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1.
Cell ; 186(10): 2219-2237.e29, 2023 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-37172566

RESUMO

The Commander complex is required for endosomal recycling of diverse transmembrane cargos and is mutated in Ritscher-Schinzel syndrome. It comprises two sub-assemblies: Retriever composed of VPS35L, VPS26C, and VPS29; and the CCC complex which contains twelve subunits: COMMD1-COMMD10 and the coiled-coil domain-containing (CCDC) proteins CCDC22 and CCDC93. Combining X-ray crystallography, electron cryomicroscopy, and in silico predictions, we have assembled a complete structural model of Commander. Retriever is distantly related to the endosomal Retromer complex but has unique features preventing the shared VPS29 subunit from interacting with Retromer-associated factors. The COMMD proteins form a distinctive hetero-decameric ring stabilized by extensive interactions with CCDC22 and CCDC93. These adopt a coiled-coil structure that connects the CCC and Retriever assemblies and recruits a 16th subunit, DENND10, to form the complete Commander complex. The structure allows mapping of disease-causing mutations and reveals the molecular features required for the function of this evolutionarily conserved trafficking machinery.


Assuntos
Anormalidades Múltiplas , Anormalidades Craniofaciais , Complexos Multiproteicos , Humanos , Endossomos/metabolismo , Transporte Proteico , Proteínas/metabolismo , Complexos Multiproteicos/metabolismo
2.
Proc Natl Acad Sci U S A ; 121(33): e2405041121, 2024 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-39116126

RESUMO

Endosomal membrane trafficking is mediated by specific protein coats and formation of actin-rich membrane domains. The Retromer complex coordinates with sorting nexin (SNX) cargo adaptors including SNX27, and the SNX27-Retromer assembly interacts with the Wiskott-Aldrich syndrome protein and SCAR homolog (WASH) complex which nucleates actin filaments establishing the endosomal recycling domain. Crystal structures, modeling, biochemical, and cellular validation reveal how the FAM21 subunit of WASH interacts with both Retromer and SNX27. FAM21 binds the FERM domain of SNX27 using acidic-Asp-Leu-Phe (aDLF) motifs similar to those found in the SNX1 and SNX2 subunits of the ESCPE-1 complex. Overlapping FAM21 repeats and a specific Pro-Leu containing motif bind three distinct sites on Retromer involving both the VPS35 and VPS29 subunits. Mutation of the major VPS35-binding site does not prevent cargo recycling; however, it partially reduces endosomal WASH association indicating that a network of redundant interactions promote endosomal activity of the WASH complex. These studies establish the molecular basis for how SNX27-Retromer is coupled to the WASH complex via overlapping and multiplexed motif-based interactions required for the dynamic assembly of endosomal membrane recycling domains.


Assuntos
Endossomos , Nexinas de Classificação , Proteínas de Transporte Vesicular , Humanos , Endossomos/metabolismo , Nexinas de Classificação/metabolismo , Nexinas de Classificação/genética , Nexinas de Classificação/química , Proteínas de Transporte Vesicular/metabolismo , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/química , Proteínas dos Microfilamentos/metabolismo , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/química , Ligação Proteica , Cristalografia por Raios X , Sítios de Ligação , Modelos Moleculares
3.
J Cell Sci ; 137(8)2024 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-38533689

RESUMO

Primary cilia are essential eukaryotic organelles required for signalling and secretion. Dynein-2 is a microtubule-motor protein complex and is required for ciliogenesis via its role in facilitating retrograde intraflagellar transport (IFT) from the cilia tip to the cell body. Dynein-2 must be assembled and loaded onto IFT trains for entry into cilia for this process to occur, but how dynein-2 is assembled and how it is recycled back into a cilium remain poorly understood. Here, we identify centrosomal protein of 170 kDa (CEP170) as a dynein-2-interacting protein in mammalian cells. We show that loss of CEP170 perturbs intraflagellar transport and hedgehog signalling, and alters the stability of dynein-2 holoenzyme complex. Together, our data indicate a role for CEP170 in supporting cilia function and dynein-2 assembly.


Assuntos
Cílios , Proteínas Associadas aos Microtúbulos , Cílios/metabolismo , Humanos , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Animais , Dineínas/metabolismo , Dineínas/genética , Proteínas Hedgehog/metabolismo , Proteínas Hedgehog/genética , Transdução de Sinais , Camundongos , Flagelos/metabolismo
4.
PLoS Pathog ; 20(6): e1012235, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38843111

RESUMO

Amikacin and piperacillin/tazobactam are frequent antibiotic choices to treat bloodstream infection, which is commonly fatal and most often caused by bacteria from the family Enterobacterales. Here we show that two gene cassettes located side-by-side in and ancestral integron similar to In37 have been "harvested" by insertion sequence IS26 as a transposon that is widely disseminated among the Enterobacterales. This transposon encodes the enzymes AAC(6')-Ib-cr and OXA-1, reported, respectively, as amikacin and piperacillin/tazobactam resistance mechanisms. However, by studying bloodstream infection isolates from 769 patients from three hospitals serving a population of 1.2 million people in South West England, we show that increased enzyme production due to mutation in an IS26/In37-derived hybrid promoter or, more commonly, increased transposon copy number is required to simultaneously remove these two key therapeutic options; in many cases leaving only the last-resort antibiotic, meropenem. These findings may help improve the accuracy of predicting piperacillin/tazobactam treatment failure, allowing stratification of patients to receive meropenem or piperacillin/tazobactam, which may improve outcome and slow the emergence of meropenem resistance.


Assuntos
Antibacterianos , Elementos de DNA Transponíveis , Humanos , Antibacterianos/farmacologia , Elementos de DNA Transponíveis/genética , Farmacorresistência Bacteriana Múltipla/genética , Piperacilina/farmacologia , Amicacina/farmacologia , Testes de Sensibilidade Microbiana , Infecções por Enterobacteriaceae/microbiologia , Infecções por Enterobacteriaceae/tratamento farmacológico , Infecções por Enterobacteriaceae/genética , Enterobacteriaceae/genética , Enterobacteriaceae/efeitos dos fármacos , Integrons/genética , Bacteriemia/microbiologia , Bacteriemia/tratamento farmacológico , Bacteriemia/genética
5.
J Cell Sci ; 136(5)2023 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-36632779

RESUMO

The dynein-2 complex must be transported anterogradely within cilia to then drive retrograde trafficking of the intraflagellar transport (IFT) machinery containing IFT-A and IFT-B complexes. Here, we screened for potential interactions between the dynein-2 and IFT-B complexes and found multiple interactions among the dynein-2 and IFT-B subunits. In particular, WDR60 (also known as DYNC2I1) and the DYNC2H1-DYNC2LI1 dimer from dynein-2, and IFT54 (also known as TRAF3IP1) and IFT57 from IFT-B contribute to the dynein-2-IFT-B interactions. WDR60 interacts with IFT54 via a conserved region N-terminal to its light chain-binding regions. Expression of the WDR60 constructs in WDR60-knockout (KO) cells revealed that N-terminal truncation mutants lacking the IFT54-binding site fail to rescue abnormal phenotypes of WDR60-KO cells, such as aberrant accumulation of the IFT machinery around the ciliary tip and on the distal side of the transition zone. However, a WDR60 construct specifically lacking just the IFT54-binding site substantially restored the ciliary defects. In line with the current docking model of dynein-2 with the anterograde IFT trains, these results indicate that extensive interactions involving multiple subunits from the dynein-2 and IFT-B complexes participate in their connection.


Assuntos
Cílios , Dineínas , Cílios/metabolismo , Dineínas/genética , Dineínas/metabolismo , Transporte Biológico , Citoesqueleto/metabolismo , Domínios Proteicos , Flagelos/metabolismo
6.
Blood ; 141(25): 3039-3054, 2023 06 22.
Artigo em Inglês | MEDLINE | ID: mdl-37084386

RESUMO

Red blood cell disorders can result in severe anemia. One such disease congenital dyserythropoietic anemia IV (CDA IV) is caused by the heterozygous mutation E325K in the transcription factor KLF1. However, studying the molecular basis of CDA IV is severely impeded by the paucity of suitable and adequate quantities of material from patients with anemia and the rarity of the disease. We, therefore, took a novel approach, creating a human cellular disease model system for CDA IV that accurately recapitulates the disease phenotype. Next, using comparative proteomics, we reveal extensive distortion of the proteome and a wide range of disordered biological processes in CDA IV erythroid cells. These include downregulated pathways the governing cell cycle, chromatin separation, DNA repair, cytokinesis, membrane trafficking, and global transcription, and upregulated networks governing mitochondrial biogenesis. The diversity of such pathways elucidates the spectrum of phenotypic abnormalities that occur with CDA IV and impairment to erythroid cell development and survival, collectively explaining the CDA IV disease phenotype. The data also reveal far more extensive involvement of KLF1 in previously assigned biological processes, along with novel roles in the regulation of intracellular processes not previously attributed to this transcription factor. Overall, the data demonstrate the power of such a model cellular system to unravel the molecular basis of disease and how studying the effects of a rare mutation can reveal fundamental biology.


Assuntos
Anemia Diseritropoética Congênita , Humanos , Anemia Diseritropoética Congênita/genética , Mutação , Regulação da Expressão Gênica , Fenótipo , Fatores de Transcrição/genética
7.
Proc Natl Acad Sci U S A ; 119(25): e2201980119, 2022 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-35696571

RESUMO

Endosomal sorting maintains cellular homeostasis by recycling transmembrane proteins and associated proteins and lipids (termed "cargoes") from the endosomal network to multiple subcellular destinations, including retrograde traffic to the trans-Golgi network (TGN). Viral and bacterial pathogens subvert retrograde trafficking machinery to facilitate infectivity. Here, we develop a proteomic screen to identify retrograde cargo proteins of the endosomal SNX-BAR sorting complex promoting exit 1 (ESCPE-1). Using this methodology, we identify Neuropilin-1 (NRP1), a recently characterized host factor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, as a cargo directly bound and trafficked by ESCPE-1. ESCPE-1 mediates retrograde trafficking of engineered nanoparticles functionalized with the NRP1-interacting peptide of the SARS-CoV-2 spike (S) protein. CRISPR-Cas9 deletion of ESCPE-1 subunits reduces SARS-CoV-2 infection levels in cell culture. ESCPE-1 sorting of NRP1 may therefore play a role in the intracellular membrane trafficking of NRP1-interacting viruses such as SARS-CoV-2.


Assuntos
COVID-19 , Endossomos , Interações Hospedeiro-Patógeno , Neuropilina-1 , SARS-CoV-2 , COVID-19/metabolismo , COVID-19/virologia , Sistemas CRISPR-Cas , Endossomos/virologia , Deleção de Genes , Humanos , Nanopartículas , Neuropilina-1/genética , Neuropilina-1/metabolismo , Proteômica , SARS-CoV-2/metabolismo , Nexinas de Classificação/metabolismo , Glicoproteína da Espícula de Coronavírus/metabolismo
8.
Antimicrob Agents Chemother ; 68(7): e0024224, 2024 Jul 09.
Artigo em Inglês | MEDLINE | ID: mdl-38767379

RESUMO

Nitrofurantoin resistance in Escherichia coli is primarily caused by mutations damaging two enzymes, NfsA and NfsB. Studies based on small isolate collections with defined nitrofurantoin MICs have found significant random genetic drift in nfsA and nfsB, making it extremely difficult to predict nitrofurantoin resistance from whole-genome sequence (WGS) where both genes are not obviously disrupted by nonsense or frameshift mutations or insertional inactivation. Here, we report a WGS survey of 200 oqxAB-negative E. coli from community urine samples, of which 34 were nitrofurantoin resistant. We characterized individual non-synonymous mutations seen in nfsA and nfsB among this collection using complementation cloning and NfsA/B enzyme assays in cell extracts. We definitively identified R203C, H11Y, W212R, A112E, and A112T in NfsA and R121C, Q142H, F84S, P163H, W46R, K57E, and V191G in NfsB as amino acid substitutions that reduce enzyme activity sufficiently to cause resistance. In contrast, E58D, I117T, K141E, L157F, A172S, G187D, and A188V in NfsA and G66D, M75I, V93A, and A174E in NfsB are functionally silent in this context. We identified that 9/166 (5.4%) nitrofurantoin-susceptible isolates were "pre-resistant," defined as having loss of function mutations in nfsA or nfsB. Finally, using NfsA/B enzyme assays and proteomics, we demonstrated that 9/34 (26.5%) ribE wild-type nitrofurantoin-resistant isolates also carried functionally wild-type nfsB or nfsB/nfsA. In these cases, NfsA/B activity was reduced through downregulated gene expression. Our biological understanding of nitrofurantoin resistance is greatly improved by this analysis but is still insufficient to allow its reliable prediction from WGS data.


Assuntos
Farmacorresistência Bacteriana , Proteínas de Escherichia coli , Escherichia coli , Nitrofurantoína , Nitrorredutases , Humanos , Antibacterianos/farmacologia , Anti-Infecciosos Urinários/farmacologia , Farmacorresistência Bacteriana/genética , Escherichia coli/genética , Escherichia coli/efeitos dos fármacos , Proteínas de Escherichia coli/genética , Genoma Bacteriano/genética , Testes de Sensibilidade Microbiana , Mutação , Nitrofurantoína/farmacologia , Nitrorredutases/genética , Nitrorredutases/metabolismo , Sequenciamento Completo do Genoma/métodos
9.
Am J Physiol Cell Physiol ; 324(1): C113-C132, 2023 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-36374169

RESUMO

Metastasis and recurrence of breast cancer remain major causes of patient mortality, and there is an ongoing need to identify new therapeutic targets relevant to tumor invasion. Protein disulfide isomerase A3 (PDIA3) is a disulfide oxidoreductase and isomerase of the endoplasmic reticulum that has known extracellular substrates and has been correlated with aggressive breast cancers. We show that either prior PDIA3 inhibition by the disulfide isomerase inhibitor 16F16 or depletion of heparin-binding proteins strongly reduces the activity of conditioned medium (CM) of MDA-MB-231 human breast cancer cells to support promigratory cell spreading and F-actin organization by newly adherent MDA-MB-231 cells. Quantitative proteomics to investigate effects of 16F16 inhibition on heparin-binding proteins in the CM of MDA-MB-231 cells identified 80 proteins reproducibly decreased at least twofold (at q ≤ 0.05) after 16F16 treatment. By Gene Ontology analysis, many of these have roles in extracellular matrix (ECM) structure and function and cell adhesion; ribosomal proteins that also correlate with extracellular vesicles were also identified. Protein-protein interaction analysis showed that many of the extracellular proteins have known network interactions with each other. The predominant types of disulfide-bonded domains in the extracellular proteins contained ß-hairpin folds, with the knottin fold the most common. From human breast cancer data sets, the extracellular proteins were found to correlate specifically with the basal subtype of breast cancer and their high expression in tumors correlated with reduced distant metastasis-free survival. These data provide new evidence that PDIA3 may be a relevant therapeutic target to alter properties of the ECM-associated microenvironment in basal breast cancer.


Assuntos
Neoplasias da Mama , Isomerases de Dissulfetos de Proteínas , Humanos , Feminino , Isomerases de Dissulfetos de Proteínas/genética , Isomerases de Dissulfetos de Proteínas/metabolismo , Isomerases de Dissulfetos de Proteínas/farmacologia , Neoplasias da Mama/patologia , Adesão Celular , Comunicação Celular , Heparina/farmacologia , Linhagem Celular Tumoral , Microambiente Tumoral
10.
Immunol Cell Biol ; 101(10): 947-963, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37694300

RESUMO

Macrophages have previously been characterized based on phenotypical and functional differences into suggested simplified subtypes of MØ, M1, M2a and M2c. These macrophage subtypes can be generated in a well-established primary monocyte culture model that produces cells expressing accepted subtype surface markers. To determine how these subtypes retain functional similarities and better understand their formation, we generated all four subtypes from the same donors. Comparative whole-cell proteomics confirmed that four distinct macrophage subtypes could be induced from the same donor material, with > 50% of 5435 identified proteins being significantly altered in abundance between subtypes. Functional assessment highlighted that these distinct protein expression profiles are primed to enable specific cell functions, indicating that this shifting proteome is predictive of meaningful changes in cell characteristics. Importantly, the 2552 proteins remained consistent in abundance across all macrophage subtypes examined, demonstrating maintenance of a stable core proteome that likely enables swift polarity changes. We next explored the cross-polarization capabilities of preactivated M1 macrophages treated with dexamethasone. Importantly, these treated cells undergo a partial repolarization toward the M2c surface markers but still retain the M1 functional phenotype. Our investigation of polarized macrophage subtypes therefore provides evidence of a sliding scale of macrophage functionality, with these data sets providing a valuable benchmark resource for further studies of macrophage polarity, with relevance for cell therapy development and drug discovery.


Assuntos
Proteoma , Proteômica , Proteoma/metabolismo , Células Cultivadas , Macrófagos/metabolismo , Monócitos/fisiologia
11.
Respir Res ; 24(1): 191, 2023 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-37474963

RESUMO

INTRODUCTION: Although different phenotypes of lung disease after preterm birth have recently been described, the underlying mechanisms associated with each phenotype are poorly understood. We, therefore, compared the urinary proteome for different spirometry phenotypes in preterm-born children with preterm- and term-born controls. METHODS: Preterm and term-born children aged 7-12 years, from the Respiratory Health Outcomes in Neonates (RHiNO) cohort, underwent spirometry and urine collection. Urine was analysed by Nano-LC Mass-Spectrometry with Tandem-Mass Tag labelling. The preterm-born children were classified into phenotypes of prematurity-associated preserved ratio impaired spirometry (pPRISm, FEV1 < lower limit of normal (LLN), FEV1/FVC ≥ LLN), prematurity-associated obstructive lung disease (POLD, FEV1 < LLN, FEV1/FVC < LLN) and preterm controls (FEV1 ≥ LLN,). Biological relationships between significantly altered protein abundances were analysed using Ingenuity Pathways Analysis software, and receiver operator characteristic curves were calculated. RESULTS: Urine was analysed from 160 preterm-born children and 44 term controls. 27 and 21 were classified into the pPRISm and POLD groups, respectively. A total of 785 proteins were detected. Compared to preterm-born controls, sixteen significantly altered proteins in the pPRISm group were linked to six biological processes related to upregulation of inflammation and T-cell biology. In contrast, four significantly altered proteins in the POLD group were linked with neutrophil accumulation. Four proteins (DNASE1, PGLYRP1, B2M, SERPINA3) in combination had an area under the curve of 0.73 for pPRISm and three combined proteins (S100A8, MMP9 and CTSC) had AUC of 0.76 for POLD. CONCLUSIONS: In this exploratory study, we demonstrate differential associations of the urinary proteome with pPRISm and POLD. TRIAL REGISTRATION: EudraCT: 2015-003712-20.


Assuntos
Pneumopatias , Nascimento Prematuro , Doença Pulmonar Obstrutiva Crônica , Recém-Nascido , Humanos , Feminino , Proteoma , Volume Expiratório Forçado , Testes de Função Respiratória , Espirometria/métodos , Capacidade Vital/fisiologia , Pulmão
12.
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
13.
Am J Physiol Cell Physiol ; 322(4): C624-C644, 2022 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-35196163

RESUMO

The matricellular glycoprotein thrombospondin-1 (TSP1) has complex roles in the extracellular matrix (ECM) and at cell surfaces, but relatively little is known about its intracellular associations prior to secretion. To search for novel intracellular interactions of TSP1 in situ, we carried out a biotin ligase-based TSP1 interactome screen and identified protein disulfide isomerase A3 (PDIA3/ERp57) as a novel candidate binding protein. In validation, TSP1 and PDIA3 were established to bind in vitro and to colocalize in the endoplasmic reticulum of human dermal fibroblasts (HDF). Loss of PDIA3 function, either by pharmacological inhibition in HDF or in Pdia3-/- mouse embryo fibroblasts (Pdia3-/- MEFs), led to alterations in the composition of cell-derived extracellular matrix, involving changed abundance of fibronectin and TSP1, was correlated with reduced cell spreading, altered organization of F-actin, and reduced focal adhesions. These cellular phenotypes of Pdia3-/- MEFs were normalized by exposure to conditioned medium (WTCM) or extracellular matrix (WTECM) from wild-type (WT)-MEFs. Rescue depended on PDIA3 activity in WT-MEFs and was not prevented by immunodepletion of fibronectin. Heparin-binding proteins in WTCM were found to be necessary for rescue. Comparative quantitative tandem-mass-tag proteomics and functional assays on the heparin-binding secretomes of WT-MEFs and Pdia3-/- MEFs identified multiple ECM and growth factor proteins to be downregulated in the CM of Pdia3-/- MEFs. Of these, cell communication network 2 (CCN2) was identified to be necessary for the adhesion-promoting activity of WTCM on Pdia3-/- MEFs and to bind TSP1. Thus, PDIA3 coordinates fibroblast production of an ECM-rich, proadhesive microenvironment, with implications for PDIA3 as a translational target.


Assuntos
Fibronectinas , Isomerases de Dissulfetos de Proteínas , Animais , Comunicação Celular , Células Cultivadas , Fibroblastos/metabolismo , Fibronectinas/metabolismo , Heparina , Camundongos , Isomerases de Dissulfetos de Proteínas/genética , Isomerases de Dissulfetos de Proteínas/metabolismo , Secretoma
14.
Ann Hematol ; 101(10): 2179-2193, 2022 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-35941390

RESUMO

Patients within the WHO-subgroup of t(6;9)-positive acute myeloid leukemia (AML) differ from other AML subgroups as they are characterised by younger age and a grim prognosis. Leukemic transformation can often be attributed to single chromosomal aberrations encoding oncogenes, in the case of t(6;9)-AML to the fusion protein DEK-CAN (also called DEK-NUP214). As being a rare disease there is the urgent need for models of t(6;9)-AML. The only cell line derived from a t(6;9)-AML patient currently available is FKH1. By using phospho-proteomics on FKH1 cells, we found a strongly activated ABL1 kinase. Further investigation revealed the presence of ETV6-ABL1. This finding renders necessary to determine DEK-CAN- and ETV6-ABL1-related features when using FKH1. This can be done as ETV6-ABL1 activity in FKH1 is responsive to imatinib. Nevertheless, we provided evidence that both SFK and mTOR activation in FKH1 are DEK-CAN-related features as they were activated also in other t(6;9) and DEK-CAN-positive models. The activation of STAT5 previously shown to be strong in t(6;9)-AML and activated by DEK-CAN is regulated in FKH1 by both DEK-CAN and ETV6-ABL1. In conclusion, FKH1 cells still represent a model for t(6;9)-AML and could serve as model for ETV6-ABL1-positive AML if the presence of these leukemia-inducing oncogenes is adequately considered.Taken together, all our results provide clear evidence of novel and specific interdependencies between leukemia-inducing oncogenes and cancer signaling pathways which will influence the design of therapeutic strategies to better address the complexity of cancer signaling.


Assuntos
Leucemia Mieloide Aguda , Proteínas de Fusão Oncogênica , Proteínas Cromossômicas não Histona/genética , Humanos , Mesilato de Imatinib , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Proteínas Oncogênicas/genética , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Transdução de Sinais , Translocação Genética
15.
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
16.
Antimicrob Agents Chemother ; 65(11): e0100421, 2021 10 18.
Artigo em Inglês | MEDLINE | ID: mdl-34460299

RESUMO

Cefalexin is a widely used first-generation cephalosporin, and resistance in Escherichia coli is caused by extended-spectrum (e.g., CTX-M) and AmpC ß-lactamase production and therefore frequently coincides with third-generation cephalosporin resistance. However, we have recently identified large numbers of E. coli isolates from human infections, and from cattle, where cefalexin resistance is not ß-lactamase mediated. Here, we show, by studying laboratory-selected mutants, clinical isolates, and isolates from cattle, that OmpF porin disruption or downregulation is a major cause of cefalexin resistance in E. coli. Importantly, we identify multiple regulatory mutations that cause OmpF downregulation. In addition to mutation of ompR, already known to downregulate OmpF and OmpC porin production, we find that rseA mutation, which strongly activates the sigma E regulon, greatly increases DegP production, which degrades OmpF, OmpC, and OmpA. Furthermore, we reveal that mutations affecting lipopolysaccharide structure, exemplified by the loss of GmhB, essential for lipopolysaccharide heptosylation, also modestly activate DegP production, resulting in OmpF degradation. Remarkably, given the critical importance attached to such systems for normal E. coli physiology, we find evidence for DegP-mediated OmpF downregulation and gmhB and rseA loss-of-function mutation in E. coli isolates derived from human infections. Finally, we show that these regulatory mutations enhance the ability of group 1 CTX-M ß-lactamase to confer reduced carbapenem susceptibility, particularly those mutations that cause OmpC in addition to OmpF downregulation.


Assuntos
Proteínas da Membrana Bacteriana Externa , Cefalexina , Farmacorresistência Bacteriana/genética , Escherichia coli , Porinas/genética , Animais , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Carbapenêmicos , Bovinos , Cefalexina/farmacologia , Regulação para Baixo , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , beta-Lactamases/genética , beta-Lactamases/metabolismo
17.
Antimicrob Agents Chemother ; 65(8): e0241220, 2021 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-33972250

RESUMO

Meropenem is a clinically important antibacterial reserved for treatment of multiresistant infections. In meropenem-resistant bacteria of the family Enterobacterales, NDM-1 is considerably more common than IMP-1, despite both metallo-ß-lactamases (MBLs) hydrolyzing meropenem with almost identical kinetics. We show that blaNDM-1 consistently confers meropenem resistance in wild-type Enterobacterales, but blaIMP-1 does not. The reason is higher blaNDM-1 expression because of its stronger promoter. However, the cost of meropenem resistance is reduced fitness of blaNDM-1-positive Enterobacterales. In parallel, from a clinical case, we identified multiple Enterobacter spp. isolates carrying a plasmid-encoded blaNDM-1 having a modified promoter region. This modification lowered MBL production to a level associated with zero fitness cost, but, consequently, the isolates were not meropenem resistant. However, we identified a Klebsiella pneumoniae isolate from this same clinical case carrying the same blaNDM-1 plasmid. This isolate was meropenem resistant despite low-level NDM-1 production because of a ramR mutation reducing envelope permeability. Overall, therefore, we show how the resistance/fitness trade-off for MBL carriage can be resolved. The result is sporadic emergence of meropenem resistance in a clinical setting.


Assuntos
Microbioma Gastrointestinal , beta-Lactamases , Antibacterianos/farmacologia , Carbapenêmicos/farmacologia , Klebsiella pneumoniae , Testes de Sensibilidade Microbiana , beta-Lactamases/genética
18.
J Antimicrob Chemother ; 76(3): 587-595, 2021 02 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.


Assuntos
Acinetobacter baumannii , Proteínas de Bactérias , beta-Lactamases , Acinetobacter baumannii/genética , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Cromatografia Líquida , Farmacorresistência Bacteriana , Testes de Sensibilidade Microbiana , Espectrometria de Massas em Tandem , beta-Lactamases/genética
19.
Artigo em Inglês | MEDLINE | ID: mdl-33139281

RESUMO

Fluoroquinolone resistance in Stenotrophomonas maltophilia is multifactorial, 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 SmaRS). The sensor kinase Smlt2646 (SmaS) is activated by mutation in K M6 LEVr causing overproduction of two novel ABC transporters and the known aminoglycoside efflux pump SmeYZ. Overproduction of one ABC transporter, Smlt1651-4 (renamed SmaCDEF), causes levofloxacin resistance in K M6 LEVr Overproduction 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.


Assuntos
Stenotrophomonas maltophilia , Amicacina , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Farmacorresistência Bacteriana Múltipla/genética , Levofloxacino/farmacologia , Testes de Sensibilidade Microbiana , Stenotrophomonas maltophilia/genética
20.
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.


Assuntos
Colistina , Klebsiella pneumoniae , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Colistina/farmacologia , Farmacorresistência Bacteriana/genética , Klebsiella pneumoniae/genética , Testes de Sensibilidade Microbiana , Proteômica , Transdução de Sinais
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