Tuyển tập các báo cáo nghiên cứu về y học được đăng trên tạp chí y học Wertheim cung cấp cho các bạn kiến thức về ngành y đề tài: Alkylating HIV-1 Nef - a potential way of HIV intervention. | Jin et al. AIDS Research and Therapy 2010 7 26 http content 7 1 26 AIDS RESEARCH AND THERAPY RESEARCH Open Access Alkylating HIV-1 Nef - a potential way of HIV intervention Yong-Jiu Jin1 Xiaoping Zhang2 Catherine Yi Cai1 Steven J Burakoff1 3 Abstract Background Nef is a 27 KDa HIV-1 accessory protein. It downregulates CD4 from infected cell surface a mechanism critical for efficient viral replication and pathogenicity. Agents that antagonize the Nef-mediated CD4 downregulation may offer a new class of drug to combat HIV infection and disease. TPCK N-a-p-tosyl-L-phenylalanine chloromethyl ketone and TLCK N-a-p-tosyl-L-lysine chloromethyl ketone are alkylation reagents that chemically modify the side chain of His or Cys residues in a protein. In search of chemicals that inhibit Nef function we discovered that TPCK and TLCK alkylated HIV Nef. Methods Nef modification by TPCK was demonstrated on reducing SDS-PAGE. The specific cysteine residues modified were determined by site-directed mutagenesis and mass spectrometry MS . The effect of TPCK modification on Nef-CD4 interaction was studied using fluorescence titration of a synthetic CD4 tail peptide with recombinant Nef-His protein. The conformational change of Nef-His protein upon TPCK-modification was monitored using CD spectrometry Results Incubation of Nef-transfected T cells or recombinant Nef-His protein with TPCK resulted in mobility shift of Nef on SDS-PAGE. Mutagenesis analysis indicated that the modification occurred at Cys55 and Cys206 in Nef. Mass spectrometry demonstrated that the modification was a covalent attachment alkylation of TPCK at Cys55 and Cys206. Cys55 is next to the CD4 binding motif A56W57L58 in Nef required for Nef-mediated CD4 downregulation and for AIDS development. This implies that the addition of a bulky TPCK molecule to Nef at Cys55 would impair Nef function and reduce HIV pathogenicity. As expected Cys55 modification reduced the strength of the interaction .
