胰岛素样生长因子1降低tau蛋白磷酸化作用机制的研究

目的探讨胰岛素样生长因子1(IGF-1)对6样淀粉蛋白(A6)导致大鼠PC1 2细胞tau蛋白磷酸化的保护作用及其机制。方法实验分为预处理1组、2组、3组、模型组、对照组、保护组和氯化锂组。MTT法观察不同浓度IGF-1对PC12细胞存活率的影响。Western blot法检测Aβ_(1-40)处理后PC12细胞tau蛋白磷酸化、总tau蛋白和糖原合成激酶3β(GSK-36)及GSK-3β Ser~9的表达情况。结果 tau蛋白Ser~(396)、Ser~(199/202)位点磷酸化水平在Aβ_(1-40)诱导3 h开始增高,1 2 h达高峰。与对照组比较,模型组GSK-3β明显升高,GSK-3β Ser~9明显降低(P0.05);与模型组比较,氯化锂组及保护组tau蛋白Ser~(396)、Ser~(199/202)位点磷酸化水平和总tau蛋白明显降低(P0.05);GSK 36明显降低,GSK-3β Ser~9明显升高(P0.05,P0.01)。结论 Aβ_(1-40)主要通过激活GSK-3β使tau蛋白的磷酸化水平增高。IGF-1有抑制GSK-3β的活性,降低Aβ_(1-40)所诱导的PC12细胞tau蛋白过度磷酸化的作用。     

Effect of inhibiting melatonin biosynthesis on spatial memory retention and tau phosphorylation in rat

We have found recently that melatonin protects SH-SY5Y neuroblastoma cells from calyculin A-induced neurofilament impairment and neurotoxicity. In the present study, we further investigated the in vivo effect of inhibiting melatonin biosynthesis on spatial memory retention and tau phosphorylation in rats and the potential underlying mechanisms by using haloperidol, a specific inhibitor of 5-hydroxyindole-O-methyltransferase, and a key enzyme in melatonin biosynthesis. We have found that injection of haloperidol into the lateral ventricle and into peritoneal cavity compromises spatial memory retention of rats and induces hyperphosphorylation of microtubule-associated protein tau at tau-1 (Ser199/Ser202) and PHF-1 (Ser396/Ser404) epitopes. At mean time, the activity of protein phosphatase-2A (PP-2A), a deficit phosphatase in the Alzheimer's disease brain and superoxide dismutase decreases with an elevated level of malondialdehyde. Supplementation with melatonin by prior injection for 1 wk and reinforcement during the haloperidol administration significantly improves memory retention deficits, arrests tau hyperphosphorylation and oxidative stress, and restores PP-2A activity. These results strongly support the involvement of decreased melatonin in Alzheimer-like spatial memory impairment and tau hyperphosphorylation, and PP-2A may play a role in mediating aberrant melatonin-induced lesions.     

Activity-dependent NMDA receptor degradation mediated by retrotranslocation and ubiquitination

The extracellular N-terminal domain (NTD) is the largest region of NMDA receptors; however, biological roles for this ectodomain remain unknown. Here, we determined that the F-box protein, Fbx2, bound to high-mannose glycans of the NR1 ectodomain. F-box proteins specify ubiquitination by linking protein substrates to the terminal E3 ligase. Indeed, ubiquitination of NR1 was increased by Fbx2 and diminished by an Fbx2 dominant-negative mutant. When expressed in hippocampal neurons, this Fbx2 dominant-negative mutant augmented NR1 subunit levels and NMDA receptor-mediated currents in an activity-dependent fashion. These results suggest that homeostatic control of synaptic NR1 involves receptor retrotranslocation and degradation by the ubiquitin-proteasome pathway.     

Akt and CHIP coregulate tau degradation through coordinated interactions

A hallmark of the pathology of Alzheimer's disease is the accumulation of the microtubule-associated protein tau into fibrillar aggregates. Recent studies suggest that they accumulate because cytosolic chaperones fail to clear abnormally phosphorylated tau, preserving a pool of toxic tau intermediates within the neuron. We describe a mechanism for tau clearance involving a major cellular kinase, Akt. During stress, Akt is ubiquitinated and degraded by the tau ubiquitin ligase CHIP, and this largely depends on the Hsp90 complex. Akt also prevents CHIP-induced tau ubiquitination and its subsequent degradation, either by regulating the Hsp90/CHIP complex directly or by competing as a client protein with tau for binding. Akt levels tightly regulate the expression of CHIP, such that, as Akt levels are suppressed, CHIP levels also decrease, suggesting a potential stress response feedback mechanism between ligase and kinase activity. We also show that Akt and the microtubule affinity-regulating kinase 2 (PAR1/MARK2), a known tau kinase, interact directly. Akt enhances the activity of PAR1 to promote tau hyperphosphorylation at S262/S356, a tau species that is not recognized by the CHIP/Hsp90 complex. Moreover, Akt1 knockout mice have reduced levels of tau phosphorylated at PAR1/MARK2 consensus sites. Hence, Akt serves as a major regulator of tau biology by manipulating both tau kinases and protein quality control, providing a link to several common pathways that have demonstrated dysfunction in Alzheimer's disease.     

Postconditioning inhibits mPTP opening independent of oxidative phosphorylation and membrane potential

Mitochondrial permeability transition pore (mPTP) inhibition plays a relevant role in postconditioning (PostC). Ischemia damages the electron transport chain, and the potential contribution of additional modifications in mitochondrial function caused by PostC remains unknown. We sought to determine which mitochondrial functions are involved in the inhibition of mPTP opening during the first minutes of reperfusion. Anesthetized New Zealand White rabbits underwent 30-min ischemia followed by 10-min reperfusion. At reperfusion, they received either no intervention (Control, C), PostC with 4 cycles of 1-min ischemia followed by 1-min reperfusion, or an IV injection of 5 mg/kg cyclosporine A (CsA: a powerful inhibitor of mPTP opening). Sham rabbits underwent no ischemia throughout the 40-min experiment. At the end of the 10-min reperfusion, mitochondria were isolated from the area at risk by differential centrifugations. Calcium retention capacity (CRC) and mitochondrial membrane potential (ΔΨ_m) were assessed by fluorimetry in subsarcolemmal (SSM) and interfibrillar (IFM) mitochondria. Oxidative phosphorylation was assessed using a Clark-type electrode, and oxidative stress via protein carbonylation by Western blotting. PostC and CsA treatments improved CRC when compared to the C group. Control, PostC and CsA mitochondria exhibited a comparable significant dissipation of ΔΨ_m, together with a comparable significant decrease in state 3 and an increase in state 4 respiration, in both SSM and IFM. However, PostC but not CsA treatment reduced total heart oxidative stress. These data suggest that during the early minutes of reperfusion, PostC reduces oxidative stress and inhibits mPTP opening, independent of alteration of oxidative phosphorylation or of ΔΨ_m.     

Increasing Cu bioavailability inhibits Aβ oligomers and tau phosphorylation

Cognitive decline in Alzheimer's disease (AD) involves pathological accumulation of synaptotoxic amyloid-β (Aβ) oligomers and hyperphosphorylated tau. Because recent evidence indicates that glycogen synthase kinase 3β (GSK3β) activity regulates these neurotoxic pathways, we developed an AD therapeutic strategy to target GSK3β. The strategy involves the use of copper-bis(thiosemicarbazonoto) complexes to increase intracellular copper bioavailability and inhibit GSK3β through activation of an Akt signaling pathway. Our lead compound Cu^II(gtsm) significantly inhibited GSK3β in the brains of APP/PS1 transgenic AD model mice. Cu^II(gtsm) also decreased the abundance of Aβ trimers and phosphorylated tau, and restored performance of AD mice in the Y-maze test to levels expected for cognitively normal animals. Improvement in the Y-maze correlated directly with decreased Aβ trimer levels. This study demonstrates that increasing intracellular copper bioavailability can restore cognitive function by inhibiting the accumulation of neurotoxic Aβ trimers and phosphorylated tau.     

α-Synuclein phosphorylation at serine 129 occurs after initial protein deposition and inhibits seeded fibril formation and toxicity

α-Synuclein (α-syn) phosphorylation at serine 129 (pS129–α-syn) is substantially increased in Lewy body disease, such as Parkinson’s disease (PD) and dementia with Lewy bodies (DLB). However, the pathogenic relevance of pS129–α-syn remains controversial, so we sought to identify when pS129 modification occurs during α-syn aggregation and its role in initiation, progression and cellular toxicity of disease. Using diverse aggregation assays, including real-time quaking-induced conversion (RT-QuIC) on brain homogenates from PD and DLB cases, we demonstrated that pS129–α-syn inhibits α-syn fibril formation and seeded aggregation. We also identified lower seeding propensity of pS129–α-syn in cultured cells and correspondingly attenuated cellular toxicity. To build upon these findings, we developed a monoclonal antibody (4B1) specifically recognizing nonphosphorylated S129–α-syn (WT–α-syn) and noted that S129 residue is more efficiently phosphorylated when the protein is aggregated. Using this antibody, we characterized the time-course of α-syn phosphorylation in organotypic mouse hippocampal cultures and mice injected with α-syn preformed fibrils, and we observed aggregation of nonphosphorylated α-syn followed by later pS129–α-syn. Furthermore, in postmortem brain tissue from PD and DLB patients, we observed an inverse relationship between relative abundance of nonphosphorylated α-syn and disease duration. These findings suggest that pS129–α-syn occurs subsequent to initial protein aggregation and apparently inhibits further aggregation. This could possibly imply a potential protective role for pS129–α-syn, which has major implications for understanding the pathobiology of Lewy body disease and the continued use of reduced pS129–α-syn as a measure of efficacy in clinical trials.

Parkinson’s disease (PD) and dementia with Lewy bodies (DLB) are both associated with underlying Lewy body disease, which represents the second most common neurodegenerative disorder after Alzheimer’s disease (1, 2). The neuropathological hallmark of Lewy body disease is the intracellular aggregation of the protein α-synuclein (α-syn) into spherical cytoplasmic inclusions, termed Lewy bodies, but are also observed in neuronal processes as Lewy neurites (LNs) (3).α-Syn is thought to play a central role in the pathobiology of Lewy body disease. Single-point mutations and genetic modifications affecting α-syn expression—through duplications, triplications, or polymorphisms in its promoter—have been linked to both idiopathic and familial forms of Lewy body disease (4–6). Nevertheless, neuropathological studies utilizing pan–α-syn antibodies, recognizing both physiological and pathological forms of the protein, do not consistently report a relationship between the load of Lewy body pathology and clinical disease severity (2). To reconcile the apparent importance of α-syn in Lewy body disease with the difficulty relating Lewy body burdens in the brain to phenotypic severity, continued research has focused on the identification of particularly disease-relevant forms of α-syn. α-Syn undergoes various posttranslational modifications (PTMs)—including acetylation, nitration, ubiquitination, and glycosylation and phosphorylation at serine 129 (pS129)—increases from ∼4% under physiological conditions to 90% in Lewy body disease, suggesting it is associated with the disease state (7–9).Previous studies have reported that pS129 enhances intracellular aggregate formation in SH-SY5Y cells (10), and mediates cell death through activation of the unfolded protein response pathway (11). Furthermore, studies in rodent models have suggested that pS129 exacerbates the rate of pathological protein aggregation and deposition, with subsequent negative effects on neuronal functioning (12). However, these studies are counterbalanced by others reporting a potentially neuroprotective function of phosphorylation in animal models (13, 14) and cellular model systems (15). Additionally, studies have reported neutral findings regarding pS129 modification as neither enhancing nor diminishing cellular toxicity and α-syn aggregation (16, 17). Despite the uncertain pathogenic role of pS129 in Lewy body disease, antibodies against pS129 are widely used, based on the putative view that they label a species of α-syn that is particularly disease-relevant. These studies often employ pS129–α-syn as a marker of the abundance of protein inclusions to stage disease severity and evaluate the relationship between its abundance and important clinical or pathological variables, such as disease duration, phenotypic severity, or cell loss (18). Such studies typically identify that pS129 abundance throughout the brain correlates with disease severity (19–21), though it remains uncertain whether phosphorylation precedes protein aggregation or occurs secondarily to deposition of nonphosphorylated α-syn, and whether pS129 is a key driver of pathogenicity or simply a useful marker of a neurodegenerative process (22, 23). Therefore, although there is a substantial literature on pS129 in Lewy body disease, there is continued controversy regarding its potential contribution to disease states, with numerous studies reporting discordant findings. Despite contradictory findings regarding the disease-relevance of pS129, it is widely viewed as a particularly disease-associated modification, thus necessitating further research to address its importance for Lewy body disease.To address the key questions regarding the pathogenic relevance of pS129–α-syn, the present study aimed to undertake a comprehensive and multidisciplinary project to address this important and pressing question. The key aim of the study was to better understand the role of pS129 in the natural history of Lewy body disease, by determining when pS129 occurs in the development of α-syn aggregates and how it affects the aggregation-propensity and cytotoxicity of α-syn     

RETRACTED ARTICLE: PEDF inhibits VEGF- and EPO- induced angiogenesis in retinal endothelial cells through interruption of PI3K/Akt phosphorylation

Retinal angiogenesis in diabetes may lead to visual impairment and even irreversible blindness in people of working age group worldwide. The main pathological feature of proliferative diabetic retinopathy (PDR) is hypoxia, and overproduction of growth factors like vascular endothelial growth factor (VEGF) and erythropoietin (Epo). This results in pathological proliferation of retinal endothelial cells (RECs), leading to new vessel formation (angiogenesis). Inhibition of angiogenesis is a promising strategy for treatment of PDR and other retinal neovascular disorders. Pigment epithelium-derived factor (PEDF), a 50-kDa protein secreted by retinal pigment epithelium, inhibits the growth of new blood vessel induced in the eye in a variety of ways with a yet elusive mechanism. Here, we investigated the possible mechanism by which PEDF inhibits VEGF- and Epo-induced angiogenic effects in RECs is mediated through PI3K/Akt pathway. PEDF treatment induced the apoptosis in RECs by activating caspase-3 and DNA fragmentation. We found a dose-dependent increase in cell survival with VEGF or Epo, which was attenuated in the presence of PEDF. In addition, PEDF significantly (P < 0.05) inhibited migration and in vitro tube formation in RECs in the presence of VEGF as like PI3K/Akt inhibitor. Of interest, PEDF effectively abrogated VEGF-mediated phosphorylation of PI3K/Akt. Further studies using RECs transfected with constitutively active and dominant-negative forms of Akt suggest that PEDF could inhibit VEGF- and also Epo-induced angiogenesis by disruption of PI3K/Akt signaling.     

M phase phosphorylation of the epigenetic regulator UHRF1 regulates its physical association with the deubiquitylase USP7 and stability

UHRF1 (Ubiquitin-like, with PHD and RING finger domains 1) plays an important role in DNA CpG methylation, heterochromatin function and gene expression. Overexpression of UHRF1 has been suggested to contribute to tumorigenesis. However, regulation of UHRF1 is largely unknown. Here we show that the deubiquitylase USP7 interacts with UHRF1. Using interaction-defective and catalytic mutants of USP7 for complementation experiments, we demonstrate that both physical interaction and catalytic activity of USP7 are necessary for UHRF1 ubiquitylation and stability regulation. Mass spectrometry analysis identified phosphorylation of serine (S) 652 within the USP7-interacting domain of UHRF1, which was further confirmed by a UHRF1 S652 phosphor (S652ph)-specific antibody. Importantly, the S652ph antibody identifies phosphorylated UHRF1 in mitotic cells and consistently S652 can be phosphorylated by the M phase-specific kinase CDK1-cyclin B in vitro. UHRF1 S652 phosphorylation significantly reduces UHRF1 interaction with USP7 in vitro and in vivo, which is correlated with a decreased UHRF1 stability in the M phase of the cell cycle. In contrast, UHRF1 carrying the S652A mutation, which renders UHRF1 resistant to phosphorylation at S652, is more stable. Importantly, cells carrying the S652A mutant grow more slowly suggesting that maintaining an appropriate level of UHRF1 is important for cell proliferation regulation. Taken together, our findings uncovered a cell cycle-specific signaling event that relieves UHRF1 from its interaction with USP7, thus exposing UHRF1 to proteasome-mediated degradation. These findings identify a molecular mechanism by which cellular UHRF1 level is regulated, which may impact cell proliferation.     

Insulin degradation: radioimmunoassay for glutathione-insulin transhy drogenase and its application

Summary A double-antibody radioimmunoassay for the insulin-degrading enzyme, glutathione-insulin transhydrogenase (GIT), has been developed with the use of rabbit antiserum against human liver GIT and [¹²⁵I]-GIT. The method can determine as little as 32 fmol of GIT, thus allowing measurements in needle tissue biopsy samples and in plasma, which have not been possible with previous enzymatic procedures. Relative competition in the radioimmunossay by unlabelled GITs purified from other sources are in agreement with homologies in GITs previously found using the enzymatic assay. No competition was observed with pork insulin, bovine ribonuclease, human albumin or human -globulin, indicating that the radioimmunoassay is highly specific for GIT. Similar competition curves were observed for native GIT; active, reduced GIT; or for the inactive, S-(ethylsuccinimido) derivative of GIT. The radioimmunoassay thus measures total (active + inactive) GIT and permits determinations in the presence of materials which react with the active site and render the enzymatic methods unusable. Radioimmunoassay of plasma and extracts of liver, muscle and adipose tissues from diabetic and non-diabetic subjects showed parallel competition curves with standard purified human GIT indicating that GITs of non-diabetic and diabetic persons are immunologically very similar or identical. Concentrations of GIT in plasma determined by radioimmunoassay were significantly higher in diabetic than those in non-diabetic subjects (1620±80 versus 1070±30 fmol/l, p<0.001). Tissue GIT levels found by the radioimmunoassay as well as by the enzyme assay, both in non-diabetic and diabetic subjects, were highest in the liver, intermediate in the adipose tissue und lowest in the muscle.     

RSK1 drives p27Kip1 phosphorylation at T198 to promote RhoA inhibition and increase cell motility

p90 ribosomal S6 kinase (RSK1) is an effector of both Ras/MEK/MAPK and PI3K/PDK1 pathways. We present evidence that RSK1 drives p27 phosphorylation at T198 to increase RhoA-p27 binding and cell motility. RSK1 activation and p27pT198 both increase in early G₁. As for many kinase–substrate pairs, cellular RSK1 coprecipitates with p27. siRNA to RSK1 and RSK1 inhibition both rapidly reduce cellular p27pT198. RSK1 overexpression increases p27pT198, p27-cyclin D1-Cdk4 complexes, and p27 stability. Moreover, RSK1 transfectants show mislocalization of p27 to cytoplasm, increased motility, and reduced RhoA-GTP, phospho-cofilin, and actin stress fibers, all of which were reversed by shRNA to p27. Phosphorylation by RSK1 increased p27pT198 binding to RhoA in vitro, whereas p27T157A/T198A bound poorly to RhoA compared with WTp27 in cells. Coprecipitation of cellular p27-RhoA was increased in cells with constitutive PI3K activation and increased in early G₁. Thus T198 phosphorylation not only stabilizes p27 and mislocalizes p27 to the cytoplasm but also promotes RhoA-p27 interaction and RhoA pathway inhibition. These data link p27 phosphorylation at T198 and cell motility. As for other PI3K effectors, RSK1 phosphorylates p27 at T198. Because RSK1 is also activated by MAPK, the increased cell motility and metastatic potential of cancer cells with PI3K and/or MAPK pathway activation may result in part from RSK1 activation, leading to accumulation of p27T198 in the cytoplasm, p27:RhoA binding, inhibition of RhoA/Rock pathway activation, and loss of actomyosin stability.     

cAMP-induced phosphorylation of 26S proteasomes on Rpn6/PSMD11 enhances their activity and the degradation of misfolded proteins

Although rates of protein degradation by the ubiquitin-proteasome pathway (UPS) are determined by their rates of ubiquitination, we show here that the proteasome’s capacity to degrade ubiquitinated proteins is also tightly regulated. We studied the effects of cAMP-dependent protein kinase (PKA) on proteolysis by the UPS in several mammalian cell lines. Various agents that raise intracellular cAMP and activate PKA (activators of adenylate cyclase or inhibitors of phosphodiesterase 4) promoted degradation of short-lived (but not long-lived) cell proteins generally, model UPS substrates having different degrons, and aggregation-prone proteins associated with major neurodegenerative diseases, including mutant FUS (Fused in sarcoma), SOD1 (superoxide dismutase 1), TDP43 (TAR DNA-binding protein 43), and tau. 26S proteasomes purified from these treated cells or from control cells and treated with PKA degraded ubiquitinated proteins, small peptides, and ATP more rapidly than controls, but not when treated with protein phosphatase. Raising cAMP levels also increased amounts of doubly capped 26S proteasomes. Activated PKA phosphorylates the 19S subunit, Rpn6/PSMD11 (regulatory particle non-ATPase 6/proteasome subunit D11) at Ser14. Overexpression of a phosphomimetic Rpn6 mutant activated proteasomes similarly, whereas a nonphosphorylatable mutant decreased activity. Thus, proteasome function and protein degradation are regulated by cAMP through PKA and Rpn6, and activation of proteasomes by this mechanism may be useful in treating proteotoxic diseases.In mammalian cells, the bulk of cell proteins are degraded by the ubiquitin-proteasome system (UPS) (1, 2). Misfolded proteins, which arise from mutations or postsynthetic damage, and normal proteins with regulatory functions tend to be degraded more rapidly than average cell proteins (2). To be degraded by the UPS, proteins are first modified by ubiquitination (2). In this highly selective process, ubiquitin moieties are conjugated to individual proteins by one of the cell’s many ubiquitin ligases (E3s) (3). Protein ubiquitination is generally assumed to be the rate-limiting step in the degradation pathway, and once ubiquitinated, proteins are believed to be efficiently hydrolyzed by the 26S proteasome. This 2.5-MDa proteolytic complex is composed of about 60 subunits (3). Proteins are digested within the core 20S proteasome, a hollow cylindrical particle containing three types of peptidase activities: chymotrypsin-like, trypsin-like, and caspase-like (3). This particle can be associated with one or two 19S regulatory particles forming a 26S proteasome (3). The 19S complex serves multiple key functions: it binds the ubiquitinated substrate, removes the ubiquitin chain, unfolds the protein substrate, and translocates it through a narrow gated channel into the 20S particle (3). This multistep process is coupled to ATP hydrolysis by the hexameric ATPase ring at the base of the 19S complex adjacent to the core particle (3, 5). These various steps are tightly coordinated; for example, gate opening into the 20S particle and ATP hydrolysis are activated upon binding of the ubiquitin (Ub) chain to the deubiquitinating enzymes, Usp14 or Uch37 (5, 6).The development of inhibitors of proteasome function have advanced our knowledge of cell regulation and proven very valuable in treating hematological cancers (7). In principle, agents that enhance proteasome function could be valuable in combating the various diseases resulting from the toxic accumulation of misfolded proteins. In the major neurodegenerative diseases [amyotrophic lateral sclerosis (ALS), Alzheimer’s, Parkinson’s, and Huntington’s diseases (8, 9)], aggregation-prone proteins build up, often as protein inclusions that contain Ub and proteasomes (10). One factor that may contribute to the pathogenesis of these diseases is the progressive impairment of the capacity of the UPS to degrade misfolded proteins (11). In fact, several studies of neurodegenerative disease models have suggested that proteasome function is impaired when these misfolded proteins (e.g., huntingtin aggregates, mutant tau, or PrP^Sc prions) accumulate in cells (11–14).A number of postsynthetic modifications of 26S proteasome subunits have been reported, including O-GlcNAc modification (15), ADP ribosylation (16), and especially phosphorylation (17–19). The subunit phosphorylation may influence the localization (20), activity (17), and formation (18, 21) of the 26S proteasome. For example, phosphorylation of one of the 19S ATPases, Rpt6, in neurons by Ca²⁺/calmodulin-dependent protein kinase II (CaMKII), has been reported to cause proteasome entry into dendrites and promote synaptic plasticity (22, 23). In addition, phosphorylation of Rpt6 by cAMP-dependent proteins kinase (PKA) was reported to increase proteasome activity against small peptides (17, 24, 25). However, the effects of this modification on the proteasome’s capacity to degrade ubiquitin conjugates and on protein degradation in cells were not examined. Although raising cAMP levels and phosphorylation by PKA alter many cellular functions, effects on protein breakdown by the UPS have not been reported, aside from a suppression of overall proteolysis in skeletal muscle (26). Here we demonstrate that PKA directly phosphorylates the 19S subunit Rpn6/PSMD11 (regulatory particle non-ATPase 6/proteasome subunit D11), and that this modification stimulates several key proteasomal processes and enhances its capacity to degrade ubiquitinated proteins. As a result, pharmacological agents that raise cAMP levels and activate PKA promote the breakdown of short-lived cell proteins by the ubiquitin proteasome pathway, and can accelerate the degradation of aggregation-prone proteins that cause major neurodegenerative diseases.     

老年不稳定型心绞痛患者纤维蛋白、纤维蛋白原及其降解产物的变化

目的探讨凝血系统在老年人不稳定型心绞痛（ＵＡ）发生和发展中的作用。方法采用一组抗纤维蛋白单克隆抗体（ＳＺ－５８、６４、６５）酶免疫分析法测定２６例老年ＵＡ患者、２４例稳定型心绞痛（ＳＡ）患者和２０例健康老年人（对照组）血浆纤维蛋白原（Ｆｇ）、可溶性纤维蛋白复合物（ＳＦＣ）及血清纤维蛋白降解产物（ＦＤＰ）的浓度。结果ＵＡ患者心绞痛发作时血浆Ｆｇ、ＳＦＣ及血清ＦＤＰ浓度（分别为３．７±０．６ｇ／Ｌ、４９．６±１９．３ｍｇ／Ｌ、３２５．６±７９．４μｇ／Ｌ）显著高于ＳＡ患者心绞痛发作时（分别为３．２±０．６ｇ／Ｌ、２０．９±１０．４ｍｇ／Ｌ、２２４．４±４７．４μｇ／Ｌ）和健康对照组（均为Ｐ＜０．０１），后两者间差异无显著性。ＵＡ发作终止后血浆Ｆｇ和血清ＦＤＰ浓度高于健康对照组，但差异无显著性。结论血栓形成是老年人ＵＡ发生和发展的重要因素之一。     

Phosphorylation of Hdmx mediates its Hdm2- and ATM-dependent degradation in response to DNA damage

Maintenance of genomic stability depends on the DNA damage response, an extensive signaling network that is activated by DNA lesions such as double-strand breaks (DSBs). The primary activator of the mammalian DSB response is the nuclear protein kinase ataxia-telangiectasia, mutated (ATM), which phosphorylates key players in various arms of this network. The activation and stabilization of the p53 protein play a major role in the DNA damage response and are mediated by ATM-dependent posttranslational modifications of p53 and Mdm2, a ubiquitin ligase of p53. p53's response to DNA damage also depends on Mdm2-dependent proteolysis of Mdmx, a homologue of Mdm2 that represses p53's transactivation function. Here we show that efficient damage-induced degradation of human Hdmx depends on functional ATM and at least three sites on the Hdmx that are phosphorylated in response to DSBs. One of these sites, S403, is a direct ATM target. Accordingly, each of these sites is important for Hdm2-mediated ubiquitination of Hdmx after DSB induction. These results demonstrate a sophisticated mechanism whereby ATM fine-tunes the optimal activation of p53 by simultaneously modifying each player in the process.     

老年2型糖尿病患者血清外泌体中神经元特异性烯醇化酶和磷酸化tau的表达及其与继发轻度认知功能障碍的相关性

目的 检测老年2型糖尿病(T2DM)患者血清外泌体神经元特异性烯醇化酶(NSE)和磷酸化tau(P-tau)的表达,并探讨其与患者发生轻度认知功能障碍(MCI)的相关性。方法 选取2019年1月至2020年12月于联勤保障部队第921医院就诊的114例老年T2DM患者为研究对象(研究组),另选取同期健康体检者100名为对照组。采用ExoQuick-TC提取研究对象血清源性外泌体。采用酶联免疫吸附测定法(ELISA)检测研究对象血清源性外泌体中NSE和P-tau的表达。使用简易认知状态评价量表(MMSE)和蒙特利尔认知评估量表(MoCA)对研究对象进行认知评估。采用SPSS 19.0统计软件进行数据分析。根据数据类型,分别采用t检验或χ²检验进行组间比较。应用Spearman秩相关分析血清外泌体中NSE及P-tau表达水平与认知受损严重程度的相关性。应用受试者工作特征(ROC)曲线分析血清源性外泌体中NSE和P-tau表达水平对MCI的预测价值。结果 研究组老年T2DM患者出现认知受损的比例高于对照组;MMSE和MoCA分值明显低于对照组;发生MCI的比例高于对照组,差异均有统计学意义(P<0.05)。根据MoCA分值将研究组患者分为MCI组(n=71)和非MCI组(n=43),MCI组血清外泌体中NSE和P-tau表达分别为(13.27±1.61)μg/L和(17.14±2.45)pg/ml,明显高于非MCI组的(10.86±1.43)μg/L和(14.49±2.25)pg/ml(t=5.728,6.154;P<0.05)。相关性分析显示,老年T2DM患者血清外泌体中NSE和P-tau表达水平与MMSE(r=-0.547,-0.562;P<0.05)和MoCA分值(r=-0.583,-0.597;P<0.05)存在负相关。ROC曲线分析结果显示,血清外泌体中NSE表达预测老年T2DM患者发生MCI的曲线下面积(AUC)为0.729,血清外泌体中P-tau表达预测老年T2DM患者发生MCI的AUC为0.741,而两者联合预测老年T2DM患者发生MCI的AUC为0.827,高于NSE和P-tau单个指标的预测价值(t=3.836,3.478;P<0.05)。结论 血清外泌体中高表达的NSE和P-tau与老年T2DM患者MCI的发生密切相关,二者联合的预测价值更高。