Substrate specificity of the human proteasome

BACKGROUND: Regulated proteolysis by the proteasome is crucial for a broad array of cellular processes, from control of the cell cycle to production of antigens. RESULTS: The rules governing the N-terminal primary and extended substrate specificity of the human 20S proteasome in the presence or absence of 11S proteasome activators (REGalpha/beta and REGgamma) have been elaborated using activity-based proteomic library tools. CONCLUSIONS: The 11S proteasome activators are shown to be important for both increasing the activity of the 20S proteasome and for altering its cleavage pattern and substrate specificity. These data also establish that the extended substrate specificity is an important factor for proteasomal cleavage. The specificities observed have features in common with major histocompatibility complex (MHC) class I ligands and can be used to improve the prediction of MHC class I restricted cytotoxic T-cell responses.     

Substrate binding and sequence preference of the proteasome revealed by active-site-directed affinity probes

Background: The proteasome is a multicatalytic protease complex responsible for most cytosolic protein breakdown. The complex has several distinct proteolytic activities that are defined by the preference of each for the carboxyterminal (P1) amino acid residue. Although mutational studies in yeast have begun to define substrate specificities of individual catalytically active β subunits, little is known about the principles that govern substrate hydrolysis by the proteasome.Results: A series of tripeptide and tetrapeptide vinyl sulfones were used to study substrate binding and specificity of the proteasome. Removal of the aromatic amino-terminal cap of the potent tripeptide vinyl sulfone proteasome inhibitor 4-hydroxy-3-iodo-2-nitrophenyl-leucinyl-leucinyl-leucine vinyl sulfone resulted in the complete loss of binding and inhibition. Addition of a fourth amino acid (P4) to the tri-leucine core sequence fully restored inhibitory potency. 1251-labeled peptide vinyl sulfones were also used to examine inhibitor binding and to determine the correlation of subunit modification with inhibition of peptidase activity. Changing the amino acid in the P4 position resulted in dramatically different profiles of β-subunit modification.Conclusions: The P4 position, distal to the site of hydrolysis, is important in defining substrate processing by the proteasome. We observed direct correlations between subunit modification and inhibition of distinct proteolytic activities, allowing the assignment of activities to individual β subunits. The ability of tetrapeptides, but not tripeptide vinyl sulfones, to act as substrates for the proteasome suggests there could be a minimal length requirement for hydrolysis by the proteasome. These studies indicate that it is possible to generate inhibitors that are largely specific for individual β subunits of the proteasome by modulation of the P4 and carboxy-terminal vinyl sulfone moieties.     

Extended peptide-based inhibitors efficiently target the proteasome and reveal overlapping specificities of the catalytic beta-subunits

BACKGROUND: The 26S proteasome is responsible for most cytosolic proteolysis, and is an important protease in major histocompatibility complex class I-mediated antigen presentation. Constitutively expressed proteasomes from mammalian sources possess three distinct catalytically active species, beta1, beta2 and beta5, which are replaced in the gamma-interferon-inducible immunoproteasome by a different set of catalytic subunits, beta1i, beta2i and beta5i, respectively. Based on preferred cleavage of short fluorogenic peptide substrates, activities of the proteasome have been assigned to individual subunits and classified as 'chymotryptic-like' (beta5), 'tryptic-like' (beta2) and 'peptidyl-glutamyl peptide hydrolyzing' (beta1). Studies with protein substrates indicate a far more complicated, less strict cleavage preference. We reasoned that inhibitors of extended size would give insight into the extent of overlapping substrate specificity of the individual activities and subunits. RESULTS: A new class of proteasome inhibitors, considerably extended in comparison with the commonly used fluorescent substrates and peptide-based inhibitors, has been prepared. Application of the safety catch resin allowed the generation of the target compounds using a solid phase protocol. Evaluation of the new compounds revealed a set of highly potent proteasome inhibitors that target all individual active subunits with comparable affinity, unlike the other inhibitors described to date. Modification of the most active compound, adamantane-acetyl-(6-aminohexanoyl)(3)-(leucinyl)(3)-vinyl-(methyl)-sulfone (AdaAhx(3)L(3)VS), itself capable of proteasome inhibition in living cells, afforded a new set of radio- and affinity labels. CONCLUSIONS: N-terminal extension of peptide vinyl sulfones has a profound influence on both their efficiency and selectivity as proteasome inhibitors. Such extensions greatly enhance inhibition and largely obliterate selectivity towards the individual catalytic activities. We conclude that for the interaction with larger substrates, there appears to be less discrimination of different substrate sequences for the catalytic activities than is normally assumed based on the use of small peptide-based substrates and inhibitors. The compounds described here are readily accessible synthetically, and are more potent inhibitors in living cells than their shorter peptide vinyl sulfone counterparts.     

Probing structural determinants distal to the site of hydrolysis that control substrate specificity of the 20S proteasome

The 20S proteasome is a large multicomponent protease complex. Relatively little is known about the mechanisms that control substrate specificity of its multiple active sites. We present here the crystal structure at 2.95 A resolution of a beta2-selective inhibitor (MB1) bound to the yeast 20S proteasome core particle (CP). This structure is compared to the structure of the CP bound to a general inhibitor (MB2) that covalently modified all three (beta1, beta2, beta5) catalytic subunits. These two inhibitors differ only in their P3 and P4 residues, thereby highlighting binding interactions distal to the active site threonine that control absolute substrate specificity of the complex. Comparisons of the CP-bound structures of MB1, MB2, and the natural products epoxomycin and TMC-95A also provide information regarding general binding modes for several classes of proteasome inhibitors.     

Quantitative analysis of a ubiquitin‐dependent substrate using capillary electrophoresis with dual laser‐induced fluorescence

Protein degradation by the ubiquitin‐proteasome system (UPS) affects many biological processes. Inhibition of the proteasome has emerged as a potential therapeutic target for cancer treatment. In this study, we developed a method for monitoring the degradation and accumulation of UPS‐dependent substrates in cells using CE with dual LIF. We used a green fluorescent protein (GFP)‐fusion of the ubiquitin substrate ribophorin 1 (GFP‐RPN1) along with red fluorescent protein (RFP) as an internal control to normalize transfection efficiency. Determination of GFP‐RPN1 and RFP in cell lysates were performed in an untreated capillary (75 μm × 50 cm) and 100 mM Tris‐CHES buffer (pH 9.0) containing 10 mM SDS. GFP‐RPN1 and RFP fluorescence were detected at excitation wavelengths of 488 and 635 nm, and emission wavelengths of 520 and 675 nm, respectively, without any interference or crosstalk. The intensity of GFP‐RPN1 fluorescence was normalized to that of RFP. Additionally, the proposed approach was used successfully to detect the degradation of GFP‐RPN1 and evaluate proteasome inhibitors. These results show that the developed method is effective and promising for rapid and quantitative monitoring of UPS‐dependent substrates compared to the current common methods, such as immunoblotting and pulse chase assays.     

Deubiquitinating enzymes are IN/(trinsic to proteasome function)

Covalent conjugation of the ubiquitin tag to cellular proteins plays a central role in a number of processes, the most notable among them being degradation by the 26S proteasome. A fundamental property of this process is that ubiquitination, in contrast to subsequent degradation, is reversible due to a number of deubiquitinating enzymes that mediate the disassembly of ubiquitin-protein conjugates. The uniqueness of ubiquitin as a reversible tag necessitates mechanisms to guarantee its efficiency. Interestingly, some deubiquitinating enzymes are associated with the 26S proteasome itself. We include a brief overview of the key proteasome-associated deubiquitinating enzymes such as Rpn11/POH1, UCH37/Uch2, Ubp6/Usp14 and Doa4/Ubp4. We go on to discuss how these enzymes may contribute to, or possibly counteract, proteolysis by the proteasome. For example, cumulative evidence points to a partitioning of proteasome action between proteolysis and deubiquitination. On the one hand, inhibition of proteolysis promotes deubiquitination, while on the other hand, inhibition of deubiquitination can promote proteolysis. The plethora of deubiquitinating enzymes may serve as proof reading devices altering the equilibrium between these two processes and allowing for reversal of fortune at various stages of the process. To promote degradation over deubiquitination, certain polyubiquitin conformations could be stabilized or protected from deubiquitinating enzymes in order that they can serve as efficient targeting signals leading to the proteasome. We hypothesize that polvubiquitin chains could also serve as "timers": by slowing down chain disassembly, longer chains allow ample time for unfolding and proteolysis of the substrate.     

Alpha,beta-unsaturated beta-silyl imide substrates for catalytic, enantioselective conjugate additions: a total synthesis of (+)-lactacystin and the discovery of a new proteasome inhibitor

Chiral (salen)Al mu-oxo dimer 1 catalyzes the highly enantioselective conjugate addition of carbon-centered nucleophiles to alpha,beta-unsaturated silyl imides. Allyldimethylsilane-substituted imide 4 was identified as an optimal substrate, undergoing addition reactions with a variety of nitrile nucleophiles in high yield and enantiomeric excess. The silicon-containing products are synthetically useful chiral building blocks, as demonstrated by their application to an enantioselective total synthesis of the potent proteasome inhibitor (+)-lactacystin (2). Elaboration of lactam 5a to the natural product was effected in 12 steps and in 11% overall yield and proceeded through an unusual spiro beta-lactone intermediate (11). This compound was found to inhibit the chymotrypsin-like site of the 26S proteasome at similar levels to known inhibitor clasto-lactacystin beta-lactone (omuralide).     

Proteasome function in antigen presentation: immunoproteasome complexes, Peptide production, and interactions with viral proteins

Proteasomes are the major nonlysosomal protein degradation machinery in eukaryotic cells and they are largely responsible for the processing of antigens for presentation by the MHC class I pathway. This review concentrates on recent developments in the area of antigen processing. Specialized proteasomes called immunoproteasomes and an 11S regulator of proteasomes (PA28) are induced by interferon-gamma, but it is not entirely clear why changes in proteasome structure are beneficial for antigen presentation. Different proteasome complexes have distinct subcellular distributions and subtle differences in cleavage specificity. Thus it is likely that the efficiency of production of MHC class I binding peptides varies in different locations. Immunoproteasome subunits are enriched at the ER where TAP transports peptides for association with newly synthesized MHC class I molecules. There is recent evidence to suggest that antigen presentation from viral expression vectors, or from peptides that are either delivered by bacterial toxins or derived from signal peptides, require proteasome activity for generation of the correct C-terminus of the epitope. The correct N-terminus may be generated by recently identified ER associated aminopeptidases. A number of viral protein interactions with proteasome subunits have been reported and such interactions may interfere with host anti-viral defenses and also contribute to mechanisms of cell transformation.     

Indolo‐Phakellins as β5‐Specific Noncovalent Proteasome Inhibitors

The proteasome represents an invaluable target for the treatment of cancer and autoimmune disorders. The application of proteasome inhibitors, however, remains limited to blood cancers because their reactive headgroups and peptidic scaffolds convey unfavorable pharmacodynamic properties. Thus, the discovery of more drug‐like lead structures is indispensable. In this study, we present the first structure of the proteasome in complex with an indolo‐phakellin that exhibits a unique noncovalent binding mode unparalleled by all hitherto reported inhibitors. The natural product inspired pentacyclic alkaloid binds solely and specificially into the spacious S3 subpocket of the proteasomal β5 substrate binding channel, gaining major stabilization through halogen bonding with the protein backbone. The presented compound provides an ideal scaffold for the structure‐based design of subunit‐specific nonpeptidic proteasome‐blockers.     

Wrenches in the works: drug discovery targeting the SCF ubiquitin ligase and APC/C complexes

Recently, the ubiquitin proteasome system (UPS) has matured as a drug discovery arena, largely on the strength of the proven clinical activity of the proteasome inhibitor Velcade in multiple myeloma. Ubiquitin ligases tag cellular proteins, such as oncogenes and tumor suppressors, with ubiquitin. Once tagged, these proteins are degraded by the proteasome. The specificity of this degradation system for particular substrates lies with the E3 component of the ubiquitin ligase system (ubiquitin is transferred from an E1 enzyme to an E2 enzyme and finally, thanks to an E3 enzyme, directly to a specific substrate). The clinical effectiveness of Velcade (as it theoretically should inhibit the output of all ubiquitin ligases active in the cell simultaneously) suggests that modulating specific ubiquitin ligases could result in an even better therapeutic ratio. At present, the only ubiquitin ligase leads that have been reported inhibit the degradation of p53 by Mdm2, but these have not yet been developed into clinical therapeutics. In this review, we discuss the biological rationale, assays, genomics, proteomics and three-dimensional structures pertaining to key targets within the UPS (SCFSkp2 and APC/C) in order to assess their drug development potential. Publication history: Republished from Current BioData's Targeted Proteins database (TPdb; http://www.targetedproteinsdb.com).     

基于VHSE结构表征的蛋白酶体酶切位点预测及酶切特异性研究

泛素-蛋白酶体在真核生物的抗原呈递、细胞周期调控和转录因子激活等生理过程中发挥着极为重要的作用,其核心就是蛋白酶体对底物的选择性酶切作用,因此对选择性酶切位点的预测一直是计算生物学的一个重点研究内容.针对现有酶切位点预测方法的非线性和物理意义不明确等问题,借鉴定量构效关系研究方法,采用基于氨基酸物理化学性质的描述子——VHSE(Principal component score vector of hydrophobic,steric,and electronic properties)对收集的2650个MHC-I配体的源蛋白序列进行了结构表征,在此基础上利用支持向量机建立了蛋白酶体酶切位点的预测模型,其最优线性模型的灵敏度(Sensitivity)、特异性(Specificity)、接受者操作特征曲线下面积(area under receiver operatingcharacteristics curve,AUC)和马休斯相关系数(Matthews coefficient of correlation,MCC)分别为90.18%,69.63%,0.8797和0.6131.模型分析结果表明:影响酶切位点选择性的氨基酸性质由大到小依次为:疏水性、电性和立体特征;P9,P8,P4,P1,P3’,P4’和P5’位氨基酸对酶切位点的选择有重要影响,研究亦显示酶切位点上游P1位和下游P1’～P5’的"疏水势差"有利于蛋白酶体的切割作用.     

Downregulation of COP9 signalosome subunits differentially affects the CSN complex and target protein stability

Background  

The COP9 signalosome (CSN) is a conserved protein complex in eukaryotic cells consisting of eight subunits (CSN1 to CSN8). Recent data demonstrate that the CSN is a regulator of the ubiquitin (Ub) proteasome system (UPS). It controls substrate ubiquitination by cullin-RING Ub ligases (CRLs), a process that determines substrate specificity of the UPS. The intrinsic deneddylating activity localized to CSN5 as well as the associated kinases and deubiquitinating activity are involved in the regulatory function of CSN. The exact mechanisms are unclear. In this study we knocked down CSN1 (siCSN1), CSN3 (siCSN3) and CSN5 (siCSN5) by specific siRNA oligos permanently expressed in HeLa cells. The analysis and comparison of siRNA cells revealed differential impact of individual subunits on CSN structure and function.     

Chaperone activities of the 26S and 20S proteasome

The accumulation of misfolded or damaged proteins causes the failure of normal cell structure and functions necessary for growth and viability. To abort this adverse development, defective proteins must be rapidly repaired by molecular chaperones or destroyed by energy-dependent cytoplasmic proteases. A balance among these processes ultimately maintains cellular homeostasis. In eukaryotes, the 26S proteasome, a protease/chaperone complex, is a central component in the protein triage decision process. The 26S proteasome generally acts as a ubiquitination system, though it also selectively degrades structurally abnormal proteins in an ubiquitin-independent manner. In either case, all substrate proteins must undergo structural changes and stabilization necessary for their rapid degradation. It has, therefore, often been suggested that several chaperone functions are closely related to the stimulation of proteasomal degradation. This review summarizes recent discoveries pertaining to chaperone activities in the proteasomal degradation pathway, and to their regulation of protein breakdown mediated by the proteasome.     

The 11S proteasome activator: Isolation from mouse brain and the influence on peptide substrate hydrolysis of the 20S and 26S proteasomes

Purification of the 11S proteasome regulator from mouse brain was optimized; the subunit composition of the isolated protein was determined by Western blot. The dependency of 20S proteasome peptidase activity on the molar concentration of the 11S regulator was examined. The Michaelis constants of hydrolysis of the specific fluorescent substrates Suc–Leu–Leu–Val–Tyr–AMC, Ac–Arg–Leu–Arg–AMC, and Z–Leu–Leu–Glu–AMC by the 20S proteasome from BALB/c mouse brain and the 20S–11S complex were determined. It was shown that the 11S particle has almost no influence on binding of specific fluorescent substrates to the 20S proteasome, but strongly accelerates hydrolysis of all three substrates, while not affecting the rate of peptide substrate hydrolysis by the 26S proteasome.     

Cationic porphyrins are reversible proteasome inhibitors

The aim of this study is to verify if water-soluble porphyrins can be used as proteasome inhibitors. We have found that cationic porphyrins inhibit proteasome peptidase activities much more effectively than the corresponding anionic derivatives. The relevance of electrostatics in driving porphyin-proteasome interactions has been confirmed by the observation that the inhibitory efficiency of the cationic macrocycles decreases with the number of positive substituents. We have also investigated various metalloporphyrins, which differ due to the different propension of the central metal ion toward axial coordination. Our experimental results indicate that the naked cationic porphyrins are the most active in reversibly inhibiting the three main protease activities of the proteasome in the micromolar range. A spectroscopic characterization of porphyrin-proteasome interactions by UV-vis spectra parallels the results of inhibition assays: the higher the inhibitory effect the stronger the spectroscopic variations are. To interpret the action of porphyrins at a molecular level, we have performed calculations evidencing that cationic porphyrins may hinder the access to the canonical proteolytic site on the proteasome β5 subunit. In particular, an inspection of the top-scoring docking modes shows that the tetracationic porphyrin blocks the catalytic pocket, close to the N termini of the β5 proteasome subunit, more efficiently than its anionic counterpart. Proteasome inhibition activity of porphyrins unites their known anticancer properties making them suitable as a scaffold for the design of novel multitargeted molecules.     

Specificity of the proteasome cleavage to the antigen protein

In the MHC classⅠmolecule binding antigenic peptides processing and presentation pathway,the ubiquitin-proteasome system plays a key role in degrading the protein substrate.For the purpose of studying the specificities of proteasomal cleavage sites,partial least squares method is used to predict the proteasomal cleavage sites,and the predictive accuracy of the model is 82.8%.The specificities of the cleavage sites and the adjacent positions come from the contribution of the amino acids of the samples to the...     

Electrophoretic analysis of phosphorylation of the yeast 20S proteasome

The 26S proteasome complex, consisting of two multisubunit complexes, a 20S proteasome and a pair of 19S regulatory particles, plays a major role in the nonlysosomal degradation of intracellular proteins. The 20S proteasome was purified from yeast and separated by two-dimensional gel electrophoresis (2-DE). A total of 18 spots separated by 2-DE were identified as the 20S proteasome subunits by peptide mass fingerprinting with matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS). The alpha2-, alpha4- and alpha7-subunits gave multiple spots, which converged into one spot for each subunit when treated with alkaline phosphatase. The difference of pI between phosphorylated and dephosphorylated spots and their reaction against anti-phosphotyrosine antibody suggested that the alpha2- and alpha4-subunits are phosphorylated either at Ser or at Thr residue, and the alpha7-subunit is phosphorylated at Tyr residue(s). These phosphorylated subunits were analyzed by electrospray ionization-quadrupole time of flight-tandem MS (ESI-QTOF-MS/MS) to deduce the phosphorylation sites. The 20S proteasome has three different protease activities: chymotrypsin-like, trypsin-like and peptidylglutamyl peptide-hydrolyzing activities. The phosphatase treatment increased K(m) value for chymotrypsin-like activity of the 20S proteasome, indicating that phosphorylation may play an important role in regulating the proteasome activity.     

金诺芬衍生物的合成及抗肿瘤活性

设计合成了一类新型金诺芬衍生物, 采用核磁共振波谱(NMR)和高分辨质谱(HRMS)确认了其结构. 以目标化合物处理肿瘤细胞后, 采用四氮唑蓝盐(MTS)法检测细胞增殖情况, 用流式细胞仪检测细胞凋亡情况, 采用蛋白质免疫印迹(Western blot)法检测总的泛素化蛋白(Ub-Prs)、 K48 位链接多聚泛素化蛋白以及蛋白酶体外源性特异性底物(GFPu)的表达情况. 结果表明, 金诺芬衍生物可通过抑制蛋白酶体功能来发挥抗肿瘤效果.     

Specific inhibition of the chymotrypsin-like activity of the proteasome induces a bipolar morphology in neuroblastoma cells

Background: Lactacystin inhibits cell proliferation and induces a distinctive, predominantly bipolar (two-neurite-bearing) morphology in Neuro 2A murine neuroblastoma cells. It binds with high specificity to the multicatalytic 20S proteasome and inhibits at least three of its peptidase activities (chymotrypsin-like, trypsin-like and peptidylglutamyl-peptide hydrolyzing), each at a different rate, without inhibiting other known proteases. The chymotrypsin-like and trypsin-like activities of the proteasome are inhibited most rapidly, and irreversibly. In an effort to determine which of the peptidase activities needs to be inhibited for neurite outgrowth to occur, we treated Neuro 2A cells with peptide aldehydes that selectively inhibit different proteasome activities.Results: Treatment with peptide aldehydes ending in a hydrophobic residue, all of which inhibit the chymotrypsin-like activity, results in a bipolar morphology in Neuro 2A cells, whereas treatment with a peptide aidehyde inhibitor of the trypsin-like activity does not lead to a detectable change in morphology. One of the inhibitors that induces neurite outgrowth has been previously shown to inhibit the chymotrypsin-like activity of the proteasome without inhibiting the other apparently distinct peptidase activities that cleave after neutral residues, the so-called ‘branched chain amino acid preferring’ (BrAAP) and ‘small neutral amino acid preferring’ (SNAAP) activities, or the peptidylglutamyl-peptide hydrolyzing (PGPH) activity.Conclusions: The chymotrypsin-like activity appears to antagonize bipolar-type neurite outgrowth in Neuro 2A cells, while the trypsin-like, PGPH, BrAAP and SNAAP appear not to do so. Selective inhibition of a single peptidase activity, as opposed to general inhibition of the proteasome, appears sufficient to induce a specific cellular process. Selective inhibition might be useful in managing diseases where only one activity is involved without completely inhibiting the proteasome. It is also possible that endogenous regulators of the proteasome could affect cellular processes and that certain peptidase activities of the proteasome may have roles in specifying a given cell fate,