Proteasome
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Proteasomes are large protein complexes inside both bacterial, archeal and eukaryotic cells that break down proteins via ATP-dependent proteolysis. Proteasomes are localized in both the nucleus and the cytoplasm<ref name="Peters">Peters JM, Franke WW, Kleinschmidt JA. (1994) Distinct 19S and 20S subcomplexes of the 26S proteasome and their distribution in the nucleus and the cytoplasm. J Biol Chem, Mar 11;269(10):7709-18. PMID 8125997</ref> and represent one of the major mechanisms by which cells regulate the concentration of particular proteins and degrade misfolded proteins. Proteins that need to be degraded are covalently modified by ubiquitin ligases with a 76-amino acid protein called ubiquitin, which itself signals other ligases to attach additional ubiquitin molecules, forming a polyubiquitin chain that serves as a chemical signal targeting the protein for degradation.<ref name="Lodish"> Lodish, H, Berk A, Matsudaira P, Kaiser CA, Krieger M, Scott MP, Zipursky SL, Darnell J. (2004). Molecular Cell Biology', 5th, New York: WH Freeman.</ref>
The proteasome is a large barrel-shaped structure consisting of a core ring-shaped complex that is the site of the proteasome's protease activity. Tightly associated with this core is a "cap" structure that contains the ATPase active sites as well as binding sites that recognize polyubiquitin. Proteins that are not tagged with ubiquitin are not bound by this structure and therefore are not susceptible to proteasomal degradation. The products of the proteolysis reaction are 7-8 residue peptides and intact ubiquitin molecules.<ref name="Lodish"/> The ubiquitin can be recycled for tagging other proteins, and the peptides can be further degraded to amino acids that can be reused in protein synthesis. The overall system of proteasomal degradation is sometimes known as the ubiquitin proteasome system.
The proteasomal degradation pathway is essential for many cellular processes, including the cell cycle, signal transduction, and regulation of gene expression. The importance of proteolytic degradation inside cells and the role of ubiquitin in proteolytic pathways was acknowledged in the awarding of the 2004 Nobel Prize in Chemistry to Aaron Ciechanover, Avram Hershko and Irwin Rose.
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[edit] 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 responsible for the degradation and recycling of exogenous proteins and aged or damaged organelles.<ref name="Lodish" /> However, work on ATP-dependent protein degradation in reticulocytes, which lack lysosomes, suggested the presence of a second intracellular degradation mechanism that 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 branched bond between a histone lysine residue and the N-terminal glycine residue of the protein ubiquitin, which was not yet associated with its function in protein degradation. A previously identified protein designated ATP-dependent proteolysis factor 1 (APF-1) was then shown to be identical to the histone-modifying ubiquitin.<ref name="Ciechanover">Ciechanover A. (2000). Early work on the ubiquitin proteasome system, an interview with Aaron Ciechanover. Cell Death Differ Sep;12(9):1167-77. PMID 16094393</ref>
[edit] Structure and organization
The proteasome subcomponents are often referred to by their Svedberg sedimentation coefficient: an assembled 26S proteasome, which is about 2000 kDa, consists of one 20S core particle structure and two 19S regulatory caps. 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 recognized polyubiquitinated proteins and transfers them to the catalytic core.
[edit] 20S core particle
The number and diversity of subunits represented 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 layers of a heptameric ring structures that are themselves composed of two different types of subunits; α subunits are structural in nature, while β subunits are predominantly catalytic. The size of the proteasome is relatively conserved and is about 150Å by 115Å. The interior chamber is at most 53Å wide, though the entrance can be as narrow as 13Å, suggesting that entering substrate proteins may be at least partially unfolded.<ref name="Nandi">Nandi D, Tahiliani P, Kumar A, Chandu D. (2006). The ubiquitin-proteasome system. J Biosci Mar;31(1):137-55. PMID 16595883</ref>
In archaea such as T. acidophilum, all the α and all the β subunits are identical, while yeast proteasomes 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)-like.<ref name="Heinemeyer">Heinemeyer W, Fischer M, Krimmer T, Stachon U, Wolf DH. (1997). The Active Sites of the Eukaryotic 20 S Proteasome and Their Involvement in Subunit Precursor Processing J Biol Chem 272(40):25200-25209.</ref> Alternative β forms denoted β1i, β2i, and β5i can be expressed in hematopoietic cells in response to exposure to pro-inflammatory signals such ascytokines, in particular, interferon gamma. The proteasome assembled with these alternative subunits is known as the "immunoproteasome", whose substrate specificity is altered relative to the constitutively expressed proteasome.<ref name="Nandi" />
[edit] 19S regulatory particle
The 19S particle in eukaryotes consists of a total of 19 individual proteins and is divisible into two subunits, a 10-protein base that binds directly to the α ring of the 20S core particle, and a 9-protein lid. Six of the ten base proteins have ATPase activity. The association of the 19S and 20S particles requires the binding of ATP to the 19S ATP-binding sites.<ref name="Liu">Liu CW, Li X, Thompson D, Wooding K, Chang TL, Tang Z, Yu H, Thomas PJ, DeMartino GN. (2006). ATP binding and ATP hydrolysis play distinct roles in the function of 26S proteasome. Mol Cell Oct 6;24(1):39-50. PMID 17018291</ref> ATP hydrolysis is required for the assembled complex to degrade a folded and ubiquitinated protein, although it is not yet clear whether that energy is used mainly for substrate unfolding,<ref name="Lam">Lam YA, Lawson TG, Velayutham M, Zweier JL, Pickart CM. (2002). A proteasomal ATPase subunit recognizes the polyubiquitin degradation signal. Nature 416(6882):763-7.</ref> opening of the core channel,<ref name="Sharon">Sharon M, Taverner T, Ambroggio XI, Deshaies RJ, Robinson CV. (2006). Structural Organization of the 19S Proteasome Lid: Insights from MS of Intact Complexes. PLoS Biol Aug;4(8):e267. PMID 16869714</ref> or some combination of processes.<ref name="Liu"/>
[edit] 11S regulatory particle
20S proteasomes can also associate with a second type of regulatory particle, a heptameric structure that does not contain any ATPases and can promote the degradation of short peptides, but not of complete proteins, presumably because it cannot unfold larger substrates. The structure is also known as PA28 or REG. The mechanisms by which it binds to the core particle via its subunits' C-terminal tails and induces α-ring conformational changes to open the 20S gate suggest a similar mechanism for the 19S particle.<ref name="Forster">Forster A, Masters EI, Whitby FG, Robinson H, Hill CP. (2005). The 1.9 Å Structure of a Proteasome-11S Activator Complex and Implications for Proteasome-PAN/PA700 Interactions. Mol Cell May 27;18(5):589-99. PMID 15916965</ref> 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 proteolytic byproducts appropriate for binding to the major histocompatibility complex.<ref name="Wang">Wang J, Maldonado MA. (2006). The Ubiquitin-Proteasome System and Its Role in Inflammatory and Autoimmune Diseases. Cell Mol Immunol 3(4): 255.</ref>
[edit] 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 28-mer 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.<ref name="Witt">Witt S, Kwon YD, Sharon M, Felderer K, Beuttler M, Robinson CV, Baumeister W, Jap BK. (2006). Proteasome assembly triggers a switch required for active-site maturation. Structure Jul;14(7):1179-88. PMID 16843899</ref> The assembly of the half-proteasomes, in turn, is initiated by the apparently spontaneous assembly of the α subunits into their heptameric ring, forming a template for the association of the corresponding pro-β ring.<ref name="Kruger">Kruger E, Kloetzel PM, Enenkel C. (2001). 20S proteasome biogenesis. Biochimie Mar-Apr;83(3-4):289-93. PMID 11295488</ref>
In general, less is known about the assembly and maturation of the 19S regulatory particles. They are believed to assemble as two distinct subcomponents, the ATPase-containing base and the ubiquitin-recognizing lid. The six ATPases in the base have been reported to assemble in a pairwise manner mediated by coiled-coil interactions.<ref name="Gorbea">Gorbea C, Taillandier D, Rechsteiner M. (1999). Assembly of the regulatory complex of the 26S proteasome. Mol Biol Rep Apr;26(1-2):15-9. PMID 10363641</ref> The order in which the nineteen subunits of the regulatory particle are bound is a likely regulatory mechanism that prevents exposure of the active site before assembly is complete.<ref name="Sharon"/>
[edit] The protein degradation process
[edit] Ubiquitination and targeting
Proteins are targeted for degradation by the proteasome by covalent modification of a lysine residue that requires the coordinated reactions of three distinct enzymes binding the extremely concerved ubiquitin tag to a protein. First a ubiquitin-activating enzyme (known as E1) hydrolyzes ATP and adenylates a ubiquitin molecule that is then transferred to E1's active-site cysteine residue in concert with the adenylation of a second ubiquitin.<ref name="Haas">Haas AL, Warms JV, Hershko A, Rose IA. Ubiquitin-activating enzyme: Mechanism and role in protein-ubiquitin conjugation. J Biol Chem. 1982 Mar 10;257(5):2543-8. PMID 6277905</ref> This adenylated ubiquitin is then transferred to a cysteine of a second enzyme, ubiquitin-conjugating enzyme (E2). Lastly, a third highly diverse class of enzymes known as ubiquitin ligases (E3) recognize the specific protein to be ubiquitinated and catalyze the transfer of ubiquitin from E2 to the target protein. It is thus the E3 that confers substrate specificity.<ref name="Risseeuw">Risseeuw EP, Daskalchuk TE, Banks TW, Liu E, Cotelesage J, Hellmann H, Estelle M, Somers DE, Crosby WL. Protein interaction analysis of SCF ubiquitin E3 ligase subunits from Arabidopsis. Plant J. 2003 Jun;34(6):753-67. PMID 12795696</ref> 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 the huge range of targets of the ubiquitin proteasome system. A target protein must be labelled with at least four ubiquitin monomers (in the form of a polyubiquitin chain) before it is recognized by the proteasome lid.<ref name="Thrower">Thrower JS, Hoffman L, Rechsteiner M, Pickart CM. (200). Recognition of the polyubiquitin proteolytic signal. EMBO J 19, 94–102.</ref>
The mechanism by which a polyubiquitinated protein is targeted to the proteasome is not yet fully elucidated. Ubiquitin-receptor proteins that 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 have been hypothesized to escort polyubiquitinated proteins to the proteasome, though the specifics of this interaction and its regulation are unclear.<ref name="Elasser">Elsasser S, Finley D. (2005). Delivery of ubiquitinated substrates to protein-unfolding machines. Nat Cell Biol 7(8):742-9.</ref>
[edit] Unfolding and translocation
The narrow central channel of the proteasome requires that substrates be at least partially unfolded before entry. The polyubiquitin chain that targeted a particular protein to the proteasome must also be removed before translocation can proceed. For some proteins, the unfolding process is rate-limiting, while the deubiquitination process is the slow step for others.<ref name="Lam" /> Deubiquitination and unfolding are necessarily preceded by an ATP-binding-dependent recognition step at the 19S ATPase ring.<ref name="Liu" /> Although the extent to which the substrate must be unfolded is not yet clear, it appears that substantial tertiary structure, and in particular nonlocal interactions such as disulfide bonds, are sufficient to inhibit degradation.<ref name="Wenzel">Wenzel T, Baumeister W. (1995). Conformational constraints in protein degradation by the 20S proteasome. Nat Struct Biol 2: 199-204.</ref>
Structurally, the entrance into the 20S core is blocked by the N-terminal regions of the α subunits, which form a "gate" through which peptides longer than about four residues cannot pass unaided. Energy from ATP hydrolysis is used in the process of substrate unfolding<ref name="Lam" /> or in gate opening,<ref name="Sharon" /> though the precise mechanism for either process is not yet clear. 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 required for substrate unfolding.<ref name="Smith">Smith DM, Kafri G, Cheng Y, Ng D, Walz T, Goldberg AL. (2005). ATP binding to PAN or the 26S ATPases causes association with the 20S proteasome, gate opening, and translocation of unfolded proteins. Mol Cell 20(5):687-98.</ref> Passage of the unfolded substrate through the opened gate occurs via facilitated diffusion if the 19S cap is in the ATP-bound state.<ref name="Smith2">Smith DM, Benaroudj N, Goldberg A. (2006). Proteasomes and their associated ATPases: a destructive combination. J Struct Biol 156(1):72-83.</ref>
The general mechanism for globular protein unfolding itself is not well characterized; however, it is not entirely independent of the amino acid sequence. Repeats of glycine and alanine have been shown to inhibit substrate unfolding and thus decrease the efficiency of proteasomal degradation, resulting in the release of partially degraded byproducts, possibly due to the decoupling of the ATP hydrolysis and unfolding steps.<ref name="Hoyt">Hoyt MA, Zich J, Takeuchi J, Zhang M, Govaerts C, Coffino P. (2006). Glycine-alanine repeats impair proper substrate unfolding by the proteasome. EMBO J 25(8):1720-9.</ref> Related sequences have been found in nature, for example in Epstein-Barr virus gene products, that stall the proteasome.<ref name="Zhang">Zhang M, Coffino P. (2004). Repeat sequence of Epstein-Barr virus-encoded nuclear antigen 1 protein interrupts proteasome substrate processing. J Biol Chem 279(10):8635-41.</ref>
[edit] Proteolysis
The β subunits of the 20S core particle perform the proteolytic degradation function of the proteasome via a threonine-dependent nucleophilic attack mechanism that is likely also dependent 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 determining product length is not fully characterized.<ref name="Voges">Voges D, Zwickl P, Baumeister W. (1999). The 26S proteasome: a molecular machine designed for controlled proteolysis. Annu Rev Biochem 68: 1015-1068</ref> Although the three catalytic β subunits share a common mechanism, they have slightly different substrate specificities considered chymotrypsin-like, trypsin-like, and peptidyl-glutamyl peptide-hydrolyzing (PHGH)-like; the variations in specificity are derived from 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.<ref name="Heinemeyer" />
Although the proteasome normally produces very short peptide fragments, in some cases its products are themselves biologically active and functional molecules. Certain transcription factors, including one component of the mammalian complex NF-kB, are synthesized as inactive precursors whose ubiquitination and subsequent proteasomal degradation converts them to their active form. Such activity requires the proteasome to cleave the substrate protein internally rather than processively degrading it from one terminus; it has been suggested that long loops on the target protein's surface serve as the proteasomal substrate and enter the central cavity while the remainder of the protein remains outside.<ref name="Rape">Rape M, Jentsch S. (2002). Taking a bite: proteasomal protein processing. Nat Cell Biol 4(5):E113-6. </ref> Similar effects have been observed in yeast proteins; this mechanism of selective degradation is known as regulated ubiquitin/proteasome dependent processing (RUP).<ref name="Rape2">Rape M, Jentsch S. (2004). Productive RUPture: activation of transcription factors by proteasomal processing. Biochim Biophys Acta 1695(1-3):209-13.</ref>
[edit] Ubiquitin-independent degradation
Although most proteasomal substrates must be ubiquitinated before being degraded, there are some exceptions to this general rule, especially when the proteasome plays a normal role in the post-translational processing of the protein. The proteasomal activation of NF-kB by processing p105 into p50 via internal proteolysis is one major example.<ref name="Rape" /> Some proteins, hypothesized to be inherently more unstable than most due to internal intrinsically unstructured regions,<ref name="Asher">Asher G, Reuven N, Shaul Y. (2006). 20S proteasomes and protein degradation "by default". Bioessays 28(8):844-9.</ref> are degraded in a ubiquitin-independent manner as part of their normal cellular life cycle. The most well-known example of a ubiquitin-independent proteasome substrate is the enzyme ornithine decarboxylase.<ref name="Zhang">Zhang M, Pickart CM, Coffino P. (2003). Determinants of proteasome recognition of ornithine decarboxylase, a ubiquitin-independent substrate. EMBO J 22(7):1488-96.</ref> Ubiquitin-independent mechanisms targeting certain key cell cycle regulators such as p53, which is also subject to ubiquitin-dependent degradation, has also been reported.<ref name="Asher2">Asher G, Shaul Y. (2005). p53 proteasomal degradation: poly-ubiquitination is not the whole story. Cell Cycle 4(8):1015-8.</ref> Finally, structurally abnormal, misfolded, or highly oxidized proteins are also subject to ubiquitin-independent and 19S-independent degradation under conditions of cellular stress.<ref name="Shringarpure">Shringarpure R, Grune T, Mehlhase J, Davies KJ. (2003). Ubiquitin conjugation is not required for the degradation of oxidized proteins by proteasome. J Biol Chem 278(1):311-8.</ref>
[edit] Evolution
The 20S proteasome is both ubiquitous and essential in eukaryotes. Some prokaryotes, including many archaea and the bacterial order Actinomycetales also share homologs of the 20S proteasome, while most bacteria possess heat shock genes hslV and hslU, whose gene products are a multimeric protease arranged in a two-layered ring and an ATPase.<ref name="Gille">Gille C, Goedel A, Schloetelburg C, Preißner R, Kloetzell PM, Gobel UB, Frommell C. (2003). A Comprehensive View on Proteasomal Sequences: Implications for the Evolution of the Proteasome. J Mol Biol 326: 1437–1448.</ref> The hslV protein has been hypothesized to resemble the likely ancestor of the 20S proteasome.<ref name="Bochtler">Bochtler, M., Ditzel, L., Groll, M., Hartmann, C., Huber, R. (1999). The proteasome. Annu. Rev. Biophys. Biomol. Struct. 28: 295–317.</ref> HslV is generally not essential in bacteria, and not all bacteria possess it, while some protists possess both 20S and hslV systems.<ref name="Gille" />
Sequence analysis suggests that the catalytic β subunits diverged earlier in evolution than the predominantly structural α subunits. In bacteria that express a 20S proteasome, the β subunits have high sequence identity to archaeal and eukaryotic β subunits, while the α sequence identity is much lower. The presence of 20S proteasomes in bacteria has been ascribed to lateral gene transfer, while the diversification of subunits among eukaryotes is ascribed to multiple gene duplication events.<ref name="Gille" />
[edit] Cell cycle control
Cell cycle progression is controlled by ordered action of cyclin-dependent kinases (CDKs), activated by specific cyclins that demarcate phases of the cell cycle. Mitotic cyclins, which persist in the cell for only a few minutes, have one of the shortest life spans of all intracellular proteins.<ref name="Lodish" /> After a CDK-cyclin complex has performed its function, the associated cyclin is polyubiquitinated and destroyed by the proteasome, which provides directionality for the cell cycle. In particular, exit from mitosis requires the proteasome-dependent dissociation of the regulatory component cyclin B from the mitosis promoting factor complex.<ref name="Chesnel">Chesnel F, Bazile F, Pascal A, Kubiak JZ. (2006). Cyclin B dissociation from CDK1 precedes its degradation upon MPF inactivation in mitotic extracts of Xenopus laevis embryos. Cell Cycle 5(15):1687-98.</ref> In vertebrate cells, "slippage" through the mitotic checkpoint leading to premature M phase exit can occur despite the delay of this exit by the spindle checkpoint.<ref name="Brito">Brito DA, Rieder CL. (2006). Mitotic checkpoint slippage in humans occurs via cyclin B destruction in the presence of an active checkpoint. Curr Biol 16(12):1194-200. </ref>
Earlier cell cycle checkpoints such a post-restriction point check between G1 phase and S phase similarly involve proteasomal degradation of cyclin A, whose ubiquitination is promoted by the anaphase promoting complex (APC), an E3 ubiquitin ligase.<ref name="Havens">Havens CG, Ho A, Yoshioka N, Dowdy SF. (2006). Regulation of late G1/S phase transition and APC Cdh1 by reactive oxygen species. Mol Cell Biol 26(12):4701-11.</ref> The APC and the Skp1/Cul1/F-box protein complex (SCF complex) are the two key regulators of cyclin degradation and checkpoint control; the SCF itself is regulated by the APC via ubiquitination of the Skp1 component, which prevents SCF activity before the G1-S transition.<ref name="Bashir">Bashir T, Dorrello NV, Amador V, Guardavaccaro D, Pagano M. (2004). Control of the SCF(Skp2-Cks1) ubiquitin ligase by the APC/C(Cdh1) ubiquitin ligase. Nature 428(6979):190-3.</ref>
[edit] Regulation of plant growth
In plants, signaling by auxins, or phytohormones that order the direction and tropism of plant growth, induces the targeting of a class of transcription factor repressors known as Aux/IAA proteins for proteasomal degradation. These proteins are ubiquitinated by SCFTIR1, or SCF in complex with the auxin receptor TIR1. Degradation of Aux/IAA proteins derepresses transcription factors in the auxin-response factor (ARF) family and induces ARF-directed gene expression.<ref name="Dharmasiri">Dharmasiri S, Estelle M. (2002). The role of regulated protein degradation in auxin response. Plant Mol Biol 49(3-4):401-9.</ref> The cellular consequences of ARF activation depend on the plant type and developmental stage, but are involved in directing growth in roots and leaf veins. The specific response to ARF derepression is thought to be mediated by specificity in the pairing of individual ARF and Aux/IAA proteins.<ref name="Weijers">Weijers D, Benkova E, Jager KE, Schlereth A, Hamann T, Kientz M, Wilmoth JC, Reed JW, Jurgens G. (2005). Developmental specificity of auxin response by pairs of ARF and Aux/IAA transcriptional regulators. EMBO J 24(10):1874-85.</ref>
[edit] Apoptosis
Both internal and external signals can lead to the induction of apoptosis, or programmed cell death. The resulting deconstruction of cellular components is primarily carried out by specialized proteases known as caspases, but the proteasome also plays important and diverse roles in the apoptotic process. The involvement of the proteasome in apoptotic proteolysis was first surmised from the increase in protein ubiquitination observed well in advance of the morphological changes that are diagnostic of apoptosis.<ref name="Schwartz">Schwartz LM, Myer A, Kosz L, Engelstein M and Maier C (1990) Activation of polyubiquitin gene expression during developmentally programmed cell death. Neuron 5: 411-419.</ref> Although apoptotic processes vary among organisms and cell types, the heightened expression of ATPase proteasome subunits<ref name="Low">Low P, Bussell K, Dawson SP, Billett MA, Mayer RJ and Reynolds SE (1997) Expression of a 26S proteasome ATPase subunit, MS73, in muscles that undergo developmentally programmed cell death, and its control by ecdysteroid hormones in the insect Manduca sexta. FEBS Lett. 400: 345-349.</ref> and of E1, E2, and E3 enzymes<ref name="Haas">Haas AL, Baboshina O, Williams B and Schwartz LM (1995) Coordinated induction of the ubiquitin conjugation pathway accompanies the developmentally programmed death of insect skeletal muscle. J. Biol. Chem. 270: 9407-9412.</ref> further supports the involvement of the proteasome in apoptosis at least in some cell types. During apoptosis, proteasomes localized to the nucleus have also been observed to translocate to outer membrane blebs characteristic of apoptosis.<ref name="Pitzer">Pitzer F, Dantes A, Fuchs T, BaumeisterWand Amsterdam A (1996) Removal of proteasomes from the nucleus and their accumulation in apoptotic blebs during programmed cell death. FEBS Lett. 394: 47-50.</ref>
Proteasome inhibition has different effects on apoptosis induction in different cell types. The proteasome is not generally required for apoptosis, because inhibiting it is pro-apoptotic in most cell types that have been studied. However, some cell lines - in particular, primary cultures of quiescent and differentiated cells such as thymocytes and neurons - are induced to undergo apoptosis on exposure to proteasome inhibitors. The mechanism for this effect is not clear, but is hypothesized to specifically relate to cells in quiescent states or to the differential activity of the pro-apoptotic kinase JNK.<ref name="Orlowski">Orlowski RZ. (1999). The role of the ubiquitin-proteasome pathway in apoptosis. Cell Death Differ 6: 303-313.</ref> The ability of proteasome inhibitors to induce apoptosis in rapidly dividing cells has been exploited in several recently developed chemotherapy agents.
[edit] Response to cellular stress
In response to cellular stress, such as infection, heat shock, or oxidative damage, heat shock proteins are expressed that identify misfolded or unfolded proteins and target them for proteasomal degradation. Both Hsp27 and Hsp90 - both chaperone proteins that aid in other proteins' folding and inhibit apoptosis - have been implicated in increasing the activity of the ubiquitin-proteasome system, though they are not direct participants in the process.<ref name="Garrido">Garrido C, Brunet M, Didelot C, Zermati Y, Schmitt E, Kroemer G. (2006). Heat Shock Proteins 27 and 70: Anti-Apoptotic Proteins with Tumorigenic Properties. Cell Cycle 5(22).</ref> Hsp70, on the other hand, specifically recognizes exposed hydrophobic patches on the surface of misfolded proteins and recruits E3 ubiquitin ligases such as CHIP to tag the proteins for proteasomal degradation.<ref name="Park">Park SH, Bolender N, Eisele F, Kostova Z, Takeuchi J, Coffino P, Wolf DH. (2006). The Cytoplasmic Hsp70 Chaperone Machinery Subjects Misfolded and ER Import Incompetent Proteins to Degradation via the Ubiquitin-Proteasome System. Mol Biol Cell Epub.</ref> The CHIP protein (carboxyl terminus of Hsp70-interacting protein) is itself regulated by BAG2 via inhibition of interactions between the E3 enzyme CHIP and its E2 binding partner.<ref name="Dai">Dai Q, Qian SB, Li HH, McDonough H, Borchers C, Huang D, Takayama S, Younger JM, Ren HY, Cyr DM, Patterson C. (2005). Regulation of the cytoplasmic quality control protein degradation pathway by BAG2. J Biol Chem 280(46):38673-81. </ref>
Similar mechanisms exist to promote the degradation of oxidatively damaged proteins via the proteasome system. In particular, proteasomes localized to the nucleus are regulated by PARP and actively degrade inappropriately oxidized histones.<ref name="Bader">Bader N, Grune T. (2006). Protein oxidation and proteolysis. Biol Chem 387(10-11):1351-5.</ref> Oxidized proteins, which often form large amorphous aggregates in the cell, can be degraded directly by the 20S core particle without the 19S regulatory cap and do not require ATP hydrolysis or tagging with ubiquitin.<ref name="Shringarpure" /> Increasing levels of oxidative damage increases the degree of cross-linking between protein fragments, rendering the aggregates increasingly resistant to proteolysis. Larger numbers and sizes of such highly oxidized aggregates are associated with aging.<ref name="Davies">Davies KJ. (2003). Degradation of oxidized proteins by the 20S proteasome. Biochimie 83(3-4):301-10.</ref>
Impaired proteasomal activity has been suggested as an explanation for some of the late-onset neurodegenerative diseases that share aggregation of misfolded proteins as a common feature, such as Parkinson's disease and Alzheimer's disease. In these diseases large insoluble aggregates of misfolded proteins can form and result in neurotoxicity through mechanisms that are not yet well understood. Decreased proteasome activity in has been suggested as a cause of aggregation and Lewy body formation in Parkinson's,<ref name="McNaught">McNaught KS, Jackson T, JnoBaptiste R, Kapustin A, Olanow CW. (2006). Proteasomal dysfunction in sporadic Parkinson's disease. Neurology 66(10 Suppl 4):S37-49.</ref> a hypothesis supported by the observation that yeast models are more susceptible to toxicity from alpha synuclein under conditions of low proteasome activity.<ref name="Sharma">Sharma N, Brandis KA, Herrera SK, Johnson BE, Vaidya T, Shrestha R, Debburman SK. (2006). Alpha-Synuclein budding yeast model: toxicity enhanced by impaired proteasome and oxidative stress. J Mol Neurosci 28(2):161-78.</ref>
[edit] Role in the immune system
The proteasome plays a straightforward but critical role in the function of the adaptive immune system. The peptide antigens displayed by the major histocompatibility complex class I (MHC) proteins on the surface of antigen-presenting cells are products of proteasomal degradation of proteins originated by the invading pathogen. Although constitutively expressed proteasomes can participate in this process, a specialized complex composed of proteins whose expression is induced by interferon gamma specifically promotes the production of degradation products of the optimal size and composition for MHC binding. These proteins whose expression increases during the immune response include the 11S regulatory particle, whose main known biological role is regulating the production of MHC ligands, and specialized β subunits called β1i, β2i, and β5i with altered substrate specificity. The complex formed with the specialized β subunits is known as the immunoproteasome.<ref name="Wang" />
MHC class I ligand binding is highly dependent on the composition of the ligand C-terminus, which is bound by hydrogen bonding and by close contacts with the "B pocket" on the MHC surface. Optimal residues for the C-terminal end are leucine and valine.<ref name="Sliz">Sliz P, Michielin O, Cerottini JC, Luescher I, Romero P, Karplus M, Wiley DC. (2001). Crystal structures of two closely related but antigenically distinct HLA-A2/melanocyte-melanoma tumor-antigen peptide complexes. J Immunol. 167:3276–3284.</ref><ref name="Davies">Davies MN, Hattotuwagama CK, Moss DS, Drew MG, Flower DR. (2006). Statistical deconvolution of enthalpic energetic contributions to MHC-peptide binding affinity. 'BMC Struct Biol 6:5.</ref> The N-terminal residues, particularly the second residue in the peptide, also play a key role in determining binding affinity.<ref name="Davies" /> The immunoproteasome complex generates the correct C-terminal tails; later processing of the products by interferon gamma-induced aminopeptidases trims the N-termini for optimal MHC ligand production.<ref name="Wang" />
Due to its role in generating activated form of NF-kB, an anti-apoptotic and pro-inflammatory regulator of cytokine expression, proteasomal activity has been linked to inflammatory and autoimmune diseases. Increased levels of proteasome activity, which correlate with disease activity, have been implicated in autoimmune diseases including systemic lupus erythematosus and rheumatoid arthritis.<ref name="Wang" />
[edit] Proteasome inhibitors
Image:Bortezomib.svg Proteasome inhibitors have been noted in cell culture for their effective anti-tumor activity, inducing apoptosis by disrupting the regulated degradation of pro-growth cell cycle proteins.<ref name="Adams">Adams J, Palombella VJ, Sausville EA, Johnson J, Destree A, Lazarus DD, Maas J, Pien CS, Prakash S, Elliott PJ. (1999). Proteasome inhibitors: a novel class of potent and effective antitumor agents. Cancer Res 59(11):2615-22.</ref> The mechanism of selectively inducing apoptosis in tumor cells has proven effective with the inhibitor bortezomib in animal models of pancreatic cancer<ref name="Shah">Shah SA, Potter MW, McDade TP, Ricciardi R, Perugini RA, Elliott PJ, Adams J, Callery MP. (2001). 26S proteasome inhibition induces apoptosis and limits growth of human pancreatic cancer. J Cell Biochem 82(1):110-22.</ref><ref name="Nawrocki">Nawrocki ST, Sweeney-Gotsch B, Takamori R, McConkey DJ. (2004). The proteasome inhibitor bortezomib enhances the activity of docetaxel in orthotopic human pancreatic tumor xenografts. Mol Cancer Ther 3(1):59-70.</ref> and with the inhibitor gemcitabine in models of certain types of non-small cell lung cancer.<ref name="Denlinger">Denlinger CE, Rundall BK, Keller MD, Jones DR. (2004). Proteasome inhibition sensitizes non-small-cell lung cancer to gemcitabine-induced apoptosis. Ann Thoracic Surg 78(4):1207-14.</ref> Both are currently in use as chemotherapy agents; bortezomib in particular has shown promise in the treatment of multiple myeloma.<ref name="Fisher">Fisher RI, Bernstein SH, Kahl BS, Djulbegovic B, Robertson MJ, de Vos S, Epner E, Krishnan A, Leonard JP, Lonial S, Stadtmauer EA, O'Connor OA, Shi H, Boral AL, Goy A. (2006). Multicenter phase II study of bortezomib in patients with relapsed or refractory mantle cell lymphoma. J Clin Oncol 24(30):4867-74.</ref> Notably, multiple myeloma has been observed to result in increased proteasome levels in blood serum that decrease to normal levels in response to successful chemotherapy.<ref name="Jakob">Jakob C, Egerer K, Liebisch P, Turkmen S, Zavrski I, Kuckelkorn U, Heider U, Kaiser M, Fleissner C, Sterz J, Kleeberg L, Feist E, Burmester GR, Kloetzel PM, Sezer O. (2006). Circulating proteasome levels are an independent prognostic factor for survival in multiple myeloma. Blood Epub.</ref>
Proteasome inhibitors have also shown promise in treating autoimmune diseases in animal models. For example, studies in mice bearing human skin grafts found a reduction in the size of lesions from psoriasis after treatment with a proteasome inhibitor.<ref name="Zollner">Zollner TM, Podda M, Pien C, Elliott PJ, Kaufmann R, Boehncke WH. (2002). Proteasome inhibition reduces superantigenmediated T cell activation and the severity of psoriasis in a SCID-hu model. J Clin Invest. 109:671-679.</ref> Inhibitors also show positive effects in rodent models of asthma.<ref name="Elliott">Elliott PJ, Pien CS, McCormack TA, Chapman ID, Adams J. (1999). Proteasome inhibition: A novel mechanism to combat asthma. J Allergy Clin Immunol. 104:294-300.</ref>
Labeling and inhibition of the proteasome is also of interest in laboratory settings for both in vitro and in vivo study of proteasomal activity in cells. The most commonly used laboratory inhibitor is lactacystin, a natural product synthesized by Streptomyces bacteria.<ref name="Orlowski" /> Fluorescent inhibitors have also been developed to specifically label the active sites of the assembled proteasome.<ref name="Verdoes">Verdoes M, Florea BI, Menendez-Benito V, Maynard CJ, Witte MD, van der Linden WA, van den Nieuwendijk AM, Hofmann T, Berkers CR, van Leeuwen FW, Groothuis TA, Leeuwenburgh MA, Ovaa H, Neefjes JJ, Filippov DV, van der Marel GA, Dantuma NP, Overkleeft HS. (2006). A fluorescent broad-spectrum proteasome inhibitor for labeling proteasomes in vitro and in vivo. Chem Biol 13(11):1217-26.</ref>
[edit] References
[edit] External links
- PLOS Primer: The Proteasome and the Delicate Balance between Destruction and Rescue
- The Yeast 26S Proteasome with list of subunits and pictures
es:Proteasoma fr:Protéasome ko:프로테아좀 it:Proteasoma ja:プロテアソーム pt:Proteassoma ru:Протеасома sl:Proteasom fi:Proteasomi pl:Proteasom
