Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 4 de 4
Filtrar
Mais filtros










Base de dados
Tipo de estudo
Intervalo de ano de publicação
1.
Mol Biol Evol ; 40(3)2023 03 04.
Artigo em Inglês | MEDLINE | ID: mdl-36798991

RESUMO

Mutations can have deleterious fitness effects when they decrease protein specific activity or decrease active protein abundance. Mutations will also be deleterious when they cause misfolding or misinteractions that are toxic to the cell (i.e., independent of whether the mutations affect specific activity and abundance). The extent to which protein evolution is shaped by these and other collateral fitness effects is unclear in part because little is known of their frequency and magnitude. Using deep mutational scanning (DMS), we previously found at least 42% of missense mutations in the TEM-1 ß-lactamase antibiotic resistance gene cause deleterious collateral fitness effects. Here, we used DMS to comprehensively determine the collateral fitness effects of missense mutations in three genes encoding the antibiotic resistance proteins New Delhi metallo-ß-lactamase (NDM-1), chloramphenicol acetyltransferase I (CAT-I), and 2″-aminoglycoside nucleotidyltransferase (AadB). AadB (20%), CAT-I (0.9%), and NDM-1 (0.2%) were less susceptible to deleterious collateral fitness effects than TEM-1 (42%) indicating that genes have different propensities for these effects. As was observed with TEM-1, all the studied deleterious aadB mutants increased aggregation. However, aggregation did not correlate with collateral fitness effects for many of the deleterious mutants of CAT-I and NDM-1. Select deleterious mutants caused unexpected phenotypes to emerge. The introduction of internal start codons in CAT-1 caused loss of the episome and a mutation in aadB made its cognate antibiotic essential for growth. Our study illustrates how the complexity of the cell provides a rich environment for collateral fitness effects and new phenotypes to emerge.


Assuntos
Mutação de Sentido Incorreto , beta-Lactamases , Mutação , beta-Lactamases/genética , Antibacterianos/farmacologia , Proteínas/genética , Resistência Microbiana a Medicamentos
2.
Methods Enzymol ; 643: 203-224, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32896282

RESUMO

Knowledge of the distribution of fitness effects (DFE) of mutations is critical to the understanding of protein evolution. Here, we describe methods for large-scale, systematic measurements of the DFE using growth competition and deep mutational scanning. We discuss techniques for producing comprehensive libraries of gene variants as well as provide necessary considerations for designing these experiments. Using these methods, we have constructed libraries containing over 18,000 variants, measured fitness effects of these mutations by deep mutational scanning, and verified the presence of fitness effects in individual variants. Our methods provide a high-throughput protocol for measuring biological fitness effects of mutations and the dependence of fitness effects on the environment.


Assuntos
Modelos Genéticos , Mutação
3.
Proc Natl Acad Sci U S A ; 117(21): 11597-11607, 2020 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-32385156

RESUMO

The distribution of fitness effects of mutation plays a central role in constraining protein evolution. The underlying mechanisms by which mutations lead to fitness effects are typically attributed to changes in protein specific activity or abundance. Here, we reveal the importance of a mutation's collateral fitness effects, which we define as effects that do not derive from changes in the protein's ability to perform its physiological function. We comprehensively measured the collateral fitness effects of missense mutations in the Escherichia coli TEM-1 ß-lactamase antibiotic resistance gene using growth competition experiments in the absence of antibiotic. At least 42% of missense mutations in TEM-1 were deleterious, indicating that for some proteins collateral fitness effects occur as frequently as effects on protein activity and abundance. Deleterious mutations caused improper posttranslational processing, incorrect disulfide-bond formation, protein aggregation, changes in gene expression, and pleiotropic effects on cell phenotype. Deleterious collateral fitness effects occurred more frequently in TEM-1 than deleterious effects on antibiotic resistance in environments with low concentrations of the antibiotic. The surprising prevalence of deleterious collateral fitness effects suggests they may play a role in constraining protein evolution, particularly for highly expressed proteins, for proteins under intermittent selection for their physiological function, and for proteins whose contribution to fitness is buffered against deleterious effects on protein activity and protein abundance.


Assuntos
Evolução Molecular , Aptidão Genética/genética , Mutação de Sentido Incorreto/genética , Mutação de Sentido Incorreto/fisiologia , Escherichia coli/enzimologia , Escherichia coli/genética , Escherichia coli/fisiologia , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , beta-Lactamases/química , beta-Lactamases/genética , beta-Lactamases/metabolismo
4.
J Biol Chem ; 292(27): 11485-11498, 2017 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-28487367

RESUMO

Cystatin C (CysC) is a versatile and ubiquitously-expressed member of the cysteine protease inhibitor family that is present at notably high concentrations in cerebrospinal fluid. Under mildly denaturing conditions, CysC forms inactive domain-swapped dimers. A destabilizing mutation, L68Q, increases the rate of domain-swapping and causes a fatal amyloid disease, hereditary cystatin C amyloid angiopathy. Wild-type (wt) CysC will also aggregate into amyloid fibrils under some conditions. Propagated domain-swapping has been proposed as the mechanism by which CysC fibrils grow. We present evidence that a CysC mutant, V57N, stabilized against domain-swapping, readily forms fibrils, contradicting the propagated domain-swapping hypothesis. Furthermore, in physiological buffer, wt CysC can form oligomers without undergoing domain-swapping. These non-swapped oligomers are identical in secondary structure to CysC monomers and completely retain protease inhibitory activity. However, unlike monomers or dimers, the oligomers bind fluorescent dyes that indicate they have characteristics of pre-amyloid aggregates. Although these oligomers appear to be a pre-amyloid assembly, they are slower than CysC monomers to form fibrils. Fibrillation of CysC therefore likely initiates from the monomer and does not require domain-swapping. The non-swapped oligomers likely represent a dead-end offshoot of the amyloid pathway and must dissociate to monomers prior to rearranging to amyloid fibrils. These prefibrillar CysC oligomers were potent inhibitors of aggregation of the Alzheimer's-related peptide, ß-amyloid. This result illustrates an example where heterotypic interactions between pre-amyloid oligomers prevent the homotypic interactions that would lead to mature amyloid fibrils.


Assuntos
Peptídeos beta-Amiloides/antagonistas & inibidores , Peptídeos beta-Amiloides/química , Cistatina C/química , Mutação de Sentido Incorreto , Multimerização Proteica , Substituição de Aminoácidos , Peptídeos beta-Amiloides/genética , Peptídeos beta-Amiloides/metabolismo , Cistatina C/genética , Cistatina C/metabolismo , Humanos
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...