RESUMEN
The domestic Bactrian camels were treated as one of the principal means of locomotion between the eastern and western cultures in history. However, whether they originated from East Asia or Central Asia remains elusive. To address this question, we perform whole-genome sequencing of 128 camels across Asia. The extant wild and domestic Bactrian camels show remarkable genetic divergence, as they were split from dromedaries. The wild Bactrian camels also contribute little to the ancestry of domestic ones, although they share close habitat in East Asia. Interestingly, among the domestic Bactrian camels, those from Iran exhibit the largest genetic distance and the earliest split from all others in the phylogeny, despite evident admixture between domestic Bactrian camels and dromedaries living in Central Asia. Taken together, our study support the Central Asian origin of domestic Bactrian camels, which were then immigrated eastward to Mongolia where native wild Bactrian camels inhabit.
Asunto(s)
Camelus/clasificación , Camelus/genética , Genoma , Genómica , Migración Animal , Animales , Asia , Evolución Molecular , Variación Genética , Genética de Población , Genómica/métodos , Filogenia , Polimorfismo de Nucleótido Simple , Secuenciación Completa del GenomaRESUMEN
Here we describe a general method to synthesize and use a panel of reagents with selectivity for deubiquitinating enzymes exhibiting specificity for each ubiquitin-like protein. A substrate (Ubl-AMC), a reversible inhibitor (Ubl-aldehyde), and an active-site-directed irreversible inhibitor (Ubl-vinylsulfone) are described for each Ubl. Because of space constraints, we give details for only the Nedd8 derivatives, but these methods have been used in our laboratory to produce these derivatives of Ubiquitin, Nedd8, Sumo-1, Sumo-2, and Isg15. These reagents are useful in defining the specificity of DUBs, as well as in purifying and identifying these important proteins. The reagents are selective and useful in crude extracts. The reactivity of these reagents reveals differences in both the S1 and S1' sites of deubiquitinating enzymes. Only active enzymes are efficiently detected with these reagents. Published results indicate that specificity is not strictly defined by the evolutionary relationships of these DUBs.
Asunto(s)
Inteínas , Ubiquitina/química , Secuencia de Bases , Catálisis , Línea Celular , Cromatografía Líquida de Alta Presión , Cartilla de ADN , Hidrólisis , Proteína NEDD8 , Empalme del ARN , Especificidad por Sustrato , Ubiquitina/metabolismo , Ubiquitinas/metabolismoRESUMEN
Free propeptides are known to function as inhibitors of the parental mature cysteine cathepsins. This general rule, however, does not apply to the aminopeptidase cathepsin H. Screening of propeptide fragments for their inhibitory potency revealed no significant effect on the native mature cathepsin H. On the other hand, inhibitory interaction was established with recombinant cathepsin H that displays endopeptidase activity due to a lack of the mini-chain. This finding suggests that the propeptide-binding region is structurally rearranged during maturation processing and mini-chain formation, which impairs the effective recognition of mature cathepsin H by its own propeptide.
Asunto(s)
Catepsinas/metabolismo , Cisteína Endopeptidasas/metabolismo , Precursores Enzimáticos/metabolismo , Secuencia de Aminoácidos , Catepsina H , Catepsinas/antagonistas & inhibidores , Catepsinas/química , Dicroismo Circular , Cisteína Endopeptidasas/química , Activación Enzimática , Precursores Enzimáticos/química , Modelos Moleculares , Datos de Secuencia Molecular , Péptidos/química , Péptidos/metabolismo , Estructura Terciaria de ProteínaRESUMEN
To identify deneddylases, proteases with specificity for hydrolysis of Nedd8 derivatives, a facile method was developed for the synthesis of Nedd8 amidomethylcoumarin (a substrate) and Nedd8 vinyl sulfone (an inhibitor). Deneddylase activity is necessary to reverse the conjugation of Nedd8 to cullin, a modification that regulates at least some ubiquitin ligases. The reaction of Nedd8 vinyl sulfone with L-M(TK-) mouse fibroblast lysates identified two deneddylases. The deubiquitinating enzyme UCH-L3 is labeled by both ubiquitin vinyl sulfone and Nedd8 vinyl sulfone. In contrast, a second and more selective enzyme is labeled only by Nedd8 vinyl sulfone. This protein, DEN1, is a 221-amino acid thiol protease that is encoded by an open reading frame previously annotated as SENP8. Recombinant human DEN1 shows significant specificity for Nedd8 and catalyzes the hydrolysis of Nedd8 amidomethylcoumarin with a Km of 51 nm and a kcat of7s-1. The catalytic efficiency of DEN1 acting upon ubiquitin amidomethylcoumarin is 6 x 10-4 that of Nedd8 amidomethylcoumarin and its activity on SUMO-1 amidomethylcoumarin is undetectable. This selectivity was unexpected as DEN1 is most closely related to enzymes that catalyze desumoylation. This observation expands to four the number of DUB families with members that can process the C terminus of Nedd8.
Asunto(s)
Endopeptidasas/análisis , Endopeptidasas/metabolismo , Ubiquitinas/metabolismo , Secuencia de Aminoácidos , Animales , Catálisis , Cumarinas/química , Endopeptidasas/química , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , Fibroblastos/enzimología , Expresión Génica , Humanos , Ratones , Datos de Secuencia Molecular , Proteína NEDD8 , Oligopéptidos , Fragmentos de Péptidos/metabolismo , Péptidos/genética , Proteínas Recombinantes de Fusión , Proteínas Recombinantes/metabolismo , Proteína SUMO-1/metabolismo , Especificidad por Sustrato , Sulfonas/química , Tioléster Hidrolasas/metabolismo , Transfección , Ubiquitina , Ubiquitina Tiolesterasa , Ubiquitinas/química , Ubiquitinas/genéticaRESUMEN
Nedd8 activates ubiquitination by increasing the efficiency of polyubiquitin chain assembly through its covalent conjugation to cullin molecules. Here we report the isolation, cloning, and characterization of a novel human Nedd8-specific protease called DEN1. Human DEN1 is encoded by AAH31411.1, a previously uncharacterized protein of 212 amino acids that shares homology with the Ulp1 cysteinyl SUMO deconjugating enzyme family. Recombinant human DEN1, purified from bacteria, selectively binds to Nedd8 and hydrolyzes C-terminal derivatives of Nedd8. Interestingly, DEN1 deconjugates cullin 1 (CUL1)-Nedd8 in a concentration-dependent manner. At a low concentration, DEN1 processes hyper-neddylated CUL1 to yield a mononeddylated form, which presumably contains the Lys-720CUL1-Nedd8 linkage. At elevated concentrations, DEN1 is able to complete the removal of Nedd8 from CUL1. These activities distinguish DEN1 from the COP9 signalosome, which is capable of efficiently cleaving the Lys-720CUL1-Nedd8 conjugate, but lacks Nedd8 C-terminal hydrolytic activity and poorly processes hyperneddylated CUL1. These results suggest a unique role for DEN1 in regulating the modification of cullins by Nedd8.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proteínas Cullin , Endopeptidasas/metabolismo , Ubiquitinas/metabolismo , Secuencia de Aminoácidos , Electroforesis en Gel de Poliacrilamida , Endopeptidasas/química , Endopeptidasas/genética , Células HeLa , Humanos , Datos de Secuencia Molecular , Proteína NEDD8 , Proteínas Recombinantes/metabolismo , Ubiquitina/metabolismoRESUMEN
The ubiquitin (Ub)-proteasome system includes a large family of deubiquitinating enzymes (DUBs). Many members are assigned to this enzyme class by sequence similarity but without evidence for biological activity. A panel of novel DUB-specific probes was generated by a chemical ligation method. These probes allowed identification of DUBs and associated components by tandem mass spectrometry, as well as rapid demonstration of enzymatic activity for gene products whose functions were inferred from primary structure. We identified 23 active DUBs in EL4 cells, including the tumor suppressor CYLD1. At least two DUBs tightly interact with the proteasome 19S regulatory complex. An OTU domain-containing protein, with no sequence homology to any known DUBs, was isolated. We show that this polypeptide reacts with the C terminus of Ub, thus demonstrating DUB-like enzymatic activity for this novel superfamily of proteases.