Eukaryotic Proteases Research Articles

Modulation of PC1/3 Activity by Self-Interaction and Substrate Binding

Prohormone convertase (PC)1/3 is a eukaryotic serine protease in the subtilase family that participates in the proteolytic maturation of prohormone and neuropeptide precursors such as proinsulin and proopiomelanocortin. Despite the important role of this enzyme in peptide synthesis, how PC1/3 activity is regulated is still poorly understood. Using ion exchange chromatography and two-dimensional gel electrophoresis we found that natural PC1/3 present in AtT-20 cells and bovine chromaffin granules, as well as recombinant PC1/3 secreted from overexpressing Chinese hamster ovary cells, exists as multiple ionic forms. Gel filtration and cross-linking studies revealed that protein oligomerization and aggregation contribute greatly to variability in surface charge. The most acidic forms of PC1/3 contained both inactive aggregates as well as oligomerized 87-kDa PC1/3 that exhibited stable activity which was partially latent and could be revealed by dilution. No such latency was observed for the more basic, 66/74-kDa forms of PC1/3. Fractions containing these species were stabilized by preincubation with micromolar concentrations of either fluorogenic substrate or peptides containing pairs of basic residues. In addition, the most active form of 87-kDa PC1/3, a probable homodimer, was activated by preincubation with these same peptides. Cleavage by PC1/3 is often the initiating step in the biosynthetic pathway for peptide hormones, implying that this is a natural step for regulation. Our data suggest that enzyme oligomerization and peptide stabilization represent important contributing factors for the control of PC1/3 activity within secretory granules.

Structural basis for the removal of ubiquitin and interferon-stimulated gene 15 by a viral ovarian tumor domain-containing protease

The attachment of ubiquitin (Ub) and the Ub-like (Ubl) molecule interferon-stimulated gene 15 (ISG15) to cellular proteins mediates important innate antiviral responses. Ovarian tumor (OTU) domain proteases from nairoviruses and arteriviruses were recently found to remove these molecules from host proteins, which inhibits Ub and ISG15-dependent antiviral pathways. This contrasts with the Ub-specific activity of known eukaryotic OTU-domain proteases. Here we describe crystal structures of a viral OTU domain from the highly pathogenic Crimean-Congo haemorrhagic fever virus (CCHFV) bound to Ub and to ISG15 at 2.5-Å and 2.3-Å resolution, respectively. The complexes provide a unique structural example of ISG15 bound to another protein and reveal the molecular mechanism of an ISG15 cross-reactive deubiquitinase. To accommodate structural differences between Ub and ISG15, the viral protease binds the β-grasp folds of Ub and C-terminal Ub-like domain of ISG15 in an orientation that is rotated nearly 75° with respect to that observed for Ub bound to a representative eukaryotic OTU domain from yeast. Distinct structural determinants necessary for binding either substrate were identified and allowed the reengineering of the viral OTU protease into enzymes with increased substrate specificity, either for Ub or for ISG15. Our findings now provide the basis to determine in vivo the relative contributions of deubiquitination and deISGylation to viral immune evasion tactics, and a structural template of a promiscuous deubiquitinase from a haemorrhagic fever virus that can be targeted for inhibition using small-molecule-based strategies.

Molecular characterization and expression analysis of Cathepsin B and L cysteine proteases from rock bream ( Oplegnathus fasciatus)

Cathepsins are lysosomal cysteine proteases of the papain family that play an important role in intracellular protein degradation and turn over within the lysosomal system. In the present study, full-length sequences of cathepsin B (RbCathepsin B) and L (RbCathepsin L) were identified after transcriptome sequencing of rock bream Oplegnathus fasciatus mixed tissue cDNA. Cathepsin B was composed of 330 amino acid residues with 36 kDa predicted molecular mass. RbCathepsin L contained 336 amino acid residues encoding for a 38 kDa predicted molecular mass protein. The sequencing analysis results showed that both cathepsin B and L contain the characteristic papain family cysteine protease signature and active sites for the eukaryotic thiol proteases of cysteine, asparagine and histidine. In addition, RbCathepsin L contained EF hand Ca 2+ binding and cathepsin propeptide inhibitor domains. The rock bream cathepsin B and L showed the highest amino acid identity of 90 and 95% to Lutjanus argentimaculatus cathepsin B and Lates calcarifer cathepsin L, respectively. By phylogenetic analysis, cathepsin B and L exhibited a high degree of evolutionary relationship to respective cathepsin family members of the papain superfamily. Quantitative real-time RT-PCR analysis results confirmed that the expression of cathepsin B and L genes was constitutive in all examined tissues isolated from un-induced rock bream. Moreover, activation of RbCathepsin B and L mRNA was observed in both lipopolysaccharide (LPS) and Edwardsiella tarda challenged liver and blood cells, indicating a role of immune response in rock bream.

Isolation, cDNA Cloning, and Structure-based Functional Characterization of Oryctin, a Hemolymph Protein from the Coconut Rhinoceros Beetle, Oryctes rhinoceros, as a Novel Serine Protease Inhibitor

We isolated oryctin, a 66-residue peptide, from the hemolymph of the coconut rhinoceros beetle Oryctes rhinoceros and cloned its cDNA. Oryctin is dissimilar to any other known peptides in amino acid sequence, and its function has been unknown. To reveal that function, we determined the solution structure of recombinant (13)C,(15)N-labeled oryctin by heteronuclear NMR spectroscopy. Oryctin exhibits a fold similar to that of Kazal-type serine protease inhibitors but has a unique additional C-terminal α-helix. We performed protease inhibition assays of oryctin against several bacterial and eukaryotic proteases. Oryctin does inhibit the following serine proteases: α-chymotrypsin, endopeptidase K, subtilisin Carlsberg, and leukocyte elastase, with K(i) values of 3.9 × 10(-10) m, 6.2 × 10(-10) m, 1.4 × 10(-9) m, and 1.2 × 10(-8) m, respectively. Although the target molecule of oryctin in the beetle hemolymph remains obscure, our results showed that oryctin is a novel single domain Kazal-type inhibitor and could play a key role in protecting against bacterial infections.

Hybrid molecular structure of the giant protease tripeptidyl peptidase II.

Tripeptidyl peptidase II (TPP II) is the largest known eukaryotic protease (6MDa). It is believed to act downstream of the 26S proteasome cleaving tripeptides from the N– termini of longer peptides and it is implicated in numerous cellular processes. Here we report the structure of Drosophila TPP II determined by a hybrid approach: The structure of the dimer was solved by x–ray crystallography and docked into the three– dimensional map of the holocomplex obtained by single-particle cryo-electron microscopy. The resulting structure reveals the compartmentalization of the active sites inside a system of chambers and suggests the existence of a molecular ruler determining the size of the cleavage products. Furthermore, the structure suggests a model for activation of TPP II involving the relocation of a flexible loop and a repositioning of the active–site serine, coupling it to holocomplex assembly and active site sequestration.

Translation Driven by Picornavirus IRES Is Hampered from Sindbis Virus Replicons: Rescue by Poliovirus 2A Protease

Alphavirus replicons are very useful for analyzing different aspects of viral molecular biology. They are also useful tools in the development of new vaccines and highly efficient expression of heterologous genes. We have investigated the translatability of Sindbis virus (SV) subgenomic mRNA bearing different 5′-untranslated regions, including several viral internal ribosome entry sites (IRESs) from picornaviruses, hepatitis C virus, and cricket paralysis virus. Our findings indicate that all these IRES-containing mRNAs are initially translated in culture cells transfected with the corresponding SV replicon but their translation is inhibited in the late phase of SV replication. Notably, co-expression of different poliovirus (PV) non-structural genes reveals that the protease 2A (2A pro) is able to increase translation of subgenomic mRNAs containing the PV or encephalomyocarditis virus IRESs but not of those of hepatitis C virus or cricket paralysis virus. A PV 2A pro variant deficient in eukaryotic initiation factor (eIF) 4GI cleavage or PV protease 3C, neither of which cleaves eIF4GI, does not increase picornavirus IRES-driven translation, whereas L protease from foot-and-mouth disease virus also rescues translation. These findings suggest that the replicative foci of SV-infected cells where translation takes place are deficient in components necessary to translate IRES-containing mRNAs. In the case of picornavirus IRESs, cleavage of eIF4GI accomplished by PV 2A pro or foot-and-mouth disease virus protease L rescues this inhibition. eIF4GI co-localizes with ribosomes both in cells electroporated with SV replicons bearing the picornavirus IRES and in cells co-electroporated with replicons that express PV 2A pro. These findings support the idea that eIF4GI cleavage is necessary to rescue the translation driven by picornavirus IRESs in baby hamster kidney cells that express SV replicons.

The Prodomain of Ssy5 Protease Controls Receptor-Activated Proteolysis of Transcription Factor Stp1

Extracellular amino acids induce the yeast SPS sensor to endoproteolytically cleave transcription factors Stp1 and Stp2 in a process termed receptor-activated proteolysis (RAP). Ssy5, the activating endoprotease, is synthesized with a large N-terminal prodomain and a C-terminal chymotrypsin-like catalytic (Cat) domain. During biogenesis, Ssy5 cleaves itself and the prodomain and Cat domain remain associated, forming an inactive primed protease. Here we show that the prodomain is a potent inhibitor of Cat domain activity and that its inactivation is a requisite for RAP. Accordingly, amino acid-induced signals trigger proteasome-dependent degradation of the prodomain. A mutation that stabilizes the prodomain prevents Stp1 processing, whereas destabilizing mutations lead to constitutive RAP-independent Stp1 processing. We fused a conditional degron to the prodomain to synthetically reprogram the amino acid-responsive SPS signaling pathway, placing it under temperature control. Our results define a regulatory mechanism that is novel for eukaryotic proteases functioning within cells.

Some assembly required: dedicated chaperones in eukaryotic proteasome biogenesis

The 26S proteasome is the key eukaryotic protease responsible for the degradation of intracellular proteins. Protein degradation by the 26S proteasome plays important roles in numerous cellular processes, including the cell cycle, differentiation, apoptosis, and the removal of damaged or misfolded proteins. How this 2.5-MDa complex, composed of at least 32 different polypeptides, is assembled in the first place is not well understood. However, it has become evident that this complicated task is facilitated by a framework of protein factors that chaperone the nascent proteasome through its various stages of assembly. We review here the known proteasome-specific assembly factors, most only recently discovered, and describe their potential roles in proteasome assembly, with an emphasis on the many remaining unanswered questions about this intricate process of assisted self-assembly.

Eukaryotic and retroviral aspartic proteases: similarities and differences

The superfamily of aspartic proteases consists of two large families, eukaryotic and retroviral. Eukaryotic enzymes are encoded as single chains with two‐domain structures, whereas retroviral enzymes are active as homodimers. Both families include enzymes of considerable medical relevance, thus a vast number of structures of their inhibitor complexes have been reported. Whereas some of these inhibitors are very specific for particular members of each family, others are pleuripotent. For example, recent findings of cross‐reactivity of potent inhibitors of HIV‐1 PR and plasmepsin emphasized the need to evaluate the inhibitor complexes of other aspartic proteases from both families in an effort to create novel inhibitors. Such a task is hindered by the large differences in size between proteins from two families, and additionally complicated by the deviation in the symmetry of the functional subunits. We succeeded in generating very accurate overall superposition of these enzymes, including the ligands bound in their active sites. That allows us to evaluate an overall fit and the interactions of any inhibitor, which has been developed for one family member (e.g. HIV‐1 PR), in respect to the other family (e.g. renin). We are also able to characterize the structural similarities and differences between the two families with much higher accuracy, addressing their functional and evolutionary significance.

Activation of Inhibitors by Sortase Triggers Irreversible Modification of the Active Site

Sortases anchor surface proteins to the cell wall of Gram-positive pathogens through recognition of specific motif sequences. Loss of sortase leads to large reductions in virulence, which identifies sortase as a target for the development of antibacterials. By screening 135,625 small molecules for inhibition, we report here that aryl (beta-amino)ethyl ketones inhibit sortase enzymes from staphylococci and bacilli. Inhibition of sortases occurs through an irreversible, covalent modification of their active site cysteine. Sortases specifically activate this class of molecules via beta-elimination, generating a reactive olefin intermediate that covalently modifies the cysteine thiol. Analysis of the three-dimensional structure of Bacillus anthracis sortase B with and without inhibitor provides insights into the mechanism of inhibition and reveals binding pockets that can be exploited for drug discovery.

拡大するユビキチンの世界：基礎から病態へ

Ubiquitin, an 8.6-kDa highly conserved protein, is a landmark molecule functioning as a posttranslational modifier. It is covalently attached to target proteins in a fashion of polymerization, resulting in a poly-ubiquitin chain that serves as a degradation signal. The 26S proteasome is an eukaryotic ATP-dependent protease responsible for selective degradation of the polyubiquitin-tagged proteins in eukaryotic cells. It appears to act as an elegantly organized apparatus designed for efficient and exhaustive hydrolysis of proteins, and can in fact be regarded as a protein-destroying machinery. To date, there are growing lines of evidence addressing the importance of the ubiquitin-proteasome system that catalyzes various biological reactions rapidly, orderly, exhaustively, and unidirectionally. On the other hand, autophagy (Greek for “self-eating”) is an evolutionarily conserved pathway in which the cytoplasm and organelles are engulfed within double-membraned vesicles, known as autophagosomes, which rapidly fuse with lysosomes and their contents together with the inner membrane are degraded by a variety of lysosomal digestive enzymes. While autophagy has been thought to contribute to bulk degradation non-selectively, but recent genetic analaysis with mice reveals that ablation of autophagy leads to accumulation of ubiquitin-positive inclusions, implying that the novel role of ubiquitin is to provide a signal that shuttles ubiquitinated proteins for autophagy. Currently, the roles of ubiqutin are expanding in various fields of life science. In this review, I will review the ubiquitin-mediated proteolysis pathway with a special reference to proteasomes and autophagy, focusing on how ubiquitin is linked to the pathogenesis of neurodegenerative diseases.

Ddi1, a Eukaryotic Protein With the Retroviral Protease Fold

Retroviral aspartyl proteases are homodimeric, whereas eukaryotic aspartyl proteases tend to be large, monomeric enzymes with 2-fold internal symmetry. It has been proposed that contemporary monomeric aspartyl proteases evolved by gene duplication and fusion from a primordial homodimeric enzyme. Recent sequence analyses have suggested that such “fossil” dimeric aspartyl proteases are still encoded in the eukaryotic genome. We present evidence for retention of a dimeric aspartyl protease in eukaryotes. The X-ray crystal structure of a domain of the Saccharomyces cerevisiae protein Ddi1 shows that it is a dimer with a fold similar to that of the retroviral proteases. Furthermore, the double Asp-Thr-Gly-Ala amino acid sequence motif at the active site of HIV protease is found with identical geometry in the Ddi1 structure. However, the putative substrate binding groove is wider in Ddi1 than in the retroviral proteases, suggesting that Ddi1 accommodates bulkier substrates. Ddi1 belongs to a family of proteins known as the ubiquitin receptors, which have in common the ability to bind ubiquitinated substrates and the proteasome. Ubiquitin receptors contain an amino-terminal ubiquitin-like (UBL) domain and a carboxy-terminal ubiquitin-associated (UBA) domain, but Ddi1 is the only representative in which the UBL and UBA domains flank an aspartyl protease-like domain. The remarkable structural similarity between the central domain of Ddi1 and the retroviral proteases, in the global fold and in active-site detail, suggests that Ddi1 functions proteolytically during regulated protein turnover in the cell.

Down-Regulation of the 26S Proteasome Subunit RPN9 Inhibits Viral Systemic Transport and Alters Plant Vascular Development

Plant viruses utilize the vascular system for systemic movement. The plant vascular network also transports water, photosynthates, and signaling molecules and is essential for plant growth. However, the molecular mechanisms governing vascular development and patterning are still largely unknown. From viral transport suppressor screening using virus-induced gene silencing, we identified a 26S proteasome subunit, RPN9, which is required for broad-spectrum viral systemic transport. Silencing of RPN9 in Nicotiana benthamiana inhibits systemic spread of two taxonomically distinct viruses, Tobacco mosaic virus and Turnip mosaic virus. The 26S proteasome is a highly conserved eukaryotic protease complex controlling many fundamental biochemical processes, but the functions of many 26S proteasome regulatory subunits, especially in plants, are still poorly understood. We demonstrate that the inhibition of viral systemic transport after RPN9 silencing is largely due to alterations in the vascular tissue. RPN9-silenced plants display extra leaf vein formation with increased xylem and decreased phloem. We further illustrate that RPN9 functions at least in part through regulation of auxin transport and brassinosteroid signaling, two processes that are crucial for vascular formation. We propose that RPN9 regulates vascular formation by targeting a subset of regulatory proteins for degradation. The brassinosteroid-signaling protein BZR1 is one of the targets.

A Serpin from the Gut Bacterium Bifidobacterium longum Inhibits Eukaryotic Elastase-like Serine Proteases

Serpins form a large class of protease inhibitors involved in regulation of a wide spectrum of physiological processes. Recently identified prokaryotic members of this protein family may provide a key to the evolutionary origins of the unique serpin fold and the associated inhibitory mechanism. We performed a biochemical characterization of a serpin from Bifidobacterium longum, an anaerobic Gram-positive bacterium that naturally colonizes human gastrointestinal tract. The B. longum serpin was shown to efficiently inhibit eukaryotic elastase-like proteases with a stoichiometry of inhibition close to 1. Porcine pancreatic elastase and human neutrophil elastase were inhibited with the second order association constants of 4.7 x 10(4) m(-1) s(-1) and 2.1 x 10(4) m(-1) s(-1), respectively. The B. longum serpin is expected to be active in the gastrointestinal tract, because incubation of the purified recombinant serpin with mouse feces produces a stable covalent serpin-protease adduct readily detectable by SDS-PAGE. Bifidobacteria may encounter both pancreatic elastase and neutrophil elastase in their natural habitat and protection against exogenous proteolysis may play an important role in the interaction between these commensal bacteria and their host.

Chlamydia trachomatis‐derived deubiquitinating enzymes in mammalian cells during infection

Chlamydia trachomatis is an obligate intracellular bacterium that causes a variety of diseases in humans. C. trachomatis has a complex developmental cycle that depends on host cells for replication, during which gene expression is tightly regulated. Here we identify two C. trachomatis proteases that possess deubiquitinating and deneddylating activities. We have designated these proteins ChlaDub1 and ChlaDub2. The genes encoding ChlaDub1 and ChlaDub2 are present in all Chlamydia species except for Chlamydia pneumoniae, and their catalytic domains bear similarity to the catalytic domains of other eukaryotic ubiquitin-like proteases (Ulp). The C. trachomatis DUBs react with activity-based probes and hydrolyse ubiquitinated and neddylated substrates. ChlaDub1 and ChlaDub2 represent the first known bacterial DUBs that possess both deubiquitinating and deneddylating activities.

Delimitation of the rice wide compatibility gene S5 n to a 40-kb DNA fragment

Wide compatibility varieties (WCVs) are a special class of rice (Oryza sativa L.) germplasm that produces hybrids with normal pollen and spikelet fertility when crossed with both indica and japonica subspecies. The wide compatibility gene S5 ( n ) has been used extensively in inter-subspecific hybrid breeding programs. We previously mapped the S5 locus to a 2.2-cM genomic region between RM253 and R2349 on chromosome 6, using a population of 356 F(1) plants derived from the three-way cross 02428/Nanjing11//Balilla. In this study, a chromosome walking strategy was employed to construct a physical map covering this genomic region using these two closest markers as the starting points. A physical map consisting of six overlapping BAC clones was formed, spanning a genomic region of 540-kb in length. By analyzing recombination events from a population of 8,000 F(1) plants derived from a three-way cross based on near isogenic lines of the S5 locus, the S5 locus was localized to a DNA fragment of 40-kb in length, flanked by two shotgun subclones, 7B1 and 15D2. Sequence analysis of this fragment predicted five open reading frames, encoding xyloglucan fucosyltransferases, dnak-type molecular chaperone BiP, a putative eukaryotic aspartyl protease, and a hypothetical protein. This result will be very useful in molecular cloning of the S5 ( n ) allele and marker-assisted transferring of the wide compatibility gene in rice breeding programs.

Theileria orientalis: Cloning a cDNA encoding a protein similar to thiol protease with haemoglobin-binding activity

A gene encoding a protein (Tocp1) from Theileria orientalis was isolated from a cDNA library and the deduced amino acid sequence of Tocp1 has 476 amino acids. The primary structure of Tocp1 is similar to eukaryotic thiol proteases (EC 3.4.22.-), but no enzymatic activity was observed with the substitution of essential cysteine at the cysteine active site for glycine. Southern blot analysis showed that multiple genes similar to Tocp1 were present in the parasite genome. Sequence analysis of the genome of the parasite showed that there are at least five different genes similar to Tocp1. Tocp1 transcripts were detected in the T. orientalis piroplasma by Northern blot analysis. Western blot analysis showed that Tocp1 was expressed in the piroplasm of T. orientalis. To address the role of Tocp1 in the life cycle of T. orientalis, Tocp1 was expressed using pET32 expression system. Binding affinity to haemoglobin was demonstrated by enzyme-linked immunosorbent assay.

Aspartic proteases from Plasmodium chabaudi: a rodent model for human malaria

Intraerythrocytic malaria parasites degrade haemoglobin to provide nutrients for their own growth and maturation. Plasmodium aspartic proteases known as plasmepsins play an important role on haemoglobin degradation and are being studied as drug targets for chemotherapy of malaria. The rodent model for human malaria, Plasmodium chabaudi, is an experimentally good model for therapy drug design. The gene encoding an aspartic protease precursor (proplasmepsin) from the rodent malaria parasite P. chabaudi was cloned and sequenced. A theoretical 3D structure model was constructed by comparative homology and used for superimposition with other known models. Analysis of the P. chabaudi and Plasmodium yoelli genomes revealed in both the presence of at least seven plasmepsins and each one has sequence similarity to its plasmepsin counterpart of the human malaria Plasmodium falciparum. The predicted proteins were confirmed as plasmepsins by detection on Blocks Database of three characteristic blocks of the eukaryotic and viral aspartic protease family. Analysis of the proline-rich loop amino acid sequence of these plasmepsins suggests that they constitute characteristic motifs of each plasmepsin group suggesting that these sequence variations are related with different substrate specificities.

Folding Pathway Mediated by an Intramolecular Chaperone: A FUNCTIONAL PEPTIDE CHAPERONE DESIGNED USING SEQUENCE DATABASES

Catalytic domains of several prokaryotic and eukaryotic protease families require dedicated N-terminal propeptide domains or "intramolecular chaperones" to facilitate correct folding. Amino acid sequence analysis of these families establishes three important characteristics: (i) propeptides are almost always less conserved than their cognate catalytic domains, (ii) they contain a large number of charged amino acids, and (iii) propeptides within different protease families display insignificant sequence similarity. The implications of these findings are, however, unclear. In this study, we have used subtilisin as our model to redesign a peptide chaperone using information databases. Our goal was to establish the minimum sequence requirements for a functional subtilisin propeptide, because such information could facilitate subsequent design of tailor-made chaperones. A decision-based computer algorithm that maintained conserved residues but varied all non-conserved residues from a multiple protein sequence alignment was developed and utilized to design a novel peptide sequence (ProD). Interestingly, despite a difference of 5 pH units between their isoelectric points and despite displaying only 16% sequence identity with the wild-type propeptide (ProWT), ProD chaperones folding and functions as a potent subtilisin inhibitor. The computed secondary structures and hydrophobic patterns within these two propeptides are similar. However, unlike ProWT, ProD adopts a well defined alpha-beta conformation as an isolated peptide and forms a stoichiometric complex with mature subtilisin. The CD spectra of this complex is similar to ProWT.subtilisin. Our results establish that despite low sequence identity and dramatically different charge distribution, both propeptides adopt similar structural scaffolds. Hence, conserved scaffolds and hydrophobic patterns, but not absolute charge, dictate propeptide function.

Eubacterial HslV and HslU subunits homologs in primordial eukaryotes.

ATP-dependent protease complexes are present in all three kingdoms of life, where they rid the cell of misfolded or damaged proteins and control the level of certain regulatory proteins. They include the proteasome in Eukaryotes, Archea, and Actinomycetales and the HslVU (ClpQY) complex in other eubacteria. We showed that genes homologous to eubacterial HslV (ClpQ) and HslU (ClpY) are present in the genome of trypanosomatid protozoa and are expressed. The features of the cDNAs indicated that bona fide trypanosomatid messengers had been cloned and ruled out bacterial contamination as the source of the material. The N-terminal microsequence of HslV from Leishmania infantum (Protozoa: Kinetoplastida) permitted the identification of the propeptide cleavage site and indicated that an active protease is present. High similarities (> or =57.5%) with the prototypical HslV and HslU from Escherichia coli and conservation of residues essential for biochemical activity suggested that a functional HslVU complex is present in trypanosomatid protozoa. The structure of the N-termini of HslV and HslU further suggested mitochondrial localization. Phylogenetic analysis indicated that HslV and HslU from trypanosomatids clustered with eubacterial homologs but did not point to any particular bacterial lineage. Because typical eukaryotic 20S proteasomes are present in trypanosomatids, we concluded that the eubacterial HslVU and the eukaryotic multicatalytic protease are simultaneously present in these organisms. To our knowledge this is the first report of a eubacterial HslVU complex in eukaryotes and, consequently, of the simultaneous occurrence of both a proteasome and HslVU in living cells.