Sivakolundu Sivashankar G, Nourse Amanda, Moshiach Simon, Bothner Brian, Ashley Chimere, Satumba John, Lahti Jill, Kriwacki Richard W
Department of Structural Biology, St. Jude Children's Research Hospital, 332 North Lauderdale Street, Memphis, TN 38105, USA.
J Mol Biol. 2008 Dec 5;384(1):240-54. doi: 10.1016/j.jmb.2008.09.019. Epub 2008 Sep 16.
Arf, Hdm2, and p53 regulate the tumor-suppressor pathway that is most frequently disrupted in human cancer. In the absence of tumorigenic stress, Hdm2 actively attenuates p53-dependent cell cycle arrest and apoptosis by mediating ubiquitination-dependent degradation of p53. Mitogenic stress activates Arf, which indirectly activates p53 by binding to and nullifying the anti-p53 activities of Hdm2. Small conserved domains within Arf and Hdm2 mediate their direct interaction. Individually, these domains are intrinsically unstructured and, when combined in vitro, cofold into bimolecular oligomeric structures that resemble amyloid fibrils in some features. Detailed structural characterization of Hdm2/Arf complexes has previously been hampered by their heterogeneity and large size. Here, we report that a nine-residue fragment of the N-terminus of mouse Arf (termed "A1-mini") cofolds specifically with the Arf-binding domain of Hdm2 to form bimolecular oligomers. We characterized these unprecedented structures using analytical ultracentrifugation and NMR spectroscopy, providing insights into their structural organization. The A1-mini peptide not only binds specifically to Hdm2 in vitro but also recapitulates the nucleolar localization features of full-length Arf in cells. Furthermore, larger fragments of Arf that contain the A1-mini segment have previously been shown to activate p53 in mouse and human cells. Our studies provide the first insights into the molecular basis through which Arf nullifies the p53-inhibiting activity of Hdm2, indirectly activating the tumor-suppressor function of p53 in mammalian cells.
Arf、Hdm2和p53调节在人类癌症中最常被破坏的肿瘤抑制途径。在没有致瘤应激的情况下,Hdm2通过介导p53的泛素化依赖性降解来积极减弱p53依赖性细胞周期停滞和凋亡。有丝分裂应激激活Arf,Arf通过结合并消除Hdm2的抗p53活性来间接激活p53。Arf和Hdm2内的小保守结构域介导它们的直接相互作用。这些结构域单独来看本质上是无结构的,在体外组合时会共同折叠成在某些特征上类似于淀粉样原纤维的双分子寡聚结构。Hdm2/Arf复合物的详细结构表征此前因它们的异质性和大尺寸而受阻。在这里,我们报告小鼠Arf N端的一个九残基片段(称为“A1-迷你”)与Hdm2的Arf结合结构域特异性共同折叠形成双分子寡聚体。我们使用分析超速离心和核磁共振光谱对这些前所未有的结构进行了表征,深入了解了它们的结构组织。A1-迷你肽不仅在体外与Hdm2特异性结合,还重现了全长Arf在细胞中的核仁定位特征。此外,包含A1-迷你片段的Arf更大片段此前已被证明能在小鼠和人类细胞中激活p53。我们的研究首次深入了解了Arf消除Hdm2对p53抑制活性从而间接激活哺乳动物细胞中p53肿瘤抑制功能的分子基础。