Adel F. M. Ibrahim

Identification of Protor as a novel Rictor-binding component of mTOR complex-2

The mTOR (mammalian target of rapamycin) protein kinase is an important regulator of cell growth. Two complexes of mTOR have been identified: complex 1, consisting of mTOR-Raptor (regulatory associated protein of mTOR)-mLST8 (termed mTORC1), and complex 2, comprising mTOR-Rictor (rapamycininsensitive companion of mTOR)-mLST8-Sin1 (termed mTORC2). mTORC1 phosphorylates the p70 ribosomal S6K (S6 kinase) at its hydrophobic motif (Thr389), whereas mTORC2 phosphorylates PKB (protein kinase B) at its hydrophobic motif (Ser473). In the present study, we report that widely expressed isoforms of unstudied proteins termed Protor-1 (protein observed with Rictor-1) and Protor-2 interact with Rictor and are components of mTORC2. We demonstrate that immunoprecipitation of Protor-1 or Protor-2 results in the co-immunoprecipitation of other mTORC2 subunits, but not Raptor, a specific component of mTORC1. We show that detergents such as Triton X-100 or n-octylglucoside dissociate mTOR and mLST8 from a complex of Protor-1, Sin1 and Rictor. We also provide evidence that Rictor regulates the expression of Protor-1, and that Protor-1 is not required for the assembly of other mTORC2 subunits into a complex. Protor-1 is a novel Rictor-binding subunit of mTORC2, but further work is required to establish its role.

Regulation of multisite phosphorylation and 14-3-3 binding of AS160 in response to IGF-1, EGF, PMA and AICAR.

AS160 (Akt substrate of 160 kDa) mediates insulin-stimulated GLUT4 (glucose transporter 4) translocation, but is widely expressed in insulin-insensitive tissues lacking GLUT4. Having isolated AS160 by 14-3-3-affinity chromatography, we found that binding of AS160 to 14-3-3 isoforms in HEK (human embryonic kidney)-293 cells was induced by IGF-1 (insulin-like growth factor-1), EGF (epidermal growth factor), PMA and, to a lesser extent, AICAR (5-aminoimidazole-4-carboxamide-1-b-D-ribofuranoside). AS160-14-3-3 interactions were stabilized by chemical cross-linking and abolished by dephosphorylation. Eight residues on AS160 (Ser318, Ser341, Thr568, Ser570, Ser588, Thr642, Ser666 and Ser751) were differentially phosphorylated in response to IGF-1, EGF, PMA and AICAR. The binding of 14-3-3 proteins to HA-AS160 (where HA is haemagglutinin) was markedly decreased by mutation of Thr642 and abolished in a Thr642Ala/Ser341Ala double mutant. The AGC (protein kinase A/protein kinase G/protein kinase C-family) kinases RSK1 (p90 ribosomal S6 kinase 1), SGK1 (serum- and glucocorticoid-induced protein kinase 1) and PKB (protein kinase B) displayed distinct signatures of AS160 phosphorylation in vitro: all three kinases phosphorylated Ser318, Ser588 and Thr642; RSK1 also phosphorylated Ser341, Ser751 and to a lesser extent Thr568; and SGK1 phosphorylated Thr568 and Ser751. AMPK (AMP-activated protein kinase) preferentially phosphorylated Ser588, with less phosphorylation of other sites. In cells, the IGF-1-stimulated phosphorylations, and certain EGF-stimulated phosphorylations, were inhibited by PI3K (phosphoinositide 3-kinase) inhibitors, whereas the RSK inhibitor BI-D1870 inhibited the PMA-induced phosphorylations. The expression of LKB1 in HeLa cells and the use of AICAR in HEK-293 cells promoted phosphorylation of Ser588, but only weak Ser341 and Thr642 phosphorylations and binding to 14-3-3s. Paradoxically however, phenformin activated AMPK without promoting AS160 phosphorylation. The IGF-1-induced phosphorylation of the novel phosphorylated Ser666-Pro site was suppressed by AICAR, and by combined mutation of a TOS (mTOR signalling)-like sequence (FEMDI) and rapamycin. Thus, although AS160 is a common target of insulin, IGF-1, EGF, PMA and AICAR, these stimuli induce distinctive patterns of phosphorylation and 14-3-3 binding, mediated by at least four protein kinases.

Proteome-Wide Identification of SUMO2 Modification Sites

A novel method for the enrichment of SUMO2-modified peptides reveals more than 1000 sites of modification in human cells. Sleuthing SUMO Sites Covalent attachment to small ubiquitin-like modifiers (SUMOs) alters the stability, localization, and function of diverse proteins. SUMO-specific enzymes transfer SUMO to lysines on target proteins in a process known as sumoylation. Tammsalu et al. present a method to discover the exact sites to which SUMOs conjugate. Overexpression of a mutated form of SUMO2 and subsequent cleavage with a sequence-specific endoproteinase resulted in tagged sumoylated peptides. Antibody-based enrichment of tagged peptides and analyses by proteomics identified more than 1000 sumoylated sites in the human proteome, thus providing a wealth of information for future studies. Posttranslational modification with small ubiquitin-like modifiers (SUMOs) alters the function of proteins involved in diverse cellular processes. SUMO-specific enzymes conjugate SUMOs to lysine residues in target proteins. Although proteomic studies have identified hundreds of sumoylated substrates, methods to identify the modified lysines on a proteomic scale are lacking. We developed a method that enabled proteome-wide identification of sumoylated lysines that involves the expression of polyhistidine (6His)–tagged SUMO2 with Thr90 mutated to Lys. Endoproteinase cleavage with Lys-C of 6His-SUMO2T90K–modified proteins from human cell lysates produced a diGly remnant on SUMO2T90K-conjugated lysines, enabling immunoprecipitation of SUMO2T90K–modified peptides and producing a unique mass-to-charge signature. Mass spectrometry analysis of SUMO-enriched peptides revealed more than 1000 sumoylated lysines in 539 proteins, including many functionally related proteins involved in cell cycle, transcription, and DNA repair. Not only can this strategy be used to study the dynamics of sumoylation and other potentially similar posttranslational modifications, but also, these data provide an unprecedented resource for future research on the role of sumoylation in cellular physiology and disease.

Structural insights into mechanism and specificity of O ‐GlcNAc transferase

Post‐translational modification of protein serines/threonines with N‐acetylglucosamine (O‐GlcNAc) is dynamic, inducible and abundant, regulating many cellular processes by interfering with protein phosphorylation. O‐GlcNAcylation is regulated by O‐GlcNAc transferase (OGT) and O‐GlcNAcase, both encoded by single, essential, genes in metazoan genomes. It is not understood how OGT recognises its sugar nucleotide donor and performs O‐GlcNAc transfer onto proteins/peptides, and how the enzyme recognises specific cellular protein substrates. Here, we show, by X‐ray crystallography and mutagenesis, that OGT adopts the (metal‐independent) GT‐B fold and binds a UDP‐GlcNAc analogue at the bottom of a highly conserved putative peptide‐binding groove, covered by a mobile loop. Strikingly, the tetratricopeptide repeats (TPRs) tightly interact with the active site to form a continuous 120 Å putative interaction surface, whereas the previously predicted phosphatidylinositide‐binding site locates to the opposite end of the catalytic domain. On the basis of the structure, we identify truncation/point mutants of the TPRs that have differential effects on activity towards proteins/peptides, giving first insights into how OGT may recognise its substrates.

The Vibrio cholerae colonization factor GbpA possesses a modular structure that governs binding to different host surfaces

Vibrio cholerae is a bacterial pathogen that colonizes the chitinous exoskeleton of zooplankton as well as the human gastrointestinal tract. Colonization of these different niches involves an N-acetylglucosamine binding protein (GbpA) that has been reported to mediate bacterial attachment to both marine chitin and mammalian intestinal mucin through an unknown molecular mechanism. We report structural studies that reveal that GbpA possesses an unusual, elongated, four-domain structure, with domains 1 and 4 showing structural homology to chitin binding domains. A glycan screen revealed that GbpA binds to GlcNAc oligosaccharides. Structure-guided GbpA truncation mutants show that domains 1 and 4 of GbpA interact with chitin in vitro, whereas in vivo complementation studies reveal that domain 1 is also crucial for mucin binding and intestinal colonization. Bacterial binding studies show that domains 2 and 3 bind to the V. cholerae surface. Finally, mouse virulence assays show that only the first three domains of GbpA are required for colonization. These results explain how GbpA provides structural/functional modular interactions between V. cholerae, intestinal epithelium and chitinous exoskeletons.

O ‐GlcNAcylation of TAB1 modulates TAK1‐mediated cytokine release

Transforming growth factor (TGF)‐β‐activated kinase 1 (TAK1) is a key serine/threonine protein kinase that mediates signals transduced by pro‐inflammatory cytokines such as transforming growth factor‐β, tumour necrosis factor (TNF), interleukin‐1 (IL‐1) and wnt family ligands. TAK1 is found in complex with binding partners TAB1–3, phosphorylation and ubiquitination of which has been found to regulate TAK1 activity. In this study, we show that TAB1 is modified with N‐acetylglucosamine (O‐GlcNAc) on a single site, Ser395. With the help of a novel O‐GlcNAc site‐specific antibody, we demonstrate that O‐GlcNAcylation of TAB1 is induced by IL‐1 and osmotic stress, known inducers of the TAK1 signalling cascade. By reintroducing wild‐type or an O‐GlcNAc‐deficient mutant TAB1 (S395A) into Tab1−/− mouse embryonic fibroblasts, we determined that O‐GlcNAcylation of TAB1 is required for full TAK1 activation upon stimulation with IL‐1/osmotic stress, for downstream activation of nuclear factor κB and finally production of IL‐6 and TNFα. This is one of the first examples of a single O‐GlcNAc site on a signalling protein modulating a key innate immunity signalling pathway.

Molecular Mechanisms of Yeast Cell Wall Glucan Remodeling

Yeast cell wall remodeling is controlled by the equilibrium between glycoside hydrolases, glycosyltransferases, and transglycosylases. Family 72 glycoside hydrolases (GH72) are ubiquitous in fungal organisms and are known to possess significant transglycosylase activity, producing elongated β(1–3) glucan chains. However, the molecular mechanisms that control the balance between hydrolysis and transglycosylation in these enzymes are not understood. Here we present the first crystal structure of a glucan transglycosylase, Saccharomyces cerevisiae Gas2 (ScGas2), revealing a multidomain fold, with a (βα)8 catalytic core and a separate glucan binding domain with an elongated, conserved glucan binding groove. Structures of ScGas2 complexes with different β-glucan substrate/product oligosaccharides provide “snapshots” of substrate binding and hydrolysis/transglycosylation giving the first insights into the mechanisms these enzymes employ to drive β(1–3) glucan elongation. Together with mutagenesis and analysis of reaction products, the structures suggest a “base occlusion” mechanism through which these enzymes protect the covalent protein-enzyme intermediate from a water nucleophile, thus controlling the balance between hydrolysis and transglycosylation and driving the elongation of β(1–3) glucan chains in the yeast cell wall.

The P-body component USP52/PAN2 is a novel regulator of HIF1A mRNA stability.

HIF1A (hypoxia-inducible factor 1α) is the master regulator of the cellular response to hypoxia and is implicated in cancer progression. Whereas the regulation of HIF1A protein in response to oxygen is well characterized, less is known about the fate of HIF1A mRNA. In the present study, we have identified the pseudo-DUB (deubiquitinating enzyme)/deadenylase USP52 (ubiquitin-specific protease 52)/PAN2 [poly(A) nuclease 2] as an important regulator of the HIF1A-mediated hypoxic response. Depletion of USP52 reduced HIF1A mRNA and protein levels and resulted in reduced expression of HIF1A-regulated hypoxic targets due to a 3′-UTR (untranslated region)-dependent poly(A)-tail-length-independent destabilization in HIF1A mRNA. MS analysis revealed an association of USP52 with several P-body (processing body) components and we confirmed further that USP52 protein and HIF1A mRNA co-localized with cytoplasmic P-bodies. Importantly, P-body dispersal by knockdown of GW182 or LSM1 resulted in a reduction of HIF1A mRNA levels. These data uncover a novel role for P-bodies in regulating HIF1A mRNA stability, and demonstrate that USP52 is a key component of P-bodies required to prevent HIF1A mRNA degradation.

E3 ubiquitin ligase HOIP attenuates apoptotic cell death induced by cisplatin.

The genotoxin cisplatin is commonly used in chemotherapy to treat solid tumors, yet our understanding of the mechanism underlying the drug response is limited. In a focused siRNA screen, using an siRNA library targeting genes involved in ubiquitin and ubiquitin-like signaling, we identified the E3 ubiquitin ligase HOIP as a key regulator of cisplatin-induced genotoxicity. HOIP forms, with SHARPIN and HOIL-1L, the linear ubiquitin assembly complex (LUBAC). We show that cells deficient in the HOIP ligase complex exhibit hypersensitivity to cisplatin. This is due to a dramatic increase in caspase-8/caspase-3-mediated apoptosis that is strictly dependent on ATM-, but not ATR-mediated DNA damage checkpoint activation. Moreover, basal and cisplatin-induced activity of the stress response kinase JNK is enhanced in HOIP-depleted cells and, conversely, JNK inhibition can increase cellular resistance to cisplatin and reverse the apoptotic hyperactivation in HOIP-depleted cells. Furthermore, we show that HOIP depletion sensitizes cancer cells, derived from carcinomas of various origins, through an enhanced apoptotic cell death response. We also provide evidence that ovarian cancer cells classified as cisplatin-resistant can regain sensitivity following HOIP downregulation. Cumulatively, our study identifies a HOIP-regulated antiapoptotic signaling pathway, and we envisage HOIP as a potential target for the development of combinatorial chemotherapies to potentiate the efficacy of platinum-based anticancer drugs.

Natural Product–Guided Discovery of a Fungal Chitinase Inhibitor

Natural products are often large, synthetically intractable molecules, yet frequently offer surprising inroads into previously unexplored chemical space for enzyme inhibitors. Argifin is a cyclic pentapeptide that was originally isolated as a fungal natural product. It competitively inhibits family 18 chitinases by mimicking the chitooligosaccharide substrate of these enzymes. Interestingly, argifin is a nanomolar inhibitor of the bacterial-type subfamily of fungal chitinases that possess an extensive chitin-binding groove, but does not inhibit the much smaller, plant-type enzymes from the same family that are involved in fungal cell division and are thought to be potential drug targets. Here we show that a small, highly efficient, argifin-derived, nine-atom fragment is a micromolar inhibitor of the plant-type chitinase ChiA1 from the opportunistic pathogen Aspergillus fumigatus. Evaluation of the binding mode with the first crystal structure of an A. fumigatus plant-type chitinase reveals that the compound binds the catalytic machinery in the same manner as observed for argifin with the bacterial-type chitinases. The structure of the complex was used to guide synthesis of derivatives to explore a pocket near the catalytic machinery. This work provides synthetically tractable plant-type family 18 chitinase inhibitors from the repurposing of a natural product.