Off-target inhibition of NGLY1 by the polycaspase inhibitor Z-VAD-fmk induces cellular autophagy.

The polycaspase inhibitor Z-VAD-fmk acts as an inhibitor of peptide: N-glycanase (NGLY1), an endoglycosidase which cleaves N-linked glycans from glycoproteins exported from the endoplasmic reticulum (ER) during ER-associated degradation (ERAD). Both pharmacological N-glycanase inhibition by Z-VAD-fmk and siRNA-mediated knockdown (KD) of NGLY1 induce GFP-LC3-positive puncta in HEK 293 cells. The activation of ER stress markers or induction of reactive oxygen species (ROS) is not observed under either condition. Moreover, Ca2+ handling is unaffected when observing release from intracellular stores. Under conditions of pharmacological NGLY1 inhibition or NGLY1 KD, upregulation of autophagosome formation without impairment of autophagic flux is observed. Enrichment of autophagosomes by immunoprecipitation (IP) and mass spectrometry-based proteomic analysis reveals comparable autophagosomal protein content. Gene ontology analysis of proteins enriched in autophagosome IPs shows overrepresentation of factors involved in protein translation, localization and targeting, RNA degradation and protein complex disassembly. Upregulation of autophagy represents a cellular adaptation to NGLY1 inhibition or KD, and ATG13-deficient mouse embryonic fibroblasts (MEFs) show reduced viability under these conditions. In contrast, treatment with pan-caspase inhibitor, Q-VD-OPh, does not induce cellular autophagy. Therefore, experiments with Z-VAD-fmk are complicated by the effects of NGLY1 inhibition, including induction of autophagy, and Q-VD-OPh represents an alternative caspase inhibitor free from this limitation. ENZYMES: Peptide:N-glycanase1, Peptide-N(4)-(N-acetyl-beta-glucosaminyl)asparagine amidase [EC:3.5.1.52].

Systemic muscle wasting and coordinated tumour response drive tumourigenesis.

Cancer cells demand excess nutrients to support their proliferation, but how tumours exploit extracellular amino acids during systemic metabolic perturbations remain incompletely understood. Here, we use a Drosophila model of high-sugar diet (HSD)-enhanced tumourigenesis to uncover a systemic host-tumour metabolic circuit that supports tumour growth. We demonstrate coordinate induction of systemic muscle wasting with tumour-autonomous Yorkie-mediated SLC36-family amino acid transporter expression as a proline-scavenging programme to drive tumourigenesis. We identify Indole-3-propionic acid as an optimal amino acid derivative to rationally target the proline-dependency of tumour growth. Insights from this whole-animal Drosophila model provide a powerful approach towards the identification and therapeutic exploitation of the amino acid vulnerabilities of tumourigenesis in the context of a perturbed systemic metabolic network.

Probing enzymatic activity - a radical approach.

Deubiquitinating enzymes (DUBs) are known to have numerous important interactions with the ubiquitin cascade and their dysregulation is associated with several diseases, including cancer and neurodegeneration. They are an important class of enzyme, and activity-based probes have been developed as an effective strategy to study them. Existing activity-based probes that target the active site of these enzymes work via nucleophilic mechanisms. We present the development of latent ubiquitin-based probes that target DUBs via a site selective, photoinitiated radical mechanism. This approach differs from existing photocrosslinking probes as it requires a free active site cysteine. In contrast to existing cysteine reactive probes, control over the timing of the enzyme-probe reaction is possible as the alkene warhead is completely inert under ambient conditions, even upon probe binding. The probe's reactivity has been demonstrated against recombinant DUBs and to capture endogenous DUB activity in cell lysate. This allows more finely resolved investigations of DUBs.

Use of Modified Clostridium perfringens Enterotoxin Fragments for Claudin Targeting in Liver and Skin Cells.

Claudins regulate paracellular permeability in different tissues. The claudin-binding domain of Clostridium perfringens enterotoxin (cCPE) is a known modulator of a claudin subset. However, it does not efficiently bind to claudin-1 (Cldn1). Cldn1 is a pharmacological target since it is (i) an essential co-receptor for hepatitis C virus (HCV) infections and (ii) a key element of the epidermal barrier limiting drug delivery. In this study, we investigated the potential of a Cldn1-binding cCPE mutant (i) to inhibit HCV entry into hepatocytes and (ii) to open the epidermal barrier. Inhibition of HCV infection by blocking of Cldn1 with cCPE variants was analyzed in the Huh7.5 hepatoma cell line. A model of reconstructed human epidermis was used to investigate modulation of the epidermal barrier by cCPE variants. In contrast to cCPEwt, the Cldn1-binding cCPE-S305P/S307R/S313H inhibited infection of Huh7.5 cells with HCV in a dose-dependent manner. In addition, TJ modulation by cCPE variant-mediated targeting of Cldn1 and Cldn4 opened the epidermal barrier in reconstructed human epidermis. cCPE variants are potent claudin modulators. They can be applied for mechanistic in vitro studies and might also be used as biologics for therapeutic claudin targeting including HCV treatment (host-targeting antivirals) and improvement of drug delivery.

Proteomics Analysis of Ovarian Cancer Cell Lines and Tissues Reveals Drug Resistance-associated Proteins.

BACKGROUND: Carboplatin and paclitaxel form the cornerstone of chemotherapy for epithelial ovarian cancer, however, drug resistance to these agents continues to present challenges. Despite extensive research, the mechanisms underlying this resistance remain unclear. MATERIALS AND METHODS: A 2D-gel proteomics method was used to analyze protein expression levels of three human ovarian cancer cell lines and five biopsy samples. Representative proteins identified were validated via western immunoblotting. Ingenuity pathway analysis revealed metabolomic pathway changes. RESULTS: A total of 189 proteins were identified with restricted criteria. Combined treatment targeting the proteasome-ubiquitin pathway resulted in re-sensitisation of drug-resistant cells. In addition, examination of five surgical biopsies of ovarian tissues revealed α-enolase (ENOA), elongation factor Tu, mitochondrial (EFTU), glyceraldehyde-3-phosphate dehydrogenase (G3P), stress-70 protein, mitochondrial (GRP75), apolipoprotein A-1 (APOA1), peroxiredoxin (PRDX2) and annexin A (ANXA) as candidate biomarkers of drug-resistant disease. CONCLUSION: Proteomics combined with pathway analysis provided information for an effective combined treatment approach overcoming drug resistance. Analysis of cell lines and tissues revealed potential prognostic biomarkers for ovarian cancer.

Systemic Administration of Glibenclamide Fails to Achieve Therapeutic Levels in the Brain and Cerebrospinal Fluid of Rodents.

Activating mutations in the Kir6.2 (KCNJ11) subunit of the ATP-sensitive potassium channel cause neonatal diabetes (ND). Patients with severe mutations also suffer from neurological complications. Glibenclamide blocks the open KATP channels and is the treatment of choice for ND. However, although glibenclamide successfully restores normoglycaemia, it has a far more limited effect on the neurological problems. To assess the extent to which glibenclamide crosses the blood-brain barrier (BBB) in vivo, we quantified glibenclamide concentrations in plasma, cerebrospinal fluid (CSF), and brain tissue of rats, control mice, and mice expressing a human neonatal diabetes mutation (Kir6.2-V59M) selectively in neurones (nV59M mice). As only small sample volumes can be obtained from rodents, we developed a highly sensitive method of analysis, using liquid chromatography tandem mass spectrometry acquisition with pseudo-selected reaction monitoring, achieving a quantification limit of 10ng/ml (20nM) glibenclamide in a 30µl sample. Glibenclamide was not detectable in the CSF or brain of rats after implantation with subcutaneous glibenclamide pellets, despite high plasma concentrations. Further, one hour after a suprapharmacological glibenclamide dose was administered directly into the lateral ventricle of the brain, the plasma concentration was twice that of the CSF. This suggests the drug is rapidly exported from the CSF. Elacridar, an inhibitor of P-glycoprotein and breast cancer resistance protein (major multidrug resistance transporters at the BBB), did not affect glibenclamide levels in CSF and brain tissue. We also identified a reduced sensitivity to volatile anaesthetics in nV59M mice and showed this was not reversed by systemic delivery of glibenclamide. Our results therefore suggest that little glibenclamide reaches the central nervous system when given systemically, that glibenclamide is rapidly removed across the BBB when given intracranioventricularly, and that any glibenclamide that does enter (and is below our detection limit) is insufficient to influence neuronal function as assessed by anaesthesia sensitivity.

The human otubain2-ubiquitin structure provides insights into the cleavage specificity of poly-ubiquitin-linkages.

Ovarian tumor domain containing proteases cleave ubiquitin (Ub) and ubiquitin-like polypeptides from proteins. Here we report the crystal structure of human otubain 2 (OTUB2) in complex with a ubiquitin-based covalent inhibitor, Ub-Br2. The ubiquitin binding mode is oriented differently to how viral otubains (vOTUs) bind ubiquitin/ISG15, and more similar to yeast and mammalian OTUs. In contrast to OTUB1 which has exclusive specificity towards Lys48 poly-ubiquitin chains, OTUB2 cleaves different poly-Ub linked chains. N-terminal tail swapping experiments between OTUB1 and OTUB2 revealed how the N-terminal structural motifs in OTUB1 contribute to modulating enzyme activity and Ub-chain selectivity, a trait not observed in OTUB2, supporting the notion that OTUB2 may affect a different spectrum of substrates in Ub-dependent pathways.

Deubiquitinating enzyme specificity for ubiquitin chain topology profiled by di-ubiquitin activity probes.

Posttranslational modification with ubiquitin (Ub) controls many cellular processes, and aberrant ubiquitination can contribute to cancer, immunopathology, and neurodegeneration. The versatility arises from the ability of Ub to form polymer chains with eight distinct linkages via lysine side chains and the N terminus. In this study, we engineered Di-Ub probes mimicking all eight different poly-Ub linkages and profiled the deubiquitinating enzyme (DUB) selectivity for recognizing Di-Ub moieties in cellular extracts. Mass spectrometric profiling revealed that most DUBs examined have broad selectivity, whereas a subset displays a clear preference for recognizing noncanonical over K48/K63 Ub linkages. Our results expand knowledge of Ub processing enzyme functions in cellular contexts that currently depends largely on using recombinant enzymes and substrates.

Protein profiling in hepatocellular carcinoma by label-free quantitative proteomics in two west African populations.

BACKGROUND: Hepatocellular Carcinoma is the third most common cause of cancer related death worldwide, often diagnosed by measuring serum AFP; a poor performance stand-alone biomarker. With the aim of improving on this, our study focuses on plasma proteins identified by Mass Spectrometry in order to investigate and validate differences seen in the respective proteomes of controls and subjects with LC and HCC. METHODS: Mass Spectrometry analysis using liquid chromatography electro spray ionization quadrupole time-of-flight was conducted on 339 subjects using a pooled expression profiling approach. ELISA assays were performed on four significantly differentially expressed proteins to validate their expression profiles in subjects from the Gambia and a pilot group from Nigeria. Results from this were collated for statistical multiplexing using logistic regression analysis. RESULTS: Twenty-six proteins were identified as differentially expressed between the three subject groups. Direct measurements of four; hemopexin, alpha-1-antitrypsin, apolipoprotein A1 and complement component 3 confirmed their change in abundance in LC and HCC versus control patients. These trends were independently replicated in the pilot validation subjects from Nigeria. The statistical multiplexing of these proteins demonstrated performance comparable to or greater than ALT in identifying liver cirrhosis or carcinogenesis. This exercise also proposed preliminary cut offs with achievable sensitivity, specificity and AUC statistics greater than reported AFP averages. CONCLUSIONS: The validated changes of expression in these proteins have the potential for development into high-performance tests usable in the diagnosis and or monitoring of HCC and LC patients. The identification of sustained expression trends strengthens the suggestion of these four proteins as worthy candidates for further investigation in the context of liver disease. The statistical combinations also provide a novel inroad of analyses able to propose definitive cut-offs and combinations for evaluation of performance.

Activity-based chemical proteomics accelerates inhibitor development for deubiquitylating enzymes.

Converting lead compounds into drug candidates is a crucial step in drug development, requiring early assessment of potency, selectivity, and off-target effects. We have utilized activity-based chemical proteomics to determine the potency and selectivity of deubiquitylating enzyme (DUB) inhibitors in cell culture models. Importantly, we characterized the small molecule PR-619 as a broad-range DUB inhibitor, and P22077 as a USP7 inhibitor with potential for further development as a chemotherapeutic agent in cancer therapy. A striking accumulation of polyubiquitylated proteins was observed after both selective and general inhibition of cellular DUB activity without direct impairment of proteasomal proteolysis. The repertoire of ubiquitylated substrates was analyzed by tandem mass spectrometry, identifying distinct subsets for general or specific inhibition of DUBs. This enabled identification of previously unknown functional links between USP7 and enzymes involved in DNA repair.

Studies on the reaction of nitric oxide with the hypoxia-inducible factor prolyl hydroxylase domain 2 (EGLN1).

The hypoxic response in animals is mediated via the transcription factor hypoxia-inducible factor (HIF). An oxygen-sensing component of the HIF system is provided by Fe(II) and 2-oxoglutarate-dependent oxygenases that catalyse the posttranslational hydroxylation of the HIF-α subunit. It is proposed that the activity of the HIF hydroxylases can be regulated by their reaction with nitric oxide. We describe biochemical and biophysical studies on the reaction of prolyl hydroxylase domain-containing enzyme (PHD) isoform 2 (EGLN1) with nitric oxide and a nitric oxide transfer reagent. The combined results reveal the potential for the catalytic domain of PHD2 to react with nitric oxide both at its Fe(II) and at cysteine residues. Although the biological significance is unclear, the results suggest that the reaction of PHD2 with nitric oxide has the potential to be complex and are consistent with proposals based on cellular studies that nitric oxide may regulate the hypoxic response by direct reaction with the HIF hydroxylases.

The chemoselective one-step alkylation and isolation of thiophosphorylated cdk2 substrates in the presence of native cysteine.

The elucidation of signalling pathways relies heavily upon the identification of protein kinase substrates. Recent investigations have demonstrated the efficacy of chemical genetics using ATP analogues and modified protein kinases for specific substrate labelling. Here we combine N(6) -(cyclohexyl)ATPγS with an analogue-sensitive cdk2 variant to thiophosphorylate its substrates and demonstrate a pH-dependent, chemoselective, one-step alkylation to facilitate the detection or isolation of thiophosphorylated peptides.

Differential sensitivity of hypoxia inducible factor hydroxylation sites to hypoxia and hydroxylase inhibitors.

Hypoxia inducible factor (HIF) is regulated by dual pathways involving oxygen-dependent prolyl and asparaginyl hydroxylation of its α-subunits. Prolyl hydroxylation at two sites within a central degradation domain promotes association of HIF-α with the von Hippel-Lindau ubiquitin E3 ligase and destruction by the ubiquitin-proteasome pathways. Asparaginyl hydroxylation blocks the recruitment of p300/CBP co-activators to a C-terminal activation domain in HIF-α. These hydroxylations are catalyzed by members of the Fe(II) and 2-oxoglutarate (2-OG) oxygenase family. Activity of the enzymes is suppressed by hypoxia, increasing both the abundance and activity of the HIF transcriptional complex. We have used hydroxy residue-specific antibodies to compare and contrast the regulation of each site of prolyl hydroxylation (Pro(402), Pro(564)) with that of asparaginyl hydroxylation (Asn(803)) in human HIF-1α. Our findings reveal striking differences in the sensitivity of these hydroxylations to hypoxia and to different inhibitor types of 2-OG oxygenases. Hydroxylation at the three sites in endogenous human HIF-1α proteins was suppressed by hypoxia in the order Pro(402) > Pro(564) > Asn(803). In contrast to some predictions from in vitro studies, prolyl hydroxylation was substantially more sensitive than asparaginyl hydroxylation to inhibition by iron chelators and transition metal ions; studies of a range of different small molecule 2-OG analogues demonstrated the feasibility of selectively inhibiting either prolyl or asparaginyl hydroxylation within cells.

Post-translational modification of the deubiquitinating enzyme otubain 1 modulates active RhoA levels and susceptibility to Yersinia invasion.

Microbial pathogens exploit the ubiquitin system to facilitate infection and manipulate the immune responses of the host. In this study, susceptibility to Yersinia enterocolitica and Yersinia pseudotuberculosis invasion was found to be increased upon overexpression of the deubiquitinating enzyme otubain 1 (OTUB1), a member of the ovarian tumour domain-containing protein family. Conversely, OTUB1 knockdown interfered with Yersinia invasion in HEK293T cells as well as in primary monocytes. This effect was attributed to a modulation of bacterial uptake. We demonstrate that the Yersinia-encoded virulence factor YpkA (YopO) kinase interacts with a post-translationally modified form of OTUB1 that contains multiple phosphorylation sites. OTUB1, YpkA and the small GTPase ras homologue gene family member A (RhoA) were found to be part of the same protein complex, suggesting that RhoA levels are modulated by OTUB1. Our results show that OTUB1 is able to stabilize active RhoA prior to invasion, which is concomitant with an increase in bacterial uptake. This effect is modulated by post-translational modifications of OTUB1, suggesting a new entry point for manipulating Yersinia interactions with the host.

Small-molecule-based inhibition of histone demethylation in cells assessed by quantitative mass spectrometry.

Post-translational modifications on histones are an important mechanism for the regulation of gene expression and are involved in all aspects of cell growth and differentiation, as well as pathological processes including neurodegeneration, autoimmunity, and cancer. A major challenge within the chromatin field is to develop methods for the quantitative analysis of histone modifications. Here we report a mass spectrometry (MS) approach based on ultraperformance liquid chromatography high/low collision switching (UPLC-MS(E)) to monitor histone modifications in cells. This approach is exemplified by the analysis of trimethylated lysine-9 levels in histone H3, following a simple chemical derivatization procedure with d(6)-acetic anhydride. This method was used to study the inhibition of histone demethylases with pyridine-2,4-dicarboxylic acid (PDCA) derivatives in cells. Our results show that the PDCA-dimethyl ester inhibits JMJD2A catalyzed demethylation of lysine-9 on histone H3 in human HEK 293T cells. Demethylase inhibition, as observed by MS analyses, was supported by immunoblotting with modification-specific antibodies. The results demonstrate that PDCA derived small molecules are cell permeable demethylase inhibitors and reveal that quantitative MS is a useful tool for measuring post-translational histone modifications in cells.

Proteomics-based identification of novel factor inhibiting hypoxia-inducible factor (FIH) substrates indicates widespread asparaginyl hydroxylation of ankyrin repeat domain-containing proteins.

Post-translational hydroxylation has been considered an unusual modification on intracellular proteins. However, following the recognition that oxygen-sensitive prolyl and asparaginyl hydroxylation are central to the regulation of the transcription factor hypoxia-inducible factor (HIF), interest has centered on the possibility that these enzymes may have other substrates in the proteome. In support of this certain ankyrin repeat domain (ARD)-containing proteins, including members of the IkappaB and Notch families, have been identified as alternative substrates of the HIF asparaginyl hydroxylase factor inhibiting HIF (FIH). Although these findings imply a potentially broad range of substrates for FIH, the precise extent of this range has been difficult to determine because of the difficulty of capturing transient enzyme-substrate interactions. Here we describe the use of pharmacological "substrate trapping" together with stable isotope labeling by amino acids in cell culture (SILAC) technology to stabilize and identify potential FIH-substrate interactions by mass spectrometry. To pursue these potential FIH substrates we used conventional data-directed tandem MS together with alternating low/high collision energy tandem MS to assign and quantitate hydroxylation at target asparaginyl residues. Overall the work has defined 13 new FIH-dependent hydroxylation sites with a degenerate consensus corresponding to that of the ankyrin repeat and a range of ARD-containing proteins as actual and potential substrates for FIH. Several ARD-containing proteins were multiply hydroxylated, and detailed studies of one, Tankyrase-2, revealed eight sites that were differentially sensitive to FIH-catalyzed hydroxylation. These findings indicate that asparaginyl hydroxylation is likely to be widespread among the approximately 300 ARD-containing species in the human proteome.

Structural basis and specificity of human otubain 1-mediated deubiquitination.

OTUB (otubain) 1 is a human deubiquitinating enzyme that is implicated in mediating lymphocyte antigen responsiveness, but whose molecular function is generally not well defined. A structural analysis of OTUB1 shows differences in accessibility to the active site and in surface properties of the substrate-binding regions when compared with its close homologue, OTUB2, suggesting variations in regulatory mechanisms and substrate specificity. Biochemical analysis reveals that OTUB1 has a preference for cleaving Lys(48)-linked polyubiquitin chains over Lys(63)-linked polyubiquitin chains, and it is capable of cleaving NEDD8 (neural-precursor-cell-expressed developmentally down-regulated 8), but not SUMO (small ubiquitin-related modifier) 1/2/3 and ISG15 (interferon-stimulated gene 15) conjugates. A functional comparison of OTUB1 and OTUB2 indicated a differential reactivity towards ubiquitin-based active-site probes carrying a vinyl methyl ester, a 2-chloroethyl or a 2-bromoethyl group at the C-terminus. Mutational analysis suggested that a narrow P1' site, as observed in OTUB1, correlates with its ability to preferentially cleave Lys(48)-linked ubiquitin chains. Analysis of cellular interaction partners of OTUB1 by co-immunoprecipitation and MS/MS (tandem mass spectrometry) experiments demonstrated that FUS [fusion involved in t(12;6) in malignant liposarcoma; also known as TLS (translocation in liposarcoma) or CHOP (CCAAT/enhancer-binding protein homologous protein)] and RACK1 [receptor for activated kinase 1; also known as GNB2L1 (guanine-nucleotide-binding protein beta polypeptide 2-like 1)] are part of OTUB1-containing complexes, pointing towards a molecular function of this deubiquitinating enzyme in RNA processing and cell adhesion/morphology.