Title
Identification of amino acid residues critical for the B cell growth-promoting activity of HIV-1 matrix protein p17 variants
Author
Wangxiao He, Pietro Mazzuca, Weirong Yuan, Kristen Varney, Antonella Bugatti, Alfredo Cagnotto, Cinzia Giagulli, Marco Rusnati, Stefania Marsico, Luisa Diomede, Mario Salmon, Arnaldo Caruso, Wuyuan Lu, Francesca Caccuri
Year
2019
Journal
Biochimica et Biophysica Acta (BBA) - General Subjects
Abstract
HIV-1 matrix protein p17 variants (vp17s) detected in HIV-1-infected patients with non-Hodgkin's lymphoma (HIV-NHL) display, differently from the wild-type protein (refp17), B cell growth-promoting activity. Biophysical analysis revealed that vp17s are destabilized as compared to refp17, motivating us to explore structure-function relationships. We used: biophysical techniques (circular dichroism (CD), nuclear magnetic resonance (NMR) and thermal/GuHCL denaturation) to study protein conformation and stability; Surface plasmon resonance (SPR) to study interactions; Western blot to investigate signaling pathways; and Colony Formation and Soft Agar assays to study B cell proliferation and clonogenicity. By forcing the formation of a disulfide bridge between Cys residues at positions 57 and 87 we obtained a destabilized p17 capable of promoting B cell proliferation. This finding prompted us to dissect refp17 to identify the functional epitope. A synthetic peptide (F1) spanning from amino acid (aa) 2 to 21 was found to activate Akt and promote B cell proliferation and clonogenicity. Three positively charged aa (Arg15, Lys18 and Arg20) proved critical for sustaining the proliferative activity of both F1 and HIV-NHL-derived vp17s. Lack of any interaction of F1 with the known refp17 receptors suggests an alternate one involved in cell proliferation. The molecular reasons for the proliferative activity of vp17s, compared to refp17, relies on the exposure of a functional epitope capable of activating Akt.
Instrument
J-810
Keywords
Circular dichroism, Secondary structure, Thermal stability, Protein folding, Protein denaturation, Chemical stability, Biochemistry