Porcine reproductive and respiratory syndrome(PRRS) is characterized by reproductive failures in sows and respiratory diseases in pigs of all ages. PRRS virus(PRRSV) is its causative agent and has caused huge economic...Porcine reproductive and respiratory syndrome(PRRS) is characterized by reproductive failures in sows and respiratory diseases in pigs of all ages. PRRS virus(PRRSV) is its causative agent and has caused huge economic losses in the swine industry. Porcine sialoadhesin(pSn) is a putative receptor of PRRSV. Previous studies have shown that a pSn V-set Ig-like domain is signi ficant in PRRSV infection. However, its structural details are not fully known, hindering our deep understanding of PRRSV infection. In this study, we successfully cloned, expressed and puri fied the p Sn V-set Ig-like domain in Drosophila S2 cells. Then we tried to crystallize the target protein and predicted its structure. This will establish the foundation for the further structural study of p Sn, deepen our understanding of the invasion mechanism of PRRSV,and support the structural information for the development of clinical drugs and vaccines against PRRSV.展开更多
Myosin light chain kinases （MLCK） phosphorylate the regulatory light chain of myosin II in thick filaments and bind to F-actin-containing thin filaments with high affinity. The ability of short myosin light chain ki...Myosin light chain kinases （MLCK） phosphorylate the regulatory light chain of myosin II in thick filaments and bind to F-actin-containing thin filaments with high affinity. The ability of short myosin light chain kinase （S-MLCK） to bind F-actin is structurally attributed to the DFRXXL regions in its N-terminus. The long myosin light chain kinase （L-MLCK） has two additional DFRXXL motifs and six Ig-like modules in its N-terminal extension. The six Ig-like modules are capable of binding to stress fibers independently. Our results from the imaging analysis demonstrated that the first two intact Ig-like modules （2Ig） in N-terminal extension of L-MLCK is the minimal binding module required for microfilament binding. Binding assay confirmed that F-actin was able to bind 2Ig. Stoichiometries of 2Ig peptide were similar for myofilament or pure F-actin. The binding affinities were slightly lower than 5DFRXXL peptide as reported previously. Similar to DFRXXL peptides, the 2Ig peptide also caused efficient F-actin bundle formation in vitro. In the living cell, over-expression of 2Ig fragment increased ＂spike＂-like protrusion formation with over-bundled F-actin. Our results suggest that L-MLCK may act as a potent F-actin bundling protein via its DFRXXL region and the 2Ig region, implying that L-MLCK plays a role in cytoskeleton organization.展开更多
基金supported by the National Natural Science Foundation of China (31490601)the National Program on the Key Basic Research Project (2014CB542702)the Basic and Advanced Technology Research Program of Henan Province (162300410252)
文摘Porcine reproductive and respiratory syndrome(PRRS) is characterized by reproductive failures in sows and respiratory diseases in pigs of all ages. PRRS virus(PRRSV) is its causative agent and has caused huge economic losses in the swine industry. Porcine sialoadhesin(pSn) is a putative receptor of PRRSV. Previous studies have shown that a pSn V-set Ig-like domain is signi ficant in PRRSV infection. However, its structural details are not fully known, hindering our deep understanding of PRRSV infection. In this study, we successfully cloned, expressed and puri fied the p Sn V-set Ig-like domain in Drosophila S2 cells. Then we tried to crystallize the target protein and predicted its structure. This will establish the foundation for the further structural study of p Sn, deepen our understanding of the invasion mechanism of PRRSV,and support the structural information for the development of clinical drugs and vaccines against PRRSV.
基金This work was supported by National Naturcal Science Foundation of China （No. 30470852）The National Gongguan Project of China （21001BA710B）.
文摘Myosin light chain kinases （MLCK） phosphorylate the regulatory light chain of myosin II in thick filaments and bind to F-actin-containing thin filaments with high affinity. The ability of short myosin light chain kinase （S-MLCK） to bind F-actin is structurally attributed to the DFRXXL regions in its N-terminus. The long myosin light chain kinase （L-MLCK） has two additional DFRXXL motifs and six Ig-like modules in its N-terminal extension. The six Ig-like modules are capable of binding to stress fibers independently. Our results from the imaging analysis demonstrated that the first two intact Ig-like modules （2Ig） in N-terminal extension of L-MLCK is the minimal binding module required for microfilament binding. Binding assay confirmed that F-actin was able to bind 2Ig. Stoichiometries of 2Ig peptide were similar for myofilament or pure F-actin. The binding affinities were slightly lower than 5DFRXXL peptide as reported previously. Similar to DFRXXL peptides, the 2Ig peptide also caused efficient F-actin bundle formation in vitro. In the living cell, over-expression of 2Ig fragment increased ＂spike＂-like protrusion formation with over-bundled F-actin. Our results suggest that L-MLCK may act as a potent F-actin bundling protein via its DFRXXL region and the 2Ig region, implying that L-MLCK plays a role in cytoskeleton organization.
