Center for Translational Medicine, Xi'an Jiaotong University School of Life Science and Technology, Xi'an, China.
Department of Molecular and Translational Medicine, University of Brescia Medical School, Brescia, Italy.
Biochim Biophys Acta Gen Subj. 2019 Jan;1863(1):13-24. doi: 10.1016/j.bbagen.2018.09.016. Epub 2018 Sep 21.
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.
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.
在感染 HIV-1 的非霍奇金淋巴瘤（HIV-NHL）患者中检测到的 HIV-1 基质蛋白 p17 变体（vp17s）与野生型蛋白（refp17）不同，具有促进 B 细胞生长的活性。生物物理分析表明，vp17s 比 refp17 更不稳定，这促使我们探索结构-功能关系。
我们使用：生物物理技术（圆二色性（CD）、核磁共振（NMR）和热/胍盐酸变性）研究蛋白质构象和稳定性；表面等离子体共振（SPR）研究相互作用；Western blot 研究信号通路；集落形成和软琼脂测定研究 B 细胞增殖和克隆形成。
通过迫使 Cys 残基 57 和 87 之间形成二硫键，我们得到了一种不稳定的 p17，能够促进 B 细胞增殖。这一发现促使我们剖析 refp17 以确定功能表位。一个跨越从氨基酸（aa）2 到 21 的合成肽（F1）被发现能激活 Akt 并促进 B 细胞增殖和克隆形成。三个正电荷的 aa（Arg15、Lys18 和 Arg20）被证明对维持 F1 和 HIV-NHL 衍生的 vp17s 的增殖活性至关重要。F1 与已知的 refp17 受体没有任何相互作用，表明存在一种参与细胞增殖的替代受体。
与 refp17 相比，vp17s 的增殖活性的分子原因依赖于暴露一个能够激活 Akt 的功能表位。
我们的发现为鉴定负责 B 细胞增殖的受体铺平了道路，并为识别新的治疗策略提供了新的机会，以对抗与 HIV 相关的 NHL。
