Cell type-specific proteasomal processing of HIV-1 Gag-p24 results in an altered epitope repertoire

Proteasomes are critical for the processing of antigens for presentation through the major histocompatibility complex (MHC) class I pathway. HIV-1 Gag protein is a component of several experimental HIV-1 vaccines. Therefore, understanding the processing of HIV-1 Gag protein and the resulting epitope repertoire is essential. Purified proteasomes from mature dendritic cells (DC) and activated CD4(+) T cells from the same volunteer were used to cleave full-length Gag-p24 protein, and the resulting peptide fragments were identified by mass spectrometry. Distinct proteasomal degradation patterns and peptide fragments were unique to either mature DC or activated CD4(+) T cells. Almost half of the peptides generated were cell type specific. Two additional differences were observed in the peptides identified from the two cell types. These were in the HLA-B35-Px epitope and the HLA-B27-KK10 epitope. These epitopes have been linked to HIV-1 disease progression. Our results suggest that the source of generation of precursor MHC class I epitopes may be a critical factor for the induction of relevant epitope-specific cytotoxic T cells.     

Eukaryotic proteasomes cannot digest polyglutamine sequences and release them during degradation of polyglutamine-containing proteins

Long glutamine sequences (polyQ) occur in many cell proteins, and several neurodegenerative diseases result from expansion of these sequences. PolyQ-containing proteins are degraded by proteasomes, whose three active sites prefer to cleave after hydrophobic, basic, or acidic residues. We tested whether these particles can digest a polyQ chain. Eukaryotic 26S and 20S proteasomes failed to cut within stretches of 9-29Q residues in peptides. While digesting a myoglobin Q(35) fusion protein, the proteasomes spared the polyQ sequence. In contrast, archaeal proteasomes, whose 14 active sites are less specific, rapidly digested such polyQ repeats. Therefore, when degrading polyQ proteins, eukaryotic proteasomes must release aggregation-prone polyQ-containing fragments for further hydrolysis by unidentified peptidases. In polyQ diseases, such polyQ sequences (38-300Qs) exceed the lengths of normal proteasome products (2-25 residues). Occasional failure of these long undegradable sequences to exit may interfere with proteasome function and help explain why longer polyQ expansions promote early disease onset.     

Adrm1, a putative cell adhesion regulating protein, is a novel proteasome-associated factor

We have identified Adrm1 as a novel component of the regulatory ATPase complex of the 26 S proteasome: Adrm1 was precipitated with an antibody to proteasomes and vice versa. Adrm1 co-migrated with proteasomes on gel-filtration chromatography and non-denaturing polyacrylamide gel electrophoresis. Adrm1 has been described as an interferon-gamma-inducible, heavily glycosylated membrane protein of 110 kDa. However, we found Adrm1 in mouse tissues only as a 42 kDa peptide, corresponding to the mass of the non-glycosylated peptide chain, and it could not be induced in HeLa cells with interferon. Adrm1 was present almost exclusively in soluble 26 S proteasomes, albeit a small fraction was membrane-associated, like proteasomes. Adrm1 was found in cells in amounts equimolar with S6a, a 26 S proteasome subunit. HeLa cells contain no pool of free Adrm1 but recombinant Adrm1 could bind to pre-existing 26 S proteasomes in cell extracts. Adrm1 may be distantly related to the yeast proteasome subunit Rpn13, mutants of which are reported to display no obvious phenotype. Accordingly, knock-down of Adrm1 in HeLa cells had no effect on the amount of proteasomes, or on degradation of bulk cell protein, or accumulation of polyubiquitinylated proteins. This indicates that Adrm1 has a specialised role in proteasome function.     

Proteasomes Can Degrade a Significant Proportion of Cellular Proteins Independent of Ubiquitination

The critical role of the ubiquitin-26S proteasome system in regulation of protein homeostasis in eukaryotes is well established. In contrast, the impact of the ubiquitin-independent proteolytic activity of proteasomes is poorly understood. Through biochemical analysis of mammalian lysates, we find that the 20S proteasome, latent in peptide hydrolysis, specifically cleaves more than 20% of all cellular proteins. Thirty intrinsic proteasome substrates (IPSs) were identified and in vitro studies of their processing revealed that cleavage occurs at disordered regions, generating stable products encompassing structured domains. The mechanism of IPS recognition is remarkably well conserved in the eukaryotic kingdom, as mammalian and yeast 20S proteasomes exhibit the same target specificity. Further, 26S proteasomes specifically recognize and cleave IPSs at similar sites, independent of ubiquitination, suggesting that disordered regions likely constitute the universal structural signal for IPS proteolysis by proteasomes. Finally, we show that proteasomes contribute to physiological regulation of IPS levels in living cells and the inactivation of ubiquitin-activating enzyme E1 does not prevent IPS degradation. Collectively, these findings suggest a significant contribution of the ubiquitin-independent proteasome degradation pathway to the regulation of protein homeostasis in eukaryotes.     

Thiostrepton interacts covalently with Rpt subunits of the 19S proteasome and proteasome substrates

Here, we report a novel mechanism of proteasome inhibition mediated by Thiostrepton (Thsp), which interacts covalently with Rpt subunits of the 19S proteasome and proteasome substrates. We identified Thsp in a cell‐based high‐throughput screen using a fluorescent reporter sensitive to degradation by the ubiquitin–proteasome pathway. Thiostrepton behaves as a proteasome inhibitor in several paradigms, including cell‐based reporters, detection of global ubiquitination status, and proteasome‐mediated labile protein degradation. In vitro, Thsp does not block the chymotrypsin activity of the 26S proteasome. In a cell‐based IκBα degradation assay, Thsp is a slow inhibitor and 4 hrs of treatment achieves the same effects as MG‐132 at 30 min. We show that Thsp forms covalent adducts with proteins in human cells and demonstrate their nature by mass spectrometry. Furthermore, the ability of Thsp to interact covalently with the cysteine residues is essential for its proteasome inhibitory function. We further show that a Thsp modified peptide cannot be degraded by proteasomes in vitro. Importantly, we demonstrate that Thsp binds covalently to Rpt subunits of the 19S regulatory particle and forms bridges with a proteasome substrate. Taken together, our results uncover an important role of Thsp in 19S proteasome inhibition.     

Assessment of proteasomal cleavage probabilities from kinetic analysis of time-dependent product formation

Proteasomes are multicatalytic cellular protease complexes that degrade intracellular proteins into smaller peptides. Proteasomal in vitro digests have revealed that the various peptide bonds of a given substrate are cleaved in a highly selective manner. Regarding the key role of proteasomes as the main supplier of antigenic peptides for MHC class I-mediated antigen presentation, it is important to know to what extent these preferences for specific peptide bonds may vary among proteasomes of different cellular origin and of different subunit composition. Here, we quantify such cleavage rates by means of a kinetic proteasome model that relates the time-dependent changes of the amount of any generated peptide to the rates with which this peptide can be either generated from longer precursor peptides or degraded into smaller successor peptides. Numerical values for these rates are estimated by minimizing the distance between simulated and measured time-courses. The proposed method is applied to kinetic data obtained by combining HPLC fractionation and mass spectrometry (MS) to trace the degradation of two model peptides (pp89-25mer and LLO-27mer) by either the constitutive (T2) or immunoproteasome (T2.27). To convert the intensity of the MS signals into the respective peptide amounts, we use two methods leading to similar results: experimental calibration curves and theoretically determined linear scaling functions based on a novel approach using mass conservation rules. Comparison of the cleavage probabilities and procession rates obtained for the two types of proteasomes reveals that the striking differences between the time-dependent peptide profiles can be accounted for mainly by a generally higher turnover rate of the immunoproteasome. For the pp89-25mer, there is no significant change of the cleavage probabilities for any of the ten observed cleavage sites. For the LLO-27mer, there appears to be a significant change in the cleavage probabilities for four of the nine observed cleavage sites when switching from the constitutive to the immunoproteasome.     

Antitopes define preferential proteasomal cleavage site usage

Protein degradation by proteasomes is a major source of peptides presented by major histocompatibility v complex class I proteins. Importantly, interferon gamma-induced immunoproteasomes in many cases strongly enhance the generation of antigenic peptides both in vitro and in vivo. Whether this is due to enhanced substrate turnover or to a change in proteasomal cleavage specificity is, however, largely unresolved. To overcome the problems of peptide quantification inherent to mass spectrometry, we introduced the "antitope" as substrate-specific internal standard. The antitope is a non-functional peptide that is generated by proteasomal cleavage within the epitope, resulting in partial overlaps with the functional epitope. Using antitopes as internal standards we demonstrate that the observed enhanced immunoproteasome-dependent presentation of the bacterial listeriolysin O T-cell epitope LLO(296-304) is indeed due to altered cleavage preferences. This method is also applicable to other major histocompatibility class I epitopes as is shown for two potential epitopes derived from Coxsackievirus.     

Isolation of human proteasomes and putative proteasome-interacting proteins using a novel affinity chromatography method

The proteasome is the primary subcellular organelle responsible for protein degradation. It is a dynamic assemblage of 34 core subunits and many differentially expressed, transiently interacting, modulatory proteins. This paper describes a novel affinity chromatography method for the purification of functional human holoproteasome complexes using mild conditions. Human proteasomes purified by this simple procedure maintained the ability to proteolytically process synthetic peptide substrates and degrade ubiquitinated parkin. Furthermore, the entire purification fraction was analyzed by mass spectrometry in order to identify proteasomal proteins and putative proteasome-interacting proteins. The mild purification conditions maintained transient physical interactions between holoproteasomes and a number of known modulatory proteins. In addition, several classes of putative interacting proteins co-purified with the proteasomes, including proteins with a role in the ubiquitin proteasome system for protein degradation or DNA repair. These results demonstrate the efficacy of using this affinity purification strategy for isolating functional human proteasomes and identifying proteins that may physically interact with human proteasomes.     

Expression of antihypertensive peptide, His-His-Leu, as tandem repeats in Escherichia coli

His-His-Leu (HHL), a tripeptide derived from a Korean soybean paste, is an angiotensin-I-converting enzyme (ACE) inhibitor. We report here a method of producing this tripeptide efficiently by expressing tandem multimers of the codons encoding the peptide in E. coli and purifying the HHL after hydrolysis of the peptide multiners. The HHL gene, tandemly multimerized to a 40-mer, was ligated with ubiquitin as a fusion gene (UH40). UH40 was inserted into vector pET29b; the UH40 fusion protein was then produced in E. coli BL21. The recombinant UH40 protein was purified by cation-exchange chromatography with a yield of 17.3 mg/l and analyzed by matrix-assisted laser desorption ionization (MALDI) time-of-flight (TOF) mass spectrometry and protein N-terminal sequencing. Leucine aminopeptidase was used to cleave a 405-Da HHL monomer from the UH40 fusion protein and the peptide was purified using reverse-phase high-performance liquid chromatography (HPLC) on a C18 HPLC column, with a final yield of 6.2 mg/l. The resulting peptide was confirmed to be HHL with the aid of MALDI-TOF mass spectrometry, glutamine-TOF mass spectrometry, N-terminal sequencing, and measurement of ACE inhibiting activity. These results suggest that our production method is useful for obtaining a large quantity of recombinant HHL for functional antihypertensive peptide studies.     

Altered -3 substrate specificity of Escherichia coli signal peptidase 1 mutants as revealed by screening a combinatorial peptide library

Signal peptidase functions to cleave signal peptides from preproteins at the cell membrane. It has a substrate specificity for small uncharged residues at -1 (P1) and aliphatic residues at the -3 (P3) position. Previously, we have reported that certain alterations of the Ile-144 and Ile-86 residues in Escherichia coli signal peptidase I (SPase) can change the specificity such that signal peptidase is able to cleave pro-OmpA nuclease A in vitro after phenylalanine or asparagine residues at the -1 position (Karla, A., Lively, M. O., Paetzel, M. and Dalbey, R. (2005) J. Biol. Chem. 280, 6731-6741). In this study, screening of a fluorescence resonance energy transfer-based peptide library revealed that the I144A, I144C, and I144C/I86T SPase mutants have a more relaxed substrate specificity at the -3 position, in comparison to the wild-type SPase. The double mutant tolerated arginine, glutamine, and tyrosine residues at the -3 position of the substrate. The altered specificity of the I144C/I86T mutant was confirmed by in vivo processing of pre-beta-lactamase containing non-canonical arginine and glutamine residues at the -3 position. This work establishes Ile-144 and Ile-86 as key P3 substrate specificity determinants for signal peptidase I and demonstrates the power of the fluorescence resonance energy transfer-based peptide library approach in defining the substrate specificity of proteases.     

Different substrate recognition requirements for cleavage of synaptobrevin-2 by Clostridium baratii and Clostridium botulinum type F neurotoxins

Botulinum neurotoxins (BoNTs) cause botulism, which can be fatal if it is untreated. BoNTs cleave proteins necessary for nerve transmission, resulting in paralysis. The in vivo protein target has been reported for all seven serotypes of BoNT, i.e., serotypes A to G. Knowledge of the cleavage sites has led to the development of several assays to detect BoNT based on its ability to cleave a peptide substrate derived from its in vivo protein target. Most serotypes of BoNT can be subdivided into subtypes, and previously, we demonstrated that three of the currently known subtypes of BoNT/F cleave a peptide substrate, a shortened version of synaptobrevin-2, between Q58 and K59. However, our research indicated that Clostridium baratii type F toxin did not cleave this peptide. In this study, we detail experiments demonstrating that Clostridium baratii type F toxin cleaves recombinant synaptobrevin-2 in the same location as that cleaved by proteolytic F toxin. In addition, we demonstrate that Clostridium baratii type F toxin can cleave a peptide substrate based on the sequence of synaptobrevin-2. This peptide substrate is an N-terminal extension of the original peptide substrate used for detection of other BoNT/F toxins and can be used to detect four of the currently known BoNT/F subtypes by mass spectrometry.     

Early degradation of paternal mitochondria in domestic pig (Sus scrofa) is prevented by selective proteasomal inhibitors lactacystin and MG132

Ubiquitin-dependent proteolysis has been implicated in the recognition and selective elimination of paternal mitochondria and mitochondrial DNA (mtDNA) after fertilization in mammals. Initial evidence suggests that this process is contributed to by lysosomal degradation of the ubiquitinated sperm mitochondrial membrane proteins. The present study examined the role of the proteasome-dependent protein degradation pathway of the ubiquitin system, as opposed to lysosomal proteolysis of the ubiquitinated proteins, in the regulation of sperm mitochondrion elimination after fertilization. Boar spermatozoa prelabeled with vital fluorescent mitochondrial probes MitoTracker were used to trace the degradation of paternal mitochondria after in vitro fertilization (IVF) of porcine oocytes. The degradation of sperm mitochondria in the cytoplasm of fertilized oocytes started very rapidly, i.e., within 12-20 h after insemination. Four stages of paternal mitochondrial degradation were distinguished, ranging from an intact mitochondrial sheath (type 1) to complete degradation (type 4). At 27-30 h postinsemination, 96% of zygotes contained the partially (type 3) or completely (type 4) degraded sperm mitochondria. Highly specific peptide inhibitors of the ubiquitin-proteasome pathway, lactacystin (10 and 100 microM) and MG132 (10 microM), efficiently blocked the degradation of the sperm mitochondria inside the fertilized egg when applied 6 h after insemination. Using 10 microM MG132, only 13.6% of fertilized oocytes screened 27-30 h after IVF displayed type 3 sperm mitochondria, and there was no incidence of type 4, completely degraded mitochondria. Although lactacystin is not a reversible agent, the effect of MG132 was fully reversible: zygotes transferred to regular culture medium after 24 h of culture with 10 microM MG132 resumed development and degraded sperm mitochondria within the next cell cycle. Surprisingly, penetration of the zona pellucida (ZP) was also inhibited by MG-132 and lactacystin when the inhibitors were added at insemination. Altogether, these data provide the first evidence of the participation of proteasomes in the control of mammalian mitochondrial inheritance and suggest a new role of the ubiquitin-proteasome pathway in mammalian fertilization.     

The caspase-like sites of proteasomes,their substrate specificity,new inhibitors and substrates,and allosteric interactions with the trypsin-like sites

Proteasomes are the primary sites for protein degradation in mammalian cells. Each proteasome particle contains two chymotrypsin-like, two trypsin-like, and two caspase-like proteolytic sites. Previous studies suggest a complex network of allosteric interactions between these catalytic and multiple regulatory sites. We used positional scanning combinatorial substrate libraries to determine the extended substrate specificity of the caspase-like sites. Based on this analysis, several new substrates were synthesized, the use of which confirmed earlier observations that caspase-like sites (often termed postglutamyl peptide hydrolase) cleave after aspartates better than after glutamates. Highly selective inhibitors of the caspase-like sites were also generated. They stimulated trypsin-like activity of yeast 20 S proteasomes up to 3-fold but not when binding of the inhibitor to the caspase-like sites was prevented in a mutant carrying an uncleaved propeptide. Although substrates of the caspase-like sites allosterically inhibit the chymotrypsin-like activity, inhibitors of the caspase-like sites do not affect the chymotrypsin-like sites. Furthermore, when caspase-like sites were occupied by the uncleaved propeptide or inhibitor, their substrates still inhibited the chymotrypsin-like activity. Thus, occupancy of the caspase-like sites stimulates the trypsin-like activity of proteasomes, but substrates of the caspase-like sites inhibit the chymotrypsin-like activity by binding to a distinct noncatalytic site.     

Determination of disulfide bridges in natural and recombinant insect defensin A

The primary-structure comparison of natural insect defensin A from Phormia terranovae and recombinant insect defensin A from Saccharomyces cerevisiae has been accomplished using a combination of Edman degradation and liquid secondary ion mass spectrometry. The natural and recombinant proteins have the same primary structure with identical disulfide-bond designations (formula; see text) as determined from the peptides obtained after thermolysin digestion. The combined use of Edman degradation and mass spectometry allowed the disulfide-bridge structure to be determined with a total of only 40 micrograms (9.9 nmol) natural peptide. Mass spectrometry provides a rapid means of disulfide-bridge verification, requiring not more than 20 micrograms recombinant insect defensin A, which is compatible with use in batch analysis.     

A truncated form of α-tubulin detected in purified proteasome complexes

The 26S proteasome is a multisubunit protein complex responsible for selective protein degradation in the cell. A number of proteins with known and unknown functions were shown to be permanently or temporarily associated with 26S proteasomes. Identification of proteins that interact with proteasomes is an important step in the understanding of the proteasome functions in the cell and the mechanisms of their regulation. Using MALDI–ICR mass spectrometry, we have shown that some proteins of the cytoskeleton, such as actin, α-actinin 4, and α- and β-tubulins are associated with proteasomes obtained by affinity purification from the human myelogenous leukemia cell line K562. Western blot analysis showed that a truncated form of α-tubulin was associated with the purified proteasomes. The presence of the α-tubulin isoform in complex with affinity purified proteasomes was also observed in the human embryonic kidney cell line 293.     

Tissue transglutaminase-mediated glutamine deamidation of beta-amyloid peptide increases peptide solubility, whereas enzymatic cross-linking and peptide fragmentation may serve as molecular triggers for rapid peptide aggregation

Tissue transglutaminase (TGase) has been implicated in a number of cellular processes and disease states, where the enzymatic actions of TGase may serve in both, cell survival and apoptosis. To date, the precise functional properties of TGase in cell survival or cell death mechanisms still remain elusive. TGase-mediated cross-linking has been reported to account for the formation of insoluble lesions in conformational diseases. We report here that TGase induces intramolecular cross-linking of β-amyloid peptide (Aβ), resulting in structural changes of monomeric Aβ. Using high resolution mass spectrometry (MS) of cross-linked Aβ peptides, we observed a shift in mass, which is, presumably associated with the loss of NH3 due to enzymatic transamidation activity and hence intramolecular peptide cross-linking. We have observed that a large population of Aβ monomers contained an 0.984 Da increase in mass at a glutamine residue, indicating that glutamine 15 serves as an indispensable substrate in TGase-mediated deamidation to glutamate 15. We provide strong analytical evidence on TGase-mediated Aβ peptide dimerization, through covalent intermolecular cross-linking and hence the formation of Aβ1-40 dimers. Our in depth analyses indicate that TGase-induced post-translational modifications of Aβ peptide may serve as an important seed for aggregation.     

Phosphorylation of ATPase subunits of the 26S proteasome

The 26S proteasome complex plays a major role in the non-lysosomal degradation of intracellular proteins. Purified 26S proteasomes give a pattern of more than 40 spots on 2D-PAGE gels. The positions of subunits have been identified by mass spectrometry of tryptic peptides and by immunoblotting with subunit-specific antipeptide antibodies. Two-dimensional polyacrylamide gel electrophoresis of proteasomes immunoprecipitated from [³²P]phosphate-labelled human embryo lung L-132 cells revealed the presence of at least three major phosphorylated polypeptides among the regulatory subunits as well as the C8 and C9 components of the core 20S proteasome. Comparison with the positions of the regulatory polypeptides revealed a minor phosphorylated form to be S7 (MSS1). Antibodies against S4, S6 (TBP7) and S12 (MOV34) all cross-reacted at the position of major phosphorylated polypeptides suggesting that several of the ATPase subunits may be phosphorylated. The phosphorylation of S4 was confirmed by double immunoprecipitation experiments in which 26S proteasomes were immunoprecipitated as above and dissociated and then S4 was immunoprecipitated with subunit-specific antibodies. Antibodies against the non-ATPase subunit S10, which has been suggested by others to be phosphorylated, did not coincide with the position of a phosphorylated polypeptide. Some differences were observed in the 2D-PAGE pattern of proteasomes immunoprecipitated from cultured cells compared to purified rat liver 26S proteasomes suggesting possible differences in subunit compositions of 26S proteasomes.     

Functional analysis of the proteasome regulatory particle

We have developed S. cerevisiae as a model system for mechanistic studies of the 26S proteasome. The subunits of the yeast 19S complex, or regulatory particle (RP), have been defined, and are closely related to those of mammalian proteasomes. The multiubiquitin chain binding subunit (S5a/Mcb1/Rpn10) was found, surprisingly, to be nonessential for the degradation of a variety of ubiquitin-protein conjugates in vivo. Biochemical studies of proteasomes from rpn10 mutants revealed the existence of two structural subassemblies within the RP, the lid and the base. The lid and the base are both composed of 8 subunits. By electron microscopy, the base and the lid correspond to the proximal and distal masses of the RP, respectively. The base is sufficient to activate the 20S core particle for degradation of peptides, but the lid is required for ubiquitin-dependent degradation. The lid subunits share sequence motifs with components of the COP9/signalosome complex, suggesting that these functionally diverse particles have a common evolutionary ancestry. Analysis of equivalent point mutations in the six ATPases of the base indicate that they have well-differentiated functions. In particular, mutations in one ATPase gene, RPT2, result in an unexpected defect in peptide hydrolysis by the core particle. One interpretation of this result is that Rpt2 participates in gating of the channel through which substrates enter the core particle.