缺眼蛋白的结构、功能与疾病

缺眼蛋白（eyes absent,Eya）是进化上高度保守的转录因子,首次在果蝇眼发育中被发现。后被证明,缺眼蛋白参与多种生物学过程,例如器官发育、先天免疫、DNA损伤修复、光周期、血管生成与肿瘤发生发展等。上述生物学功能与缺眼蛋白具有多种不同生化活性有关,包括转录激活活性、酪氨酸磷酸酶活性和苏氨酸磷酸酶活性。缺眼蛋白不同的生化活性存在于不同的结构域中。本文主要针对缺眼蛋白3个保守的结构域ED、PST、TPM以及它们在发育和疾病中的功能作简要综述。     

染色质结构蛋白变异与人类疾病

层级复杂的三维染色质结构对于细胞命运决定和功能维持所需的多种DNA相关生物学过程的时空调控至关重要,如DNA复制、转录、重组和损伤修复等.三维染色质结构失调导致基因表达异常,被认为是肿瘤或神经发育障碍等多种疾病的主要诱因.本文重点阐述组蛋白及其变体、甲基CpG结合蛋白2在三维染色质高级结构及动态性调节中的作用,总结疾病相关突变对基因功能的影响,探讨肿瘤或神经发育障碍发生发展过程中染色质层面的病理学机制.     

人类驱动结合蛋白及其与某些疾病的相关性

驱动结合蛋白（Kinectin）是真核细胞内生物进化上的保守蛋白质,已发现的功能有（1）与驱动蛋白（Kinesin）相连参与细胞胞吐、胞质运输和有丝分裂等活动;（2）是RhoG蛋白依赖微管的细胞内活动的效应物;（3）是整合素依赖的黏附分子复合体的重要组分;（4）转录延长因子-I通过与驱动结合蛋白结合锚着于内质网,发挥相应的生物学作用。随着研究的深入,发现人类驱动结合蛋白与某些疾病如甲状腺癌、白塞病、再生障碍性贫血、肝细胞癌有一定的相关性。     

缺眼蛋白的结构、功能与疾病北大核心CSCD

缺眼蛋白(eyes absent,Eya)是进化上高度保守的转录因子,首次在果蝇眼发育中被发现。后被证明,缺眼蛋白参与多种生物学过程,例如器官发育、先天免疫、DNA损伤修复、光周期、血管生成与肿瘤发生发展等。上述生物学功能与缺眼蛋白具有多种不同生化活性有关,包括转录激活活性、酪氨酸磷酸酶活性和苏氨酸磷酸酶活性。缺眼蛋白不同的生化活性存在于不同的结构域中。本文主要针对缺眼蛋白3个保守的结构域ED、PST、TPM以及它们在发育和疾病中的功能作简要综述。     

LIM蛋白KyoT2与人类紧密连接蛋白2的相互作用

KyoT是一种LIM结构域蛋白,是以RBP－J为诱饵蛋白通过酵母双杂交系统得到的新分子,KyoT2可以抑制RBP－J介导的转录,以KyoT2为诱饵蛋白,通过酵母双杂交筛选得到了人类紧密连接蛋白2（ZO－2）,的一种新的剪接体ZO－2－i3,序列分析表明,新的剪接体有19个外显子,与已发表序列比较,见ZO－2－i3分子的激酶后区发生了改变,为排除酵母双杂交实验的假阳性,实验首先在酵母中验证KyoT2与ZO－2-i3的体外直接相互作用并得到阳性结果,进而在大肠杆菌中原核表达纯化带有His标签的KyoT2蛋白,使用抗His标签的抗体,通过GST pull-down assay验证KyoT2与ZO－2－i3的体外直接相互作用,也获得阳性结果,并通过酵母实验初步确定了其作用位点,即KyoT2通过LIM2结构域与ZO－2-i3相互作用,本实验验证了KyoT2与Z－2－i3的相互作用,并初步确定其相互作用位点,对探讨KyoT的功能具有重要意义。     

Clusterin结构、功能及其在疾病中的作用

Clusterin(CLU)是一种糖蛋白,在细胞内的存在形式多样,可分为核型、胞质型和分泌型,且相对分子质量大小也因其糖基化和剪切程度不同而差异较大。尽管CLU在哺乳动物组织和体液中普遍存在,但是近年来研究报道显示,CLU主要由肝脏组织表达并分泌,而且生理和病理情况下其表达差异明显。CLU在细胞中的存在形式、分子大小差异较大,使其功能多样,如CLU可通过调控细胞内吞、凋亡等方式参与神经性、纤维化以及代谢疾病的发生发展。本文将概述CLU蛋白的结构特点、基本功能及其在生理和病理过程中的作用和潜在调控机制。     

窖蛋白-3及其与人类肌肉疾病的关系

窖蛋白-3(caveolin-3,Cav-3)是整合在窖上的肌细胞特异性蛋白质。人cav-3基因定位于3p25,其主要突变包括跨膜区的错义突变及支架区的染色体微缺失,所导致的表现型包括肢带型肌营养不良(1imb-girdle muscular dystrophy-1C,LGMD-1C)、杜氏肌营养不良(Duchenne muscular dystrophy,DMD)、自发性和家族性高CK血症(hyper CKemia,HCK)、末端肌病(distal myopathy,DM)和波形肌肉疾病(rippling muscle disease．RMD)等。Cav-3不仅与肌肉营养不良症相关,也是维持心脏正常功能的必要因素。     

小分子热激蛋白与人类疾病的关系

小分子热激蛋白具有分子伴侣功能,它可以抑制应激诱发的变性蛋白质的聚合,并在条件适宜时促进变性的蛋白质复性,从而维持蛋白质的正常结构与功能,防止疾病的发生。但当其受干扰后又可引起细胞功能障碍,与人类多种疾病的发生密切有关,并可能成为疾病治疗的新靶点。     

人类神经Tau蛋白结构与功能研究进展

Ｔａｕ属微管结合蛋白,对神经细胞的生长发育,物质运输及信息传导起着重要作用。该分子不具有明显的二级结构,其生物学功能的调节功能主要是通过磷酸化和去磷酸化来实现。目前的研究表明,早老性痴呆等疾病与Ｔａｕ分子高级结构的异常改变和功能丧失有关。     

核糖体合成调控蛋白RRS1的功能及其与疾病的关系

1999年,Tsuno等在酵母中发现了一个核糖体合成调控蛋白,命名为RRS1。在酵母中,RRS1是一个多功能的蛋白,与核糖体产物因子2相互作用,在25S r RNA成熟过程中起重要作用,并参与核糖体60S大亚基的生物合成。在哺乳动物细胞中,RRS1参与细胞周期中染色体在赤道板的聚集和端粒的聚集。RRS1在亨廷顿病等的发生中发挥着重要作用。我们简要概述RRS1的功能及其与疾病的关系。     

PHD3 Stabilizes the Tight Junction Protein Occludin and Protects Intestinal Epithelial Barrier Function

Prolyl hydroxylase domain proteins (PHDs) control cellular adaptation to hypoxia. PHDs are found involved in inflammatory bowel disease (IBD); however, the exact role of PHD3, a member of the PHD family, in IBD remains unknown. We show here that PHD3 plays a critical role in maintaining intestinal epithelial barrier function. We found that genetic ablation of Phd3 in intestinal epithelial cells led to spontaneous colitis in mice. Deletion of PHD3 decreases the level of tight junction protein occludin, leading to a failure of intestinal epithelial barrier function. Further studies indicate that PHD3 stabilizes occludin by preventing the interaction between the E3 ligase Itch and occludin, in a hydroxylase-independent manner. Examination of biopsy of human ulcerative colitis patients indicates that PHD3 is decreased with disease severity, indicating that PHD3 down-regulation is associated with progression of this disease. We show that PHD3 protects intestinal epithelial barrier function and reveal a hydroxylase-independent function of PHD3 in stabilizing occludin. These findings may help open avenues for developing a therapeutic strategy for IBD.     

Occludin oligomeric assemblies at tight junctions of the blood–brain barrier are altered by hypoxia and reoxygenation stress

Hypoxic (low oxygen) and reperfusion (post‐hypoxic reoxygenation) phases of stroke promote an increase in microvascular permeability at tight junctions (TJs) of the blood–brain barrier (BBB) that may lead to cerebral edema. To investigate the effect of hypoxia (Hx) and reoxygenation on oligomeric assemblies of the transmembrane TJ protein occludin, rats were subjected to either normoxia (Nx, 21% O₂, 60 min), Hx (6% O₂, 60 min), or hypoxia/reoxygenation (H/R, 6% O₂, 60 min followed by 21% O₂, 10 min). After treatment, cerebral microvessels were isolated, fractionated by detergent‐free density gradient centrifugation, and occludin oligomeric assemblies associated with plasma membrane lipid rafts were solubilized by perfluoro‐octanoic acid (PFO) exclusively as high molecular weight protein complexes. Analysis by non‐reducing and reducing sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis/western blot of PFO‐solubilized occludin revealed that occludin oligomeric assemblies co‐localizing with ‘TJ‐associated’ raft domains contained a high molecular weight ‘structural core’ that was resistant to disassembly by either SDS or a hydrophilic reducing agent ex vivo, and by Hx and H/R conditions in vivo. However, exposure of PFO‐solubilized occludin oligomeric assemblies to SDS ex vivo revealed the non‐covalent association of a significant amount of dimeric and monomeric occludin isoforms to the disulfide‐bonded inner core, and dispersal of these non‐covalently attached occludin subunits to lipid rafts of higher density in vivo was differentially promoted by Hx and H/R. Our data suggest a model of isoform interaction within occludin oligomeric assemblies at the BBB that enables occludin to simultaneously perform a structural role in inhibiting paracellular diffusion, and a signaling role involving interactions of dimeric and monomeric occludin isoforms with a variety of regulatory molecules within different plasma membrane lipid raft domains.     

Occludin oligomeric assembly at tight junctions of the blood-brain barrier is disrupted by peripheral inflammatory hyperalgesia

Tight junctions (TJs) at the blood-brain barrier (BBB) dynamically alter paracellular diffusion of blood-borne substances from the peripheral circulation to the CNS in response to external stressors, such as pain, inflammation, and hypoxia. In this study, we investigated the effect of lambda-carrageenan-induced peripheral inflammatory pain (i.e., hyperalgesia) on the oligomeric assembly of the key TJ transmembrane protein, occludin. Oligomerization of integral membrane proteins is a critical step in TJ complex assembly that enables the generation of tightly packed, large multiprotein complexes capable of physically obliterating the interendothelial space to inhibit paracellular diffusion. Intact microvessels isolated from rat brains were fractionated by detergent-free density gradient centrifugation, and gradient fractions were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis/ Western blot. Injection of lambda-carrageenan into the rat hind paw produced after 3 h a marked change in the relative amounts of oligomeric, dimeric, and monomeric occludin isoforms associated with different plasma membrane lipid raft domains and intracellular compartments in endothelial cells at the BBB. Our findings suggest that increased BBB permeability (i.e., leak) associated with lambda-carrageenan-induced peripheral inflammatory pain is promoted by the disruption of disulfide-bonded occludin oligomeric assemblies, which renders them incapable of forming an impermeant physical barrier to paracellular transport.     

Occludin is overexpressed in Alzheimer's disease and vascular dementia

The tight junctions (TJs) are key players in the control of blood-brain barrier (BBB) properties, the most complex TJs in the vascular system being found in the endothelial cells of brain capillaries. One of the main TJs proteins is occludin, which anchors plasma membranes of neighbour cells and is present in large amounts in the brain endothelia. Previous studies demonstrated that disruption of BBB in various pathological situations associates with changes in occludin expression, and this change could be responsible for malfunction of BBB. Therefore in this study, applying an immunohistochemical approach, we decided to explore the occludin expression in frontal cortex (FC) and basal ganglia in ageing control, Alzheimer's disease (AD), and vascular dementia (VD) brains, as far as all these pathologies associate microangiopathy and disruption of BBB. Strikingly, we found selected neurons, astrocytes and oligodendrocytes expressing occludin, in all cases studied. To estimate the number of occludin-expressing neurons, we applied a stereological approach with random systematic sampling and the unbiased optical fractionator method. We report here a significant increase in ratio of occludin-expressing neurons in FC and basal ganglia regions in both AD and VD as compared to ageing controls. Within the cerebral cortex, occludin was selectively expressed by pyramidal neurons, which are the ones responsible for cognitive processes and affected by AD pathology. Our findings could be important in unravelling new pathogenic pathways in dementia disorders and new functions of occludin and TJs.     

四君子汤对溃疡性结肠炎小鼠模型occludin和claudin-1表达的影响

目的:探讨四君子汤对溃疡性结肠炎(UC)小鼠模型结肠粘膜中occludin、claudin-1表达的影响。方法:采用右旋葡聚糖硫酸钠(DSS)诱导UC小鼠模型,实验分为五组,即正常组、模型组、四君子汤低剂量治疗组、中剂量治疗组、高剂量治疗组、西药组,共治疗7天。对小鼠肠黏膜的大体形态和组织病理变化进行观察,使用RT-PCR和Western blot检测occludin、claudin-1 m RNA和蛋白的表达。结果:与模型组相比较,四君子汤低、中、高剂量治疗组以及西药组小鼠的饮食、体重、精神、活动度、脓血便等一般情况有所改善,黏膜层缺损、隐窝破坏、炎症细胞浸润等病理表现有所缓解。与模型组相比较,高剂量治疗组小鼠结肠组织中occludin、claudin-1蛋白和m RNA的表达升高(P0.05),低剂量和中剂量治疗组也有不同程度的升高。与西药组相比较,低、中、高剂量治疗组小鼠结肠组织中occludin、claudin-1蛋白和m RNA的表达无统计学差异(P0.05)。结论:四君子汤可以改善脓血便等症状,缓解肠粘膜的损伤,上调occludin和claudin-1的表达,对UC小鼠有治疗作用。     

Platelet endothelial cell adhesion molecule‐1 (PECAM1) plays a critical role in the maintenance of human vascular endothelial barrier function

Cardiovascular endothelial barrier dysfunction is associated with a number of cardiovascular diseases. This study aims to investigate the role of platelet endothelial cell adhesion molecule‐1 (PECAM1) in the maintenance of the vascular endothelial barrier integrate. Human umbilical vein endothelial cells (HUVECs) were cultured into monolayers using as an in vitro model to assess the endothelial barrier function. Knockdown of the gene of PECAM1 markedly reduced the transendothelial resistance and increased the permeability of the HUVEC monolayers. From the wild HUVECs, we detected a complex of PECAM1, claudin1, occluding and endothelial cell selective adhesion molecule (ESAM); such a complex was not detected in the PECAM1‐deficient HUVECs. Knockdown of either claudin1, or occludin, or ESAM, did not affect the formation of the tight junction (TJ) complex. Exposure to recombinant interleukin (IL)‐13 inhibited the expression of PECAM1 and down‐regulated the HUVEC monolayer barrier function. PECAM1 plays an important role in the formation of TJ complex. Copyright © 2015 John Wiley & Sons, Ltd.     

Cytoplasmic Dynein and Dynactin Are Required for the Transport of Microtubules into the Axon

Previous work from our laboratory suggested that microtubules are released from the neuronal centrosome and then transported into the axon (Ahmad, F.J., and P.W. Baas. 1995. J. Cell Sci. 108: 2761–2769). In these studies, cultured sympathetic neurons were treated with nocodazole to depolymerize most of their microtubule polymer, rinsed free of the drug for a few minutes to permit a burst of microtubule assembly from the centrosome, and then exposed to nanomolar levels of vinblastine to suppress further microtubule assembly from occurring. Over time, the microtubules appeared first near the centrosome, then dispersed throughout the cytoplasm, and finally concentrated beneath the periphery of the cell body and within developing axons. In the present study, we microinjected fluorescent tubulin into the neurons at the time of the vinblastine treatment. Fluorescent tubulin was not detected in the microtubules over the time frame of the experiment, confirming that the redistribution of microtubules observed with the experimental regime reflects microtubule transport rather than microtubule assembly. To determine whether cytoplasmic dynein is the motor protein that drives this transport, we experimentally increased the levels of the dynamitin subunit of dynactin within the neurons. Dynactin, a complex of proteins that mediates the interaction of cytoplasmic dynein and its cargo, dissociates under these conditions, resulting in a cessation of all functions of the motor tested to date (Echeverri, C.J., B.M. Paschal, K.T. Vaughan, and R.B. Vallee. 1996. J. Cell Biol. 132: 617–633). In the presence of excess dynamitin, the microtubules did not show the outward progression but instead remained near the centrosome or dispersed throughout the cytoplasm. On the basis of these results, we conclude that cytoplasmic dynein and dynactin are essential for the transport of microtubules from the centrosome into the axon.     

添加谷氨酰胺的肠内营养对急性重症胰腺炎大鼠肠上皮细胞间紧密结合蛋白的影响

目的探讨早期肠内营养中应用谷氨酰胺(glutine,Gln)对急性重症胰腺炎(severe acute pancreatitis,SAP)大鼠肠上皮细胞间紧密结合蛋白的作用。方法:雄性SD大鼠89只,随机分成对照组,SAP+PN(肠外营养)组,SAP+EN(肠内营养)组,SAP+EN+Gln,各组又分为4 d喂养组和7 d喂养组。分别在第4 d、第7 d剖杀大鼠检测各组各项指标。免疫组化法检测小肠组织中occludin蛋白的表达。结果:(1)空肠黏膜蛋白质含量测定:各EN组粘膜蛋白质含量显著高于PN组(p<0.01),EN+Gln组的4 d、7 d分别高于EN组的4 d、7 d(p<0.01)。(2)小肠粘膜occludin蛋白表达及含量测定:PN7 d、4 d组的平均灰度值均显著高于EN、EN+Gln组(p<0.01),EN4 d、7 d组平均灰度均显著高于EN+Gln组(p<0.05)。结论:肠内营养中应用谷氨酰胺能更有效增加occludin蛋白表达,改善肠上皮紧密连接,维护肠上皮屏障完整性。     

Maintenance of the macromolecular barrier at cell extrusion sites in intestinal epithelium: Physiological rearrangement of tight junctions

Summary All epithelia slough dying cells but the consequences of this physiological process to epithelial barrier functions is unknown. In mammalian small intestine absorptive cells are known to migrate from the villus base to the villus tip from which they slough. These villus tip extrusion zones are often envisioned as sites at which macromolecules could leak across the epithelium. However, only trace amounts of macromolecules cross this epithelium even though, based on known epithelial turnover rates, extrusion events occur millions of times daily. Here, we examine the characteristics of the epithelial barrier to macromolecular permeation at villus tip extrusion zones in rats and hamsters. Freeze-fracture, light and electron microscope studies reveal that extruding cells do not leave transient holes behind as they lift from the epithelium. Rather, as cells extrude, processes of adjacent cells extend under them. Moreover, tight junction elements proliferate between extruding cells and their neighbors and appear to move down the lateral margin of the extruding cell as it extends into the lumen. These observations suggest that newly formed junctional elements zipper the epithelium closed as extrusion proceeds thus preventing epithelial discontinuities from occurring. Correlative in vivo perfusion experiments using horseradish peroxidase as a macromolecular-tracershow that the above described dynamic alterations in tight junctions at extrusion sites are generally sufficient to prevent transepithelial leaks of macromolecules.