proteasome proteasome proteasomes are protein complexes which degrade unneeded or damaged proteins by proteolysis, a chemical reaction that breaks peptide bonds. enzymes that help such reactions are called proteases. cartoon representation of a proteasome. its active sites are sheltered inside the tube (blue). the caps (red; in this case, 11s regulatory particles) on the ends regulate entry into the destruction chamber, where the protein is degraded. top view of the proteasome above. proteasomes are part of a major mechanism by which cells regulate the concentration of particular proteins and degrade misfolded proteins. proteins are tagged for degradation with a small protein called ubiquitin. the tagging reaction is catalyzed by enzymes called ubiquitin ligases. once a protein is tagged with a single ubiquitin molecule, this is a signal to other ligases to attach additional ubiquitin molecules. the result is a polyubiquitin chain that is bound by the proteasome, allowing it to degrade the tagged protein. the degradation process yields peptides of about seven to eight amino acids long, which can then be further degraded into shorter amino acid sequences and used in synthesizing new proteins. proteasomes are found inside all eukaryotes and archaea, and in some bacteria. in eukaryotes, proteasomes are located both in the nucleus and in the cytoplasm. in structure, the proteasome is a cylindrical complex containing a "core" of four stacked rings forming a central pore. each ring is composed of seven individual proteins. the inner two rings are made of seven β subunits that contain three to seven protease active sites. these sites are located on the interior surface of the rings, so that the target protein must enter the central pore before it is degraded. the outer two rings each contain seven α subunits whose function is to maintain a "gate" through which proteins enter the barrel. these α subunits are controlled by binding to "cap" structures or regulatory particles that recognize polyubiquitin tags attached to protein substrates and initiate the degradation process. the overall system of ubiquitination and proteasomal degradation is known as the ubiquitin–proteasome system. the proteasomal degradation pathway is essential for many cellular processes, including the cell cycle, the regulation of gene expression, and responses to oxidative stress. the importance of proteolytic degradation inside cells and the role of ubiquitin in proteolytic pathways was acknowledged in the award of the 2004 nobel prize in chemistry to aaron ciechanover, avram hershko and irwin rose. discovery before the discovery of the ubiquitin–proteasome system, protein degradation in cells was thought to rely mainly on lysosomes, membrane-bound organelles with acidic and protease-filled interiors that can degrade and then recycle exogenous proteins and aged or damaged organelles. however, work by joseph etlinger and alfred l. goldberg in 1977 on atp-dependent protein degradation in reticulocytes, which lack lysosomes, suggested the presence of a second intracellular degradation mechanism. this was shown in 1978 to be composed of several distinct protein chains, a novelty among proteases at the time. later work on modification of histones led to the identification of an unexpected covalent modification of the histone protein by a bond between a lysine side chain of the histone and the c-terminal glycine residue of ubiquitin, a protein that had no known function. it was then discovered that a previously identified protein associated with proteolytic degradation, known as atp-dependent proteolysis factor 1 (apf-1), was the same protein as ubiquitin. the proteolytic activities of this system were isolated as a multi-protein complex originally called the multi-catalytic proteinase complex by sherwin wilk and marion orlowski. later, the atp-dependent proteolytic complex that was responsible for ubiquitin-dependent protein degradation was discovered and was called the 26s proteasome. much of the early work leading up to the discovery of the ubiquitin proteasome system occurred in the late 1970s and early 1980s at the technion in the laboratory of avram hershko, where aaron ciechanover worked as a graduate student. hershko's year-long sabbatical in the laboratory of irwin rose at the fox chase cancer center provided key conceptual insights, though rose later downplayed his role in the discovery. the three shared the 2004 nobel prize in chemistry for their work in discovering this system. although electron microscopy data revealing the stacked-ring structure of the proteasome became available in the mid-1980s, the first structure of the proteasome core particle was not solved by x-ray crystallography until 1994. in 2018, the first atomic structures of the human 26s proteasome holoenzyme in complex with a polyubiquitylated protein substrate were solved by cryogenic electron microscopy, revealing mechanisms by which the substrate is recognized, deubiquitylated, unfolded and degraded by the human 26s proteasome. structure and organization schematic diagram of the proteasome 20s core particle viewed from one side. the α subunits that make up the outer two rings are shown in green, and the β subunits that make up the inner two rings are shown in blue. the proteasome subcomponents are often referred to by their svedberg sedimentation coefficient (denoted s). the proteasome most exclusively used in mammals is the cytosolic 26s proteasome, which is about 2000 kilodaltons (kda) in molecular mass containing one 20s protein subunit and two 19s regulatory cap subunits. the core is hollow and provides an enclosed cavity in which proteins are degraded; openings at the two ends of the core allow the target protein to enter. each end of the core particle associates with a 19s regulatory subunit that contains multiple atpase active sites and ubiquitin binding sites; it is this structure that recognizes polyubiquitinated proteins and transfers them to the catalytic core. an alternative form of regulatory subunit called the 11s particle can associate with the core in essentially the same manner as the 19s particle; the 11s may play a role in degradation of foreign peptides such as those produced after infection by a virus. 20s core particle the number and diversity of subunits contained in the 20s core particle depends on the organism; the number of distinct and specialized subunits is larger in multicellular than unicellular organisms and larger in eukaryotes than in prokaryotes. all 20s particles consist of four stacked heptameric ring structures that are themselves composed of two different types of subunits; α subunits are structural in nature, whereas β subunits are predominantly catalytic. the α subunits are pseudoenzymes homologous to β subunits. they are assembled with their n-termini adjacent to that of the β subunits. the outer two rings in the stack consist of seven α subunits each, which serve as docking domains for the regulatory particles and the alpha subunits n-termini (pfam pf10584) form a gate that blocks unregulated access of substrates to the interior cavity. the inner two rings each consist of seven β subunits and in their n-termini contain the protease active sites that perform the proteolysis reactions. three distinct catalytic activities were identified in the purified complex: chymotrypsin-like, trypsin-like and peptidylglutamyl-peptide hydrolyzing. the size of the proteasome is relatively conserved and is about 150 angstroms (å) by 115 å. the interior chamber is at most 53 å wide, though the entrance can be as narrow as 13 å, suggesting that substrate proteins must be at least partially unfolded to enter. in archaea such as thermoplasma acidophilum, all the α and all the β subunits are identical, whereas eukaryotic proteasomes such as those in yeast contain seven distinct types of each subunit. in mammals, the β1, β2, and β5 subunits are catalytic; although they share a common mechanism, they have three distinct substrate specificities considered chymotrypsin-like, trypsin-like, and peptidyl-glutamyl peptide-hydrolyzing (phgh). alternative β forms denoted β1i, β2i, and β5i can be expressed in hematopoietic cells in response to exposure to pro-inflammatory signals such as cytokines, in particular, interferon gamma. the proteasome assembled with these alternative subunits is known as the immunoproteasome, whose substrate specificity is altered relative to the normal proteasome. recently an alternative proteasome was identified in human cells that lack the α3 core subunit. these proteasomes (known as the α4-α4 proteasomes) instead form 20s core particles containing an additional α4 subunit in place of the missing α3 subunit. these alternative 'α4-α4' proteasomes have been known previously to exist in yeast. although the precise function of these proteasome isoforms is still largely unknown, cells expressing these proteasomes show enhanced resistance to toxicity induced by metallic ions such as cadmium. 19s regulatory particle the 19s particle in eukaryotes consists of 19 individual proteins and is divisible into two subassemblies, a 9-subunit base that binds directly to the α ring of the 20s core particle, and a 10-subunit lid. six of the nine base proteins are atpase subunits from the aaa family, and an evolutionary homolog of these atpases exists in archaea, called pan (proteasome-activating nucleotidase). the association of the 19s and 20s particles requires the binding of atp to the 19s atpase subunits, and atp hydrolysis is required for the assembled complex to degrade folded and ubiquitinated proteins. note that only the step of substrate unfolding requires energy from atp hydrolysis, while atp-binding alone can support all the other steps required for protein degradation (e.g., complex assembly, gate opening, translocation, and proteolysis). in fact, atp binding to the atpases by itself supports the rapid degradation of unfolded proteins. however, while atp hydrolysis is required for unfolding only, it is not yet clear whether this energy may be used in the coupling of some of these steps. cartoon representation of the 26s proteasome. in 2012, two independent efforts have elucidated the molecular architecture of the 26s proteasome by single particle electron microscopy. in 2016, three independent efforts have determined the first near-atomic resolution structure of the human 26s proteasome in the absence of substrates by cryo-em. in 2018, a major effort has elucidated the detailed mechanisms of deubiquitylation, initiation of translocation and processive unfolding of substrates by determining seven atomic structures of substrate-engaged 26s proteasome simultaneously. in the heart of the 19s, directly adjacent to the 20s, are the aaa-atpases (aaa proteins) that assemble to a heterohexameric ring of the order rpt1/rpt2/rpt6/rpt3/rpt4/rpt5. this ring is a trimer of dimers: rpt1/rpt2, rpt6/rpt3, and rpt4/rpt5 dimerize via their n-terminal coiled-coils. these coiled-coils protrude from the hexameric ring. the largest regulatory particle non-atpases rpn1 and rpn2 bind to the tips of rpt1/2 and rpt6/3, respectively. the ubiquitin receptor rpn13 binds to rpn2 and completes the base sub-complex. the lid covers one half of the aaa-atpase hexamer (rpt6/rpt3/rpt4) and, unexpectedly, directly contacts the 20s via rpn6 and to lesser extent rpn5. the subunits rpn9, rpn5, rpn6, rpn7, rpn3, and rpn12, which are structurally related among themselves and to subunits of the cop9 complex and eif3 (hence called pci subunits) assemble to a horseshoe-like structure enclosing the rpn8/rpn11 heterodimer. rpn11, the deubiquitinating enzyme, is placed at the mouth of the aaa-atpase hexamer, ideally positioned to remove ubiquitin moieties immediately before translocation of substrates into the 20s. the second ubiquitin receptor identified to date, rpn10, is positioned at the periphery of the lid, near subunits rpn8 and rpn9. conformational changes of 19s the 19s regulatory particle within the 26s proteasome holoenzyme has been observed in six strongly differing conformational states in the absence of substrates to date. a hallmark of the aaa-atpase configuration in this predominant low-energy state is a staircase- or lockwasher-like arrangement of the aaa-domains. in the presence of atp but absence of substrate three alternative, less abundant conformations of the 19s are adopted primarily differing in the positioning of the lid with respect to the aaa-atpase module. in the presence of atp-γs or a substrate, considerably more conformations have been observed displaying dramatic structural changes of the aaa-atpase module. some of the substrate-bound conformations bear high similarity to the substrate-free ones, but they are not entirely identical, particularly in the aaa-atpase module. prior to the 26s assembly, the 19s regulatory particle in a free form has also been observed in seven conformational states. notably, all these conformers are somewhat different and present distinct features. thus, the 19s regulatory particle can sample at least 20 conformational states under different physiological conditions. three distinct conformational states of the 26s proteasome. the conformations are hypothesized to be responsible for recruitment of the substrate, its irreversible commitment, and finally processing and translocation into the core particle, where degradation occurs. regulation of the 20s by the 19s the 19s regulatory particle is responsible for stimulating the 20s to degrade proteins. a primary function of the 19s