Okuda M.,Yokohama City University |
Kinoshita M.,Biosignal Research Center |
Kinoshita M.,Kobe University |
Kakumu E.,Biosignal Research Center |
And 4 more authors.
In global genome repair (GGR), XPC detects damaged nucleotides and recruits TFIIH complex. The small acidic region of XPC binds to the pleckstrin homology (PH) domain of TFIIH subunit p62; however, the recognition mechanism remains elusive. Here, we use nuclear magnetic resonance to present the tertiary structure of XPC bound to the PH domain. The XPC acidic region forms a long string stabilized by insertion of Trp133 and Val136 into two separate hollows of the PH domain, coupled with extensive electrostatic contacts. Analysis of several XPC mutants revealed that particularly Trp133 is essential for binding to the PH domain. In cell lines stably expressing mutant XPC, alanine substitution at Trp133 or Trp133/Val136 compromised UV resistance, recruitment of TFIIH to DNA damage, and removal of UV-induced photoproducts from genomic DNA. These findings show how TFIIH complex is recruited by XPC to damaged DNA, advancing our understanding of the early stage of GGR. © 2015 Elsevier Ltd. All rights reserved. Source
Wood T.R.,Albany Medical College |
Chow R.Y.,Albany Medical College |
Hanes C.M.,Albany Medical College |
Zhang X.,Albany Medical College |
And 8 more authors.
Journal of Leukocyte Biology
In RAW 264.7 cells , PKC-ε regulates FcγR-mediated phagocytosis. BMDM behave similarly; PKC-ε concentrates at phagosomes and internalization are reduced in PKC-ε-/- cells. Two questions were asked: what is the role of PKC-ε? and what domains are necessary for PKC-ε concentration? Function was studied using BMDM and frustrated phagocytosis. On IgG surfaces, PKC-ε-/- macrophages spread less than WT. Patchclamping revealed that the spreading defect is a result of the failure of PKC-ε-/macrophages to add membrane. The defect is specific for FcγR ligation and can be reversed by expression of full-length (but not the isolated RD) PKC-ε in PKC-ε-/- BMDM. Thus, PKC-ε function in phagocytosis requires translocation to phagosomes and the catalytic domain. The expression of chimeric PKC molecules in RAW cells identified the εPS as necessary for PKC-ε targeting. When placed into (nonlocalizing) PKC-δ, εPS was sufficient for concentration, albeit to a lesser degree than intact PKC-ε. In contrast, translocation of δ(εPSC1B) resembled that of WT PKC-ε. Thus, εPS and εC1B cooperate for optimal phagosome targeting. Finally, cells expressing εK437W were significantly less phagocytic than their PKC-ε-expressing counterparts, blocked at the pseudopod-extension phase. In summary, we have shown that εPS and εC1B are necessary and sufficient for targeting PKC-ε to phagosomes, where its catalytic activity is required for membrane delivery and pseudopod extension.© Society for Leukocyte Biology. Source