Huang Ming-Bo, Tiwari Purushottam B, Üren Aykut, Shelton Martin N, Brena Dara, Wu Jennifer Y, Khan Mahfuz B, Powell Michael D, Stiles Jonathan K, Johnson Erica L, Yan Fengxia, Yang Lily, Bond Vincent C
Department of Microbiology, Biochemistry, and Immunology, Morehouse School of Medicine, Atlanta, GA 30310, USA.
Department of Oncology, Georgetown University Medical Center, Lombardi Comprehensive Cancer Center, Washington, DC 20057, USA.
Int J Mol Sci. 2025 Sep 11;26(18):8848. doi: 10.3390/ijms26188848.
Breast cancer (BC) is a major cause of cancer-related mortality. Mortalin and Vimentin-two proteins implicated in BC progression and metastasis-have been identified as binding partners of the Secretion Modification Region (SMR) peptide from the HIV Nef protein. These interactions disrupt exosome release and offer novel therapeutic strategies. This study investigates the binding interactions between the SMR peptide, Mortalin, and Vimentin using surface plasmon resonance (SPR), co-immunoprecipitation (Co-IP), and Western blot assays. We also map the SMR binding sites on Mortalin through scanning peptide mapping and then identify a similar site on the Vimentin protein. Based on these data, we propose that the SMR peptide and its analogs interact with specific amino acid sequences in Mortalin and Vimentin, thereby disrupting cellular processes essential for Epithelial-Mesenchymal Transition (EMT) and tumor progression. SPR analysis revealed that the Nef protein exhibited the highest binding affinity to Vimentin (KD = 0.75 ± 1.1 nM) and Mortalin (KD = 3.16 ± 0.03 nM). The SMRwt peptide also demonstrated direct binding to both proteins with micromolar affinities (KD = 6.63 ± 0.74 µM for Vimentin; KD = 20.73 ± 2.33 µM for Mortalin), though the binding affinity was weaker than the full Nef protein. Co-IP experiments using MDA-MB-231, MCF-7, and BT474 BC cell lines confirmed that SMRwt, but not SMRmut, co-immunoprecipitated with Mortalin. Western blot analysis validated these interactions. Further, Mortalin peptide #56, derived from the substrate-binding domain, did not bind the SMR domain or inhibit Nef function. In contrast, peptides #61 and #62 from the C-terminal domain of Mortalin bound the SMR domain and effectively inhibited Nef activity. Notably, Mortalin peptide #61 inhibited SMRwt binding to both Mortalin and Vimentin, disrupting complex formation on the SPR sensor chip. These findings suggest that specific Mortalin-derived peptides can block SMR interactions, offering a potential therapeutic mechanism.
乳腺癌（BC）是癌症相关死亡的主要原因。Mortalin和波形蛋白这两种与BC进展和转移有关的蛋白质，已被确定为HIV Nef蛋白分泌修饰区（SMR）肽的结合伙伴。这些相互作用会破坏外泌体的释放，并提供了新的治疗策略。本研究使用表面等离子体共振（SPR）、免疫共沉淀（Co-IP）和蛋白质印迹分析，研究了SMR肽、Mortalin和波形蛋白之间的结合相互作用。我们还通过扫描肽图谱绘制了Mortalin上的SMR结合位点，然后在波形蛋白上确定了一个类似的位点。基于这些数据，我们提出SMR肽及其类似物与Mortalin和波形蛋白中的特定氨基酸序列相互作用，从而破坏上皮-间质转化（EMT）和肿瘤进展所必需的细胞过程。SPR分析显示，Nef蛋白对波形蛋白（KD = 0.75 ± 1.1 nM）和Mortalin（KD = 3.16 ± 0.03 nM）表现出最高的结合亲和力。SMRwt肽也以微摩尔亲和力与这两种蛋白直接结合（波形蛋白的KD = 6.63 ± 0.74 µM；Mortalin的KD = 20.73 ± 2.33 µM），尽管其结合亲和力比完整的Nef蛋白弱。使用MDA-MB-231、MCF-7和BT474 BC细胞系进行的Co-IP实验证实，SMRwt与Mortalin发生免疫共沉淀，而SMRmut则不然。蛋白质印迹分析验证了这些相互作用。此外，源自底物结合域的Mortalin肽#56不与SMR域结合或抑制Nef功能。相反，来自Mortalin C末端域的肽#61和#62与SMR域结合并有效抑制Nef活性。值得注意的是，Mortalin肽#61抑制SMRwt与Mortalin和波形蛋白的结合，破坏了SPR传感器芯片上的复合物形成。这些发现表明，特定的源自Mortalin的肽可以阻断SMR相互作用，并提供了一种潜在的治疗机制。
