Archaea, one of three major evolutionary lineages of life, encode proteasomes highly related to those of eukaryotes. In contrast, archaeal ubiquitin-like proteins are less conserved and not known to function in protein conjugation. This has complicated our understanding of the origins of ubiquitination and its connection to proteasomes. Here we report two small archaeal modifier proteins, SAMP1 and SAMP2, with a β-grasp fold and C-terminal diglycine motif similar to ubiquitin, that form protein-conjugates in the archaeon Haloferax volcanii. SAMP-conjugates were altered by nitrogen-limitation and proteasomal gene knockout and spanned various functions including components of the Urm1 pathway. LC-MS/MS-based collision-induced dissociation demonstrated isopeptide bonds between the C-terminal glycine of SAMP2 and the ε-amino group of lysines from a number of protein targets and Lys58 of SAMP2 itself, revealing poly-SAMP chains. The widespread distribution and diversity of pathways modified by SAMPylation suggest this type of protein-conjugation is central to the archaeal lineage.
archaea; ubiquitin; proteasome; protease; AAA ATPases
Post-translational modification by ubiquitin and ubiquitin-related proteins plays critical roles in protein degradation and in regulation of essential cellular processes. In mammals, transcription grinds to a halt during late spermiogenesis due to compaction of the spermatid genome, which creates a special need for robust post-transcriptional regulation. Here we report the finding of a novel mouse ubiquitin-like protein, UBL4B. Ubl4b is a testis-specific autosomal gene. Ubl4b lacks introns and evidently arose from an X-linked intron-bearing housekeeping gene, Ubl4a, by retroposition during mammalian evolution. While Ubl4a is expressed throughout spermatogenesis, Ubl4b is restricted to post-meiotic germ cells. Ubl4a is highly conserved, but Ubl4b has undergone rapid evolution and may have evolved new functions. Our data suggest that evolution of Ubl4b is not due to meiotic sex chromosome inactivation (MSCI). Alternatively, origination of Ubl4b was due to MSCI, but Ubl4b eventually evolved to be restricted to post-meiotic germ cells.
ubiquitin; meiosis; retrogene; MSCI; sex chromosomes; XY body; spermiogenesis; testis; germ cells
Modification of proteins by ubiquitin (Ub)-like proteins (UBLs) plays an important role in many cellular processes, including cell cycle progression, nuclear transport, and autophagy. Protein modification occurs via UBL-conjugating and -deconjugating enzymes, which presumably exert a regulatory function by determining the conjugation status of the substrate proteins. To target and identify UBL-modifying enzymes, we produced Nedd8, ISG15, and SUMO-1 in Escherichia coli and equipped them with a C-terminal electrophilic trap (vinyl sulfone [VS]) via an intein-based method. These C-terminally modified UBL probes reacted with purified UBL-activating (E1), -conjugating (E2), and -deconjugating enzymes in a covalent fashion. Modified UBLs were radioiodinated and incubated with cell lysates prepared from mouse cell lines and tissues to allow visualization of polypeptides reactive with individual UBL probes. The cell type- and tissue-specific labeling patterns observed for the UBL probes reflect distinct expression profiles of active enzymes, indicating tissue-specific functions of UBLs. We identify Ub C-terminal hydrolase L1 (UCH-L1) and DEN1/NEDP1/SENP8, in addition to UCH-L3, as proteases with specificity for Nedd8. The Ub-specific protease isopeptidase T/USP5 is shown to react with ISG15-VS. Furthermore, we demonstrate that the desumoylation enzyme SuPr-1 can be modified by SUMO-1-VS, a modification that is dependent on the SuPr-1 active-site cysteine. The UBL probes described here will be valuable tools for the further characterization of the enzymatic pathways that govern modification by UBLs.
Eukaryotic protein modification by ubiquitin-like proteins (Ubls) controls an enormous range of physiological processes. Ubl attachments principally regulate interactions with other macromolecules, such as proteasome-substrate binding or recruitment of proteins to chromatin. Different Ubl systems use related enzymes to attach specific Ubls to proteins (or other molecules), and most Ubl attachments are transient. Mounting evidence suggests that Ubl-protein modification evolved from prokaryotic sulfurtransferase systems or related enzymes. Surprisingly, proteins similar to Ubl-conjugating and Ubl-deconjugating enzymes appear to have already become widespread by the time of the last universal common ancestor, suggesting that Ubl-protein conjugation is not a eukaryotic invention.
A systematic analysis of prokaryotic ubiquitin-related beta-grasp fold proteins provides new insights into the Ubiquitin family functional history.
Ubiquitin (Ub)-mediated signaling is one of the hallmarks of all eukaryotes. Prokaryotic homologs of Ub (ThiS and MoaD) and E1 ligases have been studied in relation to sulfur incorporation reactions in thiamine and molybdenum/tungsten cofactor biosynthesis. However, there is no evidence for entire protein modification systems with Ub-like proteins and deconjugation by deubiquitinating enzymes in prokaryotes. Hence, the evolutionary assembly of the eukaryotic Ub-signaling apparatus remains unclear.
We systematically analyzed prokaryotic Ub-related β-grasp fold proteins using sensitive sequence profile searches and structural analysis. Consequently, we identified novel Ub-related proteins beyond the characterized ThiS, MoaD, TGS, and YukD domains. To understand their functional associations, we sought and recovered several conserved gene neighborhoods and domain architectures. These included novel associations involving diverse sulfur metabolism proteins, siderophore biosynthesis and the gene encoding the transfer mRNA binding protein SmpB, as well as domain fusions between Ub-like domains and PIN-domain related RNAses. Most strikingly, we found conserved gene neighborhoods in phylogenetically diverse bacteria combining genes for JAB domains (the primary de-ubiquitinating isopeptidases of the proteasomal complex), along with E1-like adenylating enzymes and different Ub-related proteins. Further sequence analysis of other conserved genes in these neighborhoods revealed several Ub-conjugating enzyme/E2-ligase related proteins. Genes for an Ub-like protein and a JAB domain peptidase were also found in the tail assembly gene cluster of certain caudate bacteriophages.
These observations imply that members of the Ub family had already formed strong functional associations with E1-like proteins, UBC/E2-related proteins, and JAB peptidases in the bacteria. Several of these Ub-like proteins and the associated protein families are likely to function together in signaling systems just as in eukaryotes.
Mammalian RAD51 protein plays essential roles in DNA homologous recombination, DNA repair and cell proliferation. RAD51 activities are regulated by its associated proteins. It was previously reported that a ubiquitin-like protein, UBL1, associates with RAD51 in the yeast two-hybrid system. One function of UBL1 is to covalently conjugate with target proteins and thus modify their function. In the present study we found that non-conjugated UBL1 forms a complex with RAD51 and RAD52 proteins in human cells. Overexpression of UBL1 down-regulates DNA double-strand break-induced homologous recombination in CHO cells and reduces cellular resistance to ionizing radiation in HT1080 cells. With or without overexpressed UBL1, most homologous recombination products arise by gene conversion. However, overexpression of UBL1 reduces the fraction of bidirectional gene conversion tracts. Overexpression of a mutant UBL1 that is incapable of being conjugated retains the ability to inhibit homologous recombination. These results suggest a regulatory role for UBL1 in homologous recombination.
Ubiquitin-like proteins (Ubls) are conjugated by dynamic E1-E2-E3 enzyme cascades. E1s activate Ubls by catalyzing Ubl C-terminal adenylation, forming a covalent E1~Ubl thioester intermediate, and generating a thioester-linked E2~Ubl product, which must be released for subsequent reactions. We report structural analysis of a trapped Ubl activation complex containing NEDD8’s heterodimeric E1 (APPBP1-UBA3), two NEDD8s (one thioester-linked to E1, one noncovalently-associated for adenylation), a catalytically-inactive E2 (Ubc12), and MgATP. The results suggest that a thioester switch toggles E1-E2 affinities. Two E2 binding sites depend on NEDD8 being thioester-linked to E1. One is unmasked by a striking E1 conformational change. The other comes directly from the thioester-bound NEDD8. After NEDD8 transfer to E2, reversion to an alternate E1 conformation would facilitate release of the E2~NEDD8 thioester product. Thus, transferring the Ubl’s thioester linkage between successive conjugation enzymes can induce conformational changes and alter interaction networks to drive consecutive steps in Ubl cascades.
Post-translational covalent modification by ubiquitin and ubiquitin-like proteins (UBLs) is a major eukaryotic mechanism for regulating protein function. In general, each UBL has its own E1 that serves as the entry point for a cascade. The E1 first binds the UBL and catalyzes adenylation of the UBL’s C-terminus, prior to promoting UBL transfer to a downstream E2. Ubiquitin’s Arg 72, which corresponds to Ala72 in the UBL NEDD8, is a key E1 selectivity determinant: swapping ubiquitin and NEDD8 residue 72 identity was shown previously to swap their E1 specificity. Correspondingly, Arg190 in the UBA3 subunit of NEDD8’s heterodimeric E1 (the APPBP1-UBA3 complex), which corresponds to a Gln in ubiquitin’s E1 UBA1, is a key UBL selectivity determinant. Here we dissect this specificity with biochemical and X-ray crystallographic analysis of APPBP1-UBA3-NEDD8 complexes in which NEDD8’s residue 72 and UBA3’s residue 190 are substituted with different combinations of Ala, Arg, or Gln. APPBP1-UBA3’s preference for NEDD8’s Ala72 appears to be indirect, due to proper positioning of UBA3’s Arg190. By contrast, our data are consistent with direct positive interactions between ubiquitin’s Arg72 and an E1’s Gln. However, APPBP1-UBA3’s failure to interact with a UBL having Arg72 is not due to a lack of this favorable interaction, but rather arises from UBA3’s Arg190 acting as a negative gate. Thus, parallel residues from different UBL pathways can utilize distinct mechanisms to dictate interaction selectivity, and specificity can be amplified by barriers that prevent binding to components of different conjugation cascades.
ubiquitin; NEDD8; E1; protein interaction; specificity; gating
Post-translational covalent modification by ubiquitin and ubiquitin-like proteins (UBLs) is a major eukaryotic mechanism for regulating protein function. In general, each UBL has its own E1 that serves as the entry point for a cascade. The E1 first binds the UBL and catalyzes adenylation of the UBL’s C-terminus, prior to promoting UBL transfer to a downstream E2. Ubiquitin’s Arg 72, which corresponds to Ala72 in the UBL NEDD8, is a key E1 selectivity determinant: swapping ubiquitin and NEDD8 residue 72 identity was shown previously to swap their E1 specificity. Correspondingly, Arg190 in the UBA3 subunit of NEDD8’s heterodimeric E1 (the APPBP1-UBA3 complex), which corresponds to a Gln in ubiquitin’s E1 UBA1, is a key UBL selectivity determinant. Here, we dissect this specificity with biochemical and X-ray crystallographic analysis of APPBP1-UBA3-NEDD8 complexes in which NEDD8’s residue 72 and UBA3’s residue 190 are substituted with different combinations of Ala, Arg, or Gln. APPBP1-UBA3’s preference for NEDD8’s Ala72 appears to be indirect, due to proper positioning of UBA3’s Arg190. By contrast, our data are consistent with direct positive interactions between ubiquitin’s Arg72 and an E1’s Gln. However, APPBP1-UBA3’s failure to interact with a UBL having Arg72 is not due to a lack of this favorable interaction, but rather arises from UBA3’s Arg190 acting as a negative gate. Thus, parallel residues from different UBL pathways can utilize distinct mechanisms to dictate interaction selectivity, and specificity can be amplified by barriers that prevent binding to components of different conjugation cascades.
Ubiquitin and UBL (ubiquitin-like) modifiers are small proteins that covalently modify other proteins to alter their properties or behaviours. Ubiquitin modification (ubiquitylation) targets many substrates, often leading to their proteasomal degradation. NEDD8 (neural-precursor-cell-expressed developmentally down-regulated 8) is the UBL most closely related to ubiquitin, and its best-studied role is the activation of CRLs (cullin-RING ubiquitin ligases) by its conjugation to a conserved C-terminal lysine residue on cullin proteins. The attachment of UBLs requires three UBL-specific enzymes, termed E1, E2 and E3, which are usually well insulated from parallel UBL pathways. In the present study, we report a new mode of NEDD8 conjugation (NEDDylation) whereby the UBL NEDD8 is linked to proteins by ubiquitin enzymes in vivo. We found that this atypical NEDDylation is independent of classical NEDD8 enzymes, conserved from yeast to mammals, and triggered by an increase in the NEDD8 to ubiquitin ratio. In cells, NEDD8 overexpression leads to this type of NEDDylation by increasing the concentration of NEDD8, whereas proteasome inhibition has the same effect by depleting free ubiquitin. We show that bortezomib, a proteasome inhibitor used in cancer therapy, triggers atypical NEDDylation in tissue culture, which suggests that a similar process may occur in patients receiving this treatment.
bortezomib; MG132; MLN4924; neural-precursor-cell-expressed developmentally down-regulated 8 (NEDD8)-activating enzyme (NAE); proteasome; ubiquitinactivating enzyme; BCA3, breast cancer-associated gene 3; CHO, Chinese-hamster ovary; CRL, cullin-RING ubiquitin ligase; EGFR, epidermal growth factor receptor; FBS, fetal bovine serum; HA, haemagglutinin; HRP, horseradish peroxidase; LDS, lithium dodecyl sulfate; NAE, NEDD8-activating enzyme; NEDD8, neural-precursor-cell-expressed developmentally down-regulated 8; siRNA, small interfering RNA; SUMO, small ubiquitin-like modifier; TCA, trichloroacetic acid; UBL, ubiquitin-like; WT, wild-type
The mechanism underlying the delivery of ubiquitylated substrates to the proteasome is poorly understood. Rad23 is a putative adaptor molecule for this process because it interacts with ubiquitin chains through its ubiquitin-associated motifs (UBA) and with the proteasome through a ubiquitin-like element (UBL). Here, we demonstrate that the UBL motif of Rad23 also binds Ufd2, an E4 enzyme essential for ubiquitin chain assembly onto its substrates. Mutations in the UBL of Rad23 alter its interactions with Ufd2 and the proteasome, and impair its function in the UFD proteolytic pathway. Furthermore, Ufd2 and the proteasome subunit Rpn1 compete for the binding of Rad23, suggesting that Rad23 forms separate complexes with them. Importantly, we also find that the ability of other UBL/UBA proteins to associate with Ufd2 correlates with their differential involvement in the UFD pathway, suggesting that UBL-mediated interactions may contribute to the substrate specificity of these adaptors. We propose that the UBL motif, a protein-protein interaction module, may be used to facilitate coupling between substrate ubiquitylation and delivery, and to ensure the orderly handoff of the substrate from the ubiquitylation machinery to the proteasome.
The delivery of ubiquitinated proteins to the proteasome for degradation is a key step in the regulation of the ubiquitin-proteasome pathway, yet the mechanisms underlying this step are not understood in detail. The Rad23 family of proteins is known to bind ubiquitinated proteins through its two ubiquitin-associated (UBA) domains, and may participate in the delivery of ubiquitinated proteins to the proteasome through docking via the Rad23 ubiquitin-like (UBL) domain.
In this study, we investigate how the interaction between the UBL and UBA domains may modulate ubiquitin recognition and the delivery of ubiquitinated proteins to the proteasome by autoinhibition. We have explored a competitive binding model using specific mutations in the UBL domain. Disrupting the intramolecular UBL-UBA domain interactions in HHR23A indeed potentiates ubiquitin-binding. Additionally, the analogous surface on the Rad23 UBL domain overlaps with that required for interaction with both proteasomes and the ubiquitin ligase Ufd2. We have found that mutation of residues on this surface affects the ability of Rad23 to deliver ubiquitinated proteins to the proteasome.
We conclude that the competition of ubiquitin-proteasome pathway components for surfaces on Rad23 is important for the role of the Rad23 family proteins in proteasomal targeting.
Addition of polypeptides belonging to the ubiquitin family to selected lysines residues is a widespread post-translation modification (PTM) that controls many fundamental aspects of cell's life. Specific alterations in the normal turnover of this PTM are frequently observed in tumors. The conjugation/deconjugation cycle of ubiquitin (Ub) or ubiquitin-like (Ubl) proteins influences the activities of oncogenes and tumor suppressor genes. Two families of enzymes work in antagonizing manner to add or remove Ub and Ubl-proteins on target proteins: the E3 ligases and the isopeptidases. These enzymes are the subjects of fervent research with the ambition to comprehend their regulation, their mechanisms of action, their involvement in human diseases, and to develop specific inhibitors for therapeutic intervention. Here we will discuss of isopeptidases, the deconjugating enzymes, with particular emphasis on the proapoptotic activities of the relative inhibitors identified so far.
Apoptosis; cancer; proteasome; isopeptidase; DUBs; necrosis; caspase; proteasome; USP
Protein modification by ubiquitin (Ub) and ubiquitin-like modifiers (Ubl) requires the action of activating (E1), conjugating (E2), and ligating (E3) enzymes and is a key step in the specific destruction of proteins. Deubiquitinating enzymes (DUBs) deconjugate substrates modified with Ub/Ubls and recycle Ub inside the cell. Genome mining based on sequence homology to proteins with known function has assigned many enzymes to this pathway without confirmation of either conjugating or DUB activity. Function-dependent methodologies are still the most useful for rapid identification or assessment of biological activity of expressed proteins from cells. Activity-based protein profiling (ABPP) uses chemical probes that are active-site directed for the classification of protein activities in complex mixtures. Here we show that the design and use of an expanded set of Ub-based electrophilic probes allowed us to recover and identify members of each enzyme class in the ubiquitin-proteasome system, including E3 ligases and DUBs with previously unverified activity. We show that epitope-tagged Ub-electrophilic probes can be used as activity-based probes for E3 ligase identification by in vitro labeling and activity studies of purified enzymes identified from complex mixtures in cell lysate. Furthermore, the reactivity of our probe with the HECT domain of the E3 Ub ligase ARF-BP1 suggests that multiple cysteines may be in the vicinity of the E2-binding site and are capable of the transfer of Ub to self or to a substrate protein.
Ubiquitin (Ub) and the ubiquitin-like proteins (Ubls) comprise a remarkable assortment of polypeptides that are covalently conjugated to target proteins (or other biomolecules) to modulate their intracellular localization, half-life and/or activity. Identification of Ub/Ubl conjugation sites on a protein of interest can thus be extremely important for understanding how it is regulated. While mass spectrometry (MS) has become a powerful tool for the study of many classes of post-translational modifications, the identification of Ub/Ubl conjugation sites presents a number of unique challenges. Here, we present an improved Ub/Ubl conjugation site identification strategy, utilizing SUMmOn analysis and an additional protease (LysC), as a complement to standard approaches. As compared to standard trypsin proteolysis-database search protocols alone, the addition of SUMmOn analysis can; (a) identify Ubl conjugation sites that are not detected by standard database searching methods, (b) better preserve Ub/Ubl conjugate identity, and (c) increase the number of identifications of Ub/Ubl modifications in lysine-rich protein regions. Using this methodology, we characterize for the first time a number of novel Ubl linkages and conjugation sites, including alternative yeast (K54) and mammalian SUMO chain (SUMO-2 K42, SUMO-3 K41) assemblies, as well as previously unreported NEDD8 chain (K27, K33 and K54) topologies.
NEDD8; SUMO; SUMmOn; ubiquitin; ubiquitin-like proteins
The genome sequence of Haloferax volcanii is available and several comparative genomic in silico studies were performed that yielded novel insight for example into protein export, RNA modifications, small non-coding RNAs, and ubiquitin-like Small Archaeal Modifier Proteins. The full range of functional genomic methods has been established and results from transcriptomic, proteomic and metabolomic studies are discussed. Notably, Hfx. volcanii is together with Halobacterium salinarum the only prokaryotic species for which a translatome analysis has been performed. The results revealed that the fraction of translationally-regulated genes in haloarchaea is as high as in eukaryotes. A highly efficient genetic system has been established that enables the application of libraries as well as the parallel generation of genomic deletion mutants. Facile mutant generation is complemented by the possibility to culture Hfx. volcanii in microtiter plates, allowing the phenotyping of mutant collections. Genetic approaches are currently used to study diverse biological questions–from replication to posttranslational modification—and selected results are discussed. Taken together, the wealth of functional genomic and genetic tools make Hfx. volcanii a bona fide archaeal model species, which has enabled the generation of important results in recent years and will most likely generate further breakthroughs in the future.
Ubiquitin-like proteins play important roles in diverse biological processes. In this study, we present an unexpected finding that a ubiquitin-like small archaeal modifier protein (SAMP2) from Haloferax volcanii adopts two distinct states under low ionic condition. One of these is similar to the β-grasp structure conserved in ubiquitin-like proteins from eukaryotes; the other is disordered, like prokaryotic ubiquitin-like protein, Pup. Furthermore, our study reveals that the conformation of SAMP2 is dependent on ionic strength. With the increase of ion concentration, SAMP2 undergoes a conformational conversion from disorder to order, indicating that the ordered conformation is the functional form of SAMP2 under the physiological condition of H. volcanii.
The expression of two heat-responsive cct (chaperonin-containing Tcp-1) genes from the archaeon Haloferax volcanii was investigated at the transcription level. The cct1 and cct2 genes, which encode proteins of 560 and 557 amino acids, respectively, were identified on cosmid clones of an H. volcanii genomic library and subsequently sequenced. The deduced amino acid sequences of these genes exhibited a high degree of similarity to other archaeal and eucaryal cct family members. Expression of the cct genes was characterized in detail for the purpose of developing a model for studying transcription regulation in the domain Archaea. Northern (RNA) analysis demonstrated that the cct mRNAs were maximally induced after heat shock from 37 to 55 degrees C and showed significant heat inducibility after 30 min at 60 degrees C. Transcription of cct mRNAs was also stimulated in response to dilute salt concentrations. Transcriptional analysis of cct promoter regions coupled to a yeast tRNA reporter gene demonstrated that 5' flanking sequences up to position -233 (cct1) and position -170 (cct2) were sufficient for promoting heat-induced transcription. Transcript analysis indicated that both basal transcription and stress-induced transcription of the H. volcanii cct genes were directed by a conserved archaeal consensus TATA motif (5'-TTTATA-3') centered at -25 relative to the mapped initiation site. Comparison of the cct promoter regions also revealed a striking degree of sequence conservation immediately 5' and 3' of the TATA element.
Ubiquitin receptor proteins play an important role in delivering ubiquitylated protein substrates to the proteasome for degradation. HHR23a and hPLIC2 are two such ubiquitin receptors that contain ubiquitin-like (UBL) domains, which interact with the proteasome, and ubiquitin-associated (UBA) domains, which interact with ubiquitin. Depending on their abundance UBL/UBA family members can either promote or inhibit the degradation of other proteins, which suggests their participation in the delivery of substrates to the proteasome is highly regulated. In previous work, we determined UBL/UBA domain interactions to promote intramolecular interactions in hHR23a that are abrogated with the addition of either ubiquitin or the proteasome component S5a. In yeast, we determined the hHR23a ortholog (Rad23) to interact with another UBL/UBA family member (Ddi1) and to bind a common tetraubiquitin chain. Here, we use NMR spectroscopy to reveal that hHR23a interacts with hPLIC2 via UBL/UBA domain interactions and to map their binding surfaces. In addition, we demonstrate that these two proteins associate in mammalian cells. Intriguingly, inhibition of the proteasome mitigates hHR23a/hPLIC2 interaction.
hHR23a; hPLIC2; ubiquitin receptor proteins; proteasome-mediated protein degradation; ubiquitin-associated domains
RNase P is the ribonucleoprotein enzyme that cleaves precursor sequences from the 5' ends of pre-tRNAs. In Bacteria, the RNA subunit is the catalytic moiety. Eucaryal and archaeal RNase P activities copurify with RNAs, which have not been shown to be catalytic. We report here the analysis of the RNase P RNA from the thermoacidophilic archaeon Sulfolobus acidocaldarius. The holoenzyme was highly purified, and extracted RNA was used to identify the RNase P RNA gene. The nucleotide sequence of the gene was determined, and a secondary structure is proposed. The RNA was not observed to be catalytic by itself, but it nevertheless is similar in sequence and structure to bacterial RNase P RNA. The marked similarity of the RNase P RNA from S. acidocaldarius and that from Haloferax volcanii, the other known archael RNase P RNA, supports the coherence of Archaea as a phylogenetic domain.
Ubiquitin-like proteins (Ubls) confer diverse functions on their target proteins. The modified proteins are involved in various biological processes, including DNA replication, signal transduction, cell cycle control, embryogenesis, cytoskeletal regulation, metabolism, stress response, homeostasis and mRNA processing. Modifiers such as SUMO, ATG12, ISG15, FAT10, URM1, and UFM have been shown to modify proteins thus conferring functions related to programmed cell death, autophagy and regulation of the immune system. Putative modifiers such as Domain With No Name (DWNN) have been identified in recent times but not fully characterized. In this review, we focus on cellular processes involving human Ubls and their targets. We review current progress in targeting these modifiers for drug design strategies.
ubiquitin-like; autophagy; apoptosis; immune response; DWNN; SNAMA; p53; Ubls; ubiquitin-proteasome; cancer
Autosomal recessive juvenile parkinsonism (ARJP) is an early onset familial form of Parkinson’s disease. Approximately 50% of all ARJP cases are attributed to mutations in the gene park2, coding for the protein parkin. Parkin is a multidomain E3 ubiquitin ligase with six distinct domains including an N-terminal ubiquitin-like (Ubl) domain. In this work we examined the structure, stability, and interactions of the parkin Ubl domain containing most ARJP causative mutations. Using NMR spectroscopy we show that the Ubl domain proteins containing the ARJP substitutions G12R, D18N, K32T, R33Q, P37L, and K48A retained a similar three-dimensional fold as the Ubl domain, while at least one other (V15M) had altered packing. Four substitutions (A31D, R42P, A46P, and V56E) result in poor folding of the domain, while one protein (T55I) showed evidence of heterogeneity and aggregation. Further, of the substitutions that maintained their three-dimensional fold, we found that four of these (V15M, K32T, R33Q, and P37L) lead to impaired function due to decreased ability to interact with the 19S regulatory subunit S5a. Three substitutions (G12R, D18N, and Q34R) with an uncertain role in the disease did not alter the three-dimensional fold or S5a interaction. This work provides the first extensive characterization of the structural effects of causative mutations within the ubiquitin-like domain in ARJP.
The family of ubiquitin-like molecules (UbLs) comprises several members, each of which has sequence, structural, or functional similarity to ubiquitin. ISG15 is a homolog of ubiquitin in vertebrates and is strongly upregulated following induction by type I interferon. ISG15 can be covalently attached to proteins, analogous to ubiquitination and with actual support of ubiquitin conjugating factors. Specific proteases are able to reverse modification with ubiquitin or UbLs by hydrolyzing the covalent bond between their C-termini and substrate proteins. The tail regions of ubiquitin and ISG15 are identical and we therefore hypothesized that promiscuous deubiquitinating proteases (DUBs) might exist, capable of recognizing both ubiquitin and ISG15.
We have cloned and expressed 22 human DUBs, representing the major clades of the USP protease family. Utilizing suicide inhibitors based on ubiquitin and ISG15, we have identified USP2, USP5 (IsoT1), USP13 (IsoT3), and USP14 as ISG15-reactive proteases, in addition to the bona fide ISG15-specific protease USP18 (UBP43). USP14 is a proteasome-associated DUB, and its ISG15 isopeptidase activity increases when complexed with the proteasome.
By evolutionary standards, ISG15 is a newcomer among the UbLs and it apparently not only utilizes the conjugating but also the deconjugating machinery of its more established relative ubiquitin. Functional overlap between these two posttranslational modifiers might therefore be more extensive than previously appreciated and explain the rather innocuous phenotype of ISG15 null mice.
In ubiquitin-like protein (UBL) cascades, a thioester-linked E2~UBL complex typically interacts with an E3 enzyme for UBL transfer to the target. Here we demonstrate a variant mechanism, whereby the E2 Ubc12 functions with two E3s, Hrt1 and Dcn1, for ligation of the UBL Rub1 to Cdc53’s WHB subdomain. Hrt1 functions like a conventional RING E3, with its N-terminus recruiting Cdc53 and C-terminal RING activating Ubc12~Rub1. Dcn1’s “potentiating neddylation” domain (Dcn1P) acts as an additional E3, reducing nonspecific Hrt1-mediated Ubc12~Rub1 discharge and directing Ubc12’s active site to Cdc53. Crystal structures of Dcn1P-Cdc53WHB and Ubc12 allow modeling of a catalytic complex, supported by mutational data. We propose that Dcn1’s interactions with both Cdc53 and Ubc12 would restrict the otherwise flexible Hrt1 RING-bound Ubc12~Rub1 to a catalytically competent orientation. Our data reveal mechanisms by which two E3s function synergistically to promote UBL transfer from one E2 to a target.
Ubiquitin; Rub1; NEDD8; E3; RING; ubiquitin ligase; Ubc12; SCF; Cdc53
Ubiquitin (Ub) and ubiquitin-like (Ubl) proteins regulate a diverse array of cellular pathways through post-translational attachment to protein substrates. Ub/Ubl-mediated signaling is initiated through E1, E2, and E3-mediated conjugation, transduced by proteins that recognize Ub/Ubl-modified substrates, and terminated by proteases which remove the Ub/Ubl from the substrate. Recent structural studies have elucidated mechanisms pertinent to Ub/Ubl conjugation, recognition, and deconjugation, highlighting essential steps during Ub/Ubl modification that illustrate common and divergent mechanistic themes within this important process.