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1.
Mol Cell ; 83(17): 3140-3154.e7, 2023 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-37572670

RESUMO

Peroxiredoxins (Prdxs) utilize reversibly oxidized cysteine residues to reduce peroxides and promote H2O2 signal transduction, including H2O2-induced activation of P38 MAPK. Prdxs form H2O2-induced disulfide complexes with many proteins, including multiple kinases involved in P38 MAPK signaling. Here, we show that a genetically encoded fusion between a Prdx and P38 MAPK is sufficient to hyperactivate the kinase in yeast and human cells by a mechanism that does not require the H2O2-sensing cysteine of the Prdx. We demonstrate that a P38-Prdx fusion protein compensates for loss of the yeast scaffold protein Mcs4 and MAP3K activity, driving yeast into mitosis. Based on our findings, we propose that the H2O2-induced formation of Prdx-MAPK disulfide complexes provides an alternative scaffold and signaling platform for MAPKK-MAPK signaling. The demonstration that formation of a complex with a Prdx is sufficient to modify the activity of a kinase has broad implications for peroxide-based signal transduction in eukaryotes.


Assuntos
Peroxirredoxinas , Proteínas Quinases p38 Ativadas por Mitógeno , Humanos , Cisteína/metabolismo , Dissulfetos , Peróxido de Hidrogênio/farmacologia , Peróxido de Hidrogênio/metabolismo , Oxirredução , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Peroxirredoxinas/genética , Peroxirredoxinas/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo
2.
Nature ; 598(7880): 332-337, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34616040

RESUMO

Humans have co-evolved with a dense community of microbial symbionts that inhabit the lower intestine. In the colon, secreted mucus creates a barrier that separates these microorganisms from the intestinal epithelium1. Some gut bacteria are able to utilize mucin glycoproteins, the main mucus component, as a nutrient source. However, it remains unclear which bacterial enzymes initiate degradation of the complex O-glycans found in mucins. In the distal colon, these glycans are heavily sulfated, but specific sulfatases that are active on colonic mucins have not been identified. Here we show that sulfatases are essential to the utilization of distal colonic mucin O-glycans by the human gut symbiont Bacteroides thetaiotaomicron. We characterized the activity of 12 different sulfatases produced by this species, showing that they are collectively active on all known sulfate linkages in O-glycans. Crystal structures of three enzymes provide mechanistic insight into the molecular basis of substrate specificity. Unexpectedly, we found that a single sulfatase is essential for utilization of sulfated O-glycans in vitro and also has a major role in vivo. Our results provide insight into the mechanisms of mucin degradation by a prominent group of gut bacteria, an important process for both normal microbial gut colonization2 and diseases such as inflammatory bowel disease3.


Assuntos
Bacteroides/enzimologia , Colo/metabolismo , Colo/microbiologia , Microbioma Gastrointestinal , Mucinas/metabolismo , Sulfatases/metabolismo , Acetilgalactosamina/química , Acetilgalactosamina/metabolismo , Animais , Colo/química , Cristalografia por Raios X , Feminino , Galactose/metabolismo , Humanos , Masculino , Camundongos , Modelos Moleculares , Especificidade por Substrato , Sulfatases/química
3.
Nature ; 570(7760): 194-199, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31142841

RESUMO

Serine hydroxymethyltransferase 2 (SHMT2) regulates one-carbon transfer reactions that are essential for amino acid and nucleotide metabolism, and uses pyridoxal-5'-phosphate (PLP) as a cofactor. Apo SHMT2 exists as a dimer with unknown functions, whereas PLP binding stabilizes the active tetrameric state. SHMT2 also promotes inflammatory cytokine signalling by interacting with the deubiquitylating BRCC36 isopeptidase complex (BRISC), although it is unclear whether this function relates to metabolism. Here we present the cryo-electron microscopy structure of the human BRISC-SHMT2 complex at a resolution of 3.8 Å. BRISC is a U-shaped dimer of four subunits, and SHMT2 sterically blocks the BRCC36 active site and inhibits deubiquitylase activity. Only the inactive SHMT2 dimer-and not the active PLP-bound tetramer-binds and inhibits BRISC. Mutations in BRISC that disrupt SHMT2 binding impair type I interferon signalling in response to inflammatory stimuli. Intracellular levels of PLP regulate the interaction between BRISC and SHMT2, as well as inflammatory cytokine responses. These data reveal a mechanism in which metabolites regulate deubiquitylase activity and inflammatory signalling.


Assuntos
Enzimas Desubiquitinantes/metabolismo , Glicina Hidroximetiltransferase/metabolismo , Interferon Tipo I/imunologia , Complexos Multienzimáticos/imunologia , Complexos Multienzimáticos/metabolismo , Transdução de Sinais/imunologia , Microscopia Crioeletrônica , Enzimas Desubiquitinantes/antagonistas & inibidores , Enzimas Desubiquitinantes/química , Enzimas Desubiquitinantes/ultraestrutura , Glicina Hidroximetiltransferase/ultraestrutura , Células HEK293 , Humanos , Inflamação/imunologia , Modelos Moleculares , Complexos Multienzimáticos/química , Complexos Multienzimáticos/genética , Mutação , Ligação Proteica , Multimerização Proteica , Estrutura Quaternária de Proteína , Fosfato de Piridoxal/metabolismo
4.
Nat Chem Biol ; 18(8): 841-849, 2022 08.
Artigo em Inglês | MEDLINE | ID: mdl-35710619

RESUMO

Sulfated glycans are ubiquitous nutrient sources for microbial communities that have coevolved with eukaryotic hosts. Bacteria metabolize sulfated glycans by deploying carbohydrate sulfatases that remove sulfate esters. Despite the biological importance of sulfatases, the mechanisms underlying their ability to recognize their glycan substrate remain poorly understood. Here, we use structural biology to determine how sulfatases from the human gut microbiota recognize sulfated glycans. We reveal seven new carbohydrate sulfatase structures spanning four S1 sulfatase subfamilies. Structures of S1_16 and S1_46 represent novel structures of these subfamilies. Structures of S1_11 and S1_15 demonstrate how non-conserved regions of the protein drive specificity toward related but distinct glycan targets. Collectively, these data reveal that carbohydrate sulfatases are highly selective for the glycan component of their substrate. These data provide new approaches for probing sulfated glycan metabolism while revealing the roles carbohydrate sulfatases play in host glycan catabolism.


Assuntos
Microbioma Gastrointestinal , Sulfatases , Bactérias/metabolismo , Humanos , Polissacarídeos/química , Sulfatases/química , Sulfatos/química
5.
Biochem J ; 480(2): 141-160, 2023 Jan 31.
Artigo em Inglês | MEDLINE | ID: mdl-36520605

RESUMO

Pseudokinases, so named because they lack one or more conserved canonical amino acids that define their catalytically active relatives, have evolved a variety of biological functions in both prokaryotic and eukaryotic organisms. Human PSKH2 is closely related to the canonical kinase PSKH1, which maps to the CAMK family of protein kinases. Primates encode PSKH2 in the form of a pseudokinase, which is predicted to be catalytically inactive due to loss of the invariant catalytic Asp residue. Although the biological role(s) of vertebrate PSKH2 proteins remains unclear, we previously identified species-level adaptions in PSKH2 that have led to the appearance of kinase or pseudokinase variants in vertebrate genomes alongside a canonical PSKH1 paralog. In this paper we confirm that, as predicted, PSKH2 lacks detectable protein phosphotransferase activity, and exploit structural informatics, biochemistry and cellular proteomics to begin to characterise vertebrate PSKH2 orthologues. AlphaFold 2-based structural analysis predicts functional roles for both the PSKH2 N- and C-regions that flank the pseudokinase domain core, and cellular truncation analysis confirms that the N-terminal domain, which contains a conserved myristoylation site, is required for both stable human PSKH2 expression and localisation to a membrane-rich subcellular fraction containing mitochondrial proteins. Using mass spectrometry-based proteomics, we confirm that human PSKH2 is part of a cellular mitochondrial protein network, and that its expression is regulated through client-status within the HSP90/Cdc37 molecular chaperone system. HSP90 interactions are mediated through binding to the PSKH2 C-terminal tail, leading us to predict that this region might act as both a cis and trans regulatory element, driving outputs linked to the PSKH2 pseudokinase domain that are important for functional signalling.


Assuntos
Proteínas Quinases , Transdução de Sinais , Animais , Humanos , Proteínas Quinases/metabolismo , Fosforilação , Chaperonas Moleculares/metabolismo , Evolução Biológica , Proteínas de Choque Térmico HSP90/metabolismo
6.
Biochem J ; 480(12): 875-890, 2023 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-37306403

RESUMO

Cushing's syndrome is an endocrine disorder caused by excess production of the stress hormone cortisol. Precision medicine strategies have identified single allele mutations within the PRKACA gene that drive adrenal Cushing's syndrome. These mutations promote perturbations in the catalytic core of protein kinase A (PKAc) that impair autoinhibition by regulatory subunits and compartmentalization via recruitment into AKAP signaling islands. PKAcL205R is found in ∼45% of patients, whereas PKAcE31V, PKAcW196R, and L198insW and C199insV insertion mutants are less prevalent. Mass spectrometry, cellular, and biochemical data indicate that Cushing's PKAc variants fall into two categories: those that interact with the heat-stable protein kinase inhibitor PKI, and those that do not. In vitro activity measurements show that wild-type PKAc and W196R activities are strongly inhibited by PKI (IC50 < 1 nM). In contrast, PKAcL205R activity is not blocked by the inhibitor. Immunofluorescent analyses show that the PKI-binding variants wild-type PKAc, E31V, and W196R are excluded from the nucleus and protected against proteolytic processing. Thermal stability measurements reveal that upon co-incubation with PKI and metal-bound nucleotide, the W196R variant tolerates melting temperatures 10°C higher than PKAcL205. Structural modeling maps PKI-interfering mutations to a ∼20 Šdiameter area at the active site of the catalytic domain that interfaces with the pseudosubstrate of PKI. Thus, Cushing's kinases are individually controlled, compartmentalized, and processed through their differential association with PKI.


Assuntos
Síndrome de Cushing , Humanos , Síndrome de Cushing/genética , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Mutação , Domínio Catalítico
7.
Biochem J ; 480(9): 587-605, 2023 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-37018014

RESUMO

Innate or acquired resistance to small molecule BRAF or MEK1/2 inhibitors (BRAFi or MEKi) typically arises through mechanisms that sustain or reinstate ERK1/2 activation. This has led to the development of a range of ERK1/2 inhibitors (ERKi) that either inhibit kinase catalytic activity (catERKi) or additionally prevent the activating pT-E-pY dual phosphorylation of ERK1/2 by MEK1/2 (dual-mechanism or dmERKi). Here, we show that eight different ERKi (both catERKi or dmERKi) drive the turnover of ERK2, the most abundant ERK isoform, with little or no effect on ERK1. Thermal stability assays show that ERKi do not destabilise ERK2 (or ERK1) in vitro, suggesting that ERK2 turnover is a cellular consequence of ERKi binding. ERK2 turnover is not observed upon treatment with MEKi alone, suggesting it is ERKi binding to ERK2 that drives ERK2 turnover. However, MEKi pre-treatment, which blocks ERK2 pT-E-pY phosphorylation and dissociation from MEK1/2, prevents ERK2 turnover. ERKi treatment of cells drives the poly-ubiquitylation and proteasome-dependent turnover of ERK2 and pharmacological or genetic inhibition of Cullin-RING E3 ligases prevents this. Our results suggest that ERKi, including current clinical candidates, act as 'kinase degraders', driving the proteasome-dependent turnover of their major target, ERK2. This may be relevant to the suggestion of kinase-independent effects of ERK1/2 and the therapeutic use of ERKi.


Assuntos
Sistema de Sinalização das MAP Quinases , Complexo de Endopeptidases do Proteassoma , Fosforilação , Sistema de Sinalização das MAP Quinases/fisiologia , Processamento de Proteína Pós-Traducional , Ubiquitinação
8.
J Proteome Res ; 22(12): 3754-3772, 2023 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-37939282

RESUMO

Protein tyrosine sulfation (sY) is a post-translational modification (PTM) catalyzed by Golgi-resident tyrosyl protein sulfo transferases (TPSTs). Information on sY in humans is currently limited to ∼50 proteins, with only a handful having verified sites of sulfation. As such, the contribution of sulfation to the regulation of biological processes remains poorly defined. Mass spectrometry (MS)-based proteomics is the method of choice for PTM analysis but has yet to be applied for systematic investigation of the "sulfome", primarily due to issues associated with discrimination of sY-containing from phosphotyrosine (pY)-containing peptides. In this study, we developed an MS-based workflow for sY-peptide characterization, incorporating optimized Zr4+ immobilized metal-ion affinity chromatography (IMAC) and TiO2 enrichment strategies. Extensive characterization of a panel of sY- and pY-peptides using an array of fragmentation regimes (CID, HCD, EThcD, ETciD, UVPD) highlighted differences in the generation of site-determining product ions and allowed us to develop a strategy for differentiating sulfated peptides from nominally isobaric phosphopeptides based on low collision energy-induced neutral loss. Application of our "sulfomics" workflow to a HEK-293 cell extracellular secretome facilitated identification of 21 new sulfotyrosine-containing proteins, several of which we validate enzymatically, and reveals new interplay between enzymes relevant to both protein and glycan sulfation.


Assuntos
Fosfopeptídeos , Tirosina , Humanos , Fosfopeptídeos/análise , Células HEK293 , Fluxo de Trabalho , Tirosina/metabolismo , Proteínas , Fosfotirosina
9.
EMBO J ; 38(21): e100847, 2019 10 04.
Artigo em Inglês | MEDLINE | ID: mdl-31433507

RESUMO

Phosphorylation is a key regulator of protein function under (patho)physiological conditions, and defining site-specific phosphorylation is essential to understand basic and disease biology. In vertebrates, the investigative focus has primarily been on serine, threonine and tyrosine phosphorylation, but mounting evidence suggests that phosphorylation of other "non-canonical" amino acids also regulates critical aspects of cell biology. However, standard methods of phosphoprotein characterisation are largely unsuitable for the analysis of non-canonical phosphorylation due to their relative instability under acidic conditions and/or elevated temperature. Consequently, the complete landscape of phosphorylation remains unexplored. Here, we report an unbiased phosphopeptide enrichment strategy based on strong anion exchange (SAX) chromatography (UPAX), which permits identification of histidine (His), arginine (Arg), lysine (Lys), aspartate (Asp), glutamate (Glu) and cysteine (Cys) phosphorylation sites on human proteins by mass spectrometry-based phosphoproteomics. Remarkably, under basal conditions, and having accounted for false site localisation probabilities, the number of unique non-canonical phosphosites is approximately one-third of the number of observed canonical phosphosites. Our resource reveals the previously unappreciated diversity of protein phosphorylation in human cells, and opens up avenues for high-throughput exploration of non-canonical phosphorylation in all organisms.


Assuntos
Ânions/química , Cromatografia por Troca Iônica/métodos , Fosfopeptídeos/análise , Fosfoproteínas/análise , Proteoma/análise , Biologia Computacional , Células HeLa , Humanos , Espectrometria de Massas , Fosforilação
10.
J Proteome Res ; 21(6): 1510-1524, 2022 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-35532924

RESUMO

Public phosphorylation databases such as PhosphoSitePlus (PSP) and PeptideAtlas (PA) compile results from published papers or openly available mass spectrometry (MS) data. However, there is no database-level control for false discovery of sites, likely leading to the overestimation of true phosphosites. By profiling the human phosphoproteome, we estimate the false discovery rate (FDR) of phosphosites and predict a more realistic count of true identifications. We rank sites into phosphorylation likelihood sets and analyze them in terms of conservation across 100 species, sequence properties, and functional annotations. We demonstrate significant differences between the sets and develop a method for independent phosphosite FDR estimation. Remarkably, we report estimated FDRs of 84, 98, and 82% within sets of phosphoserine (pSer), phosphothreonine (pThr), and phosphotyrosine (pTyr) sites, respectively, that are supported by only a single piece of identification evidence─the majority of sites in PSP. We estimate that around 62 000 Ser, 8000 Thr, and 12 000 Tyr phosphosites in the human proteome are likely to be true, which is lower than most published estimates. Furthermore, our analysis estimates that 86 000 Ser, 50 000 Thr, and 26 000 Tyr phosphosites are likely false-positive identifications, highlighting the significant potential of false-positive data to be present in phosphorylation databases.


Assuntos
Fosfopeptídeos , Proteoma , Humanos , Espectrometria de Massas/métodos , Fosfopeptídeos/metabolismo , Fosfoproteínas/análise , Fosforilação , Proteoma/análise
11.
Biochem J ; 478(4): 735-748, 2021 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-33480417

RESUMO

Sulfated carbohydrate metabolism is a fundamental process, which occurs in all domains of life. Carbohydrate sulfatases are enzymes that remove sulfate groups from carbohydrates and are essential to the depolymerisation of complex polysaccharides. Despite their biological importance, carbohydrate sulfatases are poorly studied and challenges remain in accurately assessing the enzymatic activity, specificity and kinetic parameters. Most notably, the separation of desulfated products from sulfated substrates is currently a time-consuming process. In this paper, we describe the development of rapid capillary electrophoresis coupled to substrate fluorescence detection as a high-throughput and facile means of analysing carbohydrate sulfatase activity. The approach has utility for the determination of both kinetic and inhibition parameters and is based on existing microfluidic technology coupled to a new synthetic fluorescent 6S-GlcNAc carbohydrate substrate. Furthermore, we compare this technique, in terms of both time and resources, to high-performance anion exchange chromatography and NMR-based methods, which are the two current 'gold standards' for enzymatic carbohydrate sulfation analysis. Our study clearly demonstrates the advantages of mobility shift assays for the quantification of near real-time carbohydrate desulfation by purified sulfatases, and will support the search for small molecule inhibitors of these disease-associated enzymes.


Assuntos
Eletroforese Capilar/métodos , Ensaio de Desvio de Mobilidade Eletroforética/métodos , Fluorometria/métodos , Ensaios de Triagem em Larga Escala/métodos , Técnicas Analíticas Microfluídicas/métodos , Sulfotransferases/análise , Proteínas de Bactérias/análise , Proteínas de Bactérias/antagonistas & inibidores , Bacteroides thetaiotaomicron/enzimologia , Compostos de Boro/análise , Configuração de Carboidratos , Cromatografia Líquida de Alta Pressão , Cromatografia por Troca Iônica , Sistemas Computacionais , Corantes Fluorescentes/análise , Glicosaminoglicanos/metabolismo , Cinética , Ressonância Magnética Nuclear Biomolecular , Proteínas Recombinantes/análise , Especificidade por Substrato , Sulfotransferases/antagonistas & inibidores
12.
BMC Bioinformatics ; 22(1): 446, 2021 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-34537014

RESUMO

BACKGROUND: Protein kinases are among the largest druggable family of signaling proteins, involved in various human diseases, including cancers and neurodegenerative disorders. Despite their clinical relevance, nearly 30% of the 545 human protein kinases remain highly understudied. Comparative genomics is a powerful approach for predicting and investigating the functions of understudied kinases. However, an incomplete knowledge of kinase orthologs across fully sequenced kinomes severely limits the application of comparative genomics approaches for illuminating understudied kinases. Here, we introduce KinOrtho, a query- and graph-based orthology inference method that combines full-length and domain-based approaches to map one-to-one kinase orthologs across 17 thousand species. RESULTS: Using multiple metrics, we show that KinOrtho performed better than existing methods in identifying kinase orthologs across evolutionarily divergent species and eliminated potential false positives by flagging sequences without a proper kinase domain for further evaluation. We demonstrate the advantage of using domain-based approaches for identifying domain fusion events, highlighting a case between an understudied serine/threonine kinase TAOK1 and a metabolic kinase PIK3C2A with high co-expression in human cells. We also identify evolutionary fission events involving the understudied OBSCN kinase domains, further highlighting the value of domain-based orthology inference approaches. Using KinOrtho-defined orthologs, Gene Ontology annotations, and machine learning, we propose putative biological functions of several understudied kinases, including the role of TP53RK in cell cycle checkpoint(s), the involvement of TSSK3 and TSSK6 in acrosomal vesicle localization, and potential functions for the ULK4 pseudokinase in neuronal development. CONCLUSIONS: In sum, KinOrtho presents a novel query-based tool to identify one-to-one orthologous relationships across thousands of proteomes that can be applied to any protein family of interest. We exploit KinOrtho here to identify kinase orthologs and show that its well-curated kinome ortholog set can serve as a valuable resource for illuminating understudied kinases, and the KinOrtho framework can be extended to any protein-family of interest.


Assuntos
Evolução Biológica , Genômica , Humanos , Anotação de Sequência Molecular , Proteínas Quinases/genética , Proteínas Serina-Treonina Quinases , Proteínas
13.
Biochem J ; 477(13): 2451-2475, 2020 07 17.
Artigo em Inglês | MEDLINE | ID: mdl-32501498

RESUMO

Polo-like kinase 4 (PLK4) is the master regulator of centriole duplication in metazoan organisms. Catalytic activity and protein turnover of PLK4 are tightly coupled in human cells, since changes in PLK4 concentration and catalysis have profound effects on centriole duplication and supernumerary centrosomes, which are associated with aneuploidy and cancer. Recently, PLK4 has been targeted with a variety of small molecule kinase inhibitors exemplified by centrinone, which rapidly induces inhibitory effects on PLK4 and leads to on-target centrosome depletion. Despite this, relatively few PLK4 substrates have been identified unequivocally in human cells, and PLK4 signalling outside centriolar networks remains poorly characterised. We report an unbiased mass spectrometry (MS)-based quantitative analysis of cellular protein phosphorylation in stable PLK4-expressing U2OS human cells exposed to centrinone. PLK4 phosphorylation was itself sensitive to brief exposure to the compound, resulting in PLK4 stabilisation. Analysing asynchronous cell populations, we report hundreds of centrinone-regulated cellular phosphoproteins, including centrosomal and cell cycle proteins and a variety of likely 'non-canonical' substrates. Surprisingly, sequence interrogation of ∼300 significantly down-regulated phosphoproteins reveals an extensive network of centrinone-sensitive [Ser/Thr]Pro phosphorylation sequence motifs, which based on our analysis might be either direct or indirect targets of PLK4. In addition, we confirm that NMYC and PTPN12 are PLK4 substrates, both in vitro and in human cells. Our findings suggest that PLK4 catalytic output directly controls the phosphorylation of a diverse set of cellular proteins, including Pro-directed targets that are likely to be important in PLK4-mediated cell signalling.


Assuntos
Proteínas Serina-Treonina Quinases/metabolismo , Pirimidinas/farmacologia , Sulfonas/farmacologia , Linhagem Celular Tumoral , Citometria de Fluxo , Fluorometria , Humanos , Imunoprecipitação , Leupeptinas/farmacologia , Microscopia de Fluorescência , Fosforilação/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Espectrometria de Massas em Tandem
14.
EMBO J ; 35(17): 1902-22, 2016 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-27481935

RESUMO

We explore mechanisms that enable cancer cells to tolerate PI3K or Akt inhibitors. Prolonged treatment of breast cancer cells with PI3K or Akt inhibitors leads to increased expression and activation of a kinase termed SGK3 that is related to Akt. Under these conditions, SGK3 is controlled by hVps34 that generates PtdIns(3)P, which binds to the PX domain of SGK3 promoting phosphorylation and activation by its upstream PDK1 activator. Furthermore, under conditions of prolonged PI3K/Akt pathway inhibition, SGK3 substitutes for Akt by phosphorylating TSC2 to activate mTORC1. We characterise 14h, a compound that inhibits both SGK3 activity and activation in vivo, and show that a combination of Akt and SGK inhibitors induced marked regression of BT-474 breast cancer cell-derived tumours in a xenograft model. Finally, we present the kinome-wide analysis of mRNA expression dynamics induced by PI3K/Akt inhibition. Our findings highlight the importance of the hVps34-SGK3 pathway and suggest it represents a mechanism to counteract inhibition of PI3K/Akt signalling. The data support the potential of targeting both Akt and SGK as a cancer therapeutic.


Assuntos
Carcinogênese , Classe III de Fosfatidilinositol 3-Quinases/metabolismo , Complexos Multiproteicos/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Serina-Treonina Quinases TOR/metabolismo , Animais , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células , Modelos Animais de Doenças , Feminino , Xenoenxertos , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina
16.
Org Biomol Chem ; 17(6): 1487-1505, 2019 02 06.
Artigo em Inglês | MEDLINE | ID: mdl-30681118

RESUMO

Bryostatins with modified C17-C27 fragments have not been widely studied. The synthesis of 20,20-difluorinated analogues was therefore investigated. Such substitution would inhibit dehydration involving the C19-hydroxyl group and stabilise the ring-closed hemiacetal tautomers. Following preliminary studies, allyldifluorination was used to prepare difluorinated alkenols. Oxidation followed by stereoselective Wittig reactions of the resulting α,α-difluorinated ketones gave (E)-α,ß-unsaturated esters that were taken through to complete syntheses of 2-hydroxytetrahydropyrans corresponding to C17-C27 fragments of 20,20-difluorinated bryostatin. These compounds showed modest binding to protein kinase Cα isozyme. Attempts were also undertaken to synthesise macrocyclic 20,20-difluorinated analogues. During preliminary studies, allyldifluorination was carried out using a 2-alkyl-3-bromo-1,1-difluoropropene.


Assuntos
Briostatinas/química , Briostatinas/síntese química , Halogenação , Briostatinas/metabolismo , Técnicas de Química Sintética , Modelos Moleculares , Conformação Molecular , Proteína Quinase C/metabolismo
17.
Biochem J ; 475(1): 185-189, 2018 01 05.
Artigo em Inglês | MEDLINE | ID: mdl-29305429

RESUMO

The addition of phosphate groups to substrates allows protein kinases to regulate a myriad of biological processes, and contextual analysis of protein-bound phosphate is important for understanding how kinases contribute to physiology and disease. Leucine-rich repeat kinase 2 (LRRK2) is a Ser/Thr kinase linked to familial and sporadic cases of Parkinson's disease (PD). Recent work established that multiple Rab GTPases are physiological substrates of LRRK2, with Rab10 in particular emerging as a human substrate whose site-specific phosphorylation mirrors hyperactive LRRK2 lesions associated with PD. However, current assays to quantify Rab10 phosphorylation are expensive, time-consuming and technically challenging. In back-to-back studies reported in the Biochemical Journal, Alessi and colleagues teamed up with clinical colleagues and collaborators at the Michael J. Fox Foundation (MJFF) for Parkinson's research to develop, and validate, a panel of exquisitely sensitive phospho-specific Rab antibodies. Of particular interest, the monoclonal antibody-designated MJFF-pRAB10 detects phosphorylated Rab 10 on Thr73 in a variety of cells, brain extracts, PD-derived samples and human neutrophils, the latter representing a previously unrecognised biological resource for LRRK2 signalling analysis. In the future, these antibodies could become universal resources in the fight to understand and quantify connections between LRRK2 and Rab proteins, including those associated with clinical PD.


Assuntos
Fenômenos Biológicos , Doença de Parkinson , Anticorpos Fosfo-Específicos , Humanos , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina , Transdução de Sinais
18.
Biochem J ; 475(15): 2435-2455, 2018 08 14.
Artigo em Inglês | MEDLINE | ID: mdl-29934490

RESUMO

Protein tyrosine sulfation is a post-translational modification best known for regulating extracellular protein-protein interactions. Tyrosine sulfation is catalysed by two Golgi-resident enzymes termed tyrosylprotein sulfotransferases (TPSTs) 1 and 2, which transfer sulfate from the cofactor PAPS (3'-phosphoadenosine 5'-phosphosulfate) to a context-dependent tyrosine in a protein substrate. A lack of quantitative tyrosine sulfation assays has hampered the development of chemical biology approaches for the identification of small-molecule inhibitors of tyrosine sulfation. In the present paper, we describe the development of a non-radioactive mobility-based enzymatic assay for TPST1 and TPST2, through which the tyrosine sulfation of synthetic fluorescent peptides can be rapidly quantified. We exploit ligand binding and inhibitor screens to uncover a susceptibility of TPST1 and TPST2 to different classes of small molecules, including the anti-angiogenic compound suramin and the kinase inhibitor rottlerin. By screening the Published Kinase Inhibitor Set, we identified oxindole-based inhibitors of the Ser/Thr kinase RAF (rapidly accelerated fibrosarcoma) as low-micromolar inhibitors of TPST1 and TPST2. Interestingly, unrelated RAF inhibitors, exemplified by the dual BRAF/VEGFR2 inhibitor RAF265, were also TPST inhibitors in vitro We propose that target-validated protein kinase inhibitors could be repurposed, or redesigned, as more-specific TPST inhibitors to help evaluate the sulfotyrosyl proteome. Finally, we speculate that mechanistic inhibition of cellular tyrosine sulfation might be relevant to some of the phenotypes observed in cells exposed to anionic TPST ligands and RAF protein kinase inhibitors.


Assuntos
Imidazóis/química , Proteínas de Membrana , Peptídeos/química , Proteínas Proto-Oncogênicas B-raf , Piridinas/química , Sulfotransferases , Tirosina/química , Humanos , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/química , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Proteínas Proto-Oncogênicas B-raf/química , Sulfotransferases/antagonistas & inibidores , Sulfotransferases/química
19.
Biochem J ; 475(15): 2417-2433, 2018 08 14.
Artigo em Inglês | MEDLINE | ID: mdl-29934491

RESUMO

Sulfation of carbohydrate residues occurs on a variety of glycans destined for secretion, and this modification is essential for efficient matrix-based signal transduction. Heparan sulfate (HS) glycosaminoglycans control physiological functions ranging from blood coagulation to cell proliferation. HS biosynthesis involves membrane-bound Golgi sulfotransferases, including HS 2-O-sulfotransferase (HS2ST), which transfers sulfate from the cofactor PAPS (3'-phosphoadenosine 5'-phosphosulfate) to the 2-O position of α-l-iduronate in the maturing polysaccharide chain. The current lack of simple non-radioactive enzyme assays that can be used to quantify the levels of carbohydrate sulfation hampers kinetic analysis of this process and the discovery of HS2ST inhibitors. In the present paper, we describe a new procedure for thermal shift analysis of purified HS2ST. Using this approach, we quantify HS2ST-catalysed oligosaccharide sulfation using a novel synthetic fluorescent substrate and screen the Published Kinase Inhibitor Set, to evaluate compounds that inhibit catalysis. We report the susceptibility of HS2ST to a variety of cell-permeable compounds in vitro, including polyanionic polar molecules, the protein kinase inhibitor rottlerin and oxindole-based RAF kinase inhibitors. In a related study, published back-to-back with the present study, we demonstrated that tyrosyl protein sulfotranferases are also inhibited by a variety of protein kinase inhibitors. We propose that appropriately validated small-molecule compounds could become new tools for rapid inhibition of glycan (and protein) sulfation in cells, and that protein kinase inhibitors might be repurposed or redesigned for the specific inhibition of HS2ST.


Assuntos
Proteínas Aviárias/química , Heparitina Sulfato/química , Oligossacarídeos/química , Inibidores de Proteínas Quinases/química , Sulfotransferases/química , Quinases raf/antagonistas & inibidores , Animais , Proteínas Aviárias/genética , Galinhas , Heparitina Sulfato/farmacologia , Humanos , Oligossacarídeos/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Sulfotransferases/genética , Suínos , Quinases raf/química
20.
PLoS Genet ; 12(2): e1005885, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26925779

RESUMO

Protein tyrosine kinases (PTKs) are a group of closely related enzymes that have evolutionarily diverged from serine/threonine kinases (STKs) to regulate pathways associated with multi-cellularity. Evolutionary divergence of PTKs from STKs has occurred through accumulation of mutations in the active site as well as in the commonly conserved hydrophobic core. While the functional significance of active site variations is well understood, relatively little is known about how hydrophobic core variations contribute to PTK evolutionary divergence. Here, using a combination of statistical sequence comparisons, molecular dynamics simulations, mutational analysis and in vitro thermostability and kinase assays, we investigate the structural and functional significance of key PTK-specific variations in the kinase core. We find that the nature of residues and interactions in the hydrophobic core of PTKs is strikingly different from other protein kinases, and PTK-specific variations in the core contribute to functional divergence by altering the stability and dynamics of the kinase domain. In particular, a functionally critical STK-conserved histidine that stabilizes the regulatory spine in STKs is selectively mutated to an alanine, serine or glutamate in PTKs, and this loss-of-function mutation is accommodated, in part, through compensatory PTK-specific interactions in the core. In particular, a PTK-conserved phenylalanine in the I-helix appears to structurally and functionally compensate for the loss of STK-histidine by interacting with the regulatory spine, which has far-reaching effects on enzyme activity, inhibitor sensing, and stability. We propose that hydrophobic core variations provide a selective advantage during PTK evolution by increasing the conformational flexibility, and therefore the allosteric potential of the kinase domain. Our studies also suggest that Tyrosine Kinase Like kinases such as RAF are intermediates in PTK evolutionary divergence inasmuch as they share features of both PTKs and STKs in the core. Finally, our studies provide an evolutionary framework for identifying and characterizing disease and drug resistance mutations in the kinase core.


Assuntos
Evolução Molecular , Proteínas Tirosina Quinases/química , Proteínas Tirosina Quinases/metabolismo , Sequência de Aminoácidos , Aurora Quinase A/química , Aurora Quinase A/genética , Aurora Quinase A/metabolismo , Domínio Catalítico , Sequência Conservada , Interações Hidrofóbicas e Hidrofílicas , Simulação de Dinâmica Molecular , Dados de Sequência Molecular , Mutação , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/química , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/metabolismo , Receptor EphA3 , Relação Estrutura-Atividade
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