大肠杆菌（Escherichia coli）LexA蛋白的结构与功能

LexA蛋白首先在大肠杆菌（Escherichia coli）中作为SOS反应的重要调节因子之一被发现. LexA蛋白含有202个氨基酸,由N端DNA结合结构域和C端催化核心结构域构成. 细胞中LexA蛋白大都以二聚体形式存在,并且有可切割和不可切割两种构象. 在正常生理条件下,LexA特异性结合16 bp的保守序列5′-CTGTN8ACAG-3′,即SOS盒,抑制约50个基因的表达. 当发生DNA损伤时,活化的RecA蛋白通过稳定LexA蛋白可切割构象,促进LexA蛋白Ala84-Gly85间肽键的切割,产生的C端LexA85 202和N端LexA1 84被蛋白酶ClpXP和Lon快速降解. LexA蛋白切割后,SOS基因以一定的顺序开始表达,并且完成DNA损伤修复. 本文回顾和总结了LexA蛋白分子结构,自我切割分子机制和影响因素,以及在SOS反应中的作用等方面的研究进展. 同时,也讨论了LexA蛋白在原核细胞中的进化保守性.     

NLRP1, a regulator of innate immunity associated with vitiligo

Some familial forms of the dermatological condition vitiligo have recently been linked to polymorphisms in the innate immunity gene, NLRP1. Here, we review what is currently known about the mechanisms that regulate activation of the NLRP1 protein and the downstream effects of NLRP1 on pathways impacting inflammation and apoptosis. How polymorphic variants of the NLRP1 gene contribute to the pathogenesis of vitiligo remains mysterious, requiring further investigation.     

Liberation of SARS-CoV main protease from the viral polyprotein: N-terminal autocleavage does not depend on the mature dimerization mode

The main protease (M^pro) plays a vital role in proteolytic processing of the polyproteins in the replicative cycle of SARS coronavirus (SARS-CoV). Dimerization of this enzyme has been shown to be indispensable for trans-cleavage activity. However, the auto-processing mechanism of M^pro, i.e. its own release from the polyproteins through autocleavage, remains unclear. This study elucidates the relationship between the N-terminal autocleavage activity and the dimerization of “immature” M^pro. Three residues (Arg4, Glu290, and Arg298), which contribute to the active dimer conformation of mature M^pro, are selected for mutational analyses. Surprisingly, all three mutants still perform N-terminal autocleavage, while the dimerization of mature protease and trans-cleavage activity following auto-processing are completely inhibited by the E290R and R298E mutations and partially so by the R4E mutation. Furthermore, the mature E290R mutant can resume N-terminal autocleavage activity when mixed with the “immature” C145A/E290R double mutant whereas its trans-cleavage activity remains absent. Therefore, the N-terminal auto-processing of M^pro appears to require only two “immature” monomers approaching one another to form an “intermediate” dimer structure and does not strictly depend on the active dimer conformation existing in mature protease. In conclusion, an auto-release model of M^pro from the polyproteins is proposed, which will help understand the auto-processing mechanism and the difference between the autocleavage and trans-cleavage proteolytic activities of SARS-CoV M^pro.     

Study on substrate specificity at subsites for severe acute respiratory syndrome coronavirus 3CL protease

Autocleavage assay and peptide-based cleavage assay were used to study the substrate specificity of 3CL protease from the severe acute respiratory syndrome coronavirus. It was found that the recognition between the enzyme and its substrates involved many positions in the substrate, at least including residues from P4 to P2＇. The deletion of either P4 or P2＇ residue in the substrate would decrease its cleavage efficiency dramatically. In contrast to the previous suggestion that only small residues in substrate could be accommodated to the S 1＇ subsite, we have found that bulky residues such as Tyr and Trp were also acceptable. In addition, based on both peptide-based assay and autocleavage assay, Ile at the PI＇ position could not be hydrolyzed, but the mutant L27A could hydrolyze the Ile peptide fragment. It suggested that there was a stereo hindrance between the S 1＇ subsite and the side chain of Ile in the substrate. All 20 amino acids except Pro could be the residue at the P2＇ position in the substrate, but the cleavage efficiencies were clearly different. The specificity information of the enzyme is helpful for potent anti-virus inhibitor design and useful for other coronavirus studies.