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1.
Nat Chem Biol ; 15(5): 510-518, 2019 05.
Artículo en Inglés | MEDLINE | ID: mdl-30962626

RESUMEN

Protein phosphorylation regulates key processes in all organisms. In Gram-positive bacteria, protein arginine phosphorylation plays a central role in protein quality control by regulating transcription factors and marking aberrant proteins for degradation. Here, we report structural, biochemical, and in vivo data of the responsible kinase, McsB, the founding member of an arginine-specific class of protein kinases. McsB differs in structure and mechanism from protein kinases that act on serine, threonine, and tyrosine residues and instead has a catalytic domain related to that of phosphagen kinases (PhKs), metabolic enzymes that phosphorylate small guanidino compounds. In McsB, the PhK-like phosphotransferase domain is structurally adapted to target protein substrates and is accompanied by a novel phosphoarginine (pArg)-binding domain that allosterically controls protein kinase activity. The identification of distinct pArg reader domains in this study points to a remarkably complex signaling system, thus challenging simplistic views of bacterial protein phosphorylation.


Asunto(s)
Arginina/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Proteínas Quinasas/química , Proteínas Quinasas/metabolismo , Arginina/química , Modelos Moleculares , Fosforilación
2.
Elife ; 102021 07 30.
Artículo en Inglés | MEDLINE | ID: mdl-34328418

RESUMEN

In Gram-positive bacteria, the McsB protein arginine kinase is central to protein quality control, labeling aberrant molecules for degradation by the ClpCP protease. Despite its importance for stress response and pathogenicity, it is still elusive how the bacterial degradation labeling is regulated. Here, we delineate the mechanism how McsB targets aberrant proteins during stress conditions. Structural data reveal a self-compartmentalized kinase, in which the active sites are sequestered in a molecular cage. The 'closed' octamer interconverts with other oligomers in a phosphorylation-dependent manner and, unlike these 'open' forms, preferentially labels unfolded proteins. In vivo data show that heat-shock triggers accumulation of higher order oligomers, of which the octameric McsB is essential for surviving stress situations. The interconversion of open and closed oligomers represents a distinct regulatory mechanism of a degradation labeler, allowing the McsB kinase to adapt its potentially dangerous enzyme function to the needs of the bacterial cell.


Asunto(s)
Bacillus subtilis/genética , Proteínas Bacterianas/metabolismo , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Bacillus subtilis/enzimología , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Regulación Bacteriana de la Expresión Génica , Fosforilación , Proteínas Quinasas/química
3.
Nat Commun ; 12(1): 3132, 2021 05 25.
Artículo en Inglés | MEDLINE | ID: mdl-34035246

RESUMEN

The COVID-19 pandemic has demonstrated the need for massively-parallel, cost-effective tests monitoring viral spread. Here we present SARSeq, saliva analysis by RNA sequencing, a method to detect SARS-CoV-2 and other respiratory viruses on tens of thousands of samples in parallel. SARSeq relies on next generation sequencing of multiple amplicons generated in a multiplexed RT-PCR reaction. Two-dimensional, unique dual indexing, using four indices per sample, enables unambiguous and scalable assignment of reads to individual samples. We calibrate SARSeq on SARS-CoV-2 synthetic RNA, virions, and hundreds of human samples of various types. Robustness and sensitivity were virtually identical to quantitative RT-PCR. Double-blinded benchmarking to gold standard quantitative-RT-PCR performed by human diagnostics laboratories confirms this high sensitivity. SARSeq can be used to detect Influenza A and B viruses and human rhinovirus in parallel, and can be expanded for detection of other pathogens. Thus, SARSeq is ideally suited for differential diagnostic of infections during a pandemic.


Asunto(s)
Técnicas de Laboratorio Clínico , Ensayos Analíticos de Alto Rendimiento , Infecciones del Sistema Respiratorio/diagnóstico , Virus/aislamiento & purificación , COVID-19/diagnóstico , Diagnóstico Diferencial , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Reacción en Cadena de la Polimerasa , ARN Viral/genética , Infecciones del Sistema Respiratorio/virología , SARS-CoV-2/genética , SARS-CoV-2/aislamiento & purificación , Saliva/virología , Sensibilidad y Especificidad , Proteínas Virales/genética , Virus/clasificación , Virus/genética
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