USP7 small-molecule inhibitors interfere with ubiquitin binding.

The ubiquitin system regulates essential cellular processes in eukaryotes. Ubiquitin is ligated to substrate proteins as monomers or chains and the topology of ubiquitin modifications regulates substrate interactions with specific proteins. Thus ubiquitination directs a variety of substrate fates including proteasomal degradation. Deubiquitinase enzymes cleave ubiquitin from substrates and are implicated in disease; for example, ubiquitin-specific protease-7 (USP7) regulates stability of the p53 tumour suppressor and other proteins critical for tumour cell survival. However, developing selective deubiquitinase inhibitors has been challenging and no co-crystal structures have been solved with small-molecule inhibitors. Here, using nuclear magnetic resonance-based screening and structure-based design, we describe the development of selective USP7 inhibitors GNE-6640 and GNE-6776. These compounds induce tumour cell death and enhance cytotoxicity with chemotherapeutic agents and targeted compounds, including PIM kinase inhibitors. Structural studies reveal that GNE-6640 and GNE-6776 non-covalently target USP7 12 Å distant from the catalytic cysteine. The compounds attenuate ubiquitin binding and thus inhibit USP7 deubiquitinase activity. GNE-6640 and GNE-6776 interact with acidic residues that mediate hydrogen-bond interactions with the ubiquitin Lys48 side chain, suggesting that USP7 preferentially interacts with and cleaves ubiquitin moieties that have free Lys48 side chains. We investigated this idea by engineering di-ubiquitin chains containing differential proximal and distal isotopic labels and measuring USP7 binding by nuclear magnetic resonance. This preferential binding protracted the depolymerization kinetics of Lys48-linked ubiquitin chains relative to Lys63-linked chains. In summary, engineering compounds that inhibit USP7 activity by attenuating ubiquitin binding suggests opportunities for developing other deubiquitinase inhibitors and may be a strategy more broadly applicable to inhibiting proteins that require ubiquitin binding for full functional activity.

DUBs, the regulation of cell identity and disease.

The post-translational modification of proteins with ubiquitin represents a complex signalling system that co-ordinates essential cellular functions, including proteolysis, DNA repair, receptor signalling and cell communication. DUBs (deubiquitinases), the enzymes that disassemble ubiquitin chains and remove ubiquitin from proteins, are central to this system. Reflecting the complexity and versatility of ubiquitin signalling, DUB activity is controlled in multiple ways. Although several lines of evidence indicate that aberrant DUB function may promote human disease, the underlying molecular mechanisms are often unclear. Notwithstanding, considerable interest in DUBs as potential drug targets has emerged over the past years. The future success of DUB-based therapy development will require connecting the basic science of DUB function and enzymology with drug discovery. In the present review, we discuss new insights into DUB activity regulation and their links to disease, focusing on the role of DUBs as regulators of cell identity and differentiation, and discuss their potential as emerging drug targets.

SUMO-targeted ubiquitin ligase, Rad60, and Nse2 SUMO ligase suppress spontaneous Top1-mediated DNA damage and genome instability.

Through as yet undefined proteins and pathways, the SUMO-targeted ubiquitin ligase (STUbL) suppresses genomic instability by ubiquitinating SUMO conjugated proteins and driving their proteasomal destruction. Here, we identify a critical function for fission yeast STUbL in suppressing spontaneous and chemically induced topoisomerase I (Top1)-mediated DNA damage. Strikingly, cells with reduced STUbL activity are dependent on tyrosyl-DNA phosphodiesterase 1 (Tdp1). This is notable, as cells lacking Tdp1 are largely aphenotypic in the vegetative cell cycle due to the existence of alternative pathways for the removal of covalent Top1-DNA adducts (Top1cc). We further identify Rad60, a SUMO mimetic and STUbL-interacting protein, and the SUMO E3 ligase Nse2 as critical Top1cc repair factors in cells lacking Tdp1. Detection of Top1ccs using chromatin immunoprecipitation and quantitative PCR shows that they are elevated in cells lacking Tdp1 and STUbL, Rad60, or Nse2 SUMO ligase activity. These unrepaired Top1ccs are shown to cause DNA damage, hyper-recombination, and checkpoint-mediated cell cycle arrest. We further determine that Tdp1 and the nucleotide excision repair endonuclease Rad16-Swi10 initiate the major Top1cc repair pathways of fission yeast. Tdp1-based repair is the predominant activity outside S phase, likely acting on transcription-coupled Top1cc. Epistasis analyses suggest that STUbL, Rad60, and Nse2 facilitate the Rad16-Swi10 pathway, parallel to Tdp1. Collectively, these results reveal a unified role for STUbL, Rad60, and Nse2 in protecting genome stability against spontaneous Top1-mediated DNA damage.

Dual recruitment of Cdc48 (p97)-Ufd1-Npl4 ubiquitin-selective segregase by small ubiquitin-like modifier protein (SUMO) and ubiquitin in SUMO-targeted ubiquitin ligase-mediated genome stability functions.

Protein modification by SUMO and ubiquitin critically impacts genome stability via effectors that "read" their signals using SUMO interaction motifs or ubiquitin binding domains, respectively. A novel mixed SUMO and ubiquitin signal is generated by the SUMO-targeted ubiquitin ligase (STUbL), which ubiquitylates SUMO conjugates. Herein, we determine that the "ubiquitin-selective" segregase Cdc48-Ufd1-Npl4 also binds SUMO via a SUMO interaction motif in Ufd1 and can thus act as a selective receptor for STUbL targets. Indeed, we define key cooperative DNA repair functions for Cdc48-Ufd1-Npl4 and STUbL, thereby revealing a new signaling mechanism involving dual recruitment by SUMO and ubiquitin for Cdc48-Ufd1-Npl4 functions in maintaining genome stability.

Variability analysis of human plasma and cerebral spinal fluid reveals statistical significance of changes in mass spectrometry-based metabolomics data.

Analytical and biological variability are issues of central importance to human metabolomics studies. Here both types of variation are examined in human plasma and cerebrospinal fluid (CSF) using a global liquid chromatography/mass spectrometry (LC/MS) metabolomics strategy. The platform shows small analytical variation with a median coefficient of variation (CV) of 15-16% for both plasma and CSF sample matrixes when the integrated area of each peak in the mass spectra is considered. Analysis of biological variation shows that human CSF has a median CV of 35% and plasma has a median CV of 46%. To understand the difference in CV between the biofluids, we compared plasma and CSF independently obtained from different healthy humans. Additionally, we analyzed another group of patients from whom we compared matched CSF and plasma (plasma and CSF obtained from the same human subject). A similar number of features was observed in both biofluids, although the majority of features appeared with greater intensity in plasma. More than a dozen metabolites shared between the human CSF and plasma metabolomes were identified based on accurate mass measurements, retention times, and MS/MS spectra. The fold change in these metabolites was consistent with the median biological CV determined for all peaks. The measured median biological CV together with analysis of intragroup variation of healthy individuals suggests that fold changes above 2 in metabolomics studies investigating plasma or CSF are statistically relevant with respect to the inherent variability of a healthy control group. These data demonstrate the reproducibility of the global metabolomics platform using LC/MS and reveal the robustness of the approach for biomarker discovery.

Reactive-site-centric chemoproteomics identifies a distinct class of deubiquitinase enzymes.

Activity-based probes (ABPs) are widely used to monitor the activity of enzyme families in biological systems. Inferring enzyme activity from probe reactivity requires that the probe reacts with the enzyme at its active site; however, probe-labeling sites are rarely verified. Here we present an enhanced chemoproteomic approach to evaluate the activity and probe reactivity of deubiquitinase enzymes, using bioorthogonally tagged ABPs and a sequential on-bead digestion protocol to enhance the identification of probe-labeling sites. We confirm probe labeling of deubiquitinase catalytic Cys residues and reveal unexpected labeling of deubiquitinases on non-catalytic Cys residues and of non-deubiquitinase proteins. In doing so, we identify ZUFSP (ZUP1) as a previously unannotated deubiquitinase with high selectivity toward cleaving K63-linked chains. ZUFSP interacts with and modulates ubiquitination of the replication protein A (RPA) complex. Our reactive-site-centric chemoproteomics method is broadly applicable for identifying the reaction sites of covalent molecules, which may expand our understanding of enzymatic mechanisms.

DNA recognition by a σ(54) transcriptional activator from Aquifex aeolicus.

Transcription initiation by bacterial σ(54)-polymerase requires the action of a transcriptional activator protein. Activators bind sequence-specifically upstream of the transcription initiation site via a DNA-binding domain (DBD). The structurally characterized DBDs from activators all belong to the Fis (factor for inversion stimulation) family of helix-turn-helix DNA-binding proteins. We report here structures of the free and DNA-bound forms of the DBD of NtrC4 (4DBD) from Aquifex aeolicus, a member of the NtrC family of σ(54) activators. Two NtrC4-binding sites were identified upstream (-145 and -85bp) from the start of the lpxC gene, which is responsible for the first committed step in lipid A biosynthesis. This is the first experimental evidence for σ(54) regulation in lpxC expression. 4DBD was crystallized both without DNA and in complex with the -145-binding site. The structures, together with biochemical data, indicate that NtrC4 binds to DNA in a manner that is similar to that of its close homolog, Fis. The greater sequence specificity for the binding of 4DBD relative to Fis seems to arise from a larger number of base-specific contacts contributing to affinity than for Fis.

Structure and regulatory mechanism of Aquifex aeolicus NtrC4: variability and evolution in bacterial transcriptional regulation.

Genetic changes lead gradually to altered protein function, making deduction of the molecular basis for activity from a sequence difficult. Comparative studies provide insights into the functional consequences of specific changes. Here we present structural and biochemical studies of NtrC4, a sigma-54 activator from Aquifex aeolicus, and compare it with NtrC1 (a paralog) and NtrC (a homolog from Salmonella enterica) to provide insight into how a substantial change in regulatory mechanism may have occurred. Activity assays show that assembly of NtrC4's active oligomer is repressed by the N-terminal receiver domain, and that BeF3- addition (mimicking phosphorylation) removes this repression. Observation of assembly without activation for NtrC4 indicates that it is much less strongly repressed than NtrC1. The crystal structure of the unactivated receiver-ATPase domain combination shows a partially disrupted interface. NMR structures of the regulatory domain show that its activation mechanism is very similar to that of NtrC1. The crystal structure of the NtrC4 DNA-binding domain shows that it is dimeric and more similar in structure to NtrC than NtrC1. Electron microscope images of the ATPase-DNA-binding domain combination show formation of oligomeric rings. Sequence alignments provide insights into the distribution of activation mechanisms in this family of proteins.

Phosphatases, DNA damage checkpoints and checkpoint deactivation.

Cells have evolved intricate and specialized responses to DNA damage, central to which are the DNA damage checkpoints that arrest cell cycle progression and facilitate the repair process. Activation of these damage checkpoints relies heavily on the activity of Ser/Thr kinases, such as Chk1 and Chk2 (Saccharomyces cerevisiae Rad53), which are themselves activated by phosphorylation. Only more recently have we begun to understand how cells disengage the checkpoints to reenter the cell cycle. Here, we review progress toward understanding the functions of phosphatases in checkpoint deactivation in S. cerevisiae, focusing on the non-redundant roles of the type 2A phosphatase Pph3 and the PP2C phosphatases Ptc2 and Ptc3 in the deactivation of Rad53. We discuss how these phosphatases may specifically recognize different phosphorylated forms of Rad53 and how each may independently regulate different facets of the checkpoint response. In conjunction with the independent dephosphorylation of other checkpoint proteins, such regulation may allow a more tailored response to DNA damage that is coordinated with the repair process, ultimately resulting in the resumption of growth.

Crystal structure of archaeal ribonuclease P protein aRpp29 from Archaeoglobus fulgidus.

The crystal structure of ribonuclease P protein aRpp29 from the sulfate-reducing hyperthermophile Archaeoglobus fulgidus was determined at 1.7 A resolution using X-ray diffraction methods. The central feature of this archaeal protein is a sheet of six antiparallel beta-strands twisted around a conserved hydrophobic core. Residues near the N- and C-termini form helical structures that are oriented in an antiparallel manner. A comparison of conserved amino acids indicates that archaeal aRpp29 is homologous to human ribonuclease P protein Rpp29. The aRpp29 protein is structurally similar to bacterial transcription factors Hfq and NusG, as well as the Sm and Sm-like RNA-associated proteins from eukarya. The crystal structure of A. fulgidus aRpp29 differs from the previously reported solution structure, where NMR data did not detect the helices and indicated that approximately 40% of the residues are relatively flexible or disordered. Circular dichroism data indicate that the protein has less helical content than the amount observed in the crystal, suggesting that in solution the helical regions are unfolded or in equilibrium between folded and unfolded forms; this hypothesis is consistent with amide proton exchange rate data. Surface residues that are conserved from archaea to humans and are likely to interact with the ribonuclease P RNA or other protein subunits are identified in the structure. The model of the aRpp29 protein defined by this work provides an essential step toward eventually understanding the overall architecture of ribonuclease P.