Defects in DNA degradation revealed in crystal structures of TREX1 exonuclease mutations linked to autoimmune disease

Mutations within the human TREX1 3' exonuclease are associated with Aicardi-Goutières Syndrome (AGS) and familial chilblain lupus (FCL). Both AGS and FCL are autoimmune diseases that result in increased levels of interferon alpha and circulating antibodies to DNA. TREX1 is a member of the endoplasmic reticulum (ER)-associated SET complex and participates in granzyme A-mediated cell death to degrade nicked genomic DNA. The loss of TREX1 activity may result in the accumulation of double-stranded DNA (dsDNA) degradation intermediates that trigger autoimmune activation. The X-ray crystal structures of the TREX1 wt apoprotein, the dominant D200H, D200N and D18N homodimer mutants derived from AGS and FCL patients, as well as the recessive V201D homodimer mutant have been determined. The structures of the D200H and D200N mutant proteins reveal the enzyme has lost coordination of one of the active site metals, and the catalytic histidine (H195) is trapped in a conformation pointing away from the active site. The TREX1 D18N and V201D mutants are able to bind both metals in the active site, but with inter-metal distances that are larger than optimal for catalysis. Additionally, all of the mutant structures reveal a reduced mobility in the catalytic histidine, providing further explanation for the loss of catalytic activity. The structures of the mutant TREX1 proteins provide insight into the dysfunction relating to human disease. Additionally, the TREX1 apoprotein structure together with the previously determined wild type substrate and product structures allow us to propose a distinct mechanism for the TREX1 exonuclease.     

The TREX1 double-stranded DNA degradation activity is defective in dominant mutations associated with autoimmune disease

Mutations in TREX1 have been linked to a spectrum of human autoimmune diseases including Aicardi-Goutières syndrome (AGS), familial chilblain lupus (FCL), systemic lupus erythematosus, and retinal vasculopathy and cerebral leukodystrophy. A common feature in these conditions is the frequent detection of antibodies to double-stranded DNA (dsDNA). TREX1 participates in a cell death process implicating this major 3' --> 5' exonuclease in genomic DNA degradation to minimize potential immune activation by persistent self DNA. The TREX1 D200N and D18N dominant heterozygous mutations were identified in AGS and FCL, respectively. TREX1 enzymes containing the D200N and D18N mutations were compared using nicked dsDNA and single-stranded DNA (ssDNA) degradation assays. The TREX1WT/D200N and TREX1WT/D18N heterodimers are completely deficient at degrading dsDNA and degrade ssDNA at an expected approximately 2-fold lower rate than TREX1WT enzyme. Further, the D200N- and D18N-containing TREX1 homo- and heterodimers inhibit the dsDNA degradation activity of TREX1WT enzyme, providing a likely explanation for the dominant phenotype of these TREX1 mutant alleles in AGS and FCL. By comparison, the TREX1 R114H homozygous mutation causes AGS and is found as a heterozygous mutation in systemic lupus erythematosus. The TREX1R114H/R114H homodimer has dysfunctional dsDNA and ssDNA degradation activities and does not detectibly inhibit the TREX1WT enzyme, whereas the TREX1WT/R114H heterodimer has a functional dsDNA degradation activity, supporting the recessive genetics of TREX1 R114H in AGS. The dysfunctional dsDNA degradation activities of these disease-related TREX1 mutants could account for persistent dsDNA from dying cells leading to an aberrant immune response in these clinically related disorders.     

Cooperative DNA binding and communication across the dimer interface in the TREX2 3' --> 5'-exonuclease

The activity of human TREX2-catalyzed 3' --> 5'-deoxyribonuclease has been analyzed in steady-state and single turnover kinetic assays and in equilibrium DNA binding studies. These kinetic data provide evidence for cooperative DNA binding within TREX2 and for coordinated catalysis between the TREX2 active sites supporting a model for communication between the protomers of a TREX2 dimer. Mobile loops positioned adjacent to the active sites provide the major DNA binding contribution and facilitate subsequent binding into the active sites. Mutations of three arginine residues on these loops cause decreased TREX2 activities by up to 60-fold. Steady-state kinetic assays of these arginine to alanine TREX2 variants result in increased K(m) values for DNA substrate with no effect on k(cat) values indicating contributions exclusively to DNA binding by all three of the loop arginines. TREX2 heterodimers were prepared to determine whether exonuclease activity in one protomer is communicated to the opposing protomer. Evidence for communication across the dimer interface is provided by the 7-fold lower catalytic activity measured in the TREX2(WT/H188A) heterodimer compared with the TREX2(WT) homodimer, contrasting the 2-fold lower activity measured in the TREX2(WT/R163A,R165A,R167A) heterodimer. The measured activity in TREX2(WT/H188A) heterodimer indicates that defective catalysis in one protomer reduces activity in the opposing protomer. A DNA binding analysis of TREX2 and the heterodimers indicates a cooperative binding effect within the TREX2 protomer. Finally, single turnover kinetic assays identify DNA binding as the rate-limiting step in TREX2 catalysis.     

黑素皮质素受体-4的研究进展

黑素皮质素受体 4 (MC4R)是人类中枢神经系统中参与调节肥胖症发生的重要因素 ,可调节动物的体重和采食量。自MC4R基因克隆以来 ,学者们对MC4R的结构 ,生理功能 ,调控 ,作用机制及其基因突变与体重的关系等方面进行了大量的研究。     

运用稳定同位素技术分析大宁河主要鱼类营养层级

运用氮稳定同位素技术分析了大宁河静水水域和流水河段主要鱼类的氮稳定同位素比值和营养层级,并对静水水域不同水文时期相同鱼类的营养层级进行了比较研究。结果表明, 颗粒有机物(POM)氮稳定性同位素变化幅度较大, 并存在季节差异。大宁河下游静水水域鱼类15N 值范围为4.5417.51, 营养级处于1.513.88, 平均营养层级为2.49;上游流水水域鱼类的15N 值范围为2.2510.81, 营养层级范围为1.494.01, 平均营养层级为2.87。大宁河上游鱼类的平均营养层级大于下游静水水域, 可能是由于上游底栖生物丰富, 鱼类倾向摄食适口性更高的动物性食物而导致。大宁河下游静水水域汛期的鱼类营养层级较非汛期的值显著降低, 可能是因为汛期的水文扰动影响鱼类摄食中间捕食者, 以及水位的降低导致鱼类食物竞争增加迫使其摄食低营养水平的食物。重复基准生物采样建立精确充足的基线值以及确定合适的富集度,是提高营养层级评估准确性的重要手段。       

利手、优势足及扣手的遗传方式初探

采用Slater区分单基因和多基因遗传的计算模式及Smith无偏分析方法对21个家系资料的分析表明：利手、优势足、扣手特征均为常染色体单基因显性遗传,R型为显性性状。虽然环境因素对这类特征的表现也有一定的影响,但遗传因素仍起主要作用。 Abstract:The data of 21 families were analyzed by the method of Slater's calculating model to differentiate between single-gene and multi-gene heredity and by the method of non-deviation analysis.The results showed that the hereditary mode of handedness or preferential foot or hand-clasping is the dominant heredity of single gene of autosome,and the right type of all of them is the dominant character.In a way,although environmental factors affected the phenotypes of these characters,hereditary factors were also the decisive ones.     

利手、优势足及扣手的遗传方式初探

采用Slater区分单基因和多基因遗传的计算模式及Smith无偏分析方法对21个家系资料的分析表明：利手、优势足、扣手特征均为常染色体单基因显性遗传,R型为显性性状。虽然环境因素对这类特征的表现也有一定的影响,但影响因素仍起主要作用。     

新疆醉马草化学成分的研究

用95％和60％乙醇提取、溶剂萃取、硅胶柱层析、重结晶等方法从新疆醉马草提取分离得到8个活性化合物,根据IR、MS、NMR等光谱技术方法分别鉴定为adenosine(1),veratroylzygadenine(2),germerine(3),十六烷酸2,3-二羟基丙酯(4),3-O-glucoside-veratramine(5),胡萝卜甙(6),4′,6,7-三羟基-3′,5′-二甲氧基黄酮(7),蔗糖(8)。这些成分均为首次从该植物及该属中获得。     

Ca(2+)- and myristoylation-dependent association of calcineurin with phosphatidylserine

It has been proposed that N-terminal myristoylation of calcineurin B is necessary for the membrane association of calcineurin. We tested the effects of Ca(2+) and myristoylation on the binding of calcineurin B alone or heterodimeric calcineurin to phosphatidylserine or phosphatidylcholine vesicles. In the presence of excess phosphatidylserine, 50-60% of total calcineurin associated with phosphatidylserine in a Ca(2+)-sensitive manner. Calcineurin did not associate with phosphatidylcholine. Calcineurin containing both the alpha and beta catalytic subunit isoforms bound to phosphatidylserine. Calmodulin interfered with the association of calcineurin with phosphatidylserine. In the presence of Ca(2+), myristoylated calcineurin B alone did not bind to phosphatidylcholine but did bind to phosphatidylserine, although to a lesser extent than the calcineurin heterodimer. Non-myristoylated calcineurin B alone, or calcineurin containing non-myristoylated calcineurin B did not associate with phosphatidylserine in the presence of Ca(2+). These results indicate: (i) Both isoforms of calcineurin bind to phosphatidylserine. (ii) A phospholipid binding site is located on the calcineurin B subunit. (iii) Calcineurin B myristoylation is required for the Ca(2+)-sensitive binding of calcineurin to phosphatidylserine vesicles in vitro.     

拉萨河中上游夏秋季纤毛虫群落时空变动及其与环境的关系

为揭示中国西藏高原河流浮游纤毛虫群落结构特征及与水环境的关系,于2015—2016年的8月和11月,利用25号浮游生物网,分别在拉萨河中上游共8个代表性采样点,共采集64个水样。物种鉴定采用活体观察和固定染色相结合的方法。共鉴定出纤毛虫91种,夏季49种,各样点物种数由小到大依次为:S2S4S8S5S1S3=S7S6。秋季64种,各样点物种数由小到大依次为:S4S3=S1=S2=S5S8S6=S7。夏季各样点丰度为1.2×10~4—5.6×10~5个/L,秋季各样点丰度在1.2×10~4—2.6×10~5个/L之间。夏、秋季的优势种均为12种且优势种组成与分布不同,表现该流域纤毛虫存在明显的时空差异;群落结构分析显示:纤毛虫群落结构简单,物种组成多样性低而分布均匀;纤毛虫营养功能结构分析表明,夏季B、S类群的物种丰富度低于秋季;相关分析表明,总磷和总氮是影响夏季纤毛虫物种多样性的主要环境因子,并且浊度、NH_4-N和NO_3-N是影响秋季纤毛虫的主要环境因子。