RESUMEN
Mammalian SIRT6 is a well-studied histone deacetylase that was recently shown to exhibit high protein deacylation activity enabling the removal of long chain fatty acyl groups from proteins. SIRT6 was shown to play key roles in cellular homeostasis by regulating a variety of cellular processes including DNA repair and glucose metabolism. However, the link between SIRT6 enzymatic activities and its cellular functions is not clear. Here, we utilized a directed enzyme evolution approach to generate SIRT6 mutants with improved deacylation activity. We found that while two mutants show increased deacylation activity at high substrate concentration and improved glucose metabolism they exhibit no improvement and even abolished deacetylation activity on H3K9Ac and H3K56Ac in cells. Our results demonstrate the separation of function between SIRT6 catalytic activities and suggest that SIRT6 deacylation activity in cells is important for glucose metabolism and can be mediated by still unknown acylated cellular proteins.
Asunto(s)
Evolución Molecular Dirigida/métodos , Glucosa/metabolismo , Histonas/química , Ingeniería de Proteínas/métodos , Sirtuinas/química , Factor de Necrosis Tumoral alfa/química , Acilación , Animales , Sitios de Unión , Biocatálisis , Biblioteca de Genes , Células HEK293 , Histonas/genética , Histonas/metabolismo , Homeostasis/genética , Humanos , Hidrólisis , Cinética , Modelos Moleculares , Péptidos/química , Péptidos/metabolismo , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Sirtuinas/deficiencia , Sirtuinas/genética , Especificidad por Sustrato , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Upon heterologous overexpression, many proteins misfold or aggregate, thus resulting in low functional yields. Human acetylcholinesterase (hAChE), an enzyme mediating synaptic transmission, is a typical case of a human protein that necessitates mammalian systems to obtain functional expression. We developed a computational strategy and designed an AChE variant bearing 51 mutations that improved core packing, surface polarity, and backbone rigidity. This variant expressed at â¼2,000-fold higher levels in E. coli compared to wild-type hAChE and exhibited 20°C higher thermostability with no change in enzymatic properties or in the active-site configuration as determined by crystallography. To demonstrate broad utility, we similarly designed four other human and bacterial proteins. Testing at most three designs per protein, we obtained enhanced stability and/or higher yields of soluble and active protein in E. coli. Our algorithm requires only a 3D structure and several dozen sequences of naturally occurring homologs, and is available at http://pross.weizmann.ac.il.
Asunto(s)
Acetilcolinesterasa/metabolismo , Biología Computacional/métodos , Escherichia coli/enzimología , Ingeniería de Proteínas/métodos , Acetilcolinesterasa/química , Acetilcolinesterasa/genética , Algoritmos , Automatización de Laboratorios , Simulación por Computador , Diseño Asistido por Computadora , ADN (Citosina-5-)-Metiltransferasas/genética , ADN (Citosina-5-)-Metiltransferasas/metabolismo , ADN Metiltransferasa 3A , Escherichia coli/genética , Proteínas Ligadas a GPI/química , Proteínas Ligadas a GPI/genética , Proteínas Ligadas a GPI/metabolismo , Regulación Bacteriana de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Mutación , Hidrolasas de Triéster Fosfórico/genética , Hidrolasas de Triéster Fosfórico/metabolismo , Conformación Proteica , Desnaturalización Proteica , Estabilidad Proteica , Sirtuinas/genética , Sirtuinas/metabolismo , Relación Estructura-Actividad , TemperaturaRESUMEN
Proper functioning of the protein-folding quality control network depends on the network's ability to discern diverse structural perturbations to the native states of its protein substrates. Despite the centrality of the detection of misfolded states to cell home-ostasis, very little is known about the exact sequence and structural features that mark a protein as being misfolded. To investigate these features, we studied the requirements for the degradation of the yeast kinetochore protein Ndc10p. Mutant Ndc10p is a substrate of a protein-folding quality control pathway mediated by the E3 ubiquitin (Ub) ligase Doa10p at the endoplasmic reticulum (ER)/nuclear envelope membrane. Analysis of Ndc10p mutant derivatives, employing a reverse genetics approach, identified an autonomous quality control-associated degradation motif near the C-terminus of the protein. This motif is composed of two indispensable hydrophobic elements: a hydrophobic surface of an amphipathic helix and a loosely structured hydrophobic C-terminal tail. Site-specific point mutations expose these elements, triggering ubiquitin-mediated and HSP70 chaperone-dependent degradation of Ndc10p. These findings substantiate the ability of the ER quality control system to recognize subtle perturbation(s) in the native structure of a nuclear protein.
