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1.
J Antimicrob Chemother ; 76(3): 587-595, 2021 Feb 11.
Artigo em Inglês | MEDLINE | ID: mdl-33338207

RESUMO

OBJECTIVES: To measure the variability in carbapenem susceptibility conferred by different OxaAb variants, characterize the molecular evolution of oxaAb and elucidate the contribution of OxaAb and other possible carbapenem resistance factors in the clinical isolates using WGS and LC-MS/MS. METHODS: Antimicrobial susceptibility tests were performed on 10 clinical Acinetobacter baumannii isolates. Carbapenem MICs were evaluated for all oxaAb variants cloned into A. baumannii CIP70.10 and BM4547, with and without their natural promoters. Molecular evolution analysis of the oxaAb variants was performed using FastTree and SplitsTree4. Resistance determinants were studied in the clinical isolates using WGS and LC-MS/MS. RESULTS: Only the OxaAb variants with I129L and L167V substitutions, OxaAb(82), OxaAb(83), OxaAb(107) and OxaAb(110) increased carbapenem MICs when expressed in susceptible A. baumannii backgrounds without an upstream IS element. Carbapenem resistance was conferred with the addition of their natural upstream ISAba1 promoter. LC-MS/MS analysis on the original clinical isolates confirmed overexpression of the four I129L and L167V variants. No other differences in expression levels of proteins commonly associated with carbapenem resistance were detected. CONCLUSIONS: Elevated carbapenem MICs were observed by expression of OxaAb variants carrying clinically prevalent substitutions I129L and L167V. To drive carbapenem resistance, these variants required overexpression by their upstream ISAba1 promoter. This study clearly demonstrates that a combination of IS-driven overexpression of oxaAb and the presence of particular amino acid substitutions in the active site to improve carbapenem capture is key in conferring carbapenem resistance in A. baumannii and other mechanisms are not required.

2.
Artigo em Inglês | MEDLINE | ID: mdl-33318029

RESUMO

BACKGROUND: Evidence for aspirin's chemopreventative properties on colorectal cancer (CRC) is substantial, but its mechanism of action is not well-understood. We combined a proteomic approach with Mendelian randomization (MR) to identify possible new aspirin targets that decrease CRC risk. METHODS: Human colorectal adenoma cells (RG/C2) were treated with aspirin (24 hours) and a stable isotope labeling with amino acids in cell culture (SILAC) based proteomics approach identified altered protein expression. Protein quantitative trait loci (pQTLs) from INTERVAL (N = 3,301) and expression QTLs (eQTLs) from the eQTLGen Consortium (N = 31,684) were used as genetic proxies for protein and mRNA expression levels. Two-sample MR of mRNA/protein expression on CRC risk was performed using eQTL/pQTL data combined with CRC genetic summary data from the Colon Cancer Family Registry (CCFR), Colorectal Transdisciplinary (CORECT), Genetics and Epidemiology of Colorectal Cancer (GECCO) consortia and UK Biobank (55,168 cases and 65,160 controls). RESULTS: Altered expression was detected for 125/5886 proteins. Of these, aspirin decreased MCM6, RRM2, and ARFIP2 expression, and MR analysis showed that a standard deviation increase in mRNA/protein expression was associated with increased CRC risk (OR: 1.08, 95% CI, 1.03-1.13; OR: 3.33, 95% CI, 2.46-4.50; and OR: 1.15, 95% CI, 1.02-1.29, respectively). CONCLUSIONS: MCM6 and RRM2 are involved in DNA repair whereby reduced expression may lead to increased DNA aberrations and ultimately cancer cell death, whereas ARFIP2 is involved in actin cytoskeletal regulation, indicating a possible role in aspirin's reduction of metastasis. IMPACT: Our approach has shown how laboratory experiments and population-based approaches can combine to identify aspirin-targeted proteins possibly affecting CRC risk.

3.
Artigo em Inglês | MEDLINE | ID: mdl-33139281

RESUMO

Fluoroquinolone resistance in Stenotrophomonas maltophilia is multi-factorial, but the most significant factor is overproduction of efflux pumps, particularly SmeDEF, following mutation. Here we report that mutations in the glycosyl transferase gene smlt0622 in S. maltophilia K279a mutant K M6 cause constitutive activation of SmeDEF production, leading to elevated levofloxacin MIC. Selection of a levofloxacin-resistant K M6 derivative, K M6 LEVR, allowed identification of a novel two-component regulatory system, Smlt2645/6 (renamed as SmaRS). The sensor kinase Smlt2646 (SmaS) is activated by mutation in K M6 LEVR causing over-production of two novel ABC transporters and the known aminoglycoside efflux pump SmeYZ. Over-production of one ABC transporter, Smlt1651-4 (renamed as SmaCDEF) causes levofloxacin resistance in K M6 LEVR Over-production of the other ABC transporter, Smlt2642/3 (renamed SmaAB) and SmeYZ both contribute to the elevated amikacin MIC against K M6 LEVR Accordingly, we have identified two novel ABC transporters associated with antimicrobial drug resistance in S. maltophilia, and two novel regulatory systems whose mutation causes resistance to levofloxacin, clinically important as a promising drug for monotherapy against this highly resistant pathogen.

4.
Sci Rep ; 10(1): 17953, 2020 10 21.
Artigo em Inglês | MEDLINE | ID: mdl-33087848

RESUMO

Proteins involved in the spaciotemporal regulation of GLUT4 trafficking represent potential therapeutic targets for the treatment of insulin resistance and type 2 diabetes. A key regulator of insulin- and exercise-stimulated glucose uptake and GLUT4 trafficking is TBC1D1. This study aimed to identify proteins that regulate GLUT4 trafficking and homeostasis via TBC1D1. Using an unbiased quantitative proteomics approach, we identified proteins that interact with TBC1D1 in C2C12 myotubes including VPS13A and VPS13C, the Rab binding proteins EHBP1L1 and MICAL1, and the calcium pump SERCA1. These proteins associate with TBC1D1 via its phosphotyrosine binding (PTB) domains and their interactions with TBC1D1 were unaffected by AMPK activation, distinguishing them from the AMPK regulated interaction between TBC1D1 and AMPKα1 complexes. Depletion of VPS13A or VPS13C caused a post-transcriptional increase in cellular GLUT4 protein and enhanced cell surface GLUT4 levels in response to AMPK activation. The phenomenon was specific to GLUT4 because other recycling proteins were unaffected. Our results provide further support for a role of the TBC1D1 PTB domains as a scaffold for a range of Rab regulators, and also the VPS13 family of proteins which have been previously linked to fasting glycaemic traits and insulin resistance in genome wide association studies.

5.
Exp Ther Med ; 20(5): 48, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32973936

RESUMO

Neonates with coarctation of the aorta (CoA) combined with a bicuspid aortic valve (BAV) show significant structural differences compared to neonatal CoA patients with a normal tricuspid aortic valve (TAV). These effects are likely to change over time in response to growth. This study investigated proteomic differences between coarcted aortic tissue of BAV and TAV patients in children older than one month. Aortic tissue just proximal to the coarctation site was collected from 10 children (BAV; n=6, 1.9±1.7 years, TAV; n=4, 1.7±1.5 years, (mean ± SEM, P=0.92.) Tissue were snap frozen, proteins extracted, and the extracts used for proteomic and phosphoproteomic analysis using Tandem Mass Tag (TMT) analysis. A total of 1811 protein and 76 phosphoprotein accession numbers were detected, of which 40 proteins and 6 phosphoproteins were significantly differentially expressed between BAV and TAV patients. Several canonical pathways involved in inflammation demonstrated enriched protein expression, including acute phase response signalling, EIF2 signalling and macrophage production of IL12 and reactive oxygen species. Acute phase response signalling also demonstrated enriched phosphoprotein expression, as did Th17 activation. Other pathways with significantly enriched protein expression include degradation of superoxide radicals and several pathways involved in apoptosis. This work suggests that BAV CoA patients older than one month have an altered proteome consistent with changes in inflammation, apoptosis and oxidative stress compared to TAV CoA patients of the same age. There is no evidence of structural differences, suggesting the pathology associated with BAV evolves with age in paediatric CoA patients.

6.
Genome Med ; 12(1): 68, 2020 07 28.
Artigo em Inglês | MEDLINE | ID: mdl-32723359

RESUMO

BACKGROUND: SARS-CoV-2 is a recently emerged respiratory pathogen that has significantly impacted global human health. We wanted to rapidly characterise the transcriptomic, proteomic and phosphoproteomic landscape of this novel coronavirus to provide a fundamental description of the virus's genomic and proteomic potential. METHODS: We used direct RNA sequencing to determine the transcriptome of SARS-CoV-2 grown in Vero E6 cells which is widely used to propagate the novel coronavirus. The viral transcriptome was analysed using a recently developed ORF-centric pipeline. Allied to this, we used tandem mass spectrometry to investigate the proteome and phosphoproteome of the same virally infected cells. RESULTS: Our integrated analysis revealed that the viral transcripts (i.e. subgenomic mRNAs) generally fitted the expected transcription model for coronaviruses. Importantly, a 24 nt in-frame deletion was detected in over half of the subgenomic mRNAs encoding the spike (S) glycoprotein and was predicted to remove a proposed furin cleavage site from the S glycoprotein. Tandem mass spectrometry identified over 500 viral peptides and 44 phosphopeptides in virus-infected cells, covering almost all proteins predicted to be encoded by the SARS-CoV-2 genome, including peptides unique to the deleted variant of the S glycoprotein. CONCLUSIONS: Detection of an apparently viable deletion in the furin cleavage site of the S glycoprotein, a leading vaccine target, shows that this and other regions of SARS-CoV-2 proteins may readily mutate. The furin site directs cleavage of the S glycoprotein into functional subunits during virus entry or exit and likely contributes strongly to the pathogenesis and zoonosis of this virus. Our data emphasises that the viral genome sequence should be carefully monitored during the growth of viral stocks for research, animal challenge models and, potentially, in clinical samples. Such variations may result in different levels of virulence, morbidity and mortality.


Assuntos
Betacoronavirus/crescimento & desenvolvimento , Perfilação da Expressão Gênica/métodos , Proteômica/métodos , Deleção de Sequência , Glicoproteína da Espícula de Coronavírus/genética , Animais , Betacoronavirus/genética , Betacoronavirus/metabolismo , Chlorocebus aethiops , Fosforilação , Análise de Sequência de RNA , Inoculações Seriadas , Espectrometria de Massas em Tandem , Células Vero
7.
J Antimicrob Chemother ; 75(9): 2471-2479, 2020 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-32542329

RESUMO

OBJECTIVES: To characterize putative AmpC-hyperproducing third-generation cephalosporin-resistant E. coli from dairy farms and their phylogenetic relationships; to identify risk factors for their presence; and to assess evidence for their zoonotic transmission into the local human population. METHODS: Proteomics was used to explain differences in antimicrobial susceptibility. WGS allowed phylogenetic analysis. Multilevel, multivariable logistic regression modelling was used to identify risk factors. RESULTS: Increased use of amoxicillin/clavulanate was associated with an increased risk of finding AmpC hyperproducers on farms. Expansion of cephalosporin resistance in AmpC hyperproducers was seen in farm isolates with marR mutations (conferring cefoperazone resistance) or when AmpC was mutated (conferring fourth-generation cephalosporin and cefoperazone resistance). Phylogenetic analysis confirmed the dominance of ST88 amongst farm AmpC hyperproducers but there was no evidence for acquisition of farm isolates by members of the local human population. CONCLUSIONS: Clear evidence was found for recent farm-to-farm transmission of AmpC-hyperproducing E. coli and of adaptive mutations to expand resistance. Whilst there was no evidence of isolates entering the local human population, efforts to reduce third-generation cephalosporin resistance on dairy farms must address the high prevalence of AmpC hyperproducers. The finding that amoxicillin/clavulanate use was associated with an increased risk of finding AmpC hyperproducers is important because this is not currently categorized as a highest-priority critically important antimicrobial and so is not currently targeted for specific usage restrictions in the UK.

8.
Proc Natl Acad Sci U S A ; 117(27): 15862-15873, 2020 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-32561647

RESUMO

Albuminuria is an independent risk factor for the progression to end-stage kidney failure, cardiovascular morbidity, and premature death. As such, discovering signaling pathways that modulate albuminuria is desirable. Here, we studied the transcriptomes of podocytes, key cells in the prevention of albuminuria, under diabetic conditions. We found that Neuropeptide Y (NPY) was significantly down-regulated in insulin-resistant vs. insulin-sensitive mouse podocytes and in human glomeruli of patients with early and late-stage diabetic nephropathy, as well as other nondiabetic glomerular diseases. This contrasts with the increased plasma and urinary levels of NPY that are observed in such conditions. Studying NPY-knockout mice, we found that NPY deficiency in vivo surprisingly reduced the level of albuminuria and podocyte injury in models of both diabetic and nondiabetic kidney disease. In vitro, podocyte NPY signaling occurred via the NPY2 receptor (NPY2R), stimulating PI3K, MAPK, and NFAT activation. Additional unbiased proteomic analysis revealed that glomerular NPY-NPY2R signaling predicted nephrotoxicity, modulated RNA processing, and inhibited cell migration. Furthermore, pharmacologically inhibiting the NPY2R in vivo significantly reduced albuminuria in adriamycin-treated glomerulosclerotic mice. Our findings suggest a pathogenic role of excessive NPY-NPY2R signaling in the glomerulus and that inhibiting NPY-NPY2R signaling in albuminuric kidney disease has therapeutic potential.


Assuntos
Albuminúria/metabolismo , Nefropatias/metabolismo , Neuropeptídeo Y/metabolismo , Receptores de Neuropeptídeo Y/metabolismo , Transdução de Sinais/fisiologia , Animais , Arginina/análogos & derivados , Arginina/farmacologia , Benzazepinas/farmacologia , Diabetes Mellitus Experimental/metabolismo , Nefropatias Diabéticas , Modelos Animais de Doenças , Regulação para Baixo , Doxorrubicina/farmacologia , Humanos , Insulina/metabolismo , Nefropatias/patologia , Glomérulos Renais/efeitos dos fármacos , Glomérulos Renais/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Neuropeptídeo Y/farmacologia , Neuropeptídeo Y/urina , Podócitos/metabolismo , Proteômica , Receptores de Neuropeptídeo Y/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos
9.
Mol Metab ; 40: 101015, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32416313

RESUMO

OBJECTIVE: Risk alleles for type 2 diabetes at the STARD10 locus are associated with lowered STARD10 expression in the ß-cell, impaired glucose-induced insulin secretion, and decreased circulating proinsulin:insulin ratios. Although likely to serve as a mediator of intracellular lipid transfer, the identity of the transported lipids and thus the pathways through which STARD10 regulates ß-cell function are not understood. The aim of this study was to identify the lipids transported and affected by STARD10 in the ß-cell and the role of the protein in controlling proinsulin processing and insulin granule biogenesis and maturation. METHODS: We used isolated islets from mice deleted selectively in the ß-cell for Stard10 (ßStard10KO) and performed electron microscopy, pulse-chase, RNA sequencing, and lipidomic analyses. Proteomic analysis of STARD10 binding partners was executed in the INS1 (832/13) cell line. X-ray crystallography followed by molecular docking and lipid overlay assay was performed on purified STARD10 protein. RESULTS: ßStard10KO islets had a sharply altered dense core granule appearance, with a dramatic increase in the number of "rod-like" dense cores. Correspondingly, basal secretion of proinsulin was increased versus wild-type islets. The solution of the crystal structure of STARD10 to 2.3 Å resolution revealed a binding pocket capable of accommodating polyphosphoinositides, and STARD10 was shown to bind to inositides phosphorylated at the 3' position. Lipidomic analysis of ßStard10KO islets demonstrated changes in phosphatidylinositol levels, and the inositol lipid kinase PIP4K2C was identified as a STARD10 binding partner. Also consistent with roles for STARD10 in phosphoinositide signalling, the phosphoinositide-binding proteins Pirt and Synaptotagmin 1 were amongst the differentially expressed genes in ßStard10KO islets. CONCLUSION: Our data indicate that STARD10 binds to, and may transport, phosphatidylinositides, influencing membrane lipid composition, insulin granule biosynthesis, and insulin processing.

10.
Front Physiol ; 11: 357, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32411010

RESUMO

Southeast Asian Ovalocytosis results from a heterozygous deletion of 9 amino acids in the erythrocyte anion exchange protein AE1 (band 3). The report of the first successful birth of an individual homozygous for this mutation showed an association with severe dyserythropoietic anemia. Imaging of the proband's erythrocytes revealed the presence of band 3 at their surface, a reduction in Wr(b) antigen expression, and increases in glycophorin C, CD44, and CD147 immunoreactivity. Immunoblotting of membranes from heterozygous Southeast Asian Ovalocytosis red cells showed a quantitative increase in CD44, CD147, and calreticulin suggesting a defect in reticulocyte maturation, as well as an increase in phosphorylation at residue Tyr359 of band 3, and peroxiredoxin-2 at the membrane, suggesting altered band 3 trafficking and oxidative stress, respectively. In vitro culture of homozygous and heterozygous Southeast Asian Ovalocytosis erythroid progenitor cells produced bi- and multi-nucleated cells. Enucleation was severely impaired in the homozygous cells and reduced in the heterozygous cells. Large internal vesicular accumulations of band 3 formed, which co-localized with other plasma membrane proteins and with the autophagosome marker, LC3, but not with ER, Golgi or recycling endosome markers. Immunoprecipitation of band 3 from erythroblast cell lysates at the orthochromatic stage showed increased interaction of the mutant band 3 with heat shock proteins, ubiquitin and cytoskeleton proteins, ankyrin, spectrin and actin. We also found that the mutant band 3 forms a strong interaction with non-muscle myosins IIA and IIB, while this interaction could not be detected in wild type erythroblasts. Consistent with this, the localization of non-muscle myosin IIA and actin was perturbed in some Southeast Asian Ovalocytosis erythroblasts. These findings provide new insights toward understanding in vivo dyserythropoiesis caused by the expression of mutant membrane proteins.

11.
Artigo em Inglês | MEDLINE | ID: mdl-32457105

RESUMO

Colistin resistance in Klebsiella pneumoniae is predominantly caused by mutations that increase expression of the arn (also known as pbg or pmrF) operon. Expression is activated by the PhoPQ and PmrAB two-component systems. Constitutive PhoPQ activation occurs directly by mutation or following loss of MgrB. PhoPQ may also cross-activate PmrAB via the linker protein PmrD. Using proteomics, we show that MgrB loss causes a wider proteomic effect than direct PhoPQ activation, suggesting additional targets for MgrB. Different mgrB mutations cause different amounts of Arn protein production, which correlated with colistin MICs. Disruption of phoP in an mgrB mutant had a reciprocal effect to direct activation of PhoQ in a wild-type background, but the regulated proteins showed almost total overlap. Disruption of pmrD or pmrA slightly reduced Arn protein production in an mgrB mutant, but production was still high enough to confer colistin resistance; disruption of phoP conferred wild-type Arn production and colistin MIC. Activation of PhoPQ directly or through mgrB mutation did not significantly activate PmrAB or PmrC production, but direct activation of PmrAB by mutation was able to do this, and also activated Arn production and conferred colistin resistance. There was little overlap between the PmrAB and PhoPQ regulons. We conclude that under the conditions used for colistin susceptibility testing, PhoPQ-PmrD-PmrAB cross-regulation is not significant and that independent activation of PhoPQ or PmrAB is the main reason that Arn protein production increases above the threshold required for colistin resistance.

12.
Artigo em Inglês | MEDLINE | ID: mdl-31743753

RESUMO

After decades in PtdIns(3,4,5)P3's shadow, PtdIns(3,4)P2 has now emerged as a bona fide regulator of important cellular events, including endocytosis and cell migration. New understanding of PtdIns(3,4)P2's cellular roles has been possible via novel approaches to observe and quantify cellular PtdIns(3,4)P2 dynamics, alongside methods to target the kinases and phosphatases governing phosphoinositide turnover. Despite this, the mechanisms by which PtdIns(3,4)P2 orchestrates its cellular roles remain more poorly understood, most notably because, to date, few PtdIns(3,4)P2 effectors have been identified. Here, we develop and apply an affinity-proteomics strategy to conduct a global screen for PtdIns(3,4)P2 interactors in human platelets; a primary cell type with striking PtdIns(3,4)P2 accumulation. Through an integrated approach, coupling affinity capture of PtdIns(3,4)P2-binding proteins to both label-free and isobaric tag-based quantitative proteomics, we identify a diverse PtdIns(3,4)P2 interactome. Included are long-established PtdIns(3,4)P2-binding proteins such as PLEKHA1, PLEKHA2, AKT and DAPP1, and a host of potentially novel effectors, including MTMR5, PNKD, RASA3 and GAB3. The PtdIns(3,4)P2 interactome shows an enrichment of pleckstrin homology (PH) domain-containing proteins, and through bioinformatics and array analyses we characterise the PH domain of MTMR5 and define its phosphoinositide selectivity. The interactome is also diverse in function, including several proteins known to support protein trafficking and cytoskeletal mobilisation. Such proteins have the ability to drive key platelet events, and to fulfil recently-defined roles for PtdIns(3,4)P2 in a wider range of cell types. Moreover, this study will serve as a valuable resource for the future characterisation of effector-driven PtdIns(3,4)P2 function.


Assuntos
Plaquetas/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Mapeamento de Interação de Proteínas , Biologia Computacional , Conjuntos de Dados como Assunto , Voluntários Saudáveis , Humanos , Espectrometria de Massas , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Mapas de Interação de Proteínas , Proteômica
13.
Mol Microbiol ; 113(2): 492-503, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31773806

RESUMO

The ß-lactam antibiotic ceftazidime is one of the handful of drugs with proven clinical efficacy against the important opportunistic human pathogen Stenotrophomonas maltophilia. Here, we show that mutations in the energy transducer TonB, encoded by smlt0009 in S. maltophilia, confer ceftazidime resistance and smlt0009 mutants have reduced uptake of ceftazidime. This breaks the dogma that ß-lactams enter Gram-negative bacteria only by passive diffusion through outer membrane porins. We also show that ceftazidime-resistant TonB mutants are cross-resistant to fluoroquinolone antimicrobials and a siderophore-conjugated lactivicin antibiotic designed to target TonB-dependent uptake. This implies that attempts to improve the penetration of antimicrobials into S. maltophilia by conjugating them with TonB substrates will suffer from the fact that ß-lactams and fluoroquinolones coselect resistance to these novel and otherwise promising antimicrobials. Finally, we show that smlt0009 mutants already exist among S. maltophilia clinical isolates and have reduced susceptibility to siderophore-conjugated lactivicin, despite the in vitro growth impairment seen in smlt0009 mutants selected in the laboratory.


Assuntos
Proteínas de Bactérias/genética , Farmacorresistência Bacteriana , Proteínas de Membrana/genética , Stenotrophomonas maltophilia/efeitos dos fármacos , beta-Lactamas/farmacologia , Anti-Infecciosos/farmacologia , Proteínas de Bactérias/metabolismo , Ceftazidima/farmacologia , Farmacorresistência Bacteriana Múltipla , Humanos , Proteínas de Membrana/metabolismo , Testes de Sensibilidade Microbiana , Mutação , Peptídeos Cíclicos/farmacologia , Sideróforos/farmacologia
14.
Life Sci Alliance ; 2(6)2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31792063

RESUMO

Determining the exact targets and mechanisms of action of drug molecules that modulate circadian rhythms is critical to develop novel compounds to treat clock-related disorders. Here, we have used phenotypic proteomic profiling (PPP) to systematically determine molecular targets of four circadian period-lengthening compounds in human cells. We demonstrate that the compounds cause similar changes in phosphorylation and activity of several proteins and kinases involved in vital pathways, including MAPK, NGF, B-cell receptor, AMP-activated protein kinases (AMPKs), and mTOR signaling. Kinome profiling further indicated inhibition of CKId, ERK1/2, CDK2/7, TNIK, and MST4 kinases as a common mechanism of action for these clock-modulating compounds. Pharmacological or genetic inhibition of several convergent kinases lengthened circadian period, establishing them as novel circadian targets. Finally, thermal stability profiling revealed binding of the compounds to clock regulatory kinases, signaling molecules, and ubiquitination proteins. Thus, phenotypic proteomic profiling defines novel clock effectors that could directly inform precise therapeutic targeting of the circadian system in humans.


Assuntos
Relógios Circadianos/genética , Ritmo Circadiano/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos/métodos , Adenina/análogos & derivados , Adenina/farmacologia , Antracenos/farmacologia , Linhagem Celular Tumoral , Relógios Circadianos/efeitos dos fármacos , Ritmo Circadiano/genética , Humanos , Fenótipo , Fosforilação , Proteômica , Purinas/farmacologia , Roscovitina/farmacologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Fatores de Transcrição/genética
15.
Nat Cell Biol ; 21(10): 1219-1233, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31576058

RESUMO

Protein trafficking requires coat complexes that couple recognition of sorting motifs in transmembrane cargoes with biogenesis of transport carriers. The mechanisms of cargo transport through the endosomal network are poorly understood. Here, we identify a sorting motif for endosomal recycling of cargoes, including the cation-independent mannose-6-phosphate receptor and semaphorin 4C, by the membrane tubulating BAR domain-containing sorting nexins SNX5 and SNX6. Crystal structures establish that this motif folds into a ß-hairpin, which binds a site in the SNX5/SNX6 phox homology domains. Over sixty cargoes share this motif and require SNX5/SNX6 for their recycling. These include cargoes involved in neuronal migration and a Drosophila snx6 mutant displays defects in axonal guidance. These studies identify a sorting motif and provide molecular insight into an evolutionary conserved coat complex, the 'Endosomal SNX-BAR sorting complex for promoting exit 1' (ESCPE-1), which couples sorting motif recognition to the BAR-domain-mediated biogenesis of cargo-enriched tubulo-vesicular transport carriers.


Assuntos
Endossomos/metabolismo , Proteínas de Membrana/metabolismo , Nexinas de Classificação/química , Nexinas de Classificação/metabolismo , Motivos de Aminoácidos/genética , Animais , Drosophila melanogaster , Células HEK293 , Células HeLa , Humanos , Domínios Proteicos/genética , Transporte Proteico/fisiologia , Receptor IGF Tipo 2/química , Receptor IGF Tipo 2/metabolismo , Semaforinas/genética , Semaforinas/metabolismo , Nexinas de Classificação/genética
16.
J Am Soc Nephrol ; 30(10): 1910-1924, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31416818

RESUMO

BACKGROUND: Mutations in the transient receptor potential channel 6 (TRPC6) gene are associated with an inherited form of FSGS. Despite widespread expression, patients with TRPC6 mutations do not present with any other pathologic phenotype, suggesting that this protein has a unique yet unidentified role within the target cell for FSGS, the kidney podocyte. METHODS: We generated a stable TRPC6 knockout podocyte cell line from TRPC6 knockout mice. These cells were engineered to express wild-type TRPC6, a dominant negative TRPC6 mutation, or either of two disease-causing mutations of TRPC6, G109S or K874*. We extensively characterized these cells using motility, detachment, and calpain activity assays; immunofluorescence; confocal or total internal reflection fluorescence microscopy; and western blotting. RESULTS: Compared with wild-type cells, TRPC6-/- podocytes are less motile and more adhesive, with an altered actin cytoskeleton. We found that TRPC6 binds to ERK1/2 and the actin regulatory proteins, caldesmon (a calmodulin- and actin-binding protein) and calpain 1 and 2 (calcium-dependent cysteine proteases that control the podocyte cytoskeleton, cell adhesion, and motility via cleavage of paxillin, focal adhesion kinase, and talin). Knockdown or expression of the truncated K874* mutation (but not expression of the gain-of-function G019S mutation or dominant negative mutant of TRPC6) results in the mislocalization of calpain 1 and 2 and significant downregulation of calpain activity; this leads to altered podocyte cytoskeleton, motility, and adhesion-characteristics of TRPC6 -/- podocytes. CONCLUSIONS: Our data demonstrate that independent of TRPC6 channel activity, the physical interaction between TRPC6 and calpain in the podocyte is important for cell motility and detachment and demonstrates a scaffolding role of the TRPC6 protein in disease.


Assuntos
Calpaína/fisiologia , Adesão Celular , Movimento Celular , Citoesqueleto/fisiologia , Podócitos/fisiologia , Podócitos/ultraestrutura , Canal de Cátion TRPC6/fisiologia , Animais , Camundongos , Camundongos Knockout
17.
FASEB J ; 33(11): 12277-12287, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31431053

RESUMO

Endothelial cells (ECs) form an active barrier between the circulation and the body. In addition to controlling transport of molecules between these 2 compartments, the endothelium is a major secretory organ, releasing proteins both into the circulation and into the vascular matrix. Although it is clearly important that proteins are correctly sorted into these 2 spaces, we currently know little of the polarization of this secretion or how it is controlled. Here, we present an optimized system for the analysis of polarized secretion and show that it allows the derivation of deep, robust proteomes from small numbers of primary ECs. We present the first endothelial apically and basolaterally secreted proteomes, demonstrating that ECs polarize the secretion of extracellular vesicle cargoes to the apical surface. Conversely, we find that protein secretion at the basolateral surface is focused on components of the extracellular matrix (ECM). Finally, we examine the role of liprin-α1 in secretion toward the basolateral compartment and identify a subset of ECM components that share this route with fibronectin.-Wei, H., Sundararaman, A., Dickson, E., Rennie-Campbell, L., Cross, E., Heesom, K. J., Mellor, H. Characterization of the polarized endothelial secretome.


Assuntos
Polaridade Celular , Células Endoteliais/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Células Cultivadas , Proteínas da Matriz Extracelular/metabolismo , Humanos , Proteômica
18.
J Clin Med ; 8(4)2019 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-30995723

RESUMO

Coarctation of the aorta is a form of left ventricular outflow tract obstruction in paediatric patients that can be presented with either bicuspid (BAV) or normal tricuspid (TAV) aortic valve. The congenital BAV is associated with hemodynamic changes and can therefore trigger different molecular remodelling in the coarctation area. This study investigated the proteomic and phosphoproteomic changes associated with BAV for the first time in neonatal coarctation patients. Aortic tissue was collected just proximal to the coarctation site from 23 neonates (BAV; n = 10, TAV; n = 13) that were matched for age (age range 4-22 days). Tissue from half of the patients was frozen and used for proteomic and phosphoproteomic analysis whilst the remaining tissue was formalin fixed and used for analysis of elastin content using Elastic Van-Gieson (EVG) staining. A total of 1796 protein and 75 phosphoprotein accession numbers were detected, of which 34 proteins and one phosphoprotein (SSH3) were differentially expressed in BAV patients compared to TAV patients. Ingenuity Pathway Analysis identified the formation of elastin fibres as a significantly enriched function (p = 1.12 × 10-4) due to the upregulation of EMILIN-1 and the downregulation of TNXB. Analysis of paraffin sections stained with EVG demonstrated increased elastin content in BAV patients. The proteomic/phosphoproteomic analysis also suggested changes in inositol signalling pathways and reduced expression of the antioxidant SOD3. This work demonstrates for the first time that coarcted aortic tissue in neonatal BAV patients has an altered proteome/phosphoproteome consistent with observed structural vascular changes when compared to TAV patients.

19.
Haematologica ; 104(7): 1365-1377, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-30630973

RESUMO

Canonical Wnt/ß-catenin signaling is frequently dysregulated in myeloid leukemias and is implicated in leukemogenesis. Nuclear-localized ß-catenin is indicative of active Wnt signaling and is frequently observed in acute myeloid leukemia (AML) patients; however, some patients exhibit little or no nuclear ß-catenin even where cytosolic ß-catenin is abundant. Control of the subcellular localization of ß-catenin therefore represents an additional mechanism regulating Wnt signaling in hematopoietic cells. To investigate the factors mediating the nuclear-localization of ß-catenin, we carried out the first nuclear/cytoplasmic proteomic analysis of the ß-catenin interactome in myeloid leukemia cells and identified putative novel ß-catenin interactors. Comparison of interacting factors between Wnt-responsive cells (high nuclear ß-catenin) versus Wnt-unresponsive cells (low nuclear ß-catenin) suggested the transcriptional partner, LEF-1, could direct the nuclear-localization of ß-catenin. The relative levels of nuclear LEF-1 and ß-catenin were tightly correlated in both cell lines and in primary AML blasts. Furthermore, LEF-1 knockdown perturbed ß-catenin nuclear-localization and transcriptional activation in Wnt-responsive cells. Conversely, LEF-1 overexpression was able to promote both nuclear-localization and ß-catenin-dependent transcriptional responses in previously Wnt-unresponsive cells. This is the first ß-catenin interactome study in hematopoietic cells and reveals LEF-1 as a mediator of nuclear ß- catenin level in human myeloid leukemia.


Assuntos
Núcleo Celular/metabolismo , Leucemia Mieloide Aguda/metabolismo , Fator 1 de Ligação ao Facilitador Linfoide/metabolismo , Síndromes Mielodisplásicas/metabolismo , Proteoma/análise , Proteína Wnt1/metabolismo , beta Catenina/metabolismo , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Fator 1 de Ligação ao Facilitador Linfoide/antagonistas & inibidores , Fator 1 de Ligação ao Facilitador Linfoide/genética , Síndromes Mielodisplásicas/patologia , Domínios e Motivos de Interação entre Proteínas , RNA Interferente Pequeno/genética , Ativação Transcricional , Células Tumorais Cultivadas , Proteína Wnt1/genética , beta Catenina/genética
20.
J Neurochem ; 149(3): 399-412, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30664241

RESUMO

The generation of the amyloid-ß (Aß) peptides from the amyloid precursor protein (APP) through sequential proteolysis by ß- and γ-secretases is a key pathological event in the initiation and propagation of Alzheimer's disease. Aß and the transcriptionally active APP intracellular domain are generated preferentially from the APP695 isoform compared to the longer APP751 isoform. As the Aß and amyloid precursor protein intracellular domain produced from cleavage of APP695 and APP751 are identical we hypothesised that the two isoforms have differences within their interactomes which mediate the differential processing of the two isoforms. To investigate this, we applied a proteomics-based approach to identify differences in the interactomes of the APP695 and APP751 isoforms. Using stable isotope labelling of amino acids in cell culture and quantitative proteomics, we compared the interactomes of APP695 and APP751 expressed in human SH-SY5Y cells. Through this approach, we identified enrichment of proteins involved in mitochondrial function, the nuclear pore and nuclear transport specifically in the APP695 interactome. Further interrogation of the APP interactome and subsequent experimental validation (co-immunoprecipitation and siRNA knockdown) revealed GAP43 as a specific modulator of APP751 proteolysis, altering Aß generation. Our data indicate that interrogation of the APP interactome can be exploited to identify proteins which influence APP proteolysis and Aß production in an isoform dependent-manner. Cover Image for this issue: doi: 10.1111/jnc.14504.


Assuntos
Precursor de Proteína beta-Amiloide/metabolismo , Linhagem Celular Tumoral , Humanos , Mitocôndrias/metabolismo , Neurônios/metabolismo , Isoformas de Proteínas , Proteômica
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