Wangxiao He1, Pietro Mazzuca2, Weirong Yuan3, Kristen Varney4, Antonella Bugatti5, Alfredo Cagnotto6, Cinzia Giagulli7, Marco Rusnati8, Stefania Marsico9, Luisa Diomede10, Mario Salmona11, Arnaldo Caruso12, Wuyuan Lu13, Francesca Caccuri14. 1. Center for Translational Medicine, Xi'an Jiaotong University School of Life Science and Technology, Xi'an, China. Electronic address: hewangxiao5366@sina.com. 2. Department of Molecular and Translational Medicine, University of Brescia Medical School, Brescia, Italy. Electronic address: p.mazzuca@unibs.it. 3. Institute of Human Virology, University of Maryland School of Medicine, Baltimore, USA. Electronic address: wyuan@ihv.umaryland.edu. 4. Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, USA. Electronic address: kvarney@som.umaryland.edu. 5. Department of Molecular and Translational Medicine, University of Brescia Medical School, Brescia, Italy. Electronic address: antonella.bugatti@unibs.it. 6. IRCCS Istituto Ricerche Farmacologiche "Mario Negri", Milan, Italy. Electronic address: alfredo.cagnotto@marionegri.it. 7. Department of Molecular and Translational Medicine, University of Brescia Medical School, Brescia, Italy. Electronic address: cinzia.giagulli@unibs.it. 8. Department of Molecular and Translational Medicine, University of Brescia Medical School, Brescia, Italy. Electronic address: marco.rusnati@unibs.it. 9. Department of Pharmacy, Health and Nutritional Sciences, University of Calabria, Arcavacata di Rende, Italy. Electronic address: stefania.marsico@unical.it. 10. IRCCS Istituto Ricerche Farmacologiche "Mario Negri", Milan, Italy. Electronic address: luisa.diomede@marionegri.it. 11. IRCCS Istituto Ricerche Farmacologiche "Mario Negri", Milan, Italy. Electronic address: mario.salmona@marionegri.it. 12. Department of Molecular and Translational Medicine, University of Brescia Medical School, Brescia, Italy. Electronic address: arnaldo.caruso@unibs.it. 13. Center for Translational Medicine, Xi'an Jiaotong University School of Life Science and Technology, Xi'an, China; Institute of Human Virology, University of Maryland School of Medicine, Baltimore, USA; Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, USA. Electronic address: wlu@ihv.umaryland.edu. 14. Department of Molecular and Translational Medicine, University of Brescia Medical School, Brescia, Italy. Electronic address: francesca.caccuri@unibs.it.
Abstract
BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: The molecular reasons for the proliferative activity of vp17s, compared to refp17, relies on the exposure of a functional epitope capable of activating Akt. GENERAL SIGNIFICANCE: Our findings pave the way for identifying the receptor(s) responsible for B cell proliferation and offer new opportunities to identify novel treatment strategies in combating HIV-related NHL.
BACKGROUND:HIV-1 matrix protein p17 variants (vp17s) detected in HIV-1-infectedpatients 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: The molecular reasons for the proliferative activity of vp17s, compared to refp17, relies on the exposure of a functional epitope capable of activating Akt. GENERAL SIGNIFICANCE: Our findings pave the way for identifying the receptor(s) responsible for B cell proliferation and offer new opportunities to identify novel treatment strategies in combating HIV-related NHL.
Authors: Francesca Caccuri; Serena Messali; Alberto Zani; Giovanni Campisi; Marta Giovanetti; Stefania Zanussi; Emanuela Vaccher; Silvia Fabris; Antonella Bugatti; Emanuele Focà; Francesco Castelli; Massimo Ciccozzi; Riccardo Dolcetti; Robert C Gallo; Arnaldo Caruso Journal: Proc Natl Acad Sci U S A Date: 2022-06-28 Impact factor: 12.779
Authors: Francesca Caccuri; Vera Neves; Arnaldo Caruso; Miguel Castanho; Lurdes Gano; João D G Correia; Maria Cristina Oliveira; Pietro Mazzuca Journal: J Virol Date: 2021-10-20 Impact factor: 6.549
Authors: Francesca Caccuri; Pasqualina D'Ursi; Matteo Uggeri; Antonella Bugatti; Pietro Mazzuca; Alberto Zani; Federica Filippini; Mario Salmona; Domenico Ribatti; Mark Slevin; Alessandro Orro; Wuyuan Lu; Pietro Liò; Robert C Gallo; Arnaldo Caruso Journal: Proc Natl Acad Sci U S A Date: 2021-01-12 Impact factor: 11.205