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Liver-derived circulating factors deeply affect the metabolism of distal organs. Herein, we took advantage of the hepatocyte-specific PTEN knockout mice (LPTENKO), a model of hepatic steatosis associated with increased muscle insulin sensitivity and decreased adiposity, to identify potential secreted hepatic factors improving metabolic homeostasis. Our results indicated that protein factors, rather than specific metabolites, released by PTEN-deficient hepatocytes trigger an improved muscle insulin sensitivity and a decreased adiposity in LPTENKO. In this regard, a proteomic analysis of conditioned media from PTEN-deficient primary hepatocytes identified seven hepatokines whose expression/secretion was deregulated. Distinct expression patterns of these hepatokines were observed in hepatic tissues from human/mouse with NAFLD. The expression of specific factors was regulated by the PTEN/PI3K, PPAR or AMPK signaling pathways and/or modulated by classical antidiabetic drugs. Finally, loss-of-function studies identified FGF21 and the triad AHSG, ANGPTL4 and LECT2 as key regulators of insulin sensitivity in muscle cells and in adipocytes biogenesis, respectively. These data indicate that hepatic PTEN deficiency and steatosis alter the expression/secretion of hepatokines regulating insulin sensitivity in muscles and the lipid metabolism in adipose tissue. These hepatokines could represent potential therapeutic targets to treat obesity and insulin resistance.

ObjectiveHepatocellular carcinoma (HCC) development occurs with non-alcoholic fatty liver disease (NAFLD) in the absence of cirrhosis and with an increasing incidence due to the obesity pandemic. Mutations of tumour suppressor (TS) genes and oncogenes (ONC) have been widely characterised in HCC. However, mounting evidence indicates that non-genomic alterations of TS/ONC occur early with NAFLD, thereby potentially promoting hepatocarcinogenesis in an inflammatory/fibrotic context. The aim of this study was to identify and characterise these alterations.DesignThe proteome of steatotic liver tissues from mice spontaneously developing HCC was analysed. Alterations of TSs/ONCs were further investigated in various mouse models of NAFLD/HCC and in human samples. The inflammatory, fibrogenic and oncogenic functions of S100A11 were assessed through in vivo, in vitro and ex-vivo analyses.ResultsA whole set of TSs/ONCs, respectively, downregulated or upregulated was uncovered in mice and human with NAFLD. Alterations of these TSs/ONCs were preserved or even exacerbated in HCC. Among them, overexpression of S100A11 was associated with high-grade HCC and poor prognosis. S100A11 downregulation in vivo significantly restrains the development of inflammation and fibrosis in mice fed a choline/methionine-deficient diet. Finally, in vitro and ex-vivo analyses revealed that S100A11 is a marker of hepatocyte de-differentiation, secreted by cancer cells, and promoting cell proliferation and migration.ConclusionCellular stress associated with NAFLD triggers non-genomic alterations of a whole network of TSs/ONCs fostering hepatocarcinogenesis. Among those, overexpression of the oncogenic factor S100A11 promotes inflammation/fibrosis in vivo and is significantly associated with high-grade HCC with poor prognosis.

The natural product cepafungin I was recently reported to be one of the most potent covalent inhibitors of the 20S proteasome core particle through a series of in vitro activity assays. Here, we report a short chemoenzymatic total synthesis of cepafungin I featuring the use of a regioselective enzymatic oxidation to prepare a key hydroxylated amino acid building block in a scalable fashion. The strategy developed herein enabled access to a chemoproteomic probe, which in turn revealed the exceptional selectivity and potency of cepafungin I towards the b2 and b5 subunits of the proteasome. Further structure-activity relationship studies suggest the key role of the hydroxyl group in the macrocycle and the identity of the lipid tail in modulating the potency of this natural product family. This study lays the groundwork for further medicinal chemistry exploration to fully realize the anticancer potential of cepafungin I.

The disulfide dihedral angle in epidithiodiketopiperazines (ETPs) is near 0°. Application of this highest possible ring tension to strain-promoted thiol-mediated uptake results in efficient delivery to the cytosol and nucleus. Compared to the previous best asparagusic acid (AspA), ring-opening disulfide exchange with ETPs occurs more efficiently even with nonactivated thiols, and the resulting thiols exchange rapidly with nonactivated disulfides. ETP-mediated cellular uptake is more than 20 times more efficient compared to AspA, occurs without endosomal capture, depends on temperature, and is “unstoppable” by inhibitors of endocytosis and conventional thiol-mediated uptake, including siRNA against the transferrin receptor. These results suggest that ETP-mediated uptake not only maximizes delivery to the cytosol and nucleus but also opens the door to a new multitarget hopping mode of action., Epidithiodiketopiperazines are introduced for nontoxic delivery to the cytosol and nucleus, without endosomal capture, unstoppable by conventional inhibitors, opening the doors to an intriguing multitarget disulfide hopping mechanism.

Ischaemic heart disease and stroke are the most common causes of death worldwide. Anoxia, defined as the lack of oxygen, is commonly seen in both these pathologies and triggers profound metabolic and cellular changes. Sphingolipids have been implicated in anoxia injury, but the pathomechanism is unknown. Here we show that anoxia-associated injury causes accumulation of the non-canonical sphingolipid 1-deoxydihydroceramide (DoxDHCer). Anoxia causes an imbalance between serine and alanine resulting in a switch from normal serine-derived sphinganine biosynthesis to non-canonical alanine-derived 1-deoxysphinganine. 1-Deoxysphinganine is incorporated into DoxDHCer, which impairs actin folding via the cytosolic chaperonin TRiC, leading to growth arrest in yeast, increased cell death upon anoxia-reoxygenation in worms and ischaemia-reperfusion injury in mouse hearts. Prevention of DoxDHCer accumulation in worms and in mouse hearts resulted in decreased anoxia-induced injury. These findings unravel key metabolic changes during oxygen deprivation and point to novel strategies to avoid tissue damage and death.

In this study, we demonstrate that appendage of a single asparagusic acid residue (AspA tag) is sufficient to ensure efficient cellular uptake and intracellular distribution of fully unprotected peptides. We apply this new delivery method to induce apoptotic response in cancer cells using long (up to 20mer) BH3 domain peptides. Moreover, to understand the molecular mechanism of the cellular uptake, we perform chemical proteomics experiments and identify the direct molecular targets of the asparagusic acid tag. Our findings document covalent bond formation between the asparagusic acid moiety and the cysteines 556 and 558 on the surface of the transferrin receptor resulting in subsequent endocytic uptake of the payload. We believe that the small size, low cellular toxicity and the efficient transferrin receptor-mediated uptake render the AspA tag highly attractive for various life science applications.

The 2016 International Symposium on Chemical Biology was organized by the Swiss National Centre of Competence in Research (NCCR) Chemical Biology. The NCCR Chemical Biology is located at the University of Geneva (UniGE; Director: Prof. Howard Riezman) and the Ecole Polytechnique Fedé rale de Lausanne (EPFL; Co-director: Prof. ́ Kai Johnsson). The symposium was held on January 13−15, 2016 at the Campus Biotech, a new center for biotechnology and life science research that was formed through a unique partnership between the University of Geneva and EPFL and is located within walking distance from the city center of Geneva. This exciting scientific venue attracted more than 240 attendees from around the world. With the aim of providing a sound overview of key challenges in cutting edge chemical biology, as well as fostering an open dialogue with networking opportunities, the organizing committee selected 15 plenary lectures with diverse scope under the broad subject of chemical biology. The scientific program was rounded up by four poster sessions with over 60 posters.

In this study, we present a highly efficient method for proteomic profiling of cysteine residues in complex proteomes and in living cells. Our method is based on alkynylation of cysteines in complex proteomes using a "clickable" alkynyl benziodoxolone bearing an azide group. This reaction proceeds fast, under mild physiological conditions, and with a very high degree of chemoselectivity. The formed azide-capped alkynyl-cysteine adducts are readily detectable by LC-MS/MS, and can be further functionalized with TAMRA or biotin alkyne via CuAAC. We demonstrate the utility of alkynyl benziodoxolones for chemical proteomics applications by identifying the proteomic targets of curcumin, a diarylheptanoid natural product that was and still is part of multiple human clinical trials as anticancer agent. Our results demonstrate that curcumin covalently modifies several key players of cellular signaling and metabolism, most notably the enzyme casein kinase I gamma. We anticipate that this new method for cysteine profiling will find broad application in chemical proteomics and drug discovery.

In eukaryotic organisms, cysteine palmitoylation is an important reversible modification that impacts protein targeting, folding, stability, and interactions with partners. Evidence suggests that protein palmitoylation contributes to key biological processes in Apicomplexa with the recent palmitome of the malaria parasite Plasmodium falciparum reporting over 400 substrates that are modified with palmitate by a broad range of protein S-acyl transferases. Dynamic palmitoylation cycles require the action of an acyl-protein thioesterase (APT) that cleaves palmitate from substrates and conveys reversibility to this posttranslational modification. In this work, we identified candidates for APT activity in Toxoplasma gondii. Treatment of parasites with low micromolar concentrations of β-lactone- or triazole urea-based inhibitors that target human APT1 showed varied detrimental effects at multiple steps of the parasite lytic cycle. The use of an activity-based probe in combination with these inhibitors revealed the existence of several serine hydrolases that are targeted by APT1 inhibitors. The active serine hydrolase, TgASH1, identified as the homologue closest to human APT1 and APT2, was characterized further. Biochemical analysis of TgASH1 indicated that this enzyme cleaves substrates with a specificity similar to APTs, and homology modeling points toward an APT-like enzyme. TgASH1 is dispensable for parasite survival, which indicates that the severe effects observed with the β-lactone inhibitors are caused by the inhibition of non-TgASH1 targets. Other ASH candidates for APT activity were functionally characterized, and one of them was found to be resistant to gene disruption due to the potential essential nature of the protein.

A comparative analysis of bacterial and mammalian glycomes based on the statistical analysis of two major carbohydrate databases, Bacterial Carbohydrate Structure Data Base (BCSDB) and GLYCOSCIENCES.de (GS), is presented. An in-depth comparison of these two glycomes reveals both striking differences and unexpected similarities. Within the prokaryotic kingdom, we focus on the glycomes of seven classes of pathogenic bacteria with respect to (i) their most abundant monosaccharide units; (ii) disaccharide pairs; (iii) carbohydrate modifications; (iv) occurrence and use of sialic acids; and (v) class-specific monosaccharides. The aim of this work is to gain insights into unique carbohydrate patterns in bacteria. Data interpretation reveals significant trends in the composition of specific carbohydrate classes as result of evolution-driven structural adaptations of bacterial pathogens and symbionts to their mammalian hosts. The differences are discussed in light of their value for biomedical applications, such as the targeting of unique glycosyl transferases, vaccine development, and devising novel diagnostic tools. peerReviewed

Serine hydrolases (SHs) are one of the largest and most diverse enzyme classes in mammals. They play fundamental roles in virtually all physiological processes and are targeted by drugs to treat diseases such as diabetes, obesity, and neurodegenerative disorders. Despite this, we lack biological understanding for most of the 110+ predicted mammalian metabolic SHs, in large part because of a dearth of assays to assess their biochemical activities and a lack of selective inhibitors to probe their function in living systems. We show here that the vast majority (> 80%) of mammalian metabolic SHs can be labeled in proteomes by a single, active site-directed fluorophosphonate probe. We exploit this universal activity-based assay in a library-versus-library format to screen 70+ SHs against 140+ structurally diverse carbamates. Lead inhibitors were discovered for ∼40% of the screened enzymes, including many poorly characterized SHs. Global profiles identified carbamate inhibitors that discriminate among highly sequence-related SHs and, conversely, enzymes that share inhibitor sensitivity profiles despite lacking sequence homology. These findings indicate that sequence relatedness is not a strong predictor of shared pharmacology within the SH superfamily. Finally, we show that lead carbamate inhibitors can be optimized into pharmacological probes that inactivate individual SHs with high specificity in vivo.