title: The cellular isopeptidase T deubiquitinating enzyme regulates Kaposi's sarcoma-associated herpesvirus K7 degradation.
abstract: OBJECTIVE
  To understand the regulated degradation of KSHV K7.
  
  
  METHODS
  Proteomic screen and immunofluoresence microscopy identified that K7 recruits polyubiquitin chains to membrane fractions; IP and GST pulldown verified the interaction between K7 and Iso T1; Protein stability assay and RQ-PCR demonstrated Iso T1 facilitates K7 degradation.
  
  
  RESULTS
  The K7-containing membrane fraction contains a higher level of deubiquitinating (DUB) activity and K7 interacts with a cellular DUB, isopeptidase T1 (Iso T1). Mutational analyses revealed that the ubiquitin-associated domains of Iso T1 are necessary and sufficient to bind K7. Confocal microscopy and fractionation analyses indicated that K7 increases the membrane-associated Iso T1. Furthermore, the knockdown of IsoT1 by shRNA-mediated silencing greatly increased K7 ubiquitination even when proteasome activity was inhibited by lactacystin.
  
  
  CONCLUSIONS
  IsoT1 disassembles of free ubiquitin chains to facilitate K7 degradation.
authors.affiliation: Key Laboratory of Protein Chemistry and Developmental Biology of Ministry of Education of China, College of Life Science, Hunan Normal University, Changsha, China, 410081.
authors.forename: Jun
authors.initials: J
authors.lastname: Xiao
authors.forename: Hui
authors.initials: H
authors.lastname: Wu
authors.forename: Lingzhi
authors.initials: L
authors.lastname: Peng
authors.forename: Mengdie
authors.initials: M
authors.lastname: Chi
authors.forename: Hao
authors.initials: H
authors.lastname: Feng
mesh_terms: D002462:Cell Membrane / Q000201:enzymology*; D010450:Endopeptidases / Q000235:genetics / Q000378:metabolism*; D057809:HEK293 Cells; D006367:HeLa Cells; D019288:Herpesvirus 8, Human / Q000378:metabolism*; D006801:Humans; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D024101:Mitochondrial Proteins / Q000378:metabolism*; D009154:Mutation; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D025941:Protein Interaction Mapping; D011499:Protein Processing, Post-Translational; D055550:Protein Stability; D059748:Proteolysis; D040901:Proteomics / Q000379:methods; D034622:RNA Interference; D013997:Time Factors; D014162:Transfection; D064570:Ubiquitin-Specific Proteases / Q000235:genetics / Q000378:metabolism*; D014764:Viral Proteins / Q000378:metabolism*
chemical_list: C468524:K7 protein, Human herpesvirus 8; D024101:Mitochondrial Proteins; D014764:Viral Proteins; D010450:Endopeptidases; D064570:Ubiquitin-Specific Proteases; C091939:ubiquitin isopeptidase