Protein Structure, Stability, and Amyloid Formation

Ras与癌症：gtp依赖的K-Ras二聚体，钙调素在KRAS驱动的腺癌中的作用，致癌KRAS在G1细胞周期解除调控中引发癌症的关键作用，以及Ras亚型在癌症中的新观点。我们发现了gtp依赖性K-Ras二聚体的两个主要界面（GTP-dependent K-Ras二聚体）。Muratcioglu S，等。结构23(7):1325- 35,2015)。首先，高密度的β -片二聚体界面位于Switch I和效应器结合区，重叠Raf's, PI3K's, RalGDS‘ ’和其他效应器结合表面。这个界面必须对这些效应物有抑制作用。第二，螺旋界面也重叠了一些效应器的结合位点。该接口可能促进Raf的激活。我们的数据揭示了Ras自我结合如何调节效应物的结合和活性，并表明药物破坏螺旋二聚体界面可能会减弱癌症中Raf的信号传导。我们指出了钙调素在kras驱动腺癌中被忽视的关键作用（the Key role of calmodulin in kras驱动腺癌）。Muratcioglu S，等。癌症杂志，2013(9):1265- 73,2015)。钙调素（CaM）选择性结合gtp结合的K-Ras4B；但不是同工异构体。细胞的增殖和生长需要MAPK （Ras/Raf/MEK/ERK）和PI3Kalpha/Akt通路。我们提出Ca2+/CaM促进PI3K/Akt信号传导，并提出其机制。Ca2+/CaM的参与可以解释令人费解的观察结果，如腺癌中钙水平升高。我们假设CaM招募并帮助激活膜上的PI3K，这可能是腺癌中Ca2+/CaM依赖的原因。CaM可以通过PI3Kalpha/Akt和Raf/MEK/ERK途径促进导管（胰腺、结直肠癌、肺癌）的发生/进展。在K-Ras4B/CaM/PI3Kalpha三聚体中阻断K-Ras/MAPK通路和CaM/PI3Kalpha结合可能是一种有希望的腺癌特异性治疗策略。我们进一步阐明了通过G1细胞周期的解除调控，致癌KRAS在癌症起始中的关键作用（K-Ras效应物组织原理和通过G1细胞周期解除调控，致癌K-Ras在癌症起始中的作用）。Nussinov R等。专家蛋白质组学50(6):669- 82,2015)。我们还提出了Ras亚型在癌症中的新观点（a new view of Ras isoforms in cancer）。Nussinov R等。巨蟹座，2016年1月1日；76(1):18-23。我们提出小GTPase K-Ras4A具有单一或两种状态，一种类似于K-Ras4B，另一种类似于N-Ras。最近对K-Ras4A的一项研究发现，即使在没有棕榈酰的情况下，K-Ras4A也能在质膜上活跃。重要的是，这表明K-Ras4A可能存在于两种不同的信号状态。在状态1中，K-Ras4A和K-Ras4B一样只有法酰化；状态2是法酰化和棕榈酰化，就像N-Ras一样。K-Ras4A高变区（HVR）序列带正电，位于K-Ras4B和N-Ras之间。综上所述，这提出了一种可能性，即像K-Ras4B一样，法酰化但非棕榈酰化的状态1与钙调素结合，并与结直肠癌和其他腺癌（如肺癌和PDAC）相关。另一方面，状态2可能与黑色素瘤和其他N-Ras是主要因素的癌症有关，如急性髓性白血病（AML）。重要的是，H-Ras具有两种单棕榈酰化和双棕榈酰化状态，它们也可能具有不同的功能作用。棕榈酰化Ras异构体的多重信号状态质疑了GTPase Ras小异构体统计在不同癌症类型中的完整性，并呼吁对概念和方案进行重新评估。他们也可能要求重新考虑致癌的Ras疗法。此外，我们讨论了Ras与膜的相互作用，这是其激活所必需的，以及KRas上的致癌突变如何影响其行为(例如GTP结合和致癌突变可能减弱小GTPase KRAS4B中的高变区(HVR)-催化结构域相互作用，暴露效应结合位点，Lu S等。[J] .中国生物医学工程学报，2015(4):387 - 391。张海，等。J Biol Chem. 2015)等。我们的工作得益于我们与实验组的合作，包括结构组、核磁共振和晶体学。我们很幸运能有这些杰出的合作。我们进一步扩展了这项工作，研究Raf的半胱氨酸富结构域（CRD）在Raf-Ras相互作用中的作用，并在膜上探索致癌Ras亚型的信号特异性，以及PI3K脂质激酶的激活机制，尽管它在癌症中起着重要的作用，但迄今为止仍然是一个谜。PI3K脂质激酶在PI3K/Akt/mTOR通路中将PIP2磷酸化为PIP3，从而调节细胞过程。它们在癌症中经常发生突变。我们在原子水平上确定了PI3Kalpha的活化机制。与底物毗邻ATP的蛋白激酶不同，晶体结构表明，在PI3Kalpha中，ATP的γ磷酸盐与PIP2脂质底物之间的距离超过6埃，对于磷酸化转移来说太远了，这就提出了催化是如何执行的问题。PI3Kalpha有两个亚基，催化p110 α和调节p85 α。我们的模拟表明，释放p85 α的nSH2结构域施加的自抑制会触发p110 α的显着构象变化，导致激酶结构域暴露于膜相互作用。激酶结构域c叶的结构重排减少了ATP -磷酸和底物之间的距离，这就解释了磷酸基转移是如何进行的。另一种机制可能涉及ATP的重新定位。这一机制不仅解释了致癌突变如何通过促进nSH2释放或nSH2释放诱导的变构运动来促进PI3Kalpha活化；它还提供了一种创新的，PI3K亚型特异性药物发现原理。该机制表明，阻断ATP结合袋和底物结合位点之间的PI3Kalpha序列特异性空腔，而不是与ATP结合袋中的纳摩尔范围的ATP竞争，也不是与ATP结合袋中的ATP守恒和大量结合靶点竞争。靶向空腔中异构体特异性残基可能阻止PIP2磷酸化。目前，在与校内和校外研究者的合作中，我们也致力于揭示Raf的自身抑制作用。

Ras and cancer: GTP-dependent K-Ras dimers, the role of calmodulin in KRAS-driven adenocarcinomas, the critical role of oncogenic KRAS in the initiation of cancer through deregulation of the G1 cell cycle, and a new view of Ras isoforms in cancers. We discovered the two major interfaces of GTP-dependent K-Ras dimers (GTP-Dependent K-Ras Dimerization. Muratcioglu S, et al. Structure 23(7): 1325-35, 2015). The first, highly populated beta-sheet dimer interface is at the Switch I and effector binding regions, overlapping Raf's, PI3K's, RalGDS' and additional effectors' binding surfaces. This interface has to be inhibitory to such effectors. The second, helical interface also overlaps some effectors' binding sites. This interface may promote Raf's activation. Our data reveal how Ras self-association can regulate effector binding and activity, and suggest that disruption of the helical dimer interface by drugs may abate Raf's signaling in cancer. We pointed out the overlooked critical role of calmodulin in KRAS-driven adenocarcinomas (The Key Role of Calmodulin in KRAS-Driven Adenocarcinomas. Muratcioglu S, et al. Mol Cancer Res. 13(9): 1265-73, 2015). Calmodulin (CaM) selectively binds to GTP-bound K-Ras4B; but not to its isoforms. Cell proliferation and growth require the MAPK (Ras/Raf/MEK/ERK) and PI3Kalpha/Akt pathways. We proposed that Ca2+/CaM promote PI3K/Akt signaling, and suggest how. Ca2+/CaM involvement may explain puzzling observations like the elevated calcium levels in adenocarcinomas. We hypothesized that CaM recruits and helps activate PI3K at the membrane, and that this is the likely reason for Ca2+/CaM-dependence in adenocarcinomas. CaM can contribute to initiation/progression of ductal (pancreatic, colorectal, lung) cancers via both PI3Kalpha/Akt and Raf/MEK/ERK pathways. Blocking the K-Ras/MAPK pathway and CaM/PI3Kalpha binding in a K-Ras4B/CaM/PI3Kalpha trimer could be a promising adenocarcinoma-specific therapeutic strategy. We further illustrated the critical role of oncogenic KRAS in the initiation of cancer through deregulation of the G1 cell cycle (Principles of K-Ras effector organization and the role of oncogenic K-Ras in cancer initiation through G1 cell cycle deregulation. Nussinov R, et al. Expert Rev Proteomics 50(6): 669-82, 2015). We also proposed a new view of Ras isoforms in cancers (A New View of Ras Isoforms in Cancers. Nussinov R, et al. Cancer Res. 2016 Jan 1;76(1):18-23). We proposed that small GTPase K-Ras4A have a single state or two states, one resembling K-Ras4B and the other N-Ras. A recent study of K-Ras4A made the remarkable observation that even in the absence of the palmitoyl K-Ras4A can be active at the plasma membrane. Importantly, this suggests that K-Ras4A may exist in two distinct signaling states. In state 1 K-Ras4A is only farnesylated, like K-Ras4B; in state 2 farnesylated and palmitoylated, like N-Ras. The K-Ras4A hypervariable region (HVR) sequence is positively charged, in-between K-Ras4B and N-Ras. Taken together, this raises the possibility that the farnesylated but nonpalmitoylated state 1, like K-Ras4B, binds calmodulin and is associated with colorectal and other adenocarcinomas like lung cancer and PDAC (pancreatic ductal adenocarcinoma). On the other hand, state 2 may be associated with melanoma and other cancers where N-Ras is a major contributor, such as acute myeloid leukemia (AML). Importantly, H-Ras has two - single and double - palmitoylated states that may also serve distinct functional roles. The multiple signaling states of palmitoylated Ras isoforms question the completeness of small GTPase Ras isoform statistics in different cancer types and call for reevaluation of concepts and protocols. They may also call for reconsideration of oncogenic Ras therapeutics. Additionally, we addressed the interaction of Ras with the membrane which is required for its activation and how oncogenic mutations on KRas would affect its behavior (e.g. GTP Binding and Oncogenic Mutations May Attenuate Hypervariable Region (HVR)-Catalytic Domain Interactions in Small GTPase KRAS4B, Exposing the Effector Binding Site, Lu S, et al. J Biol Chem. 290(48): 28887-900, 2015) and Mechanisms of Membrane Binding of Small GTPase K-Ras4B Farnesylated Hypervariable Region. Jang H, et al. J Biol Chem. 2015) and more. Our work benefits from our collaborations with experimental groups, including structural groups, NMR and crystallography. We are fortunate to have these outstanding collaborations. We have further extended this work to work out the role of the cysteine-rich domain (CRD) of Raf in Raf-Ras interaction, and have been exploring signaling specificity of oncogenic Ras isoforms at the membrane, and the mechanism of activation of PI3K lipid kinase, which despite its important role in cancer, to date has still been a mystery. PI3K lipid kinases phosphorylate PIP2 to PIP3 in the PI3K/Akt/mTOR pathway to regulate cellular processes. They are frequently mutated in cancer. We determined the PI3Kalpha activation mechanism at the atomic level. Unlike protein kinases where the substrate abuts the ATP, crystal structures indicate that in PI3Kalpha, the distance between the gamma phosphate of the ATP and the PIP2 lipid substrate is over 6 Angstrom, much too far for the phosphoryl transfer, raising the question of how catalysis is executed. PI3Kalpha has two subunits, the catalytic p110alpha and the regulatory p85alpha. Our simulations show that release of the autoinhibition exerted by the nSH2 domain of the p85alpha triggers significant conformational change in p110alpha, leading to the exposure of the kinase domain for membrane interaction. Structural rearrangement in the C-lobe of the kinase domain reduces the distance between the ATP gamma-phosphate and the substrate, offering an explanation as to how phosphoryl transfer is executed. An alternative mechanism may involve ATP relocation. This mechanism not only explains how oncogenic mutations promote PI3Kalpha activation by facilitating nSH2 release, or nSH2-release-induced, allosteric motions; it also offers an innovative, PI3K isoform-specific drug discovery principle. Rather than competing with nanomolar range ATP in the ATP-binding pocket and contending with ATP pocket conservation and massive binding targets, this mechanism suggests blocking the PI3Kalpha sequence-specific cavity between the ATP-binding pocket and the substrate binding site. Targeting isoform-specific residues in the cavity may prevent PIP2 phosphorylation. Currently, in collaboration with intramural and extramural investigators, we also aim to uncover Raf's autoinhibition.