胃癌中CDC4和cyclin E的表达及临床意义

检测胃癌中CDC4/Fbxw7、cyclin E的表达,分析其与胃癌临床病理特征的关系和临床意义.采用逆转录多聚酶链反应(RT-PCR)检测部分胃癌和胃正常组织中CDC4、cyclin E mRNA的表达;免疫组化(SP法)检测60例胃癌组织及对应正常组织中CDC4/FBXW7、cyclin E蛋白的表达,探讨两者的临床病理特征之间的关系.胃癌中CDC4蛋白表达显著低于正常组织(P<0.05),而cyclin E蛋白的表达在胃癌组织中显著增高,阳性率与癌旁正常组织比较,差异有统计学意义(P<0.01).CDC4、cyclin E蛋白和mRNA表达均与胃癌的分化程度、TNM分期、淋巴结转移及浸润深度有关.CDC4、cyclin E在mRNA表达水平上呈负相关.CDC4的表达缺失可能导致cyclin E的过表达.CDC4的低表达可能是胃癌诊疗及预后判断的重要生物学指标.     

The protein kinases AtMAP3Kepsilon1 and BnMAP3Kepsilon1 are functional homologues of S. pombe cdc7p and may be involved in cell division

We identified an Arabidopsis thaliana gene, AtMAP3Kepsilon1, and a Brassica napus cDNA, BnMAP3Kepsilon1, encoding functional protein serine/threonine kinases closely related to cdc7p and Cdc15p from Schizosaccharomyces pombe and Saccharomyces cerevisiae, respectively. This is the first report of cdc7-related genes in non-fungal eukaryotes; no such genes have as yet been identified in Metazoans. The B. napus protein is able to partially complement a cdc7 loss of function mutation in S. pombe. RT-PCR and in situ hybridisation revealed that the A. thaliana and B. napus genes are expressed in both the sporophytic and the gametophytic tissues of the respective plant species and revealed further that expression is highest in dividing cells. Moreover, AtMAP3Kepsilon1 gene expression is cell cycle-regulated, with higher expression in G2-M phases. Our results strongly suggest that the plant cdc7p-related protein kinases are involved in a signal transduction pathway similar to the SIN pathway, which positively regulates cytokinesis in S. pombe.     

Structural Basis for the Dual Substrate Specificity of DOCK7 Guanine Nucleotide Exchange Factor

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Structural Basis for Activity and Specificity of an Anticoagulant Anti-FXIa Monoclonal Antibody and a Reversal Agent

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Structural Basis for the ATP-dependent Configuration of Adenylation Active Site in Bacillus subtilis o-Succinylbenzoyl-CoA Synthetase

o-Succinylbenzoyl-CoA synthetase, or MenE, is an essential adenylate-forming enzyme targeted for development of novel antibiotics in the menaquinone biosynthesis. Using its crystal structures in a ligand-free form or in complex with nucleotides, a conserved pattern is identified in the interaction between ATP and adenylating enzymes, including acyl/aryl-CoA synthetases, adenylation domains of nonribosomal peptide synthetases, and luciferases. It involves tight gripping interactions of the phosphate-binding loop (P-loop) with the ATP triphosphate moiety and an open-closed conformational change to form a compact adenylation active site. In MenE catalysis, this ATP-enzyme interaction creates a new binding site for the carboxylate substrate, allowing revelation of the determinants of substrate specificities and in-line alignment of the two substrates for backside nucleophilic substitution reaction by molecular modeling. In addition, the ATP-enzyme interaction is suggested to play a crucial catalytic role by mutation of the P-loop residues hydrogen-bonded to ATP. Moreover, the ATP-enzyme interaction has also clarified the positioning and catalytic role of a conserved lysine residue in stabilization of the transition state. These findings provide new insights into the adenylation half-reaction in the domain alteration catalytic mechanism of the adenylate-forming enzymes.     

Structural Basis for Glycerol Efflux and Selectivity of Human Aquaporin 7

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Structural Basis for Auto-Inhibition of the NDR1 Kinase Domain by an Atypically Long Activation Segment

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Structural Basis for the SUMO2 Isoform Specificity of SENP7

SUMO proteases or deSUMOylases regulate the lifetime of SUMO-conjugated targets in the cell by cleaving off the isopetidic bond between the substrate and the SUMO modifier, thus reversing the conjugation activity of the SUMO E3 ligases. In humans the deSUMOylating activity is mainly conducted by the SENP/ULP protease family, which is constituted of six members sharing a homologous catalytic globular domain. SENP6 and SENP7 are the most divergent members of the family and they show a unique SUMO2/3 isoform preference and a particular activity for dismantling polySUMO2 chains. Here, we present the crystal structure of the catalytic domain of human SENP7 bound to SUMO2, revealing structural key elements for the SUMO2 isoform specificity of SENP7. In particular, we describe the specific contacts between SUMO2 and a unique insertion in SENP7 (named Loop1) that is responsible for the SUMO2 isoform specificity. All the other interface contacts between SENP7 and SUMO2, including the SUMO2 C-terminal tail interaction, are conserved among members of the SENP/ULP family. Our data give insight into an evolutionary adaptation to restrict the deSUMOylating activity in SENP6 and SENP7 for the SUMO2/3 isoforms.     

Structural and Functional Characterization of the JH2 Pseudokinase Domain of JAK Family Tyrosine Kinase 2 (TYK2)

JAK (Janus family of cytoplasmic tyrosine kinases) family tyrosine kinase 2 (TYK2) participates in signaling through cytokine receptors involved in immune responses and inflammation. JAKs are characterized by dual kinase domain: a tyrosine kinase domain (JH1) that is preceded by a pseudokinase domain (JH2). The majority of disease-associated mutations in JAKs map to JH2, demonstrating its central regulatory function. JH2s were considered catalytically inactive, but JAK2 JH2 was found to have low autoregulatory catalytic activity. Whether the other JAK JH2s share ATP binding and enzymatic activity has been unclear. Here we report the crystal structure of TYK2 JH2 in complex with adenosine 5′-O-(thiotriphosphate) (ATP-γS) and characterize its nucleotide binding by biochemical and biophysical methods. TYK2 JH2 did not show phosphotransfer activity, but it binds ATP and the nucleotide binding stabilizes the protein without inducing major conformational changes. Mutation of the JH2 ATP-binding pocket increased basal TYK2 phosphorylation and downstream signaling. The overall structural characteristics of TYK2 JH2 resemble JAK2 JH2, but distinct stabilizing molecular interactions around helix αAL in the activation loop provide a structural basis for differences in substrate access and catalytic activities among JAK family JH2s. The structural and biochemical data suggest that ATP binding is functionally important for both TYK2 and JAK2 JH2s, whereas the regulatory phosphorylation appears to be a unique property of JAK2. Finally, the co-crystal structure of TYK2 JH2 complexed with a small molecule inhibitor demonstrates that JH2 is accessible to ATP-competitive compounds, which offers novel approaches for targeting cytokine signaling as well as potential therapeutic applications.     

Structural analysis of the polo‐box domain of human Polo‐like kinase 2

Polo‐like kinases (Plks) are the key regulators of cell cycle progression, the members of which share a kinase domain and a polo‐box domain (PBD) that serves as a protein‐binding module. While Plk1 is a promising target for antitumor therapy, Plk2 is regarded as a tumor suppressor even though the two Plks commonly recognize the S‐pS/T‐P motif through their PBD. Herein, we report the crystal structure of the PBD of Plk2 at 2.7 Å. Despite the overall structural similarity with that of Plk1 reflecting their high sequence homology, the crystal structure also contains its own features including the highly ordered loop connecting two subdomains and the absence of 3₁₀‐helices in the N‐terminal region unlike the PBD of Plk1. Based on the three‐dimensional structure, we furthermore could model its interaction with two types of phosphopeptides, one of which was previously screened as the optimal peptide for the PBD of Plk2. Proteins 2015; 83:1201–1208. © 2015 Wiley Periodicals, Inc.     

细胞周期和骨架在CNE-2Z细胞凋亡中的变化

采用DNA电泳、PI染色流式细胞仪(FCM)分析和激光共聚焦显微镜(LCM)观察, 检测了蛋白激酶C(PKC)抑制剂诱导CNE-2Z细胞凋亡时细胞周期和骨架的改变. PKC抑制剂staurospoine(ST)、sphingosine(SS), 终浓度分别为1×10^-6 mol/L和4×10^-5 mol/L, 诱导细胞24 h.结果发现处理组细胞均有典型的DNA亚二倍体峰, DNA电泳有梯状图谱; 细胞周期百分比SS组较对照组S期增加及G1期减少明显(P＜0.05); ST组G2期增加、G1和S期显著减少(P＜0.01). 对照组细胞染色质分布均匀; 胞质微丝呈细颗粒状, 均匀分布. 诱导细胞染色质碎裂呈不规则缺损; 胞质微丝散乱, 颗粒粗大, 排列不均. 结果表明, SS、ST可诱导CNE-2Z细胞凋亡, 细胞周期和骨架在细胞凋亡时, 均发生了明显改变.     

Structural and functional characterization of ubiquitin variant inhibitors for the JAMM-family deubiquitinases STAMBP and STAMBPL1

Ubiquitination is a crucial posttranslational protein modification involved in a myriad of biological pathways. This modification is reversed by deubiquitinases (DUBs) that deconjugate the single ubiquitin (Ub) moiety or poly-Ub chains from substrates. In the past decade, tremendous efforts have been focused on targeting DUBs for drug discovery. However, most chemical compounds with inhibitory activity for DUBs suffer from mild potency and low selectivity. To overcome these obstacles, we developed a phage display-based protein engineering strategy for generating Ub variant (UbV) inhibitors, which was previously successfully applied to the Ub-specific protease (USP) family of cysteine proteases. In this work, we leveraged the UbV platform to selectively target STAMBP, a member of the JAB1/MPN/MOV34 (JAMM) metalloprotease family of DUB enzymes. We identified two UbVs (UbV^SP.1 and UbV^SP.3) that bind to STAMBP with high affinity but differ in their selectivity for the closely related paralog STAMBPL1. We determined the STAMBPL1-UbV^SP.1 complex structure by X-ray crystallography, revealing hotspots of the JAMM-UbV interaction. Finally, we show that UbV^SP.1 and UbV^SP.3 are potent inhibitors of STAMBP isopeptidase activity, far exceeding the reported small-molecule inhibitor BC-1471. This work demonstrates that UbV technology is suitable to develop molecules as tools to target metalloproteases, which can be used to further understand the cellular function of JAMM family DUBs.     

Structural Basis for the Activation of Muscle Contraction by Troponin and Tropomyosin

The molecular regulation of striated muscle contraction couples the binding and dissociation of Ca²⁺ on troponin (Tn) to the movement of tropomyosin on actin filaments. In turn, this process exposes or blocks myosin binding sites on actin, thereby controlling myosin crossbridge dynamics and consequently muscle contraction. Using 3D electron microscopy, we recently provided structural evidence that a C-terminal extension of TnI is anchored on actin at low Ca²⁺ and competes with tropomyosin for a common site to drive tropomyosin to the B-state location, a constrained, relaxing position on actin that inhibits myosin-crossbridge association. Here, we show that release of this constraint at high Ca²⁺ allows a second segment of troponin, probably representing parts of TnT or the troponin core domain, to promote tropomyosin movement on actin to the Ca²⁺-induced C-state location. With tropomyosin stabilized in this position, myosin binding interactions can begin. Tropomyosin appears to oscillate to a higher degree between respective B- and C-state positions on troponin-free filaments than on fully regulated filaments, suggesting that tropomyosin positioning in both states is troponin-dependent. By biasing tropomyosin to either of these two positions, troponin appears to have two distinct structural functions; in relaxed muscles at low Ca²⁺, troponin operates as an inhibitor, while in activated muscles at high Ca²⁺, it acts as a promoter to initiate contraction.     

Structural Basis for EPC1-Mediated Recruitment of MBTD1 into the NuA4/TIP60 Acetyltransferase Complex

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RecQL4 is required for the association of Mcm10 and Ctf4 with replication origins in human cells

Though RecQL4 was shown to be essential for the initiation of DNA replication in mammalian cells, its role in initiation is poorly understood. Here, we show that RecQL4 is required for the origin binding of Mcm10 and Ctf4, and their physical interactions and association with replication origins are controlled by the concerted action of both CDK and DDK activities. Although RecQL4-dependent binding of Mcm10 and Ctf4 to chromatin can occur in the absence of pre-replicative complex, their association with replication origins requires the presence of the pre-replicative complex and CDK and DDK activities. Their association with replication origins and physical interactions are also targets of the DNA damage checkpoint pathways which prevent initiation of DNA replication at replication origins. Taken together, the RecQL4-dependent association of Mcm10 and Ctf4 with replication origins appears to be the first important step controlled by S phase promoting kinases and checkpoint pathways for the initiation of DNA replication in human cells.     

Structural Mechanisms Determining Inhibition of the Collagen Receptor DDR1 by Selective and Multi-Targeted Type II Kinase Inhibitors

The discoidin domain receptors (DDRs), DDR1 and DDR2, form a unique subfamily of receptor tyrosine kinases that are activated by the binding of triple-helical collagen. Excessive signaling by DDR1 and DDR2 has been linked to the progression of various human diseases, including fibrosis, atherosclerosis and cancer. We report the inhibition of these unusual receptor tyrosine kinases by the multi-targeted cancer drugs imatinib and ponatinib, as well as the selective type II inhibitor DDR1-IN-1. Ponatinib is identified as the more potent molecule, which inhibits DDR1 and DDR2 with an IC₅₀ of 9 nM. Co-crystal structures of human DDR1 reveal a DFG-out conformation (DFG, Asp-Phe-Gly) of the kinase domain that is stabilized by an unusual salt bridge between the activation loop and αD helix. Differences to Abelson kinase (ABL) are observed in the DDR1 P-loop, where a β-hairpin replaces the cage-like structure of ABL. P-loop residues in DDR1 that confer drug resistance in ABL are therefore accommodated outside the ATP pocket. Whereas imatinib and ponatinib bind potently to both the DDR and ABL kinases, the hydrophobic interactions of the ABL P-loop appear poorly satisfied by DDR1-IN-1 suggesting a structural basis for its DDR1 selectivity. Such inhibitors may have applications in clinical indications of DDR1 and DDR2 overexpression or mutation, including lung cancer.     

Activation of JNK/c-Jun is required for the proliferation, survival, and angiogenesis induced by EET in pulmonary artery endothelial cells

Pulmonary artery endothelial plexiform lesion is responsible for pulmonary vascular remodeling (PVR), a basic pathological change of pulmonary arterial hypertension (PAH). Recent evidence suggests that epoxyeicosatrienoic acid (EET), which is derived from arachidonic acid by cytochrome p450 (CYP) epoxygenase, has an essential role in PAH. However, until now, most research has focused on pulmonary vasoconstriction; it is unclear whether EET produces mitogenic and angiogenic effects in pulmonary artery endothelial cells (PAEC). Here we found that 500 nM/l 8,9-EET, 11,12-EET, and 14,15-EET markedly augmented JNK and c-Jun activation in PAECs and that the activation of c-Jun was mediated by JNK, but not the ERK or p38 MPAK pathway. Moreover, treatment with 8,9-EET, 11,12-EET, and 14,15-EET promoted cell proliferation and cell-cycle transition from the G0/G1 phase to S phase and stimulated tube formation in vitro. All these effects were reversed after blocking JNK with Sp600125 (a JNK inhibitor) or JNK1/2 siRNA. In addition, the apoptotic process was alleviated by three EET region isomers through the JNK/c-Jun pathway. These observations suggest that 8,9-EET, 11,12-EET, and 14,15-EET stimulate PAEC proliferation and angiogenesis, as well as protect the cells from apoptosis, via the JNK/c-Jun pathway, an important underlying mechanism that may promote PAEC growth and angiogenesis during PAH.     

Interaction of the non-phosphorylated peptide G7-18NATE with Grb7-SH2 domain requires phosphate for enhanced affinity and specificity

Src‐homology (SH2) domains are an attractive target for the inhibition of specific signalling pathways but pose the challenge of developing a truly specific inhibitor. The G7‐18NATE cyclic peptide is reported to specifically inhibit the growth factor receptor bound protein 7 (Grb7) adapter protein, implicated in the progression of several cancer types, via interactions with its SH2 domain. G7‐18NATE effectively inhibits the interaction of Grb7 with ErbB3 and focal adhesion kinase in cell lysates and, with the addition of a cell permeability sequence, inhibits the growth and migration of a number of breast cancer cell lines. It is thus a promising lead in the development of therapeutics targeted to Grb7. Here we investigate the degree to which G7‐18NATE is specific for the Grb7‐SH2 domain compared with closely related SH2 domains including those of Grb10, Grb14, and Grb2 using surface plasmon resonance. We demonstrate that G7‐18NATE binds with micromolar binding affinity to Grb7‐SH2 domain (K_D = 4–6 μm ) compared with 50–200 times lower affinity for Grb10, Grb14, and Grb2 but that this specificity depends critically on the presence of phosphate in millimolar concentrations. Other differences in buffer composition, including use of Tris or 2‐(N‐Morpholino)ethanesulfonic acid or varying the pH, do not impact on the interaction. This suggests that under cellular conditions, G7‐18NATE binds with highest affinity to Grb7. In addition, our findings demonstrate that the basis of specificity of G7‐18NATE binding to the Grb7‐SH2 domain is via other than intrinsic structural features of the protein, representing an unexpected mode of molecular recognition. Copyright © 2011 John Wiley & Sons, Ltd.     

Structural basis for phosphotyrosine recognition by the Src homology-2 domains of the adapter proteins SH2-B and APS

SH2-B, APS, and Lnk constitute a family of adapter proteins that modulate signaling by protein tyrosine kinases. These adapters contain an N-terminal dimerization region, a pleckstrin homology domain, and a C-terminal Src homology-2 (SH2) domain. SH2-B is recruited via its SH2 domain to various protein tyrosine kinases, including Janus kinase-2 (Jak2) and the insulin receptor. Here, we present the crystal structure at 2.35 A resolution of the SH2 domain of SH2-B in complex with a phosphopeptide representing the SH2-B recruitment site in Jak2 (pTyr813). The structure reveals a canonical SH2 domain-phosphopeptide binding mode, but with specific recognition of a glutamate at the +1 position relative to phosphotyrosine, in addition to recognition of a hydrophobic residue at the +3 position. Biochemical studies of SH2-B and APS demonstrate that, although the SH2 domains of these two adapter proteins share 79% sequence identity, the SH2-B SH2 domain binds preferentially to Jak2, whereas the APS SH2 domain has higher affinity for the insulin receptor. This differential specificity is attributable to the difference in the oligomeric states of the two SH2 domains: monomeric for SH2-B and dimeric for APS.