乳腺癌易感蛋白BRCA1的BRCT2结构域染色质伸展活性的定位

40 %～ 5 0 %的遗传性乳腺癌和至少 80 %的既有乳腺癌又有卵巢癌家族史的患者是由BRCA1突变引起的 .BRCA1C末端含有 2个BRCT结构域 (BRCT1和BRCT2 ) ,它们与BRCA1的重要功能密切相关 .许多乳腺癌易感突变发生在BRCA1的BRCT结构域中 .利用染色质结构检测技术表明 ,BRCT结构域具有染色质伸展活性 .利用缺失突变技术构建了 6种BRCT2结构域 (175 6～ 185 2位氨基酸残基 )缺失突变体并将BRCT2结构域中与染色质伸展相关的重要区域定位到 175 6～ 180 8之间的氨基酸残基 ;用丙氨酸扫描技术构建了 6种BRCT2结构域丙氨酸扫描突变体并将重要氨基酸残基序列定位到 1784～ 1788之间的VQLCG .BRCT2结构域的定位有助于预测BRCT2结构域突变后发生乳腺癌的风险 ,也为进一步研究BRCT2结构域的功能机制提供了有用的材料 .     

乳腺癌易感蛋白BRCA1的BRCT1结构域染色质伸展活性的定位

乳腺癌易感基因BRCA1（Breast cancer susceptibility gene 1）在乳腺癌的发生、发展中起重要作用。BRCA1 C末端含有2个BRCT结构域（BRCT1和BRCT2）,许多乳腺癌易感突变发生在BRCA1的BRCT结构域中。利用染色质结构检测技术表明,BRCT结构域具有染色质伸展活性。本文利用缺失突变技术构建了6种BRCT1结构域（1642-1736 aa）缺失突变体并将BRCT1结构域中与染色质伸展相关的重要区域定位到1691-1721之间的氨基酸残基;用丙氨酸扫描技术构建了10种BRCT1结构域丙氨酸扫描突变体并将重要氨基酸残基序列定位到1707-1711之间的IAGGK。利用定位的重要区域进行Blast分析,结果找到一新型同源蛋白质。BRCT1结构域的定位有助于预测BRCT1结构域突变后发生乳腺癌的风险,也为进一步研究BRCT1结构域的功能机制提供了有用的材料。     

位点2蛋白酶的结构域、功能与作用机制研究进展

膜内蛋白酶对跨膜蛋白的不可逆性切割过程在跨膜信号转导途径中起着重要作用。位点2蛋白酶(site-2 proteases, S2P)属于膜内蛋白酶中的金属蛋白酶家族。基于进化树的序列分析,S2P及其同源物可以分为亚组Ⅰ至亚组Ⅳ,亚组Ⅰ包括了绝大多数真核生物和细菌的S2P及其同源物,亚组Ⅲ则为少数原核生物如枯草芽孢杆菌和古细菌如詹氏甲烷球菌的S2P同源物。由于S2P参与的信号转导从细菌到人类均具有保守性,本文以亚组Ⅰ和亚组Ⅲ的S2P为例进行综述,阐明S2P及其同源物对跨膜蛋白的切割过程,并展望尚待研究的方向。     

Deafl的功能结构域分析和预测

目的分析转录因子Deafl的功能结构域并预测其功能。方法利用现有的数据库和软件对Deafl的转录因子结构域,核定位信号及和输出信号进行分析和预测。结果Deafl含有一个保守的SAND结构域及一个能介导蛋白质一蛋白质相互作用的MYND结构域;有核定位信号和核输出信号;在其N端还有一个富含丙氨酸结构域。结论Deafl除有典型转录因子必需的功能结构域之外,可能还有不止一个结构域能介导与其它蛋白质因子的相互作用,这对Deafl调控外周组织抗原表达至关重要。     

真核蛋白质中G-patch结构域的结构和功能研究

G-patch是广泛存在真核生物蛋白质中的保守结构域,含有六个高度保守的甘氨酸残基,推测它是一种RNA结合结构域,其功能与RNA代谢有关.     

溴结构域蛋白4的结构和功能及其抑制剂在肿瘤研究中的应用

溴结构域和超末端结构域(bromodomain and extraterminal domain, Bet)家族是表观基因组的调节因子,也是肿瘤细胞生存所依赖的肿瘤相关基因表达的关键驱动因子。溴结构域蛋白4 (bromodomain-containing protein 4, Brd4)是溴域和端外蛋白家族中的一员,通常识别乙酰化组蛋白,并定位于目的基因的启动子或增强子区域,启动并维持肿瘤相关基因的表达。Brd4与多种转录因子调控和染色质修饰密切相关,并参与DNA损伤修复、维持端粒功能,从而维持肿瘤细胞的存活。本文围绕Brd4蛋白的结构、功能及其抑制剂在肿瘤研究中的应用进行综述。     

α-淀粉酶AmyP中淀粉结合结构域的鉴定

【目的】检测具有生淀粉降解活性的新型α-淀粉酶AmyP是否具有淀粉结合结构域(SBD)。【方法】通过结构域预测和序列分析, 推测AmyP的C端是一个SBD。将这段序列克隆、表达和重组蛋白纯化后, 采用亲和电泳和生淀粉吸附两种方法对重组表达的蛋白进行研究。【结果】AmyP的C端序列是一个新型的SBD, 根据序列特征可以将其划分在碳水化合物结合结构域(CBM) 20家族。该SBD与生大米淀粉的吸附能力最强, 生玉米淀粉次之, 不能与生小麦淀粉、生马铃薯淀粉和生绿豆淀粉吸附。【结论】α-淀粉酶AmyP在蛋白C端具有一个SBD, 有助于理解AmyP快速偏好性降解生淀粉的能力。     

IV型限制酶ScoMcrA中SRA结构域介导的二聚体化对硫结合结构域功能的影响机制

[背景] 部分细菌的DNA骨架会发生磷硫酰化修饰,硫结合结构域（Sulfur Binding Domain,SBD）可以特异性识别这种生理修饰。与绝大多数SBD-HNH双结构域核酸酶不同,ScoMcrA的SBD和HNH结构域中间插入了一个特异性识别5-甲基胞嘧啶（5mC）修饰DNA的SET and RING-Associated （SRA）结构域。晶体结构显示,单独的SBD是单体,而SBD-SRA是双体。[目的] 探究ScoMcrA中SRA结构域的存在对SBD识别硫修饰DNA的影响及影响方式。[方法] 凝胶迁移实验（Electrophoresis Mobility Shift Assay,EMSA）比较SBD、SBD-SRA对硫修饰DNA结合力的差异;对参与SBD-SRA二聚体化的关键氨基酸残基突变,并检测点突变对SBD-SRA蛋白二聚体化及结合硫修饰DNA的影响。[结果] 相较于SBD结构域,SBD-SRA双结构域对磷硫酰化修饰DNA的结合能力明显增强。对SBD-SRA双体互作界面进行单点突变基本不影响其对硫修饰DNA的结合,当二聚体化界面连续的L₂₆₁LGET₂₆₅突变成A₂₆₁AAAA₂₆₅时,突变体对硫修饰DNA的结合力下降到与SBD相似的水平。[结论] 根据EMSA实验结果可以初步判断,SRA结构域介导的SBD-SRA双体化能增强SBD对硫修饰DNA的结合力;L₂₆₁LGET₂₆₅是SRA结构域上影响SBD对硫修饰DNA结合力的关键氨基酸位点。     

验证性筛选PDZ结构域结合配体文库快速研究HtrA2/Omi PDZ结构域的结合特性

HtrA2/Omi是一种线粒体丝氨酸蛋白酶, 在哺乳动物细胞中具有双重功能, 即诱导细胞凋亡和参与维持线粒体活性的动态平衡. PDZ结构域是最重要的蛋白质相互作用结构域之一, 参与多种生物学过程, 如细胞信号转导、蛋白质降解、细胞骨架组织等. 最近研究发现, HtrA2/Omi蛋白的PDZ结构域与配体的相互作用, 可以调节HtrA2/Omi蛋白自身的水解酶活性.以HtrA2/Omi PDZ结构域为研究对象, 用酵母双杂交系统验证性筛选PDZ结构域结合配体文库, 快速研究该结构域的结合特性, 并在人类全蛋白质组范围内预测并发现该结构域新的相互作用蛋白, 最后分析这些新的相互作用所能够形成的最小相互作用网络来评估其可信度. 研究结果揭示了HtrA2/Omi PDZ结构域新的结合特性, 即: 不仅能够结合已报道的II类PDZ配体而且还可以结合I类和III类PDZ配体, 并且配体-3位氨基酸具有一定范围内的可变性. 而且, 发现了7个新的HtrA2/Omi PDZ结构域相互作用蛋白, 为进一步阐明HtrA2/Omi蛋白的生物学功能提供了重要线索. 同时证明了验证性筛选目的结构域结合配体文库, 这一结构域结合特性研究新策略的实用性和高效性.     

人白介素6与其受体结合特定结构域的预测

借助人白介素6的核磁共振构象, 以人生长因子受体为模板同源模建的人白介素6受体空间构象, 通过表观静电势分析, 结合空间构象的互补性对人白介素6与其受体进行分子对接, 在分子力学优化、分子动力学动态模拟的基础上获得稳定的复合物结构, 从理论上预测人白介素6与其受体相互作用的结构域, 进一步确定影响人白介素6与其受体相互作用的特定位点, 为确定靶蛋白人白介素6的活性部位、利用计算机寻找和辅助设计新的拮抗剂提供理论依据.     

Localization of pyrodoxal phosphate binding site on the mero-receptor domain of the glucocorticoid receptor

Previous studies have demonstrated that the vitamin pyridoxal phosphate can alter the physicochemical properties of glucocorticoid receptors. We now report the localization of a pyridoxal phosphate binding site within the mero-receptor domain of this glucocorticoid receptor. Mero-glucocorticoid receptors that are generated by trypsin (10 μg/ml) or chymotrypsin (100 μg/ml) digestion of intact receptors sediment as 2.6 S species on 5–20% sucrose gradients in the presence or absence of pyridoxal phosphate. Mero-glucocoritcoid receptors prepared by exogenous proteinases are hydrophobic and show no affinity for DEAE Bio-Gel A. Treating either trypsin-generated or chymotrypsin-generated mero-receptors with pyridoxal phosphate rapidly converts the proteins (60 and 35%, respectively) into forms that bind to DEAE Bio-Gel A. Induction of DEAE binding is specific to pyridoxal phosphate, for treating mero-receptors with pyridoxal, pyridoxamine or pyridoxine phosphate is ineffective. Furthermore, DEAE binding cannot be induced by adding other pyridoxal phosphate-treated cytosols to untreated mero-receptors. High-resolution polyacrylamide gel isoelectric focussing studies indicated that treating mero-receptor generated by either proteinase with pyridoxal phosphate shifted the isoelectric points of lower pH values. The conversion of the mero-receptor to a more acidic form also occurred when the intact glucocorticoid receptor was treated with the vitamin prior to proteolysis. These studies localize at least one pyridoxal phosphate binding site on the mero-receptor domain of the rat thymocyte glucocorticoid receptor.     

Functional site plasticity in domain superfamilies

We present, to our knowledge, the first quantitative analysis of functional site diversity in homologous domain superfamilies. Different types of functional sites are considered separately. Our results show that most diverse superfamilies are very plastic in terms of the spatial location of their functional sites. This is especially true for protein–protein interfaces. In contrast, we confirm that catalytic sites typically occupy only a very small number of topological locations. Small-ligand binding sites are more diverse than expected, although in a more limited manner than protein–protein interfaces. In spite of the observed diversity, our results also confirm the previously reported preferential location of functional sites. We identify a subset of homologous domain superfamilies where diversity is particularly extreme, and discuss possible reasons for such plasticity, i.e. structural diversity. Our results do not contradict previous reports of preferential co-location of sites among homologues, but rather point at the importance of not ignoring other sites, especially in large and diverse superfamilies. Data on sites exploited by different relatives, within each well annotated domain superfamily, has been made accessible from the CATH website in order to highlight versatile superfamilies or superfamilies with highly preferential sites. This information is valuable for system biology and knowledge of any constraints on protein interactions could help in understanding the dynamic control of networks in which these proteins participate. The novelty of our work lies in the comprehensive nature of the analysis – we have used a significantly larger dataset than previous studies – and the fact that in many superfamilies we show that different parts of the domain surface are exploited by different relatives for ligand/protein interactions, particularly in superfamilies which are diverse in sequence and structure, an observation not previously reported on such a large scale. This article is part of a Special Issue entitled: The emerging dynamic view of proteins: Protein plasticity in allostery, evolution and self-assembly.     

The murine Pes1 gene encodes a nuclear protein containing a BRCT domain

Pescadillo was originally identified in the zebrafish Danio rerio as a site of a retrovirus-insertion mutation that caused severe defects during embryogenesis. In particular, growth of the fetal zebrafish liver was significantly affected by loss of pescadillo function. To begin to understand the role of pescadillo during mammalian hepatogenesis we identified the murine homologue of pescadillo and named it Pes1. A single gene localized to chromosome 11 on the mouse genome encodes Pes1. Although Pes1 mRNA was detected in all tissues examined it was present at the highest levels in both adult and fetal liver. Analysis of the predicted amino acid sequence of Pes1 found it to contain a BRCT domain, which has previously been found in several proteins involved in cell-cycle checkpoints and DNA repair. Consistent with a putative role in these processes we found that when recombinant Pes1 protein was expressed in HepG2 cells it localized to the nucleus.     

Isolation of a 5,300-dalton peptide containing a pyridoxal phosphate binding site from the 38,000-dalton domain of band 3 of human erythrocyte membranes

A hydrophobic 5,300-dalton peptide was isolated from the 38,000-dalton domain of Band 3 by sodium dodecyl sulfate polyacrylamide gel electrophoresis and reversed-phase high-performance liquid chromatography. The peptide was affinity labeled with pyridoxal phosphate and sodium [3H]borohydride when erythrocytes were incubated in vitro. The peptide was not labeled with these agents when cells were incubated in the presence of a specific inhibitor of anion transport, suggesting that the peptide contains at least a part of the active center for the anion transport system in the cell membrane. The peptide was eluted from a reversed-phase high-performance liquid chromatography column with a high concentration of acetonitrile (more than 65%), although the elution pattern of the hydrophobic peptide was not as sharp as that of the soluble peptides. However, a satisfactory separation was achieved when this procedure was employed in combination with sodium dodecyl sulfate polyacrylamide gel electrophoresis.     

Inorganic phosphate binds to the empty nucleotide binding pocket of conventional myosin II

In muscle inorganic phosphate strongly decreases force generation in the presence of millimolar MgATP, whereas phosphate slows shortening velocity only at micromolar MgATP concentrations. It is still controversial whether reduction in shortening velocity by phosphate results from phosphate binding to the nucleotide-free myosin head or from binding of phosphate to an actomyosin-ADP state as postulated for the inhibition of force generation by phosphate. Because most single-molecule studies are performed at micromolar concentrations of MgATP where phosphate effects on movement are rather prominent, clarification of the mechanisms of phosphate inhibition is essential for interpretation of data in which phosphate is used in single molecule studies to probe molecular events of force generation and movement. In in vitro assays we found that inhibition of filament gliding by inorganic phosphate was associated with increased fragmentation of actin filaments. In addition, phosphate did not extend dwell times of Cy3-EDA-ATP (2'(3')-O-[[2-[[6-[2-[3-(1-ethyl-1,3-dihydro-3,3-dimethyl-5-sulfo-2H-indol-2-ylidene)-1-propenyl]-3,3-dimethyl-5-sulfo-3H-indolio]-1-oxohexyl]amino]ethyl]carbamoyl]ATP) but reduced the number of Cy3-signals per field of view, approaching 50% at phosphate concentrations of 1-2 mM. Apparently, inhibition of movement does not result from binding of phosphate to an actomyosin-ADP intermediate as proposed by Hooft and coworkers (Hooft, A. M., Maki, E. J., Cox, K. K., and Baker, J. E. (2007) Biochemistry 46, 3513-3520) but, rather, from forming a strong-binding actomyosin-phosphate intermediate.     

Identification of the phosphoribulokinase sugar phosphate binding domain.

A recombinant form of Rhodobacter sphaeroides phosphoribulokinase (form I; NADH dependent) has been expressed in and purified to homogeneity from Escherichia coli that harbor the prkA gene in the plasmid pKP1565b. Restriction digestion of the phosphoribulokinase-encoding plasmid produces a tractable 450 bp fragment that encodes amino acid residues 28-179, which include a region (residues 42-54) highly conserved among phosphoribulokinase proteins. Using overlap extension polymerase chain reaction methodology, directed mutagenesis was performed to produce mutant proteins in which basic residues in this conserved region were replaced by neutral amino acids. Lysine-53, implicated by affinity labeling studies, has been replaced by methionine; little effect on substrate binding or catalysis is apparent. In contrast, when histidine-45 is replaced by asparagine, a 40-fold increase in the Km for ribulose 5-phosphate results; a 200-fold increase results when arginine-49 is replaced by glutamine. Implication of this region as part of the sugar phosphate binding site is compatible with previous results that indicate targeting by an ATP analogue containing a reactive functionality esterified to the gamma-phosphoryl group. The phosphoribulokinase reaction involves a single in-line phosphoryl transfer, requiring that the gamma-phosphoryl of ATP be closely juxtaposed to the bound cosubstrate. It follows that any reactive group attached to the gamma-phosphoryl in a nucleotide analogue that is bound to PRK in the absence of the cosubstrate will be favorably positioned to modify the sugar phosphate binding site.