小泛素样修饰蛋白特异性蛋白酶1介导糖尿病性血管内皮细胞损伤修复的研究

目的利用小泛素相关修饰体(SUMO)特异性蛋白酶1(SENP1)解离过氧化物酶体增殖物激活受体γ辅激活因子1α(PGC-1α)的小泛素样修饰蛋白1(SUMO1)修饰。方法人脐静脉内皮细胞培养传代后分对照组、高糖组及SENP1组;Western blot法检测SENP1、SUMO1、PGC-1α和半胱氨酸天冬氨酸蛋白酶3(Caspase-3)水平;实时荧光定量PCR检测线粒体转录因子A(TFAM)、核呼吸因子2α(NRF-2α)和雌激素受体相关受体α(ERRα)的mRNA表达;ELISA测定乳酸脱氢酶(LDH);细胞划痕愈合方法、Transwell方法和体外血管模拟形成实验分别检测细胞自愈、迁移和形成血管拟态的能力。结果与对照组比较,高糖组共价SUMO1、PGC-1α和活化Caspase-3蛋白表达明显升高,SENP1蛋白表达明显降低,TFAM、NRF-2α和ERRα的mRNA表达明显降低,细胞划痕修复能力及模拟血管形成能力下降(P<0.05,P<0.01)。与高糖组比较,SENP1组SENP1蛋白表达明显升高,共价SUMO1、PGC-1α和活化Caspase-3蛋白表达明显降低,TFAM、NRF-2α和ERRα的mRNA表达明显升高,细胞划痕修复和迁移能力及模拟血管形成能力明显改善(P<0.05,P<0.01)。对照组、高糖组和SENP1组细胞上清液中LDH水平比较,差异有统计学意义[(24.66±6.39)ng/ml vs(302.45±30.54)ng/ml vs(174.08±21.03)ng/ml,P<0.01]。结论SENP1能够诱导PGC-1α发生去SUMO修饰,解除其对PGC-1α下游转录因子的抑制作用,改善线粒体功能,抑制高糖诱导的血管内皮细胞功能损伤作用。     

SENP5, a SUMO isopeptidase,induces apoptosis and cardiomyopathy

Cardiomyopathy presents a major health issue and is a leading cause of heart failure. Although a subset of familial cardiomyopathy is associated with genetic mutations, over 50% of cardiomyopathy is defined as idiopathic, the mechanisms underlying which are under intensive investigation. SUMO conjugation is a dynamic posttranslational modification that can be readily reversed by the activity of sentrin-specific proteases (SENPs). However, whether SENPs are implicated in heart disease pathophysiology remains unexplored. We observed a significant increase in the level of SENP5, a SUMO isopeptidase, in human idiopathic failing hearts. To reveal whether it plays a role in the pathogenesis of cardiac muscle disorders, we used a gain-of-function approach to overexpress SENP5 in murine cardiomyocytes (SENP5 transgenic, SENP5-Tg). Overexpression of SENP5 led to cardiac dysfunction, accompanied by decreased cardiomyocyte proliferation and elevated apoptosis. The increase in apoptosis preceded other detectable pathological changes, suggesting its causal link to cardiomyopathy. Further examination of SENP5-Tg hearts unveiled a decrease in SUMO attachment to dynamin related protein (Drp1), a factor critical for mitochondrial fission. Correspondingly, the mitochondria of SENP5-Tg hearts at an early developmental stage were significantly larger compared with those in the control hearts, suggesting that desumoylation of Drp1 at least partially accounts for the cardiac phenotypes observed in the SENP5-Tg mice. Finally, overexpression of Bcl2 in SENP5-Tg hearts improved cardiac function of SENP5-Tg mice, further supporting the notion that SENP5 mainly targets mitochondrial function in vivo. Our findings demonstrate an important role of the desumoylation enzyme SENP5 in the development of cardiac muscle disorders, and point to the SUMO conjugation pathway as a potential target in the prevention/treatment of cardiomyopathy. This article is part of a Special Issue entitled "Mitochondria: From Basic Mitochondrial Biology to Cardiovascular Disease".     

Enhanced desumoylation in murine hearts by overexpressed SENP2 leads to congenital heart defects and cardiac dysfunction

Sumoylation is a posttranslational modification implicated in a variety of cellular activities, and its role in a number of human pathogeneses such as cleft lip/palate has been well documented. However, the importance of the SUMO conjugation pathway in cardiac development and functional disorders is newly emerging. We previously reported that knockout of SUMO-1 in mice led to congenital heart diseases (CHDs). To further investigate the effects of imbalanced SUMO conjugation on heart development and function and its underlying mechanisms, we generated transgenic (Tg) mice with cardiac-specific expression of SENP2, a SUMO-specific protease that deconjugates sumoylated proteins, to evaluate the impact of desumoylation on heart development and function. Overexpression of SENP2 resulted in premature death of mice with CHDs-atrial septal defects (ASDs) and/or ventricular septal defects (VSDs). Immunobiochemistry revealed diminished cardiomyocyte proliferation in SENP2-Tg mouse hearts compared with that in wild type (WT) hearts. Surviving SENP2-Tg mice showed growth retardation, and developed cardiomyopathy with impaired cardiac function with aging. Cardiac-specific overexpression of the SUMO-1 transgene reduced the incidence of cardiac structural phenotypes in the sumoylation defective mice. Moreover, cardiac overexpression of SENP2 in the mice with Nkx2.5 haploinsufficiency promoted embryonic lethality and severity of CHDs, indicating the functional interaction between SENP2 and Nkx2.5 in vivo. Our findings indicate the indispensability of a balanced SUMO pathway for proper cardiac development and function. This article is part of a Special Issue entitled 'Post-translational Modification SI'.     

C2株蓝氏贾第鞭毛虫SUMO特异性蛋白酶基因的克隆、生物信息学分析及其催化活性区的原核表达

目的 克隆C2株蓝氏贾第鞭毛虫(Giardia lamblia,简称贾第虫)的SUMO-Specific Protease(SENP)基因,并对其序列进行生物信息学分析,原核表达贾第虫SENP的催化活性区。方法 提取C2株贾第虫基因组DNA,以基因组DNA为模板获得SENP编码区全长片段,连入克隆载体pGM-T,测序后进行生物信息学分析;根据分析结果克隆SENP的催化活性区,构建其原核表达载体pET-28a(+)-SENPc,在E.coli Rosetta(DE3)中诱导表达,SDS-PAGE及Western blot观察表达结果。结果 成功克隆了C2株贾第虫SENP编码区全长序列,生物信息学分析显示C2株贾第虫SENP蛋白序列与WB株相同,二级结构以无规则卷曲为主,其催化活性区位于126-497aa,被一段插入序列分割成两个部分;构建了SENP催化活性区原核表达载体并在大肠杆菌中高效表达,在相对分子量约43 kD的位置出现目的 蛋白条带,与理论值相符。结论 成功克隆了贾第虫SENP基因并原核表达了其催化活性区,为贾第虫SENP蛋白功能的研究提供了基础。     

The small ubiquitin‐like modifier mediates the resistance of prosthesis‐loosening fibroblast‐like synoviocytes against fas‐induced apoptosis

Objective

To study the expression of small ubiquitin‐like modifier 1 (SUMO‐1) in aseptic loosening of prosthesis implants and to investigate its role in regulating the susceptibility of prosthesis‐loosening fibroblast‐like synoviocytes (FLS) to Fas‐induced apoptosis.

Methods

Specimens of aseptically loosened tissue were obtained at revision surgery, and the expression of SUMO‐1 was analyzed by in situ hybridization. SUMO‐1 levels in FLS were determined by quantitative polymerase chain reaction and Western blot analysis. Immunohistochemistry and confocal microscopy were used to study the subcellular localization of SUMO‐1. The functional role of SUMO‐1 in Fas‐induced apoptosis of prosthesis‐loosening FLS was investigated by small interfering RNA–mediated knockdown of SUMO‐1 and by gene transfer of the nuclear SUMO‐specific protease SENP1.

Results

SUMO‐1 was expressed strongly in aseptically loosened tissue and was found prominently at sites adjacent to bone. Prosthesis‐loosening FLS expressed levels of SUMO‐1 similar to the levels expressed by rheumatoid arthritis (RA) FLS, with SUMO‐1 being found mainly in promyelocytic leukemia protein nuclear bodies. Knockdown of SUMO‐1 had no effect on spontaneous apoptosis but significantly increased the susceptibility of prosthesis‐loosening FLS to Fas‐induced apoptosis. Gene transfer of the nuclear SUMO‐specific protease SENP1 reverted the apoptosis‐inhibiting effects of SUMO‐1.

Conclusion

These data suggest that SUMO‐1 is involved in the activation of both RA FLS and prosthesis‐loosening FLS by preventing these cells from undergoing apoptosis. Modification of nuclear proteins by SUMO‐1 contributes to the antiapoptotic effects of SUMO‐1 in prosthesis‐loosening FLS, providing evidence for the specific activation of sumoylation during their differentiation. Therefore, SUMO‐1 may be an interesting target for novel strategies to prevent aseptic prosthesis loosening.

     

Phosphoinositide isoforms determine compartment-specific ion channel activity

Phosphoinositides serve as address labels for recruiting peripheral cytoplasmic proteins to specific subcellular compartments, and as endogenous factors for modulating the activity of integral membrane proteins. Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) is a plasma-membrane (PM)-specific phosphoinositide and a positive cofactor required for the activity of most PM channels and transporters. This requirement for phosphoinositide cofactors has been proposed to prevent PM channel/transporter activity during passage through the biosynthetic/secretory and endocytic pathways. To determine whether intracellularly localized channels are similarly "inactivated" at the PM, we studied PIP(2) modulation of intracellular TRPML1 channels. TRPML1 channels are primarily localized in lysosomes, but can also be detected temporarily in the PM upon lysosomal exocytosis. By directly patch-clamping isolated lysosomes, we previously found that lysosomal, but not PM-localized, TRPML1 is active with PI(3,5)P(2), a lysosome-specific PIP(2), as the underlying positive cofactor. Here we found that "silent" PM-localized TRPML1 could be activated by depleting PI(4,5)P(2) levels and/or by adding PI(3,5)P(2) to inside-out membrane patches. Unlike PM channels, surface-expressed TRPML1 underwent a unique and characteristic run-up upon patch excision, and was potently inhibited by a low micromolar concentration of PI(4,5)P(2). Conversely, depletion of PI(4,5)P(2) by either depolarization-induced activation or chemically induced translocation of 5'-phosphatase potentiated whole-cell TRPML1 currents. PI(3,5)P(2) activation and PI(4,5)P(2) inhibition of TRPML1 were mediated by distinct basic amino acid residues in a common PIP(2)-interacting domain. Thus, PI(4,5)P(2) may serve as a negative cofactor for intracellular channels such as TRPML1. Based on these results, we propose that phosphoinositide regulation sets compartment-specific activity codes for membrane channels and transporters.     

TREK-1 K(+) channels in the cardiovascular system: their significance and potential as a therapeutic target

Potassium (K(+) ) channels are important in cardiovascular disease both as drug targets and as a cause of underlying pathology. Voltage-dependent K(+) (K(V) ) channels are inhibited by the class III antiarrhythmic agents. Certain vasodilators work by opening K(+) channels in vascular smooth muscle cells (VSMCs), and K(+) channel activation may also be a route to improving endothelial function. The two-pore domain K(+) (K(2P) ) channels form a group of 15 known channels with an expanding list of functions in the cardiovascular system. One of these K(2P) channels, TREK-1, is the focus of this review. TREK-1 channel activity is tightly regulated by intracellular and extracellular pH, membrane stretch, polyunsaturated fatty acids (PUFAs), temperature, and receptor-coupled second messenger systems. TREK-1 channels are also activated by volatile anesthetics and some neuroprotectant agents, and they are inhibited by selective serotonin reuptake inhibitors (SSRIs) as well as amide local anesthetics. Some of the clinical cardiovascular effects and side effects of these drugs may be through their actions on TREK-1 channels. It has recently been suggested that TREK-1 channels have a role in mechano-electrical coupling in the heart. They also seem important in the vascular responses to PUFAs, and this may underlie some of the beneficial cardiovascular effects of the essential dietary fatty acids. Development of selective TREK-1 openers and inhibitors may provide promising routes for intervention in cardiovascular diseases.     

Abscisic acid regulation of guard-cell K+ and anion channels in Gbeta- and RGS-deficient Arabidopsis lines

In mammals, basal currents through G protein-coupled inwardly rectifying K(+) (GIRK) channels are repressed by Galpha(i/o)GDP, and the channels are activated by direct binding of free Gbetagamma subunits released upon stimulation of Galpha(i/o)-coupled receptors. However, essentially all information on G protein regulation of GIRK electrophysiology has been gained on the basis of coexpression studies in heterologous systems. A major advantage of the model organism, Arabidopsis thaliana, is the ease with which knockout mutants can be obtained. We evaluated plants harboring mutations in the sole Arabidopsis Galpha (AtGPA1), Gbeta (AGB1), and Regulator of G protein Signaling (AtRGS1) genes for impacts on ion channel regulation. In guard cells, where K(+) fluxes are integral to cellular regulation of stomatal apertures, inhibition of inward K(+) (K(in)) currents and stomatal opening by the phytohormone abscisic acid (ABA) was equally impaired in Atgpa1 and agb1 single mutants and the Atgpa1 agb1 double mutant. AGB1 overexpressing lines maintained a wild-type phenotype. The Atrgs1 mutation did not affect K(in) current magnitude or ABA sensitivity, but K(in) voltage-activation kinetics were altered. Thus, Arabidopsis cells differ from mammalian cells in that they uniquely use the Galpha subunit or regulation of the heterotrimer to mediate K(in) channel modulation after ligand perception. In contrast, outwardly rectifying (K(out)) currents were unaltered in the mutants, and ABA activation of slow anion currents was conditionally disrupted in conjunction with cytosolic pH clamp. Our studies highlight unique aspects of ion channel regulation by heterotrimeric G proteins and relate these aspects to stomatal aperture control, a key determinant of plant biomass acquisition and drought tolerance.     

Senp2 expression was induced by chronic glucose stimulation in INS1 cells,and it was required for the associated induction of Ccnd1 and Mafa

Post-translational modification by bonding of small ubiquitin-like modifier (SUMO) peptides influences various cellular functions, and is regulated by SUMO-specific proteases (SENPs). Several proteins have been suggested to have diverse impact on insulin synthesis and secretion through SUMO modification in β cells. However, the role of SUMO modification in β cell mass has not been established. Here, we examined the changes in expression of Senp in INS1 cells and pancreatic islets under diabetes-relevant stress conditions and associated changes in β cell mass. Treatment with 25 mM glucose for 72 h induced Senp2 mRNA expression but not that of Senp1 in INS1 cells. Immunohistochemical staining with anti-SENP2 antibody on human pancreas sections revealed that SENP2 was localized in the nucleus. Moreover, in a patient with type 2 diabetes, SENP2 levels were enhanced, especially in the cytoplasm. Senp2 cytoplasmic levels were also increased in islet cells in obese diabetic mice. Cell number peaked earlier in INS1 cells cultured in high-glucose conditions compared to those cultured in control media. This finding was associated with increased Ccnd1 mRNA expression in high-glucose conditions, and siRNA-mediated Senp2 suppression abrogated it. Mafa expression, unlike Pdx1, was also dependent on Senp2 expression during high-glucose conditions. In conclusion, Senp2 may play a role in β cell mass in response to chronic high-glucose through Cyclin D1 and Mafa.     

Modulation of nucleotide sensitivity of ATP-sensitive potassium channels by phosphatidylinositol-4-phosphate 5-kinase

ATP-sensitive potassium channels (K(ATP) channels) regulate cell excitability in response to metabolic changes. K(ATP) channels are formed as a complex of a sulfonylurea receptor (SURx), a member of the ATP-binding cassette protein family, and an inward rectifier K(+) channel subunit (Kir6.x). Membrane phospholipids, in particular phosphatidylinositol (PI) 4,5-bisphosphate (PIP(2)), activate K(ATP) channels and antagonize ATP inhibition of K(ATP) channels when applied to inside-out membrane patches. To examine the physiological relevance of this regulatory mechanism, we manipulated membrane PIP(2) levels by expressing either the wild-type or an inactive form of PI-4-phosphate 5-kinase (PIP5K) in COSm6 cells and examined the ATP sensitivity of coexpressed K(ATP) channels. Channels from cells expressing the wild-type PIP5K have a 6-fold lower ATP sensitivity (K(1/2), the half maximal inhibitory concentration, approximately 60 microM) than the sensitivities from control cells (K(1/2) approximately 10 microM). An inactive form of the PIP5K had little effect on the K(1/2) of wild-type channels but increased the ATP-sensitivity of a mutant K(ATP) channel that has an intrinsically lower ATP sensitivity (from K(1/2) approximately 450 microM to K(1/2) approximately 100 microM), suggesting a decrease in membrane PIP(2) levels as a consequence of a dominant-negative effect of the inactive PIP5K. These results show that PIP5K activity, which regulates PIP(2) and PI-3,4,5-P(3) levels, is a significant determinant of the physiological nucleotide sensitivity of K(ATP) channels.     

Differential structure of atrial and ventricular KATP: atrial KATP channels require SUR1

The isoform-specific structure of the ATP-sensitive potassium (K(ATP)) channel endows it with differential fundamental properties, including physiological activation and pharmacology. Numerous studies have convincingly demonstrated that the pore-forming Kir6.2 (KCNJ11) and regulatory SUR2A (ABCC9) subunits are essential elements of the sarcolemmal K(ATP) channel in cardiac ventricular myocytes. Using a novel antibody directed against the COOH terminus of SUR1 (ABCC8), we show that this K(ATP) subunit is also expressed in mouse myocardium and is the dominant SUR isoform in the atrium. This suggests differential sarcolemmal K(ATP) composition in atria and ventricles, and, to test this, K(ATP) currents were measured in isolated atrial and ventricular myocytes from wild-type and SUR1(-/-) animals. K(ATP) conductance is essentially abolished in SUR1(-/-) atrial myocytes but is normal in SUR1(-/-) ventricular myocytes. Furthermore, pharmacological properties of wild-type atrial K(ATP) match closely the properties of heterologously expressed SUR1/Kir6.2 channels, whereas ventricular K(ATP) properties match those of heterologously expressed SUR2A/Kir6.2 channels. Collectively, the data demonstrate a previously unappreciated K(ATP) channel heterogeneity: SUR1 is an essential component of atrial, but not ventricular, K(ATP) channels. Differential molecular make-up of the 2 channels underlies differential pharmacology, with important implications when considering sulfonylurea therapy or dissecting the role of cardiac K(ATP) pharmacologically, as well as for understanding of the role of diazoxide in preconditioning.     

Prorenin accumulation and activation in human endothelial cells: importance of mannose 6-phosphate receptors

ACE inhibitors improve endothelial dysfunction, possibly by blocking endothelial angiotensin production. Prorenin, through its binding and activation by endothelial mannose 6-phosphate (M6P) receptors, may contribute to this production. Here, we investigated this possibility as well as prorenin activation kinetics, the nature of the prorenin-activating enzyme, and M6P receptor-independent prorenin binding. Human umbilical vein endothelial cells (HUVECs) were incubated with wild-type prorenin, K/A-2 prorenin (in which Lys42 is mutated to Ala, thereby preventing cleavage by known proteases), M6P-free prorenin, and nonglycosylated prorenin, with or without M6P, protease inhibitors, or angiotensinogen. HUVECs bound only M6P-containing prorenin (K(d) 0.9+/-0.1 nmol/L, maximum number of binding sites [B(max)] 1010+/-50 receptors/cell). At 37 degrees C, because of M6P receptor recycling, the amount of prorenin internalized via M6P receptors was >25 times B(max). Inside the cells, wild-type and K/A-2 prorenin were proteolytically activated to renin. Renin was subsequently degraded. Protease inhibitors interfered with the latter but not with prorenin activation, thereby indicating that the activating enzyme is different from any of the known prorenin-activating enzymes. Incubation with angiotensinogen did not lead to endothelial angiotensin generation, inasmuch as HUVECs were unable to internalize angiotensinogen. Most likely, therefore, in the absence of angiotensinogen synthesis or endocytosis, M6P receptor-mediated prorenin internalization by endothelial cells represents prorenin clearance.     

O2 sensing is preserved in mice lacking the gp91 phox subunit of NADPH oxidase.

The rapid response to hypoxia in the pulmonary artery (PA), carotid body, and ductus arteriosus is partially mediated by O2-responsive K+ channels. K+ channels in PA smooth muscle cells (SMCs) are inhibited by hypoxia, causing membrane depolarization, increased cytosolic calcium, and hypoxic pulmonary vasoconstriction. We hypothesize that the K+ channels are not themselves "O2 sensors" but rather respond to the reduced redox state created by hypoxic inhibition of candidate O2 sensors (NADPH oxidase or the mitochondrial electron transport chain). Both pathways shuttle electrons from donors, down a redox gradient, to O2. Hypoxia inhibits these pathways, decreasing radical production and causing cytosolic accumulation of unused, reduced, freely diffusible electron donors. PASMC K+ channels are redox responsive, opening when oxidized and closing when reduced. Inhibitors of NADPH oxidase (diphenyleneiodonium) and mitochondrial complex 1 (rotenone) both inhibit PASMC whole-cell K+ current but lack the specificity to identify the O2-sensor pathway. We used mice lacking the gp91 subunit of NADPH oxidase [chronic granulomatous disease (CGD) mice] to assess the hypothesis that NADPH oxidase is a PA O2-sensor. In wild-type lungs, gp91 phox and p22 phox subunits are present (relative expression: macrophages > airways and veins > PASMCs). Deletion of gp91 phox did not alter p22 phox expression but severely inhibited activated O2 species production. Nonetheless, hypoxia caused identical inhibition of whole-cell K+ current (in PASMCs) and hypoxic pulmonary vasoconstriction (in isolated lungs) from CGD vs. wild-type mice. Rotenone vasoconstriction was preserved in CGD mice, consistent with a role for the mitochondrial electron transport chain in O2 sensing. NADPH oxidase, though a major source of lung radical production, is not the pulmonary vascular O2 sensor in mice.