The prolyl isomerase Pin1 restores the function of Alzheimer-associated phosphorylated tau protein.

One of the neuropathological hallmarks of Alzheimer's disease is the neurofibrillary tangle, which contains paired helical filaments (PHFs) composed of the microtubule-associated protein tau. Tau is hyperphosphorylated in PHFs, and phosphorylation of tau abolishes its ability to bind microtubules and promote microtubule assembly. Restoring the function of phosphorylated tau might prevent or reverse PHF formation in Alzheimer's disease. Phosphorylation on a serine or threonine that precedes proline (pS/T-P) alters the rate of prolyl isomerization and creates a binding site for the WW domain of the prolyl isomerase Pin1. Pin1 specifically isomerizes pS/T-P bonds and regulates the function of mitotic phosphoproteins. Here we show that Pin1 binds to only one pT-P motif in tau and copurifies with PHFs, resulting in depletion of soluble Pin1 in the brains of Alzheimer's disease patients. Pin1 can restore the ability of phosphorylated tau to bind microtubules and promote microtubule assembly in vitro. As depletion of Pin1 induces mitotic arrest and apoptotic cell death, sequestration of Pin1 into PHFs may contribute to neuronal death. These findings provide a new insight into the pathogenesis of Alzheimer's disease.     

The prolyl isomerase Pin1 is a regulator of p53 in genotoxic response

p53 is activated in response to various genotoxic stresses resulting in cell cycle arrest or apoptosis. It is well documented that DNA damage leads to phosphorylation and activation of p53 (refs 1-3), yet how p53 is activated is still not fully understood. Here we report that DNA damage specifically induces p53 phosphorylation on Ser/Thr-Pro motifs, which facilitates its interaction with Pin1, a member of peptidyl-prolyl isomerase. Furthermore, the interaction of Pin1 with p53 is dependent on the phosphorylation that is induced by DNA damage. Consequently, Pin1 stimulates the DNA-binding activity and transactivation function of p53. The Pin1-mediated p53 activation requires the WW domain, a phosphorylated Ser/Thr-Pro motif interaction module, and the isomerase activity of Pin1. Moreover, Pin1-deficient cells are defective in p53 activation and timely accumulation of p53 protein, and exhibit an impaired checkpoint control in response to DNA damage. Together, these data suggest a mechanism for p53 regulation in cellular response to genotoxic stress.     

The prolyl isomerase Pin1 regulates amyloid precursor protein processing and amyloid-beta production

Neuropathological hallmarks of Alzheimer's disease are neurofibrillary tangles composed of tau and neuritic plaques comprising amyloid-beta peptides (Abeta) derived from amyloid precursor protein (APP), but their exact relationship remains elusive. Phosphorylation of tau and APP on certain serine or threonine residues preceding proline affects tangle formation and Abeta production in vitro. Phosphorylated Ser/Thr-Pro motifs in peptides can exist in cis or trans conformations, the conversion of which is catalysed by the Pin1 prolyl isomerase. Pin1 has been proposed to regulate protein function by accelerating conformational changes, but such activity has never been visualized and the biological and pathological significance of Pin1 substrate conformations is unknown. Notably, Pin1 is downregulated and/or inhibited by oxidation in Alzheimer's disease neurons, Pin1 knockout causes tauopathy and neurodegeneration, and Pin1 promoter polymorphisms appear to associate with reduced Pin1 levels and increased risk for late-onset Alzheimer's disease. However, the role of Pin1 in APP processing and Abeta production is unknown. Here we show that Pin1 has profound effects on APP processing and Abeta production. We find that Pin1 binds to the phosphorylated Thr 668-Pro motif in APP and accelerates its isomerization by over 1,000-fold, regulating the APP intracellular domain between two conformations, as visualized by NMR. Whereas Pin1 overexpression reduces Abeta secretion from cell cultures, knockout of Pin1 increases its secretion. Pin1 knockout alone or in combination with overexpression of mutant APP in mice increases amyloidogenic APP processing and selectively elevates insoluble Abeta42 (a major toxic species) in brains in an age-dependent manner, with Abeta42 being prominently localized to multivesicular bodies of neurons, as shown in Alzheimer's disease before plaque pathology. Thus, Pin1-catalysed prolyl isomerization is a novel mechanism to regulate APP processing and Abeta production, and its deregulation may link both tangle and plaque pathologies. These findings provide new insight into the pathogenesis and treatment of Alzheimer's disease.     

Catalysis of protein folding by prolyl isomerase

Rates of protein folding reactions vary considerably. Some denatured proteins regain the native conformation within milliseconds or seconds, whereas others refold very slowly in the time range of minutes or hours. Varying folding rates are observed not only for different proteins, but can also be detected for single polypeptide species. This originates from the co-existence of fast- and slow-folding forms of the unfolded protein, which regain the native state with different rates. The proline hypothesis provides a plausible explanation for this heterogeneity. It assumes that the slow-folding molecules possess non-native isomers of peptide bonds between proline and another residue, and that crucial steps in the refolding of the slow-folding molecules are limited in rate by the slow reisomerization of such incorrect proline peptide bonds. Recently the enzyme peptidyl-prolyl cis-trans isomerase (PPIase) was discovered and purified from pig kidney. It catalyses efficiently the cis in equilibrium trans isomerization of proline imidic peptide bonds in oligopeptides. Here we show that it also catalyses slow steps in the refolding of a number of proteins of which fast- and slow-folding species have been observed and where it was suggested that proline isomerization was involved in slow refolding. The efficiency of catalysis depends on the accessibility for the isomerase of the particular proline peptide bonds in the refolding protein chain.     

Protein-disulphide isomerase and prolyl isomerase act differently and independently as catalysts of protein folding

Two enzymes are now known that catalyse slow steps in protein folding. Peptidyl-prolyl cis-trans isomerase catalyses the cis-trans isomerization of Xaa-Pro peptide bonds in oligopeptides and during the refolding of several proteins. The other enzyme, protein-disulphide isomerase, accelerates the reactivation of reduced proteins, presumably by catalysis of thiol-disulphide exchange reactions. Recent evidence indicates that the beta-subunit of prolyl 4-hydroxylase, an enzyme involved in collagen biosynthesis, is identical with disulphide isomerase. On the basis of this important finding, it was suggested that disulphide isomerase accelerates protein folding, not by 'reshuffling' incorrect disulphide bonds, but in the same way as prolyl isomerase by catalysing proline isomerization which is known to be important for the folding of collagen and other proteins. Here we show that the catalytic activities of these two enzymes are different. Disulphide isomerase accelerates the reformation of native disulphide bonds during protein reoxidation. We find no evidence that this enzyme can catalyse the isomerization of proline peptide bonds, a reaction efficiently accelerated by prolyl isomerase. When both enzymes are present simultaneously during protein folding, they act independently of one another.     

OGG1 initiates age-dependent CAG trinucleotide expansion in somatic cells

Although oxidative damage has long been associated with ageing and neurological disease, mechanistic connections of oxidation to these phenotypes have remained elusive. Here we show that the age-dependent somatic mutation associated with Huntington's disease occurs in the process of removing oxidized base lesions, and is remarkably dependent on a single base excision repair enzyme, 7,8-dihydro-8-oxoguanine-DNA glycosylase (OGG1). Both in vivo and in vitro results support a 'toxic oxidation' model in which OGG1 initiates an escalating oxidation-excision cycle that leads to progressive age-dependent expansion. Age-dependent CAG expansion provides a direct molecular link between oxidative damage and toxicity in post-mitotic neurons through a DNA damage response, and error-prone repair of single-strand breaks.     

Pin1的细胞定位调控Rb蛋白的磷酸化状态

为了研究Pin1和Rb蛋白的细胞定位、Pin1调控Rb磷酸化的相关机制,本研究构建了慢病毒载体pLVX-Flag-HA-Pin1和Plko.1-shRNA,包装病毒感染人非小细胞肺癌(H1299),免疫印迹检测Pin1蛋白表达水平,采用免疫荧光染色、免疫组化技术研究蛋白的定位.结果表明,沉默Pin1可降低Rb的磷酸化并抑制细胞的增殖;培养的肿瘤细胞和肿瘤组织中,Pin1可定位到细胞质中,而胞质定位的Pin1也可增加Rb的磷酸化.     

Role of duplicate genes in genetic robustness against null mutations

Deleting a gene in an organism often has little phenotypic effect, owing to two mechanisms of compensation. The first is the existence of duplicate genes: that is, the loss of function in one copy can be compensated by the other copy or copies. The second mechanism of compensation stems from alternative metabolic pathways, regulatory networks, and so on. The relative importance of the two mechanisms has not been investigated except for a limited study, which suggested that the role of duplicate genes in compensation is negligible. The availability of fitness data for a nearly complete set of single-gene-deletion mutants of the Saccharomyces cerevisiae genome has enabled us to carry out a genome-wide evaluation of the role of duplicate genes in genetic robustness against null mutations. Here we show that there is a significantly higher probability of functional compensation for a duplicate gene than for a singleton, a high correlation between the frequency of compensation and the sequence similarity of two duplicates, and a higher probability of a severe fitness effect when the duplicate copy that is more highly expressed is deleted. We estimate that in S. cerevisiae at least a quarter of those gene deletions that have no phenotype are compensated by duplicate genes.     

销钉机筒挤出机混合段的混合性能

使用有限元法数值模拟有销钉和无销钉混合段流道内胶料的三维等温流场,用粒子示踪分析方法比较两者的混合性能.研究结果表明,有销钉混合段流道内,销钉局部打乱了胶料的运动轨迹,打散了滞留熔体,6个销钉所在横截面的流场被分成6个区域.料流被迫改变方向重新排列;胶料熔体的速度梯度增加;随着胶料速度梯度的增大,胶料的剪切应力增大,混合指数分布均匀.有销钉混合段的11个截面平均分离尺度小于无销钉混合段的10%.机筒销钉对于分散混合有一定的强化作用,其混合程度高于无销钉结构.     

查尔酮合酶基因在植物防御反应中的调控作用

论述了查尔酮合酶基因(chs)在植物防御反应中与病原物的相互关系,探讨了与查尔酮合酶因诱导表达相关的顺式作用元件和反式作用因子及其DNA-蛋白质间的相互作用,阐明其在植物抗病性防御反应中的重要作用。     

半浮式活塞销噪声产生的机理研究

综合采用有限差分法和基于模态压缩法的多体动力学仿真方法对活塞连杆组进行了考虑活塞销轴承混合润滑的动力学分析.研究了半浮式活塞销噪声的产生机理,并考察了活塞销轴承间隙、最大爆发压力、发动机转速对活塞销噪声的影响.结果表明:活塞销噪声主要来自于活塞销与连杆小头衬套间的碰撞,且活塞销噪声发生在其受到的活塞销座作用力方向的反转时刻附近;若活塞销轴承间隙越大、发动机转速越高,活塞销与小头衬套间的碰撞越激烈,活塞销噪声越大,最大爆发压力对活塞销噪声的影响很小.      

Caveolin-1 在肝纤维化中作用的研究

摘要:目的 通过 10%四氯化碳( CCl₄ )橄榄油溶液,分别诱导野生型小鼠和小窝蛋白-1( Caveolin-1) 敲除小鼠建立肝纤维化模型,比较分析敲除 Caveolin-1 对肝损伤和胶原沉积的影响,揭示其在肝纤维化中的作用。 方法 按剂量 1 μL / g,腹腔注射野生型小鼠和 Caveolin-1 敲除小鼠 10% CCl₄ 橄榄油溶液,2 次 / 周。 注射后,1、3、7、14 和 28 d,分 别处死 5 只小鼠,取血清和肝脏,通过苏木素-伊红( HE) 染色、血清谷丙转氨酶( ALT) 和谷草转氨酶( AST) 水平检测,分析肝脏损伤程度;利用天狼猩红染色、Real-time PCR 及 Western blot 方法,观察肝脏胶原沉积及肝纤维化标志物 α 平滑肌肌动蛋白( α-SMA) 、Ⅰ 型胶原蛋白( collagen-Ⅰ )和Ⅲ 型胶原蛋白( collagen-Ⅲ ) mRNA 及蛋白表达水平, 分析肝纤维化程度。 结果 与野生型小鼠相比,造模后 3 d,敲除小鼠的血清 ALT 和 AST 含量显著升高( P<0. 01) ;3、7 和 14 d,敲除小鼠肝脏坏死面积显著增大( P<0. 01 或 P<0. 05) ;7、14 及 28 d 时,敲除小鼠胶原染色面积明显增多( P<0. 01 或 P<0. 05) ;肝星状细胞活化标志物 α-SMA mRNA 水平在 3 d 和 14 d 时显著升高(P<0. 05),collagen-Ⅰ和collagen-Ⅲ mRNA 在 14 d 时显著上调(P<0. 01);14 d 时,敲除小鼠肝脏 α-SMA、collagen-Ⅰ及 collagen-Ⅲ 蛋白表达水平均显著升高( P<0. 01 或 P<0. 05) 。 结论 Caveolin-1 抑制 CCl₄ 诱导的小鼠肝脏炎性损伤及纤维化。     

具有柱肋阵列与分离式柱肋阵列的冷却通道内流阻与传热实验研究

在常规柱肋阵列冷却结构的基础上对一种新型分离式柱肋阵列冷却结构内的流动和传热特征进行了实验和数值计算研究.在雷诺数Re=8 200~50 500时,对具有新型分离式柱肋阵列的冷却通道和具有常规柱肋阵列的冷却通道内传热和流动压力损失性能进行了对比实验研究.对2种通道内的流动与传热进行了三维稳态数值计算,对比研究了其各自的速度场和局部传热特性,揭示了分离式柱肋阵列强化传热以及降低流动阻力的机理.实验结果表明,在所研究的Re范围内,与常规柱肋阵列通道相比,新型分离式柱肋阵列通道具有更高的平均努塞尔数(Nu)以及更小的阻力损失.     

HIV-1 Nef inhibits ASK1-dependent death signalling providing a potential mechanism for protecting the infected host cell

In vivo infection of lymphatic tissues by the human immunodeficiency virus type 1 (HIV-1) leads to enhanced apoptosis, which prominently involves uninfected bystander cells. Increased killing of such bystander cells is mediated in part through Nef induction of Fas ligand (FasL) expression on the surface of the virally infected T cells. The subsequent interaction of FasL with Fas (CD95) displayed on neighbouring cells, including HIV-1-specific cytotoxic T lymphocytes, may lead to bystander cell killing and thus forms an important mechanism of immune evasion. As HIV-1 also enhances Fas expression on virally infected cells, it is unclear how these hosts avoid rapid cell-autonomous apoptosis mediated through cis ligation of Fas by FasL. Here we show that HIV-1 Nef associates with and inhibits apoptosis signal-regulating kinase 1 (ASK1), a serine/threonine kinase that forms a common and key signalling intermediate in the Fas and tumour-necrosis factor-alpha (TNFalpha) death-signalling pathways. The interaction of Nef with ASK1 inhibits both Fas- and TNFalpha-mediated apoptosis, as well as the activation of the downstream c-Jun amino-terminal kinase. Our findings reveal a strategy by which HIV-1 Nef promotes the killing of bystander cells through the induction of FasL, while simultaneously protecting the HIV-1-infected host cell from these same pro-apoptotic signals through its interference with ASK1 function.     

Loss of autophagy in the central nervous system causes neurodegeneration in mice

Protein quality-control, especially the removal of proteins with aberrant structures, has an important role in maintaining the homeostasis of non-dividing neural cells. In addition to the ubiquitin-proteasome system, emerging evidence points to the importance of autophagy--the bulk protein degradation pathway involved in starvation-induced and constitutive protein turnover--in the protein quality-control process. However, little is known about the precise roles of autophagy in neurons. Here we report that loss of Atg7 (autophagy-related 7), a gene essential for autophagy, leads to neurodegeneration. We found that mice lacking Atg7 specifically in the central nervous system showed behavioural defects, including abnormal limb-clasping reflexes and a reduction in coordinated movement, and died within 28 weeks of birth. Atg7 deficiency caused massive neuronal loss in the cerebral and cerebellar cortices. Notably, polyubiquitinated proteins accumulated in autophagy-deficient neurons as inclusion bodies, which increased in size and number with ageing. There was, however, no obvious alteration in proteasome function. Our results indicate that autophagy is essential for the survival of neural cells, and that impairment of autophagy is implicated in the pathogenesis of neurodegenerative disorders involving ubiquitin-containing inclusion bodies.     

The roles of intracellular protein-degradation pathways in neurodegeneration

Many late-onset neurodegenerative diseases, including Parkinson's disease and Huntington's disease, are associated with the formation of intracellular aggregates by toxic proteins. It is therefore crucial to understand the factors that regulate the steady-state levels of these 'toxins', at both the synthetic and degradation stages. The degradation pathways acting on such aggregate-prone cytosolic proteins include the ubiquitin-proteasome system and macroautophagy. Dysfunction of the ubiquitin-proteasome or macroautophagy pathways might contribute to the pathology of various neurodegenerative conditions. However, enhancing macroautophagy with drugs such as rapamycin could offer a tractable therapeutic strategy for a number of these diseases.