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1.
Protein Expr Purif ; 96: 32-8, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24508568

RESUMO

TREM-2 (triggering receptor expressed on myeloid cells-2) is an innate immune receptor expressed on dendritic cells, macrophages, osteoclasts, and microglia. Recent genetic studies have reported the occurrence of point mutations in TREM-2 that correlate with a dramatically increased risk for the development of neurodegenerative diseases, including Alzheimer's disease, frontotemporal dementia, and Parkinson's disease. Structural and biophysical studies of wild-type and mutant TREM-2 ectodomains are required to understand the functional consequences of these mutations. In order to facilitate these studies, we undertook the production and crystallization of these proteins. Here we demonstrate that, unlike many single Ig domain proteins, TREM-2 could not be readily refolded from bacterially-expressed inclusion bodies. Instead, we developed a mammalian-cell based expression system for the successful production of wild-type and mutant TREM-2 proteins in milligram quantities and a single-chromatography-step purification scheme that produced diffraction-quality crystals. These crystals diffract to a resolution of 3.3 Å and produce data sufficient for structure determination. We describe herein the procedures to produce wild-type and mutant human TREM-2 Ig domains in sufficient quantities for structural and biophysical studies. Such studies are crucial to understand the functional consequences of TREM-2 point mutations linked to the development of neurodegenerative diseases and, ultimately, to develop patient-specific molecular therapies to treat them.


Assuntos
Inflamação/patologia , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/ultraestrutura , Doenças Neurodegenerativas/patologia , Receptores Imunológicos/genética , Receptores Imunológicos/ultraestrutura , Clonagem Molecular , Cristalografia por Raios X , Expressão Gênica , Humanos , Glicoproteínas de Membrana/biossíntese , Mutação , Dobramento de Proteína , Estrutura Terciária de Proteína , Receptores Imunológicos/biossíntese
2.
J Biol Chem ; 287(50): 42138-49, 2012 Dec 07.
Artigo em Inglês | MEDLINE | ID: mdl-23112050

RESUMO

The chloride channel calcium-activated (CLCA) family are secreted proteins that regulate both chloride transport and mucin expression, thus controlling the production of mucus in respiratory and other systems. Accordingly, human CLCA1 is a critical mediator of hypersecretory lung diseases, such as asthma, chronic obstructive pulmonary disease, and cystic fibrosis, that manifest mucus obstruction. Despite relevance to homeostasis and disease, the mechanism of CLCA1 function remains largely undefined. We address this void by showing that CLCA proteins contain a consensus proteolytic cleavage site recognized by a novel zincin metalloprotease domain located within the N terminus of CLCA itself. CLCA1 mutations that inhibit self-cleavage prevent activation of calcium-activated chloride channel (CaCC)-mediated chloride transport. CaCC activation requires cleavage to unmask the N-terminal fragment of CLCA1, which can independently gate CaCCs. Gating of CaCCs mediated by CLCA1 does not appear to involve proteolytic cleavage of the channel because a mutant N-terminal fragment deficient in proteolytic activity is able to induce currents comparable with that of the native fragment. These data provide both a mechanistic basis for CLCA1 self-cleavage and a novel mechanism for regulation of chloride channel activity specific to the mucosal interface.


Assuntos
Canais de Cloreto/metabolismo , Ativação do Canal Iônico/fisiologia , Metaloproteases/metabolismo , Proteólise , Linhagem Celular , Canais de Cloreto/genética , Humanos , Transporte de Íons/fisiologia , Metaloproteases/genética , Estrutura Terciária de Proteína
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