Two novel ubiquitin-fold modifier 1 (Ufm1)-specific proteases, UfSP1 and UfSP2

Ubiquitin-fold modifier 1 (Ufm1) is a recently identified new ubiquitin-like protein, whose tertiary structure displays a striking resemblance to ubiquitin. Similar to ubiquitin, it has a Gly residue conserved across species at the C-terminal region with extensions of various amino acid sequences that need to be processed in vivo prior to conjugation to target proteins. Here we report the isolation, cloning, and characterization of two novel mouse Ufm1-specific proteases, named UfSP1 and UfSP2. UfSP1 and UfSP2 are composed of 217 and 461 amino acids, respectively, and they have no sequence homology with previously known proteases. UfSP2 is present in most, if not all, of multicellular organisms including plant, nematode, fly, and mammal, whereas UfSP1 could not be found in plant and nematode upon data base search. UfSP1 and UfSP2 cleaved the C-terminal extension of Ufm1 but not that of ubiquitin or other ubiquitin-like proteins, such as SUMO-1 and ISG15. Both were also capable of releasing Ufm1 from Ufm1-conjugated cellular proteins. They were sensitive to inhibition by sulfhydryl-blocking agents, such as N-ethylmaleimide, and their active site Cys could be labeled with Ufm1-vinylmethylester. Moreover, replacement of the conserved Cys residue by Ser resulted in a complete loss of the UfSP1 and UfSP2 activities. These results indicate that UfSP1 and UfSP2 are novel thiol proteases that specifically process the C terminus of Ufm1.     

Structure of ubiquitin-fold modifier 1-specific protease UfSP2

Ubiquitin-fold modifier 1 (Ufm1)-specific protease 2 (UfSP2) is a cysteine protease that is responsible for the release of Ufm1 from Ufm1-conjugated cellular proteins, as well as for the generation of mature Ufm1 from its precursor. The 2.6 Å resolution crystal structure of mouse UfSP2 reveals that it is composed of two domains. The C-terminal catalytic domain is similar to UfSP1 with Cys²⁹⁴, Asp⁴¹⁸, His⁴²⁰, Tyr²⁸², and a regulatory loop participating in catalysis. The novel N-terminal domain shows a unique structure and plays a role in the recognition of its cellular substrate C20orf116 and thus in the recruitment of UfSP2 to the endoplasmic reticulum, where C20orf116 predominantly localizes. Mutagenesis studies were carried out to provide the structural basis for understanding the loss of catalytic activity observed in a recently identified UfSP2 mutation that is associated with an autosomal dominant form of hip dysplasia.     

An ER Complex of ODR-4 and ODR-8/Ufm1 Specific Protease 2 Promotes GPCR Maturation by a Ufm1-Independent Mechanism

Despite the importance of G-protein coupled receptors (GPCRs) their biogenesis is poorly understood. Like vertebrates, C. elegans uses a large family of GPCRs as chemoreceptors. A subset of these receptors, such as ODR-10, requires the odr-4 and odr-8 genes to be appropriately localized to sensory cilia. The odr-4 gene encodes a conserved tail-anchored transmembrane protein; the molecular identity of odr-8 is unknown. Here, we show that odr-8 encodes the C. elegans ortholog of Ufm1-specific protease 2 (UfSP2). UfSPs are cysteine proteases identified biochemically by their ability to liberate the ubiquitin-like modifier Ufm1 from its pro-form and protein conjugates. ODR-8/UfSP2 and ODR-4 are expressed in the same set of twelve chemosensory neurons, and physically interact at the ER membrane. ODR-4 also binds ODR-10, suggesting that an ODR-4/ODR-8 complex promotes GPCR folding, maturation, or export from the ER. The physical interaction between human ODR4 and UfSP2 suggests that this complex''s role in GPCR biogenesis may be evolutionarily conserved. Unexpectedly, mutant versions of ODR-8/UfSP2 lacking catalytic residues required for protease activity can rescue all odr-8 mutant phenotypes tested. Moreover, deleting C. elegans ufm-1 does not alter chemoreceptor traffic to cilia, either in wild type or in odr-8 mutants. Thus, UfSP2 proteins have protease- and Ufm1-independent functions in GPCR biogenesis.     

Solution structure and dynamics of Ufm1, a ubiquitin-fold modifier 1

The ubiquitin-fold modifier 1 (Ufm1) is one of various ubiquitin-like modifiers and conjugates to target proteins in cells through Uba5 (E1) and Ufc1 (E2). The Ufm1-system is conserved in metazoa and plants, suggesting its potential roles in various multicellular organisms. Herein, we analyzed the solution structure and dynamics of human Ufm1 (hsUfm1) by nuclear magnetic resonance spectroscopy. Although the global fold of hsUfm1 is similar to those of ubiquitin (Ub) and NEDD8, the cluster of acidic residues conserved in Ub and NEDD8 does not exist on the Ufm1 surface. 15N spin relaxation data revealed that the amino acid residues of hsUfm1 exhibiting conformational fluctuations form a cluster at the C-terminal segment and its spatial proximity, which correspond to the versatile ligand-binding sites of Ub and other ubiquitin-like proteins (Ubls). We suggest that Ub and other Ubl-modifiers share a common feature of potential conformational multiplicity, which might be associated with the broad ligand specificities of these proteins.     

ECEPE proteins: a novel family of eukaryotic cysteine proteinases

Using a variety of fold-recognition methods, a novel eukaryotic cysteine proteinase (ECEPE) family has been identified. This family encompasses sequences from an uncharacterized KOG4621, including the Arabidopsis thaliana guanylyl cyclase-related protein AtGC1. ECEPE proteins are predicted to possess the papain-like cysteine proteinase fold and are evolutionarily linked to C39 peptidases. The presence of the invariant Cys-His-Asp/Asn catalytic triad and the oxyanion-hole glutamine residue characteristic of papain-like cysteine proteases indicate that ECEPE proteins might function as proteases.     

Comparative site-directed mutagenesis in the catalytic amino acid triad in calicivirus proteases

Calicivirus proteases cleave the viral precursor polyprotein encoded by open reading frame 1 (ORF1) into multiple intermediate and mature proteins. These proteases have conserved histidine (His), glutamic acid (Glu) or aspartic acid (Asp), and cysteine (Cys) residues that are thought to act as a catalytic triad (i.e. general base, acid and nucleophile, respectively). However, is the triad critical for processing the polyprotein? In the present study, we examined these amino acids in viruses representing the four major genera of Caliciviridae: Norwalk virus (NoV), Rabbit hemorrhagic disease virus (RHDV), Sapporo virus (SaV) and Feline calicivirus (FCV). Using single amino‐acid substitutions, we found that an acidic amino acid (Glu or Asp), as well as the His and Cys in the putative catalytic triad, cannot be replaced by Ala for normal processing activity of the ORF1 polyprotein in vitro. Similarly, normal activity is not retained if the nucleophile Cys is replaced with Ser. These results showed the calicivirus protease is a Cys protease and the catalytic triad formation is important for protease activity. Our study is the first to directly compare the proteases of the four representative calicivirus genera. Interestingly, we found that RHDV and SaV proteases critically need the acidic residues during catalysis, whereas proteolytic cleavage occurs normally at several cleavage sites in the ORF1 polyprotein without a functional acid residue in the NoV and FCV proteases. Thus, the substrate recognition mechanism may be different between the SaV and RHDV proteases and the NoV and FCV proteases.     

Molecular cloning and characterization of cathepsin F gene from olive flounder Paralichthys Olivaceus

We isolated a homologue of cathepsin F from cDNA library of olive flounder liver. A 2,077 kb full-length cDNA encoding a predicted polypeptide of 474 amino acids was sequenced. The flounder cathepsin F exhibits a domain structure typical for papain-like cysteine proteases, a 17 amino acid N-terminal hydrophobic signal sequence followed by an extraordinarily long propeptide of 244 amino acids and the domain of the mature protease comprising 213 amino acids. The mature region contains all features characteristic of a papain-like cysteine protease, including the highly conserved cysteine, histidine and asparagine residues of the ‘catalytic triad’. The cathepsin F protein showed 49–99% amino acid sequence identity with other known cathepsin F sequences. An in vivo expression study showed that cathepsin F mRNA was expressed predominantly in brain, liver, eye and heart, and moderately in other tissues. The accumulation of cathepsin F mRNA in early stage of development increased with development. This expression pattern suggests that flounder cathepsin F has been implicated in the growth and reproduction regulation.     

The S8 serine, C1A cysteine and A1 aspartic protease families in Arabidopsis

The Arabidopsis thaliana genome has over 550 protease sequences representing all five catalytic types: serine, cysteine, aspartic acid, metallo and threonine (MEROPS peptidase database, http://merops.sanger.ac.uk/), which probably reflect a wide variety of as yet unidentified functions performed by plant proteases. Recent indications that the 26S proteasome, a T1 family-threonine protease, is a regulator of light and hormone responsive signal transduction highlight the potential of proteases to participate in many aspects of plant growth and development. Recent discoveries that proteases are required for stomatal distribution, embryo development and disease resistance point to wider roles for four additional multigene families that include some of the most frequently studied (yet poorly understood) plant proteases: the subtilisin-like, serine proteases (family S8), the papain-like, cysteine proteases (family C1A), the pepsin-like, aspartic proteases (family A1) and the plant matrixin, metalloproteases (family M10A). In this report, 54 subtilisin-like, 30 papain-like and 59 pepsin-like proteases from Arabidopsis, are compared with S8, C1A and A1 proteases known from other plant species at the functional, phylogenetic and gene structure levels. Examples of structural conservation between S8, C1A and A1 genes from rice, barley, tomato and soybean and those from Arabidopsis are noted, indicating that some common, essential plant protease roles were established before the divergence of monocots and eudicots. Numerous examples of tandem duplications of protease genes and evidence for a variety of restricted expression patterns suggest that a high degree of specialization exists among proteases within each family. We propose that comprehensive analysis of the functions of these genes in Arabidopsis will firmly establish serine, cysteine and aspartic proteases as regulators and effectors of a wide range of plant processes.     

A Novel Type of E3 Ligase for the Ufm1 Conjugation System

The ubiquitin fold modifier 1 (Ufm1) is the most recently discovered ubiquitin-like modifier whose conjugation (ufmylation) system is conserved in multicellular organisms. Ufm1 is known to covalently attach with cellular protein(s) via a specific E1-activating enzyme (Uba5) and an E2-conjugating enzyme (Ufc1), but its E3-ligating enzyme(s) as well as the target protein(s) remain unknown. Herein, we report both a novel E3 ligase for Ufm1, designated Ufl1, and an Ufm1-specific substrate ligated by Ufl1, C20orf116. Ufm1 was covalently conjugated with C20orf116. Although Ufl1 has no obvious sequence homology to any other known E3s for ubiquitin and ubiquitin-like modifiers, the C20orf116·Ufm1 formation was greatly accelerated by Ufl1. The C20orf116·Ufm1 conjugate was cleaved by Ufm1-specific proteases, implying the reversibility of ufmylation. The conjugation was abundant in the liver and lungs of Ufm1-transgenic mice, fractionated into membrane fraction, and impaired in Uba5 knock-out cells. Intriguingly, immunological analysis revealed localizations of Ufl1 and C20orf116 mainly to the endoplasmic reticulum. Our results provide novel insights into the Ufm1 system involved in cellular regulation of multicellular organisms.     

Crystal structure of a deubiquitinating enzyme (human UCH-L3) at 1.8 A resolution.

Ubiquitin C-terminal hydrolases catalyze the removal of adducts from the C-terminus of ubiquitin. We have determined the crystal structure of the recombinant human Ubiquitin C-terminal Hydrolase (UCH-L3) by X-ray crystallography at 1.8 A resolution. The structure is comprised of a central antiparallel beta-sheet flanked on both sides by alpha-helices. The beta-sheet and one of the helices resemble the well-known papain-like cysteine proteases, with the greatest similarity to cathepsin B. This similarity includes the UCH-L3 active site catalytic triad of Cys95, His169 and Asp184, and the oxyanion hole residue Gln89. Papain and UCH-L3 differ, however, in strand and helix connectivity, which in the UCH-L3 structure includes a disordered 20 residue loop (residues 147-166) that is positioned over the active site and may function in the definition of substrate specificity. Based upon analogy with inhibitor complexes of the papain-like enzymes, we propose a model describing the binding of ubiquitin to UCH-L3. The UCH-L3 active site cleft appears to be masked in the unliganded structure by two different segments of the enzyme (residues 9-12 and 90-94), thus implying a conformational change upon substrate binding and suggesting a mechanism to limit non-specific hydrolysis.     

Covalent inhibition of SUMO and ubiquitin-specific cysteine proteases by an in situ thiol–alkyne addition

Posttranslational modification of proteins with ubiquitin and ubiquitin-like modifiers such as SUMO can be reverted by specific proteases, also referred to as deubiquitinases and isopeptidases, most of which are cysteine-dependent. We have found that the replacement of the conserved C-terminal glycine with propargylamine converts SUMO and ubiquitin to highly efficient covalent inhibitors of their cognate cysteine proteases. Attack of the catalytic cysteine onto the terminal alkyne results in the formation of a vinyl sulfide linkage. Although this reaction is reminiscent of the inhibitory mechanism of the isosteric nitrile inhibitors it was unexpected due to the low electrophilicity of the alkyne group. We show that a precise location of the functional group in the active site of the protease is crucial for the reaction, which was not inhibited by the presence of a radical scavenger. Furthermore, a mutational study of key catalytic residues in the SUMO-protease Senp1, that is H533A and D550A of the catalytic triad and Q597A as part of the oxyanion hole, revealed that these residues are not required for the observed covalent adduct formation. We therefore propose that the reaction is an in situ thiol–alkyne addition. Due to the high chemical inertness of the alkyne moiety the respective protease inhibitors should be well-suited for cellular and therapeutic applications. In keeping with this idea, selective labeling with propargylated SUMO and Ub probes was observed in lysates of cell lines expressing the cognate proteases after transient transfection.     

Characterization of a novel otubain-like cysteine protease of Cryptosporidium parvum

Otubains are a recently discovered family of cysteine proteases that participate in the ubiquitin pathway. Here, we partially characterized the biochemical properties of a cysteine protease of Cryptosporidium parvum, which is closely related to otubains. The gene encoding otubain-like cysteine protease of C. parvum (CpOTU) contained the aspartate, cysteine and histidine residues that form the catalytic triad of otubains. The modified ubiquitin-associated domain and LxxL motif were identified in CpOTU. The recombinant CpOTU showed the isopeptidase activity at neutral pH values and its activity was effectively inhibited by ubiquitin aldehyde, N-ethylmaleimide and iodoacetic acid. Interestingly, CpOTU had an unusual C-terminal extension of 217 amino acids compared to mammalian otubains, and the C-terminal extension is essential for the activity of the enzyme. Expression of CpOTU peaked in the oocyst stage of the parasite, which suggested its potential physiological role for the oocyst stage.     

BTLCP proteins: a novel family of bacterial transglutaminase-like cysteine proteinases

Using sequence similarity searches and top-of-the-range fold-recognition methods, we have identified a novel family of bacterial transglutaminase-like cysteine proteinases (BTLCPs) with an invariant Cys-His-Asp catalytic triad and a predicted N-terminal signal sequence. This family of previously uncharacterized hypothetical proteins encompasses sequences of unknown function from DUF920 (in the Pfam database) and COG3672. BTLCPs are predicted to possess the papain-like cysteine proteinase fold and catalyze post-translational protein modification through transamidase, acetylase or hydrolase activity. Inspection of neighboring genes encoding BTLCPs suggests a link between this predicted activity and a type-I secretion system resembling ATP-binding cassette exporters of toxins and proteases involved in bacterial pathogenicity.     

Solution NMR structure of the NlpC/P60 domain of lipoprotein Spr from Escherichia coli: structural evidence for a novel cysteine peptidase catalytic triad

Escherichia coli Spr is a membrane-anchored cell wall hydrolase. The solution NMR structure of the C-terminal NlpC/P60 domain of E. coli Spr described here reveals that the protein adopts a papain-like alpha+beta fold and identifies a substrate-binding cleft featuring several highly conserved residues. The active site features a novel Cys-His-His catalytic triad that appears to be a unique structural signature of this cysteine peptidase family. Moreover, the relative orientation of these catalytic residues is similar to that observed in the analogous Ser-His-His triad, a variant of the classic Ser-His-Asp charge relay system, suggesting the convergent evolution of a catalytic mechanism in quite distinct peptidase families.     

Cysteine proteases of positive strand RNA viruses and chymotrypsin-like serine proteases. A distinct protein superfamily with a common structural fold

Evidence is presented, based on sequence comparison and secondary structure prediction, of structural and evolutionary relationship between chymotrypsin-like serine proteases, cysteine proteases of positive strand RNA viruses (3C proteases of picornaviruses and related enzymes of como-, nepo- and potyviruses) and putative serine protease of a sobemovirus. These observations lead to re-identification of principal catalytic residues of viral proteases. Instead of the pair of Cys and His, both located in the C-terminal part of 3C proteases, a triad of conserved His, Asp(Glu) and Cys(Ser) has been identified, the first two residues resident in the N-terminal, and Cys in the C-terminal beta-barrel domain. These residues are suggested to form a charge-transfer system similar to that formed by the catalytic triad of chymotrypsin-like proteases. Based on the structural analogy with chymotrypsin-like proteases, the His residue previously implicated in catalysis, together with two partially conserved Gly residues, is predicted to constitute part of the substrate-binding pocket of 3C proteases. A partially conserved ThrLys/Arg dipeptide located in the loop preceding the catalytic Cys is suggested to confer the primary cleavage specificity of 3C toward Glx/Gly(Ser) sites. These observations provide the first example of relatedness between proteases belonging, by definition, to different classes.     

The reconstituted 'humanized liver' in TK-NOG mice is mature and functional

The Marasmius oreades mushroom lectin (MOA) is well known for its exquisite binding specificity for blood group B antigens. In addition to its N-terminal carbohydrate-binding domain, MOA possesses a C-terminal domain with unknown function, which structurally resembles hydrolytic enzymes. Here we show that MOA indeed has catalytic activity. It is a calcium-dependent cysteine protease resembling papain-like cysteine proteases, with Cys215 being the catalytic nucleophile. The possible importance of MOA’s proteolytic activity for mushroom defense against pathogens is discussed.     

Full-length cDNA of human cathepsin F predicts the presence of a cystatin domain at the N-terminus of the cysteine protease zymogen

A novel human cDNA encoding a cysteine protease of the papain family named cathepsin F is reported. The mature part of the predicted protease precursor displays between 26% and 42% identity to other human cysteine proteases while the proregion is unique by means of length and sequence. The very long proregion of the cathepsin F precursor (251 amino acid residues) can be divided into three regions: a C-terminal domain similar to the pro-segment of cathepsin L-like enzymes, a 50 residue flexible linker peptide, and an N-terminal domain predicted to adopt a cystatin-like fold. Cathepsin F would therefore be the first cysteine protease zymogen containing a cystatin-like domain.     

Crystal structure of DeSI-1, a novel deSUMOylase belonging to a putative isopeptidase superfamily

Post-translational modification by small ubiquitin-like modifier (SUMO) can be reversed by sentrin/SUMO-specific proteases (SENPs), the first known class of deSUMOylase. Recently, we identified a new deSUMOylating enzyme DeSI-1, which is distinct from SENPs and belongs to the putative deubiquitinating isopeptidase PPPDE superfamily. Herein, we report the crystal structure of DeSI-1, revealing that this enzyme forms a homodimer and that the groove between the two subunits is the active site harboring two absolutely conserved cysteine and histidine residues that form a catalytic dyad. We also show that DeSI-1 exhibits an extremely low endopeptidase activity toward precursor forms of SUMO-1 and SUMO-2, unlike SENPs.     

Human rhinovirus 3C protease: a cysteine protease showing the trypsin(ogen)-like fold

Viral-encoded proteases cleave precursor polyprotein(s) leading to maturation of infectious virions. Strikingly, human rhinovirus 3C protease shows the trypsin(ogen)-like serine protease fold based on two topologically equivalent six-stranded β-barrels, but displays residue Cys147 as the active site nucleophile. By contrast, papain, which is representative of most cysteine proteases, does not display the trypsin(ogen)-like fold. Remarkably, in human rhinovirus 3C cysteine protease, the catalytic residues Cys147, His40 and Glu71 are positioned as Ser195, His57 and Asp102, respectively, building up the catalytic triad of serine proteases in the chymotrypsin–trypsin–elastase family. However, as compared to trypsin-like serine proteases and their zymogens, residue His40 and the oxyanion hole of human rhinovirus 3C cysteine protease, both key structural components of the active site, are located closer to the protein core. Human rhinovirus 3C cysteine protease cleaves preferentially GlnGly peptide bonds or, less commonly, the GlnSer, GlnAla, GluSer or GluGly pairs. Finally, human rhinovirus 3C cysteine protease and the 3CD cysteine protease–polymerase covalent complex bind the 5′ non-coding region of rhinovirus genomic RNA, an essential function for replication of the viral genome.