Effects of 2,3-diphosphoglyceric acid on the human erythrocyte membrane phosphorylation system

The effect of 2,3-diphosphoglycerate (2,3-P2-glycerate) on the phosphorylation of spectrin in solution by purified membrane cyclic AMP-independent protein kinase and in membrane preparations by the endogenous kinase was investigated. 2,3-P2-Glycerate inhibited spectrin phosphorylation both in solution and in the intact membrane. Kinetic analyses showed that 2,3-P2-glycerate had no effect on the Km for ATP but appeared to lower the Vmax of the reaction. When the effect of 2,3-P2-glycerate was examined in the presence of varying concentrations of spectrin, competitive inhibition kinetics were obtained. Interestingly, low concentrations of 2,3-P2-glycerate were found to effect the release of the membrane kinase from erythrocyte membranes. This release reaction may be related to the ability of 2,3-P2-glycerate to interfere with the interaction between the kinase and spectrin. The data suggest the possibility that the kinase may be bound to spectrin in the erythrocyte membrane. 2,3-P2-glycerate also caused the solubilization of 3-phosphoglyceraldehyde dehydrogenase, but not of cyclic AMP-dependent protein kinase. Taken together, our data indicate that 2,3-P2-glycerate may have a regulatory role in membrane protein phosphorylation and also may regulate the extent of association of the kinase with the membrane.     

Inhibition of protein phosphorylation and induction of protein cross-linking in erythrocyte membranes by diamide

This report presents studies on the effect of diamide on protein phosphorylation in erythrocyte membranes. Diamide, a thiol-oxidizing reagent, nonspecifically inhibits cyclic Amp-dependent and -independent autophosphorylation of red cell memvranes, but not the activity of the solubilized membrane cycle AMP-independent protein kinases. Analysis of diamide-treated membranes by gel electrophoresis indicates that diamide is capable of inducing cross-linking of membrane proteins. The action of diamide, both in the inhibition of membrane autophosphorylation and in the cross-linking of membrane proteins, is very similar to that of Cu2+. o-phenanthroline complex. Our data indicate that diamide inhibits erythrocyte membrane autophosphorylation by perturbing the protein substrates.     

Enhanced Orai1-mediated store-operated Ca2+ channel/calpain signaling contributes to high glucose-induced podocyte injury

Podocyte injury induced by hyperglycemia is the main cause of kidney dysfunction in diabetic nephropathy. However, the underlying mechanism is unclear. Store-operated Ca²⁺ entry (SOCE) regulates a diversity of cellular processes in a variety of cell types. Calpain, a Ca²⁺-dependent cysteine protease, was recently shown to be involved in podocyte injury. In the present study, we sought to determine whether increased SOCE contributed to high glucose (HG)–induced podocyte injury through activation of the calpain pathway. In cultured human podocytes, whole-cell patch clamp indicated the presence of functional store-operated Ca²⁺ channels, which are composed of Orai1 proteins and mediate SOCE. Western blots showed that HG treatment increased the protein abundance of Orai1 in a dose-dependent manner. Consistently, calcium imaging experiments revealed that SOCE was significantly enhanced in podocytes following HG treatment. Furthermore, HG treatment caused overt podocyte F-actin disorganization as well as a significant decrease in nephrin protein abundance, both of which are indications of podocyte injury. These podocyte injury responses were significantly blunted by both pharmacological inhibition of Orai1 using the small molecule inhibitor BTP2 or by genetic deletion of Orai1 using CRISPR-Cas9 lentivirus. Moreover, activation of SOCE by thapsigargin, an inhibitor of Ca²⁺ pump on the endoplasmic/sarcoplasmic reticulum membrane, significantly increased the activity of calpain, which was inhibited by BTP2. Finally, the calpain-1/calpain-2 inhibitor calpeptin significantly blunted the nephrin protein reduction induced by HG treatment. Taken together, our results suggest that enhanced signaling via an Orai1/SOCE/Calpain axis contributes to HG-induced podocyte injury.     

STE20 phosphorylation of AMPK-related kinases revealed by biochemical purifications combined with genetics

We have recently purified mammalian sterile 20 (STE20)–like kinase 3 (MST3) as a kinase for the multifunctional kinases, AMP-activated protein kinase–related kinases (ARKs). However, unresolved questions from this study, such as remaining phosphorylation activities following deletion of the Mst3 gene from human embryonic kidney cells and mice, led us to conclude that there were additional kinases for ARKs. Further purification recovered Ca²⁺/calmodulin-dependent protein kinase kinases 1 and 2 (CaMKK1 and 2), and a third round of purification revealed mitogen-activated protein kinase kinase kinase kinase 5 (MAP4K5) as potential kinases of ARKs. We then demonstrated that MST3 and MAP4K5, both belonging to the STE20-like kinase family, could phosphorylate all 14 ARKs both in vivo and in vitro. Further examination of all 28 STE20 kinases detected variable phosphorylation activity on AMP-activated protein kinase (AMPK) and the salt-inducible kinase 3 (SIK3). Taken together, our results have revealed novel relationships between STE20 kinases and ARKs, with potential physiological and pathological implications.     

不同血清型肉毒毒素受体结合域研究进展

肉毒毒素(botulinum neurotoxin, BoNT)是人类已知毒性最强的蛋白质之一,可以引起肌肉松弛麻痹,严重时可导致死亡。肉毒毒素共分为7种血清型(BoNT/A-BoNT/G),根据氨基酸序列差异可进一步分为40多种亚型。肉毒毒素分子结构由3个基本结构域组成：重链羧基端细胞受体结合域、氨基端的易位域和轻链催化域。在运动神经元表面,受体结合域首先与聚唾液酸神经节苷脂结合,随后与突触囊泡蛋白2或突触囊泡结合蛋白结合形成双受体复合物。每种血清型的受体结合域都必须与其相应受体结合才能发挥作用。肉毒毒素的结构功能及其对宿主的作用一直都是研究热点。近年来,因受体结合域可以促进肉毒毒素与运动神经元膜特异性结合,而成为新的研究方向。本综述将概述不同血清型肉毒毒素与受体结合过程中受体结合域结构变化和结合位点差异。通过分析不同血清型及亚型的序列以及受体结合域结构特征,可以更好地了解细胞受体结合域的序列差异和功能,并为肉毒毒素的治疗策略提供新思路。     

生物启发的细菌表面人造矿物壳:通过仿生矿化保护活细胞

【目的】生物启发的细菌表面仿生矿化人造矿物壳被用于保护活细胞。【方法】将细菌限制在坚固而完整的矿物壳中,有限的物理空间和物质交换使其暂时进行休眠,降低长期保存期间的活力损失以及提高在各种极端环境中的生存能力,并且能够通过酸去除矿物壳而重新激活细菌。【结果】相较于未仿生矿化的细菌(EcN),矿化细菌(EcN@CaCO₃)在32 d的储存实验中活力最高提升262倍;在pH 2.5的强酸环境中存活率提高837倍;在pH 12.0的强碱环境中存活率提高171倍;在80 ℃的高温条件下存活率提高59.1倍;甚至在抗生素溶液中,EcN@CaCO₃中细菌的存活率是EcN的729.7倍。【结论】本研究利用仿生矿化提高了细菌的保存稳定性,使其能在酸刺激下去除涂层恢复活性,也能在极端环境下保留细菌的活力,为微生物在环境生态、食品制造和生物医药等领域的应用提供研究基础。     

大丽轮枝菌甘油-3-磷酸脱氢酶的功能分析

大丽轮枝菌可侵染棉花、马铃薯、番茄等660余种寄主植物,引致黄萎病,造成严重的经济损失。为了深入了解大丽轮枝菌的致病机制,本研究在前期棉花提取物诱导大丽轮枝菌转录组分析的基础上,选择上调差异表达的线粒体甘油-3-磷酸脱氢酶基因VdGut2(VD592＿6958＿Chr4)和非差异表达的胞质甘油-3-磷酸脱氢酶基因VdGpd(VD592＿10256＿Chr2),进行了功能分析。结果表明,两个VdGut2敲除突变体菌株的产孢量较野生型菌株分别下降了32%和41%,病情指数分别下降了70%和51%,菌落生长速率也显著下降;VdGpd过表达菌株产孢量和病情指数较野生型菌株显著下降,但在甘油为唯一碳源的培养基上其菌落生长速率显著上升。因此,VdGut2促进了大丽轮枝菌分生孢子的形成、碳源的利用以及对寄主植物的致病过程,VdGpd抑制了大丽轮枝菌分生孢子的形成和对寄主植物的致病力,却促进了甘油的代谢。