Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 16 de 16
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
2.
Cell Rep ; 42(6): 112563, 2023 06 27.
Artigo em Inglês | MEDLINE | ID: mdl-37267104

RESUMO

It is challenging to apply traditional mutational scanning to voltage-gated sodium channels (NaVs) and functionally annotate the large number of coding variants in these genes. Using a cytosine base editor and a pooled viability assay, we screen a library of 368 guide RNAs (gRNAs) tiling NaV1.2 to identify more than 100 gRNAs that change NaV1.2 function. We sequence base edits made by a subset of these gRNAs to confirm specific variants that drive changes in channel function. Electrophysiological characterization of these channel variants validates the screen results and provides functional mechanisms of channel perturbation. Most of the changes caused by these gRNAs are classifiable as loss of function along with two missense mutations that lead to gain of function in NaV1.2 channels. This two-tiered strategy to functionally characterize ion channel protein variants at scale identifies a large set of loss-of-function mutations in NaV1.2.


Assuntos
Edição de Genes , Canal de Sódio Disparado por Voltagem NAV1.2 , Canais de Sódio Disparados por Voltagem , Edição de Genes/métodos , Mutagênese/genética , Mutação , Mutação de Sentido Incorreto/genética
3.
Cell Metab ; 35(5): 887-905.e11, 2023 05 02.
Artigo em Inglês | MEDLINE | ID: mdl-37075753

RESUMO

Cellular exposure to free fatty acids (FFAs) is implicated in the pathogenesis of obesity-associated diseases. However, there are no scalable approaches to comprehensively assess the diverse FFAs circulating in human plasma. Furthermore, assessing how FFA-mediated processes interact with genetic risk for disease remains elusive. Here, we report the design and implementation of fatty acid library for comprehensive ontologies (FALCON), an unbiased, scalable, and multimodal interrogation of 61 structurally diverse FFAs. We identified a subset of lipotoxic monounsaturated fatty acids associated with decreased membrane fluidity. Furthermore, we prioritized genes that reflect the combined effects of harmful FFA exposure and genetic risk for type 2 diabetes (T2D). We found that c-MAF-inducing protein (CMIP) protects cells from FFA exposure by modulating Akt signaling. In sum, FALCON empowers the study of fundamental FFA biology and offers an integrative approach to identify much needed targets for diverse diseases associated with disordered FFA metabolism.


Assuntos
Diabetes Mellitus Tipo 2 , Ácidos Graxos não Esterificados , Humanos , Ácidos Graxos não Esterificados/metabolismo , Ácidos Graxos , Transdução de Sinais , Biologia
4.
bioRxiv ; 2023 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-36865221

RESUMO

Cellular exposure to free fatty acids (FFA) is implicated in the pathogenesis of obesity-associated diseases. However, studies to date have assumed that a few select FFAs are representative of broad structural categories, and there are no scalable approaches to comprehensively assess the biological processes induced by exposure to diverse FFAs circulating in human plasma. Furthermore, assessing how these FFA- mediated processes interact with genetic risk for disease remains elusive. Here we report the design and implementation of FALCON (Fatty Acid Library for Comprehensive ONtologies) as an unbiased, scalable and multimodal interrogation of 61 structurally diverse FFAs. We identified a subset of lipotoxic monounsaturated fatty acids (MUFAs) with a distinct lipidomic profile associated with decreased membrane fluidity. Furthermore, we developed a new approach to prioritize genes that reflect the combined effects of exposure to harmful FFAs and genetic risk for type 2 diabetes (T2D). Importantly, we found that c-MAF inducing protein (CMIP) protects cells from exposure to FFAs by modulating Akt signaling and we validated the role of CMIP in human pancreatic beta cells. In sum, FALCON empowers the study of fundamental FFA biology and offers an integrative approach to identify much needed targets for diverse diseases associated with disordered FFA metabolism. Highlights: FALCON (Fatty Acid Library for Comprehensive ONtologies) enables multimodal profiling of 61 free fatty acids (FFAs) to reveal 5 FFA clusters with distinct biological effectsFALCON is applicable to many and diverse cell typesA subset of monounsaturated FAs (MUFAs) equally or more toxic than canonical lipotoxic saturated FAs (SFAs) leads to decreased membrane fluidityNew approach prioritizes genes that represent the combined effects of environmental (FFA) exposure and genetic risk for diseaseC-Maf inducing protein (CMIP) is identified as a suppressor of FFA-induced lipotoxicity via Akt-mediated signaling.

5.
Annu Rev Physiol ; 85: 407-423, 2023 02 10.
Artigo em Inglês | MEDLINE | ID: mdl-36763970

RESUMO

Numerous trafficking and quality control pathways evolved to handle the diversity of proteins made by eukaryotic cells. However, at every step along the biosynthetic pathway, there is the potential for quality control system failure. This review focuses on the mechanisms of disrupted proteostasis. Inspired by diseases caused by misfolded proteins in the kidney (mucin 1 and uromodulin), we outline the general principles of protein biosynthesis, delineate the recognition and degradation pathways targeting misfolded proteins, and discuss the role of cargo receptors in protein trafficking and lipid homeostasis. We also discuss technical approaches including live-cell fluorescent microscopy, chemical screens to elucidate trafficking mechanisms, multiplexed single-cell CRISPR screening platforms to systematically delineate mechanisms of proteostasis, and the advancement of novel tools to degrade secretory and membrane-associated proteins. By focusing on components of trafficking that go awry, we highlight ongoing efforts to understand fundamental mechanisms of disrupted proteostasis and implications for the treatment of human proteinopathies.


Assuntos
Rim , Humanos , Transporte Proteico , Transporte Biológico , Homeostase
6.
Front Med (Lausanne) ; 8: 721865, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34621762

RESUMO

Podocyte injury and the appearance of proteinuria are key features of several progressive kidney diseases. Genetic deletion or selective inhibition of TRPC5 channels with small-molecule inhibitors protects podocytes in rodent models of kidney disease, but less is known about the human relevance and translatability of TRPC5 inhibition. Here, we investigate the effect of TRPC5 inhibition in puromycin aminonucleoside (PAN)-treated rats, human iPSC-derived podocytes, and kidney organoids. We first established that systemic administration of the TRPC5 inhibitor AC1903 was sufficient to protect podocyte cytoskeletal proteins and suppress proteinuria in PAN-induced nephrosis rats, an established model of podocyte injury. TRPC5 current was recorded in the human iPSC-derived podocytes and was blocked by AC1903. PAN treatment caused podocyte injury in human iPSC-derived podocytes and kidney organoids. Inhibition of TRPC5 channels reversed the effects of PAN-induced injury in human podocytes in both 2D and 3D culture systems. Taken together, these results revealed the relevance of TRPC5 channel inhibition in puromycin-aminonucleoside induced nephrosis models, highlighting the potential of this therapeutic strategy for patients.

7.
Nature ; 593(7860): 607-611, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33883744

RESUMO

As organelles of the innate immune system, inflammasomes activate caspase-1 and other inflammatory caspases that cleave gasdermin D (GSDMD). Caspase-1 also cleaves inactive precursors of the interleukin (IL)-1 family to generate mature cytokines such as IL-1ß and IL-18. Cleaved GSDMD forms transmembrane pores to enable the release of IL-1 and to drive cell lysis through pyroptosis1-9. Here we report cryo-electron microscopy structures of the pore and the prepore of GSDMD. These structures reveal the different conformations of the two states, as well as extensive membrane-binding elements including a hydrophobic anchor and three positively charged patches. The GSDMD pore conduit is predominantly negatively charged. By contrast, IL-1 precursors have an acidic domain that is proteolytically removed by caspase-110. When permeabilized by GSDMD pores, unlysed liposomes release positively charged and neutral cargoes faster than negatively charged cargoes of similar sizes, and the pores favour the passage of IL-1ß and IL-18 over that of their precursors. Consistent with these findings, living-but not pyroptotic-macrophages preferentially release mature IL-1ß upon perforation by GSDMD. Mutation of the acidic residues of GSDMD compromises this preference, hindering intracellular retention of the precursor and secretion of the mature cytokine. The GSDMD pore therefore mediates IL-1 release by electrostatic filtering, which suggests the importance of charge in addition to size in the transport of cargoes across this large channel.


Assuntos
Inflamassomos/química , Interleucina-1beta/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/química , Macrófagos/metabolismo , Proteínas de Ligação a Fosfato/química , Animais , Caspase 1/metabolismo , Microscopia Crioeletrônica , Humanos , Interleucina-1/metabolismo , Camundongos Endogâmicos C57BL , Precursores de Proteínas/metabolismo , Estrutura Quaternária de Proteína , Eletricidade Estática
8.
Cell Rep Med ; 1(8): 100137, 2020 11 17.
Artigo em Inglês | MEDLINE | ID: mdl-33294858

RESUMO

Drug repurposing has the advantage of identifying potential treatments on a shortened timescale. In response to the pandemic spread of SARS-CoV-2, we took advantage of a high-content screen of 3,713 compounds at different stages of clinical development to identify FDA-approved compounds that reduce mucin-1 (MUC1) protein abundance. Elevated MUC1 levels predict the development of acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) and correlate with poor clinical outcomes. Our screen identifies fostamatinib (R788), an inhibitor of spleen tyrosine kinase (SYK) approved for the treatment of chronic immune thrombocytopenia, as a repurposing candidate for the treatment of ALI. In vivo, fostamatinib reduces MUC1 abundance in lung epithelial cells in a mouse model of ALI. In vitro, SYK inhibition by the active metabolite R406 promotes MUC1 removal from the cell surface. Our work suggests fostamatinib as a repurposing drug candidate for ALI.

9.
bioRxiv ; 2020 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-32637960

RESUMO

Drug repurposing is the only method capable of delivering treatments on the shortened time-scale required for patients afflicted with lung disease arising from SARS-CoV-2 infection. Mucin-1 (MUC1), a membrane-bound molecule expressed on the apical surfaces of most mucosal epithelial cells, is a biochemical marker whose elevated levels predict the development of acute lung injury (ALI) and respiratory distress syndrome (ARDS), and correlate with poor clinical outcomes. In response to the pandemic spread of SARS-CoV-2, we took advantage of a high content screen of 3,713 compounds at different stages of clinical development to identify FDA-approved compounds that reduce MUC1 protein abundance. Our screen identified Fostamatinib (R788), an inhibitor of spleen tyrosine kinase (SYK) approved for the treatment of chronic immune thrombocytopenia, as a repurposing candidate for the treatment of ALI. In vivo , Fostamatinib reduced MUC1 abundance in lung epithelial cells in a mouse model of ALI. In vitro , SYK inhibition by Fostamatinib promoted MUC1 removal from the cell surface. Our work reveals Fostamatinib as a repurposing drug candidate for ALI and provides the rationale for rapidly standing up clinical trials to test Fostamatinib efficacy in patients with COVID-19 lung injury.

10.
Trends Pharmacol Sci ; 40(12): 911-918, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31704171

RESUMO

Ion channels are critical to kidney function, and their dysregulation leads to several distinct kidney diseases. Of the diversity of ion channels in kidney cells, the transient receptor potential (TRP) superfamily of proteins plays important and varied roles in both maintaining homeostasis as well as in causing disease. Recent work showed that TRPC5 blockers could successfully protect critical components of the kidney filter both in vitro and in vivo, thus revealing TRPC5 as a tractable therapeutic target for focal and segmental glomerulosclerosis (FSGS), a common cause of kidney failure. Human genetics point to three additional TRP channels as plausible therapeutic targets: TRPC6 in FSGS, PKD2 in polycystic kidney disease, and TRPM6 in familial hypomagnesemia with secondary hypocalcemia (HSH). We conclude that targeting TRP channels could pave the way for much needed therapies for kidney diseases.


Assuntos
Nefropatias/tratamento farmacológico , Canais de Potencial de Receptor Transitório/antagonistas & inibidores , Animais , Humanos , Nefropatias/genética , Nefropatias/metabolismo , Terapia de Alvo Molecular , Canais de Cátion TRPC/antagonistas & inibidores , Canais de Cátion TRPC/metabolismo , Canal de Cátion TRPC6/antagonistas & inibidores , Canal de Cátion TRPC6/genética , Canais de Potencial de Receptor Transitório/genética , Proteínas rac1 de Ligação ao GTP/metabolismo
11.
Bioorg Med Chem Lett ; 29(2): 155-159, 2019 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-30538066

RESUMO

The transient receptor potential cation channel 5 (TRPC5) has been previously shown to affect podocyte survival in the kidney. As such, inhibitors of TRPC5 are interesting candidates for the treatment of chronic kidney disease (CKD). Herein, we report the synthesis and biological characterization of a series of N-heterocyclic-1-benzyl-1H-benzo[d]imidazole-2-amines as selective TRPC5 inhibitors. Work reported here evaluates the benzimidazole scaffold and substituents resulting in the discovery of AC1903, a TRPC5 inhibitor that is active in multiple animal models of CKD.


Assuntos
Aminas/farmacologia , Compostos Heterocíclicos/farmacologia , Imidazóis/farmacologia , Indazóis/farmacologia , Insuficiência Renal Crônica/tratamento farmacológico , Canais de Cátion TRPC/antagonistas & inibidores , Aminas/síntese química , Aminas/química , Animais , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Desenho de Fármacos , Compostos Heterocíclicos/síntese química , Compostos Heterocíclicos/química , Humanos , Imidazóis/síntese química , Imidazóis/química , Indazóis/síntese química , Indazóis/química , Estrutura Molecular , Insuficiência Renal Crônica/metabolismo , Relação Estrutura-Atividade , Canais de Cátion TRPC/síntese química , Canais de Cátion TRPC/química , Canais de Cátion TRPC/metabolismo , Canais de Cátion TRPC/farmacologia
13.
Proc Natl Acad Sci U S A ; 114(1): 154-159, 2017 01 03.
Artigo em Inglês | MEDLINE | ID: mdl-27994149

RESUMO

KCNQ2/3 (Kv7.2/7.3) channels and voltage-gated sodium channels (VGSCs) are enriched in the axon initial segment (AIS) where they bind to ankyrin-G and coregulate membrane potential in central nervous system neurons. The molecular mechanisms supporting coordinated regulation of KCNQ and VGSCs and the cellular mechanisms governing KCNQ trafficking to the AIS are incompletely understood. Here, we show that fibroblast growth factor 14 (FGF14), previously described as a VGSC regulator, also affects KCNQ function and localization. FGF14 knockdown leads to a reduction of KCNQ2 in the AIS and a reduction in whole-cell KCNQ currents. FGF14 positively regulates KCNQ2/3 channels in a simplified expression system. FGF14 interacts with KCNQ2 at a site distinct from the FGF14-VGSC interaction surface, thus enabling the bridging of NaV1.6 and KCNQ2. These data implicate FGF14 as an organizer of channel localization in the AIS and provide insight into the coordination of KCNQ and VGSC conductances in the regulation of membrane potential.


Assuntos
Segmento Inicial do Axônio/metabolismo , Fatores de Crescimento de Fibroblastos/metabolismo , Canal de Potássio KCNQ2/metabolismo , Canal de Potássio KCNQ3/metabolismo , Potenciais da Membrana/fisiologia , Anquirinas/metabolismo , Células Cultivadas , Fatores de Crescimento de Fibroblastos/genética , Regulação da Expressão Gênica/genética , Hipocampo/citologia , Hipocampo/metabolismo , Humanos , Canal de Sódio Disparado por Voltagem NAV1.6/metabolismo , Neurônios/metabolismo , Interferência de RNA , RNA Interferente Pequeno/genética , Canais de Sódio Disparados por Voltagem/metabolismo
14.
J Gen Physiol ; 149(1): 37-47, 2017 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-27999145

RESUMO

Mammalian cilia are ubiquitous appendages found on the apical surface of cells. Primary and motile cilia are distinct in both morphology and function. Most cells have a solitary primary cilium (9+0), which lacks the central microtubule doublet characteristic of motile cilia (9+2). The immotile primary cilia house unique signaling components and sequester several important transcription factors. In contrast, motile cilia commonly extend into the lumen of respiratory airways, fallopian tubes, and brain ventricles to move their contents and/or produce gradients. In this review, we focus on the composition of putative ion channels found in both types of cilia and in the periciliary membrane and discuss their proposed functions. Our discussion does not cover specialized cilia in photoreceptor or olfactory cells, which express many more ion channels.


Assuntos
Cílios/fisiologia , Canais Iônicos/fisiologia , Transdução de Sinais/fisiologia , Animais , Humanos , Ativação do Canal Iônico/fisiologia
15.
Proc Natl Acad Sci U S A ; 113(19): E2665-74, 2016 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-27044086

RESUMO

Clustering of voltage-gated sodium channels (VGSCs) within the neuronal axon initial segment (AIS) is critical for efficient action potential initiation. Although initially inserted into both somatodendritic and axonal membranes, VGSCs are concentrated within the axon through mechanisms that include preferential axonal targeting and selective somatodendritic endocytosis. How the endocytic machinery specifically targets somatic VGSCs is unknown. Here, using knockdown strategies, we show that noncanonical FGF13 binds directly to VGSCs in hippocampal neurons to limit their somatodendritic surface expression, although exerting little effect on VGSCs within the AIS. In contrast, homologous FGF14, which is highly concentrated in the proximal axon, binds directly to VGSCs to promote their axonal localization. Single-point mutations in FGF13 or FGF14 abrogating VGSC interaction in vitro cannot support these specific functions in neurons. Thus, our data show how the concerted actions of FGF13 and FGF14 regulate the polarized localization of VGSCs that supports efficient action potential initiation.


Assuntos
Potenciais de Ação , Canais de Sódio Disparados por Voltagem/metabolismo , Axônios/metabolismo , Humanos , Neurônios/metabolismo , Sódio/metabolismo , Canais de Sódio/genética
16.
N Biotechnol ; 28(5): 435-47, 2011 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-21477671

RESUMO

High-resolution structures of liganded and unliganded antibody molecules were analyzed in terms of the interaction between the antibody with ligand, between the residues in the contact between the variable domains, and between the framework and the complementarity-determining regions of the antibody. The solvent accessibilities of the residues in the variable domains were also analyzed. The structural information is useful in the engineering of antibodies for therapeutic and other purposes.


Assuntos
Anticorpos/química , Animais , Anticorpos/imunologia , Anticorpos/uso terapêutico , Cristalografia por Raios X , Humanos , Modelos Moleculares , Conformação Proteica , Engenharia de Proteínas
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...