基于荧光偏振的高灵敏度蛋白激酶活性分析

利用TiO₂纳米棒与磷酸化肽的特异性相互作用,基于荧光偏振检测,建立了快速、简便的高灵敏度蛋白激酶活性分析方法. 在蛋白激酶催化作用下,荧光标记的肽底物被磷酸化,磷酸化的荧光肽通过磷酸基团特异性结合在TiO₂纳米棒表面,从而使底物肽上标记的荧光分子的旋转速率发生改变,通过对荧光偏振度进行测量,可实现蛋白激酶活性的定量检测,该方法对蛋白激酶A(PKA)的检出限可达0.0004 U·μL^-1. 此外,该方法还成功用于PKA抑制剂H-89的检测,在基于蛋白激酶抑制剂的靶向药物筛选方面具有很好的应用前景.     

Cytoprotective Role of Mitogen-activated Protein Kinase Phosphatase-1 in Light-damaged Human Retinal Pigment Epithelial Cells

The role of the mitogen-activated protein (MAP) kinase phosphatases (MKPs) in light-damaged cells is unclear. Therefore we investigated the involvement of MKP-1 in the regulation of apoptosis and cell survival mediated by MAP kinase pathways in light-damaged human retinal pigment epithelial cells (ARPE-19). Light dose-dependent changes in the expression of MKP-1 and in the phosphorylation status of the MAP kinases, c-Jun-N-terminal kinase (JNK) and p38 were demonstrated. Low light doses up to 2 J cm⁻² led to an upregulation of MKP-1 which resulted in the prevention of cell death by inactivating JNK kinase. However, higher light doses (≥3 J cm⁻²) significantly reduced MKP-1 protein expression and subsequently led to an increased JNK kinase activity followed by a significant increase in cell death. JNK kinase inactivation by the JNK inhibitor SP600125 significantly reduced light-induced cell death, suggesting that the cytoprotective properties of MKP-1 are mediated mainly by the JNK MAP kinase pathway. Physiological concentrations of ascorbic acid or taurine were seen to prevent apoptosis and cell death in light-damaged ARPE-19 cells by reducing oxidative stress within cells, thus maintaining MKP-1 at high levels, leading to an inactivation of the JNK kinase pathway which resulted in an increased cell viability.     

Phosphorylation of Thr9 Affects the Folding Landscape of the N-Terminal Segment of Human AGT Enhancing Protein Aggregation of Disease-Causing Mutants

The mutations G170R and I244T are the most common disease cause in primary hyperoxaluria type I (PH1). These mutations cause the misfolding of the AGT protein in the minor allele AGT-LM that contains the P11L polymorphism, which may affect the folding of the N-terminal segment (NTT-AGT). The NTT-AGT is phosphorylated at T9, although the role of this event in PH1 is unknown. In this work, phosphorylation of T9 was mimicked by introducing the T9E mutation in the NTT-AGT peptide and the full-length protein. The NTT-AGT conformational landscape was studied by circular dichroism, NMR, and statistical mechanical methods. Functional and stability effects on the full-length AGT protein were characterized by spectroscopic methods. The T9E and P11L mutations together reshaped the conformational landscape of the isolated NTT-AGT peptide by stabilizing ordered conformations. In the context of the full-length AGT protein, the T9E mutation had no effect on the overall AGT function or conformation, but enhanced aggregation of the minor allele (LM) protein and synergized with the mutations G170R and I244T. Our findings indicate that phosphorylation of T9 may affect the conformation of the NTT-AGT and synergize with PH1-causing mutations to promote aggregation in a genotype-specific manner. Phosphorylation should be considered a novel regulatory mechanism in PH1 pathogenesis.     

Mimicking Tyrosine Phosphorylation in Human Cytochrome c by the Evolved tRNA Synthetase Technique

Phosphorylation of tyrosine 48 of cytochrome c is related to a wide range of human diseases due to the pleiotropic role of the heme‐protein in cell life and death. However, the structural conformation and physicochemical properties of phosphorylated cytochrome c are difficult to study as its yield from cell extracts is very low and its kinase remains unknown. Herein, we report a high‐yielding synthesis of a close mimic of phosphorylated cytochrome c, developed by optimization of the synthesis of the non‐canonical amino acid p‐carboxymethyl‐L ‐phenylalanine (pCMF) and its efficient site‐specific incorporation at position 48. It is noteworthy that the Y48pCMF mutation significantly destabilizes the Fe?Met bond in the ferric form of cytochrome c, thereby lowering the pK_a value for the alkaline transition of the heme‐protein. This finding reveals the differential ability of the phosphomimic protein to drive certain events. This modified cytochrome c might be an important tool to investigate the role of the natural protein following phosphorylation.     

3D-QSAR Study on the Inhibitory Activity of Flavonoids on PIM-1 Kinase

20 Typical flavonoids were selected for study on the interaction between them and PIM-1 kinase with the comparative molecular field analysis method(CoMFA) as well as the comparative molecular similarity index analysis method(CoMSIA) based on molecule docking.3D-QSAR models between these flavonoids and receptor PIM-1 kinase were established.The obtained optimal cross-validation correlation coefficient Q2 for CoMFA model was 0.582,and the non-cross-validation correlation coefficient R2 was 0.955;the corresponding values for CoMSIA model were 0.790 and 0.974,respectively.These two models showed fairly fine stability and predictive ability.In addition,molecule docking results revealed the key residues in the receptor cavity and their specific action ways with flavonoids.     

Determination of glucan phosphorylation using heteronuclear 1H,13C double and 1H,13C,31P triple‐resonance NMR spectra

Phosphorylation and dephosphorylation of starch and glycogen are important for their physicochemical properties and also their physiological functions. It is therefore desirable to reliably determine the phosphorylation sites. Heteronuclear multidimensional NMR‐spectroscopy is in principle a straightforward analytical approach even for complex carbohydrate molecules. With heterogeneous samples from natural sources, however, the task becomes more difficult because a full assignment of the resonances of the carbohydrates is impossible to obtain. Here, we show that the combination of heteronuclear ¹H,¹³C and ¹H,¹³C,³¹P techniques and information derived from spectra of a set of reference compounds can lead to an unambiguous determination of the phosphorylation sites even in heterogeneous samples. Copyright © 2013 John Wiley & Sons, Ltd.     

Structural Biology of Cisplatin Complexes with Cellular Targets: The Adduct with Human Copper Chaperone Atox1 in Aqueous Solution

Cisplatin is one of the most used anticancer drugs. Its cellular influx and delivery to target DNA may involve the copper chaperone Atox1 protein. Although the mode of binding is established by NMR spectroscopy measurements in solution—the Pt atom binds to Cys12 and Cys15 while retaining the two ammine groups—the structural determinants of the adduct are not known. Here a structural model by hybrid Car–Parrinello density functional theory‐based QM/MM simulations is provided. The platinated site minimally modifies the fold of the protein. The calculated NMR and CD spectral properties are fully consistent with the experimental data. Our in silico/in vitro approach provides, together with previous studies, an unprecedented view into the structural biology of cisplatin–protein adducts.     

Inhibitory Effect of Lithospermic Acid on the HIV-1 Nucleocapsid Protein

The HIV-1 nucleocapsid protein (NC) is a desirable target in antiretroviral therapy due to its high conservation among HIV-1 strains, and to its multiple and crucial roles in the HIV-1 replication cycle. Natural products represent a valuable source of NC inhibitors, with the catechol group being a privileged scaffold in NC inhibition. By coupling molecular modeling with NMR spectroscopy and fluorescence-based assays, we disclosed lithospermic acid, a catechol derivative extracted from Salvia miltiorrhizza, as a potent and chemically stable non-covalent inhibitor of the NC. Being different from other catechol derivative reported so far, lithospermic acid does not undergo spontaneous oxidation in physiological conditions, thus becoming a profitable starting point for the development of efficient NC inhibitors.     

糖尿病和硝化条件下胰岛素受体及底物酪氨酸磷酸化的研究

利用新颖的定量核磁共振(³¹P NMR)法和免疫印迹法研究了四氧嘧啶诱导的糖尿病状态下以及酪氨酸经过氧亚硝酸根供体SIN-1硝化条件下大鼠肝脏胰岛素受体(IR)的自磷酸化和受体底物1(IRS1)的磷酸化。结果表明,四氧嘧啶诱导的糖尿病大鼠肝脏中IR自磷酸化水平削弱了,硝化对大鼠肝脏中IR自磷酸化的影响依赖于SIN-1浓度,根据IRS1磷酸化位点基序设计的多肽的硝化完全抑制了其磷酸化,提示酪氨酸硝化可能干扰胰岛素磷酸化信号通路。     

Conformational Dynamics in the Core of Human Y145Stop Prion Protein Amyloid Probed by Relaxation Dispersion NMR

Microsecond to millisecond timescale backbone dynamics of the amyloid core residues in Y145Stop human prion protein (PrP) fibrils were investigated by using ¹⁵N rotating frame (R_1ρ) relaxation dispersion solid-state nuclear magnetic resonance spectroscopy over a wide range of spin-lock fields. Numerical simulations enabled the experimental relaxation dispersion profiles for most of the fibril core residues to be modelled by using a two-state exchange process with a common exchange rate of 1000 s⁻¹, corresponding to protein backbone motion on the timescale of 1 ms, and an excited-state population of 2 %. We also found that the relaxation dispersion profiles for several amino acids positioned near the edges of the most structured regions of the amyloid core were better modelled by assuming somewhat higher excited-state populations (∼5–15 %) and faster exchange rate constants, corresponding to protein backbone motions on the timescale of ∼100–300 μs. The slow backbone dynamics of the core residues were evaluated in the context of the structural model of human Y145Stop PrP amyloid.     

Molecular Structure of Amyloid Fibrils Formed by Residues 127 to 147 of the Human Prion Protein

Amyloid fibrils are filamentous and insoluble forms of peptides or proteins. Proline has long been considered to be incompatible with the cross‐β structural motif of amyloid fibrils. On the basis of solid‐state NMR spectroscopy data, we present a structural model of an in‐register parallel β sheet for the amyloid fibrils formed from a human prion protein fragment, huPrP_127–47. We have developed a simple solid‐state NMR spectroscopy technique to identify solvent‐protected backbone amide protons in a H/D exchange experiment without disaggregating the amyloid fibrils, from which we find that proline residue P₁₃₇ does not disrupt the β‐sheet structure from G₁₂₇ to G₁₄₂. We suggest that the resultant kink at P₁₃₇ generates a twist between adjacent peptide strands to maintain hydrogen bonding in the β‐sheet regions flanking the P₁₃₇ residue. Although proline can be well integrated into the cross‐β structure of amyloid fibrils, the kink formed at the position of the proline residue will considerably weaken the hydrogen bonding between the neighboring strands, especially when the mutation site is near the central region of a β sheet.     

The Inhibition of Gastric Cancer Cells’ Progression by 23,24-Dihydrocucurbitacin E through Disruption of the Ras/Raf/ERK/MMP9 Signaling Pathway

Gastric cancer is considered to be one of the most common causes of cancer death worldwide due to its high recurrence and metastasis rates. The molecule 23,24-Dihydrocucurbitacin E (DHCE) is a cucurbitacin-derived tetracyclic triterpenoid compound that has anti-tumor activity, but the exact mechanism remains to be elucidated. This research aimed to explore the effects of DHCE on human gastric cancer cells and the possible mechanisms. The results showed that DHCE suppressed proliferation, migration, and invasion of gastric cancer cells, as well as induced apoptosis and G2/M phase arrest. Mechanistically, the potential targets and pathways of DHCE were predicted using database screening and verified using a molecular docking study, fluorescence staining, and Western blot. The results indicated that DHCE obviously inhibited the kinase activity of ERK2 via targeting its ATP-binding domain, destroyed F-actin microfilament, and reduced the expression levels of Ras, p-c-Raf, ERK, p-ERK, and MMP9 proteins. Collectively, our study demonstrated that DHCE suppressed gastric cancer cells’ proliferation, migration, and invasion through targeting ERK2 and disrupting the Ras/Raf/ERK/MMP9 signaling pathway. These properties make DHCE a promising candidate drug for the further design and development of novel and effective Ras/Raf/ERK/MMP9 pathway inhibitors for treating gastric cancer.     

人类胞外信号调节激酶1的同源模建及其抑制剂的对接与结构改造

通过同源模建和分子动力学模拟构建了人类胞外信号调节激酶1(hERK1)的三维结构,并利用profile-3D和procheck方法评估了模型的合理性.对所得的结构使用分子对接程序Affinity和CDOCKER进行了两种抑制剂的对接.结果显示这两种抑制剂与酶的结合方式相似,它们均与残基K36,Q87之间存在氢键作用,二者取代基的不同导致了抑制能力的差别.基于对接结果分析,对已知抑制剂进行结构改造,得到了一个理论上结合能力更强的抑制剂.它在保持与K36和Q87之间氢键的同时,又与残基D93,K96,S135形成了四条氢键,显著提高了与酶的相互作用.对接相互作用能显著下降,MM-PBSA结合自由能降为负值,这些均体现了抑制能力的提高.本工作对于针对该酶的抑制剂设计和相关疾病的新药开发具有理论指导价值.