Ubiquitin Moieties Research Articles

Studying OTUD6B-OTUB1 Protein-Protein Interaction by Low-Throughput GFP-Trap Assays and High-Throughput AlphaScreen Assays.

Protein-protein interactions (PPI) are involved in a myriad of cellular processes, and their deregulation can lead to many diseases. One such process is protein ubiquitination that requires an orchestrated action of three key enzymes to add ubiquitin moieties to substrate proteins. Importantly, this process is reversible through deubiquitinating enzymes. Both ubiquitination and deubiquitination require many PPIs that once classified can be utilized to identify small molecule inhibitors counteracting these reactions. Here, we study the protein-protein interaction between the two deubiquitinating enzymes OTUB1 and OTUD6B and report for the first time that both proteins directly interact with each other. We describe the GFP-Trap immunoprecipitation as a cell-based method to analyze the OTUD6B-OTUB1 interaction in the cellular context and the AlphaScreen (amplified luminescent proximity homogeneous assay) assay as a tool to detect direct interactions and to search for PPI inhibitors.

Branched Ubiquitination: Detection Methods, Biological Functions and Chemical Synthesis.

Ubiquitination is a versatile posttranslational modification that elicits signaling roles to impact on various cellular processes and disease states. The versatility is a result of the complexity of ubiquitin conjugates, ranging from a single ubiquitin monomer to polymers with different length and linkage types. Recent studies have revealed the abundant existence of branched ubiquitin chains in which one ubiquitin molecule is connected to two or more ubiquitin moieties in the same ubiquitin polymer. Compared to the homotypic ubiquitin chain, the branched chain is recognized or processed differently by readers and erasers of the ubiquitin system, respectively, resulting in a qualitative or quantitative alteration of the functional output. Furthermore, certain types of branched ubiquitination are induced by cellular stresses, implicating their important physiological role in stress adaption. In addition, the current chemical methodologies of solid phase peptide synthesis and expanding genetic code approach have been developed to synthesize different architectures of branched ubiquitin chains. The synthesized branched ubiquitin chains have shown their significance in understanding the topologies and binding partners of the branched chains. Here, we discuss the recent progresses on the detection, functional characterization and synthesis of branched ubiquitin chains as well as the future perspectives of this emerging field.

The USP22 promotes the growth of cancer cells through the DYRK1A in pancreatic ductal adenocarcinoma.

As a member of the ubiquitin-specific protease (USP) family, USP22 could remove ubiquitin moieties from its target proteins to control the function of the target proteins. Accumulating studies show that USP22 essentially participates in diverse types of cancer as an oncogene-like protein. However, the roles of USP22 in human pancreatic ductal adenocarcinoma (PDAC) and the underlying mechanism are unknown. Here we report that USP22 promotes the growth of PDAC cells by promoting the expression of dual-specificity tyrosine regulated kinase 1A (DYRK1A). Our results showed that the expression levels of USP22 were up-regulated in human PDAC tissues and cell lines (BxPC-3, AsPC-1, MIA-PaCa-2, PANC-1, and CAPAN-1). Lentivirus-mediated knockdown of USP22 repressed the rate of proliferation and capacity of colony formation of BxPC3 and CAPAN1 cancer cells and USP22 overexpression promoted the proliferation and capacity of the colony formation of BxPC3 and CAPAN1 cancer cells. The further mechanism study showed that USP22 elevated the expression of the mRNA and protein levels of DYRK1A in PDAC cancer cells. Inhibition of DYRK1A with EHT-5732 or lentivirus-mediated knockdown of DYRK1A blocked the function of USP22 overexpression in the regulation of the proliferation and colony formation of PDAC cells. Taken together, our findings demonstrated that USP22 overexpression in PDAC promoted the growth of the cancer cells partially through upregulating the expression of DYRK1A.

Advances in Deubiquitinating Enzyme Inhibition and Applications in Cancer Therapeutics.

Since the discovery of the ubiquitin proteasome system (UPS), the roles of ubiquitinating and deubiquitinating enzymes (DUBs) have been widely elucidated. The ubiquitination of proteins regulates many aspects of cellular functions such as protein degradation and localization, and also modifies protein-protein interactions. DUBs cleave the attached ubiquitin moieties from substrates and thereby reverse the process of ubiquitination. The dysregulation of these two paramount pathways has been implicated in numerous diseases, including cancer. Attempts are being made to identify inhibitors of ubiquitin E3 ligases and DUBs that potentially have clinical implications in cancer, making them an important target in the pharmaceutical industry. Therefore, studies in medicine are currently focused on the pharmacological disruption of DUB activity as a rationale to specifically target cancer-causing protein aberrations. Here, we briefly discuss the pathophysiological and physiological roles of DUBs in key cancer-related pathways. We also discuss the clinical applications of promising DUB inhibitors that may contribute to the development of DUBs as key therapeutic targets in the future.

Specificity in Ubiquitination Triggered by Virus Infection.

Ubiquitination is a prominent posttranslational modification, in which the ubiquitin moiety is covalently attached to a target protein to influence protein stability, interaction partner and biological function. All seven lysine residues of ubiquitin, along with the N-terminal methionine, can each serve as a substrate for further ubiquitination, which effectuates a diverse combination of mono- or poly-ubiquitinated proteins with linear or branched ubiquitin chains. The intricately composed ubiquitin codes are then recognized by a large variety of ubiquitin binding domain (UBD)-containing proteins to participate in the regulation of various pathways to modulate the cell behavior. Viruses, as obligate parasites, involve many aspects of the cell pathways to overcome host defenses and subjugate cellular machineries. In the virus-host interactions, both the virus and the host tap into the rich source of versatile ubiquitination code in order to compete, combat, and co-evolve. Here, we review the recent literature to discuss the role of ubiquitin system as the infection progresses in virus life cycle and the importance of ubiquitin specificity in the regulation of virus-host relation.

Structure-Guided Mutagenesis Alters Deubiquitinating Activity and Attenuates Pathogenesis of a Murine Coronavirus.

Coronaviruses express a multifunctional papain-like protease, termed papain-like protease 2 (PLP2). PLP2 acts as a protease that cleaves the viral replicase polyprotein and as a deubiquitinating (DUB) enzyme which removes ubiquitin (Ub) moieties from ubiquitin-conjugated proteins. Previous in vitro studies implicated PLP2/DUB activity as a negative regulator of the host interferon (IFN) response, but the role of DUB activity during virus infection was unknown. Here, we used X-ray structure-guided mutagenesis and functional studies to identify amino acid substitutions within the ubiquitin-binding surface of PLP2 that reduced DUB activity without affecting polyprotein processing activity. We engineered a DUB mutation (Asp1772 to Ala) into a murine coronavirus and evaluated the replication and pathogenesis of the DUB mutant virus (DUBmut) in cultured macrophages and in mice. We found that the DUBmut virus replicates similarly to the wild-type (WT) virus in cultured cells, but the DUBmut virus activates an IFN response at earlier times compared to the wild-type virus infection in macrophages, consistent with DUB activity negatively regulating the IFN response. We compared the pathogenesis of the DUBmut virus to that of the wild-type virus and found that the DUBmut-infected mice had a statistically significant reduction (P < 0.05) in viral titer in liver and spleen at day 5 postinfection (d p.i.), although both wild-type and DUBmut virus infections resulted in similar liver pathology. Overall, this study demonstrates that structure-guided mutagenesis aids the identification of critical determinants of the PLP2-ubiquitin complex and that PLP2/DUB activity plays a role as an interferon antagonist in coronavirus pathogenesis.IMPORTANCE Coronaviruses employ a genetic economy by encoding multifunctional proteins that function in viral replication and also modify the host environment to disarm the innate immune response. The coronavirus papain-like protease 2 (PLP2) domain possesses protease activity, which cleaves the viral replicase polyprotein, and also DUB activity (deconjugating ubiquitin/ubiquitin-like molecules from modified substrates) using identical catalytic residues. To separate the DUB activity from the protease activity, we employed a structure-guided mutagenesis approach and identified residues that are important for ubiquitin binding. We found that mutating the ubiquitin-binding residues results in a PLP2 that has reduced DUB activity but retains protease activity. We engineered a recombinant murine coronavirus to express the DUB mutant and showed that the DUB mutant virus activated an earlier type I interferon response in macrophages and exhibited reduced replication in mice. The results of this study demonstrate that PLP2/DUB is an interferon antagonist and a virulence trait of coronaviruses.

How yeast cells deal with stalled replication forks.

DNA polymerases sometimes stall during DNA replication at sites where DNA is damaged, or upon encounter with proteins or secondary structures of DNA. When that happens, the polymerase clamp PCNA can become modified with a single ubiquitin moiety at lysine 164, opening DNA Damage Tolerance (DDT) mechanisms that either repair or bypass the lesions. An alternative repair mechanism is the salvage recombination (SR) pathway, which copies information from the sister chromatid. SUMOylation of PCNA at the same lysine, or at lysine 127, can recruit the Srs2 helicase, which negatively controls SR. Recently, we have dissected the relationship between SR and the DDT pathways, and showed that overexpression of either the PCNA unloader Elg1, or the Rad52 homologous recombination protein, can bypass the repression by Srs2. Our results shed light on the interactions between different DNA damage repair/bypass proteins, and underscore the importance of PCNA modifications in organizing the complex task of dealing with DNA damage during replication of the genetic material.

The deubiquitinating enzyme MoUbp8 is required for infection-related development, pathogenicity, and carbon catabolite repression in Magnaporthe oryzae.

Deubiquitination is an essential regulatory step in the Ub-dependent pathway. Deubiquitinating enzymes (DUBs) mediate the removal of ubiquitin moieties from substrate proteins, which are involved in many regulatory mechanisms. As a component of the DUB module (Ubp8/Sgf11/Sus1/Sgf73) in the SAGA (Spt-Ada-Gcn5-acetyltransferase) complex, Ubp8 plays a crucial role in both Saccharomyces cerevisiae and humans. In S. cerevisiae, Ubp8-mediated deubiquitination regulates transcriptional activation processes. To investigate the contributions of Ubp8 to physiological and pathological development of filamentous fungi, we generated the deletion mutant of ortholog MoUBP8 (MGG-03527) in Magnaporthe oryzae (syn. Pyricularia oryzae). The ΔMoubp8 strain showed reduced sporulation, pathogenicity, and resistance to distinct stresses. Even though the conidia of the ΔMoubp8 mutant were delayed in appressorium formation, the normal and abnormal (none-septum or one-septum) conidia could finally form appressoria. Reduced melanin in the ΔMoubp8 mutant is highly responsible for the attenuated pathogenicity since the appressoria of the ΔMoubp8 mutant was much more fragile than those of the wild type, due to the defective turgidity. The weakened ability to detoxify or scavenge host-derived reactive oxygen species (ROS) further restricted the invasion of the pathogen. We also showed that carbon derepression, on the one hand, rendered the ΔMoubp8 strain highly sensitive to allyl alcohol, on the other hand, it enhances the resistance of the MoUBP8 defective strain to deoxyglucose. Overall, we suggest that MoUbp8 is not only required for sporulation, melanin formation, appressoria development, and pathogenicity but also involved in carbon catabolite repression of M. oryzae.

Effects of selected deubiquitinating enzyme inhibitors on the proliferation and motility of lung cancer and mesothelioma cell lines.

The post-translational modification of proteins by ubiquitinating enzymes plays a central role in a number of cellular functions, such as cell proteolysis, DNA repair, and cell signaling and communication. Deubiquitinating enzymes (DUBs) disassemble ubiquitin chains and remove ubiquitin moieties from proteins. Targeting DUBs in cancer models has revealed an important role for these enzymes in tumorigenesis, and they therefore have emerged as attractive therapeutic targets. In the present study, the effects of three DUB inhibitors, PR-619, RA-9 and LDN-91946, on a non-small cell lung cancer cell line (A549) and a mesothelioma cell line (H2373) were investigated. PR-619 significantly inhibited cell adhesion and the proliferation of both cell lines. RA-9 exerted an inhibitory effect on the adhesion and proliferation of H2373 cells, whereas it had no effect on A549 cells. Notably, however, while PR-619 attenuated the proliferation of both cell lines, it exerted an opposite effect on cell motility; in the case of A549 cells, there was a significant increase in cell motility, while for the H2373 cells, there was a significant decrease. Furthermore, protein phosphorylation kinetic analyses revealed that the effects were cell line-specific. In H2373 cells, the phosphorylation of only one peptide corresponding to the P85A protein was significantly affected, and while LDN-91946 treatment increased phosphorylation, treatment with RA-9 or PR-619 decreased its phosphorylation compared to the DMSO control. By contrast, in the case of A549 cells, the phosphorylation of 21 peptides was significantly affected by the same compounds. In light of the potential for the negative side-effects of DUB inhibition, such as increased cancer cell motility, the data presented herein underscore the dire need for the development of specific DUB inhibitors and to elucidate the individual role of DUB family members in cancer biology before they can be specifically pharmacologically targeted.

The bacterial deubiquitinase Ceg23 regulates the association of Lys-63–linked polyubiquitin molecules on the Legionella phagosome

Legionella pneumophila is the causative agent of the lung malady Legionnaires' disease, it modulates host function to create a niche termed the Legionella-containing vacuole (LCV) that permits intracellular L. pneumophila replication. One important aspect of such modulation is the co-option of the host ubiquitin network with a panel of effector proteins. Here, using recombinantly expressed and purified proteins, analytic ultracentrifugation, structural analysis, and computational modeling, along with deubiquitinase (DUB), and bacterial infection assays, we found that the bacterial defective in organelle trafficking/intracellular multiplication effector Ceg23 is a member of the ovarian tumor (OTU) DUB family. We found that Ceg23 displays high specificity toward Lys-63-linked polyubiquitin chains and is localized on the LCV, where it removes ubiquitin moieties from proteins ubiquitinated by the Lys-63-chain type. Analysis of the crystal structure of a Ceg23 variant lacking two putative transmembrane domains at 2.80 Å resolution revealed that despite very limited homology to established members of the OTU family at the primary sequence level, Ceg23 harbors a catalytic motif resembling those associated with typical OTU-type DUBs. ceg23 deletion increased the association of Lys-63-linked polyubiquitin with the bacterial phagosome, indicating that Ceg23 regulates Lys-63-linked ubiquitin signaling on the LCV. In summary, our findings indicate that Ceg23 contributes to the regulation of the association of Lys-63 type polyubiquitin with the Legionella phagosome. Future identification of host substrates targeted by Ceg23 could clarify the roles of these polyubiquitin chains in the intracellular life cycle of L. pneumophila and Ceg23's role in bacterial virulence.

A Novel Luminescence-Based High-Throughput Approach for Cellular Resolution of Protein Ubiquitination Using Tandem Ubiquitin Binding Entities (TUBEs).

Protein turnover is highly regulated by the posttranslational process of ubiquitination. Deregulation of the ubiquitin proteasome system (UPS) has been implicated in cancer and neurodegenerative diseases, and modulating this system has proven to be a viable approach for therapeutic intervention. The development of novel technologies that enable high-throughput studies of substrate protein ubiquitination is key for UPS drug discovery. Conventional approaches for studying ubiquitination either have high protein requirements or rely on exogenous or modified ubiquitin moieties, thus limiting their utility. In order to circumvent these issues, we developed a high-throughput live-cell assay that combines the NanoBiT luminescence-based technology with tandem ubiquitin binding entities (TUBEs) to resolve substrate ubiquitination. To demonstrate the effectiveness and utility of this assay, we studied compound-induced ubiquitination of the G to S Phase Transition 1 (GSPT1) protein. Using this assay, we characterized compounds with varying levels of GSPT1 ubiquitination activity. This method provides a live-cell-based approach for assaying substrate ubiquitination that can be adapted to study the kinetics of ubiquitin transfer onto a substrate protein of interest. In addition, our results show that this approach is portable for studying the ubiquitination of target proteins with diverse functions.

The Ubiquitin System: a Regulatory Hub for Intellectual Disability and Autism Spectrum Disorder.

Intellectual disability (ID) and autism spectrum disorder (ASD) are two of the most common neurodevelopmental disorders. Both disorders are extremely heterogenous, and only ~ 40% of reported cases have so far been attributed to genetic mutations. Of the many cellular processes that are affected, the ubiquitin system (UbS) is of particular relevance in that it can rapidly regulate multiple signaling cascades simultaneously. The UbS is a post-translational modification process that revolves around the covalent attachment of a ubiquitin moiety to a substrate, thereby influencing different elements of protein biology, including trafficking, signal transduction, and degradation. Importantly, the UbS has been implicated in regulating multiple pathophysiological pathways related to ASD and ID. This review will discuss how the UbS acts as major signaling hub in the pathogenesis of ASD and ID, raising the prospect of treating broader patient cohorts by targeting the UbS as a common point of convergence of various mutations.

Usp14 is required for spermatogenesis and ubiquitin stress responses in Drosophila melanogaster.

Deubiquitylating (DUB) enzymes free covalently linked ubiquitin moieties from ubiquitin-ubiquitin and ubiquitin-protein conjugates, and thereby maintain the equilibrium between free and conjugated ubiquitin moieties and regulate ubiquitin-mediated cellular processes. Here, we performed genetic analyses of mutant phenotypes in Drosophila melanogaster and demonstrate that loss of Usp14 function results in male sterility, with defects in spermatid individualization and reduced testicular free monoubiquitin levels. These phenotypes were rescued by germline-specific overexpression of wild-type Usp14. Synergistic genetic interactions with Ubi-p63E and cycloheximide sensitivity suggest that ubiquitin shortage is a primary cause of male sterility. In addition, Usp14 is predominantly expressed in testes in Drosophila, indicating a higher demand for this DUB in testes that is also reflected by testis-specific loss-of-function Usp14 phenotypes. Collectively, these results suggest a major role of Usp14 in maintaining normal steady state free monoubiquitin levels during the later stages of Drosophila spermatogenesis.This article has an associated First Person interview with the first author of the paper.

Linear Ubiquitin Chains: Cellular Functions and Strategies for Detection and Quantification.

Ubiquitination of proteins is a sophisticated post-translational modification implicated in the regulation of an ever-growing abundance of cellular processes. Recent insights into different layers of complexity have shaped the concept of the ubiquitin code. Key players in determining this code are the number of ubiquitin moieties attached to a substrate, the architecture of polyubiquitin chains, and post-translational modifications of ubiquitin itself. Ubiquitination can induce conformational changes of substrates and alter their interactive profile, resulting in the formation of signaling complexes. Here we focus on a distinct type of ubiquitination that is characterized by an inter-ubiquitin linkage through the N-terminal methionine, called M1-linked or linear ubiquitination. Formation, recognition, and disassembly of linear ubiquitin chains are highly specific processes that are implicated in immune signaling, cell death regulation and protein quality control. Consistent with their role in influencing signaling events, linear ubiquitin chains are formed in a transient and spatially regulated manner, making their detection and quantification challenging.

Rad6/Rad18 Competes with DNA Polymerases η and δ for PCNA Encircling DNA.

Translesion DNA synthesis (TLS) bypasses DNA lesions encountered during S-phase and is critical for cell survival after exposure to DNA-damaging agents. In humans, Rad6/Rad18 attaches single ubiquitin moieties (i.e., monoubiquitination) to proliferating cell nuclear antigen (PCNA) sliding clamps encircling primer/template (P/T) junctions that are stalled at DNA lesions. TLS occurs via PCNA monoubiquitination-independent and -dependent pathways, and both contribute to cell survival. The interaction of Rad6/Rad18 with PCNA is paramount to PCNA monoubiquitination and remains poorly defined. In particular, the location of the Rad6/Rad18 binding site on PCNA is unknown. Many PCNA-binding proteins, particularly DNA polymerases (pols), converge on PCNA encircling stalled P/T junctions in human cells, and all interact in a similar manner with the universal binding sites on PCNA. We reasoned the following: if Rad6/Rad18 utilizes the universal binding sites (or nearby sites), then PCNA monoubiquitination may be suppressed by pols involved in TLS. Results from quantitative studies reveal that (1) a Y-family pol (pol η) and a B-family pol (pol δ) critical to TLS each inhibit the transfer of ubiquitin from Rad6/Rad18 to PCNA and that (2) the observed inhibitions are dependent on the interaction of these pols with PCNA encircling DNA. These studies suggest that Rad6/Rad18 utilizes the universal PCNA-binding sites or nearby sites and, hence, competes for PCNA encircling DNA with pols η and δ and possibly other PCNA-binding proteins involved in TLS. These findings provide valuable insight into the nature of the interaction between Rad6/Rad18 and PCNA and have important implications for the division of human TLS pathways.

Competitive ubiquitination activates the tumor suppressor p53

Blocking p53 ubiquitination through disrupting its interaction with MDM2 or inhibiting the MDM2 catalytic activity is the central mechanism by which the tumor suppressor p53 is activated in response to genotoxic challenges. Although MDM2 is first characterized as the major E3 ubiquitin ligase for p53, it can also catalyze the conjugation of ubiquitin moieties to other proteins (e.g., activating transcription factor 3, or ATF3). Here we report that ATF3 can act as an ubiquitin “trap” and competes with p53 for MDM2-mediated ubiquitination. While ATF3-mediated p53 stabilization required ATF3 binding to the MDM2 RING domain, we demonstrated that ATF3 ubiquitination catalyzed by MDM2 was indispensable for p53 activation in response to DNA damage. Moreover, a cancer-derived ATF3 mutant (R88G) devoid of ubiquitination failed to prevent p53 from MDM2-mediated degradation and thus was unable to activate the tumor suppressor. Therefore, we have identified a previously-unknown mechanism that can activate p53 in the genotoxic response.

Conformational and functional characterization of artificially conjugated non-canonical ubiquitin dimers

Ubiquitylation is an eminent posttranslational modification referring to the covalent attachment of single ubiquitin molecules or polyubiquitin chains to a target protein dictating the fate of such labeled polypeptide chains. Here, we have biochemically produced artificially Lys11-, and Lys27-, and Lys63-linked ubiquitin dimers based on click-chemistry generating milligram quantities in high purity. We show that the artificial linkage used for the conjugation of two ubiquitin moieties represents a fully reliable surrogate of the natural isopeptide bond by acquiring highly resolved nuclear magnetic resonance (NMR) spectroscopic data including ligand binding studies. Extensive coarse grained and atomistic molecular dynamics (MD) simulations allow to extract structures representing the ensemble of domain-domain conformations used to verify the experimental data. Advantageously, this methodology does not require individual isotopic labeling of both ubiquitin moieties as NMR data have been acquired on the isotopically labeled proximal moiety and complementary MD simulations have been used to fully interpret the experimental data in terms of domain-domain conformation. This combined approach intertwining NMR spectroscopy with MD simulations makes it possible to describe the conformational space non-canonically Lys11-, and Lys27-linked ubiquitin dimers occupy in a solution averaged ensemble by taking atomically resolved information representing all residues in ubiquitin dimers into account.

Structural insights into ubiquitin recognition and Ufd1 interaction of Npl4

Npl4 is likely to be the most upstream factor recognizing Lys48-linked polyubiquitylated substrates in the proteasomal degradation pathway in yeast. Along with Ufd1, Npl4 forms a heterodimer (UN), and functions as a cofactor for the Cdc48 ATPase. Here, we report the crystal structures of yeast Npl4 in complex with Lys48-linked diubiquitin and with the Npl4-binding motif of Ufd1. The distal and proximal ubiquitin moieties of Lys48-linked diubiquitin primarily interact with the C-terminal helix and N-terminal loop of the Npl4 C-terminal domain (CTD), respectively. Mutational analysis suggests that the CTD contributes to linkage selectivity and initial binding of ubiquitin chains. Ufd1 occupies a hydrophobic groove of the Mpr1/Pad1 N-terminal (MPN) domain of Npl4, which corresponds to the catalytic groove of the MPN domain of JAB1/MPN/Mov34 metalloenzyme (JAMM)-family deubiquitylating enzyme. This study provides important structural insights into the polyubiquitin chain recognition by the Cdc48–UN complex and its assembly.

Ubiquitin carboxyl-terminal hydrolases are required for period maintenance of the circadian clock at high temperature in Arabidopsis

Protein ubiquitylation participates in a number of essential cellular processes including signal transduction and transcription, often by initiating the degradation of specific substrates through the 26S proteasome. Within the ubiquitin-proteasome system, deubiquitylating enzymes (DUBs) not only help generate and maintain the supply of free ubiquitin monomers, they also directly control functions and activities of specific target proteins by modulating the pool of ubiquitylated species. Ubiquitin carboxyl-terminal hydrolases (UCHs) belong to an enzymatic subclass of DUBs, and are represented by three members in Arabidopsis, UCH1, UCH2 and UCH3. UCH1 and UCH2 influence auxin-dependent developmental pathways in Arabidopsis through their deubiquitylation activities, whereas biological and enzymatic functions of UCH3 remain unclear. Here, we demonstrate that Arabidopsis UCH3 acts to maintain the period of the circadian clock at high temperatures redundantly with UCH1 and UCH2. Whereas single uch1, uch2 and uch3 mutants have weak circadian phenotypes, the triple uch mutant displays a drastic lengthening of period at high temperatures that is more extreme than the uch1 uch2 double mutant. UCH3 also possesses a broad deubiquitylation activity against a range of substrates that link ubiquitin via peptide and isopeptide linkages. While the protein target(s) of UCH1-3 are not yet known, we propose that these DUBs act on one or more factors that control period length of the circadian clock through removal of their bound ubiquitin moieties, thus ensuring that the clock oscillates with a proper period even at elevated temperatures.

Double Monoubiquitination Modifies the Molecular Recognition Properties of p15PAF Promoting Binding to the Reader Module of Dnmt1.

The proliferating cell nuclear antigen (PCNA)-associated factor p15PAF is a nuclear protein that acts as a regulator of DNA repair during DNA replication. The p15PAF gene is overexpressed in several types of human cancer, and its function is regulated by monoubiquitination of two lysines (K15 and K24) at the protein N-terminal region. We have previously shown that p15PAF is an intrinsically disordered protein which partially folds upon binding to PCNA and independently contacts DNA through its N-terminal tail. Here we present an NMR conformational characterization of p15PAF monoubiquitinated at both K15 and K24 via a disulfide bridge mimicking the isopeptide bond. We show that doubly monoubiquitinated p15PAF is monomeric, intrinsically disordered, and binds to PCNA as nonubiquitinated p15PAF does but interacts with DNA with reduced affinity. Our SAXS-derived conformational ensemble of doubly monoubiquitinated p15PAF shows that the ubiquitin moieties, separated by eight disordered residues, form transient dimers because of the high local effective ubiquitin concentration. This observation and the sequence similarity with histone H3 N-terminal tail suggest that doubly monoubiquitinated p15PAF is a binding target of DNA methyl transferase Dnmt1, as confirmed by calorimetry. Therefore, doubly monoubiquitinated p15PAF directly interacts with PCNA and recruits Dnmt1 for maintenance of DNA methylation during replication.