent proteasomal degradation converts them to an active form. such activity requires the proteasome to cleave the substrate protein internally, rather than processively