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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.
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.

4.
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
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