综述与专论: 机械敏感性离子通道TMEM63A在髓鞘形成障碍相关疾病中的作用

跨膜蛋白63A（transmembrane protein 63,TMEM63A）是一种机械敏感性离子通道（mechanosensitive ion channel,MSC）,在髓鞘形成过程中发挥重要作用。TMEM63A于2019年与髓鞘形成低下性脑白质营养不良19型（hypomyelinating leukodystrophy 19,HLD19）相关联,确定为HLD19的致病基因。髓鞘是神经系统中由少突胶质细胞形成的兼具营养轴突和加速动作电位传导的结构,髓鞘形成障碍可表现为髓鞘形成低下、髓鞘囊性化和髓鞘变性。髓鞘中脂质含量丰富,不同脂质参与髓鞘形成、修复和胶质细胞与轴突识别等重要过程。TMEM63A变异导致的HLD19为髓鞘形成低下性疾病。TMEM63A变异可引起渗透压改变,细胞上TMEM63A跨膜蛋白受机械刺激产生电流,从而影响少突胶质细胞分化、成熟,导致髓鞘形成异常;同时,TMEM63A变异也可引起细胞膜脂质的分布异常,影响脂质正常功能,异常的脂质通过参与不同的髓鞘形成环节最终导致了髓鞘形成障碍。     

Theaflavin binds to a druggable pocket of TMEM16A channel and inhibits lung adenocarcinoma cell viability

As a calcium-activated chloride channel regulated by the intracellular Ca²⁺ concentration and membrane potential, TMEM16A has attracted considerable attention and has been proposed as a novel anticancer drug target. We have previously reported that the pocket above the ion conductance pore could be a nonselective inhibitor-binding pocket. However, whether this pocket is druggable remains unexplored. In this study, we performed virtual screening to target the presumed inhibitor-binding pocket and identified a highly effective TMEM16A inhibitor, theaflavin (TF: a tea polyphenol in black tea). Molecular dynamics simulations revealed that theaflavin adopts a “wedge insertion mode” to block the ion conduction pore and induces pore closure. Moreover, the binding mode showed that the TF pedestal plays an important role in pore blockade, and R515, R535, T539, K603, E623, and E633 were determined to be most likely to interact directly with the pedestal. Mutagenesis experiment results corroborated the mechanism through which TF binds to this pocket. Combined with the quantitative calculation results, our data indicated that the three hydroxyl groups on the pedestal may be the most crucial pharmacophores for TMEM16A inhibition by TF. Finally, antitumor experiments revealed that TF could target TMEM16A to inhibit the proliferation and migration of LA795 cells, indicating the potential therapeutic effect of TF on the growth of lung adenocarcinoma with high TMEM16A expression. The successful application of drug screening strategies based on this binding pocket highlights new directions for discovering superior modulators and contributes to the development of novel therapeutics for lung adenocarcinoma.     

Developmental expression of the calcium‐activated chloride channels TMEM16A and TMEM16B in the mouse olfactory epithelium

Calcium‐activated chloride channels are involved in several physiological processes including olfactory perception. TMEM16A and TMEM16B, members of the transmembrane protein 16 family (TMEM16), are responsible for calcium‐activated chloride currents in several cells. Both are present in the olfactory epithelium of adult mice, but little is known about their expression during embryonic development. Using immunohistochemistry we studied their expression in the mouse olfactory epithelium at various stages of prenatal development from embryonic day (E) 12.5 to E18.5 as well as in postnatal mice. At E12.5, TMEM16A immunoreactivity was present at the apical surface of the entire olfactory epithelium, but from E16.5 became restricted to a region near the transition zone with the respiratory epithelium, where localized at the apical part of supporting cells and in their microvilli. In contrast, TMEM16B immunoreactivity was present at E14.5 at the apical surface of the entire olfactory epithelium, increased in subsequent days, and localized to the cilia of mature olfactory sensory neurons. These data suggest different functional roles for TMEM16A and TMEM16B in the developing as well as in the postnatal olfactory epithelium. The presence of TMEM16A at the apical part and in microvilli of supporting cells is consistent with a role in the regulation of the chloride ionic composition of the mucus covering the apical surface of the olfactory epithelium, whereas the localization of TMEM16B to the cilia of mature olfactory sensory neurons is consistent with a role in olfactory signal transduction. © 2013 Wiley Periodicals, Inc. Develop Neurobiol 74: 657–675, 2014     

Calmodulin regulation of TMEM16A and 16B Ca2+-activated chloride channels

Ca²⁺-activated chloride channels encoded by TMEM16A and 16B are important for regulating epithelial mucus secretion, cardiac and neuronal excitability, smooth muscle contraction, olfactory transduction, and cell proliferation. Whether and how the ubiquitous Ca²⁺ sensor calmodulin (CaM) regulates the activity of TMEM16A and 16B channels has been controversial and the subject of an ongoing debate. Recently, using a bioengineering approach termed ChIMP (Channel Inactivation induced by Membrane-tethering of an associated Protein) we argued that Ca²⁺-free CaM (apoCaM) is pre-associated with functioning TMEM16A and 16B channel complexes in live cells. Further, the pre-associated apoCaM mediates Ca²⁺-dependent sensitization of activation (CDSA) and Ca²⁺-dependent inactivation (CDI) of some TMEM16A splice variants. In this review, we discuss these findings in the context of previous and recent results relating to Ca²⁺-dependent regulation of TMEM16A/16B channels and the putative role of CaM. We further discuss potential future directions for these nascent ideas on apoCaM regulation of TMEM16A/16B channels, noting that such future efforts will benefit greatly from the pioneering work of Dr. David T. Yue and colleagues on CaM regulation of voltage-dependent calcium channels.     

Knockdown of TMEM14A expression by RNAi inhibits the proliferation and invasion of human ovarian cancer cells

Transmembrane protein 14A (TMEM14A) is a member of TMEMs. Alterations in TMEMs expression have been identified in several types of cancer, but the expression and function of TMEM14A in ovarian cancer is still unclear. Here, analysis on the expression data of the Cancer Genome Atlas (TCGA) ovarian serous cystadenocarcinoma (OV) dataset demonstrated the overexpression of TMEM14A in ovarian cancer tissues compared with normal tissues, which was consistent with our real-time PCR analysis on ovarian cancer and normal tissues collected from 30 patients. In addition, TMEM14A knockdown in two ovarian cancer cell lines, A2780 and HO-8910, reduced cell proliferation, causes cell cycle arrest and suppressed cell invasion. Moreover, silencing of TMEM14A notably repressed G1/S cell cycle transition and cell invasion via down-regulating the expression of cell cycle related proteins (Cyclin D1, Cyclin E and PCNA) and metastasis-related proteins (MMP-2 and MMP-9), respectively. TMEM14A knockdown significantly reduced the phosphorylation status of Smad2 and Smad3, downstream effectors of TGF-β signalling. In summary, these results indicate that TMEM14A has a pro-tumorigenic effect in ovarian cancer cells, suggesting an important role of this protein in ovarian cancer oncogenesis and metastasis.     

Prostacyclin-induced rundown of N-methyl-D-aspartate receptor currents in HEK293 cells is protein kinase A-dependent and NR2 subunit-selective

Previous studies in neurons have demonstrated a rapid decrease in NMDA receptor currents following tyrosine kinase inhibition or exposure to platelet-derived growth factor (PDGF). Inhibitors of protein kinase A (PKA) block the PDGF-induced rundown suggesting a multistep pathway that leads to decreased amplitudes of NMDA-activated currents. In this study, HEK293 cells expressing different NMDA receptor subunits were used to study the effects of prostacyclin receptor-mediated PKA activation on the magnitude of glutamate-activated currents. The prostacyclin agonist iloprost induced a rapid and time-dependent depression of otherwise stable glutamate-activated currents in cells expressing NR1-2a/2A or NR1-2a/2D receptors but not NR1-2a/2B or NR1-2a/2C receptors. This rundown was prevented by treatment of cells with the PKA inhibitor H89. The iloprost effect persisted in cells coexpressing NR1-2a/2A receptors and either wild-type or mutant Src kinase (SrcS17A). Co-expression of PSD-95 with NR1-2a/2A receptors reduced but did not eliminate the extent of rundown. Iloprost also produced current rundown in cells expressing NR1-2a and a C-terminal truncated NR2A subunit (NR2A1050stop) but not in those transfected with an NR2A tyrosine mutant (Y842F). The iloprost-induced rundown of wild-type NR1-2a/2A receptors was prevented by prior exposure of cells to hypertonic sucrose. These results suggest that PKA influences the functional activity of NMDA receptors in an NR2 subunit-selective fashion.     

Deficiencies in C4A and C4B isotypes of human complement revealed by isoelectrofocusing and chemical reactivity of activated forms

An approach is proposed to detect deficiencies in isotypes A and B of the C4 component of human complement, based on the calculation of the ratio of their IEA activities and the ratio of their quantities determined by isoelectrofocusing of their desialated forms with chemiluminescent detection in an immunoblot. The ratios of the quantities and activities of C4A/C4B practically coincided when determined in blood serum of 20 patients, many of which had inherited deficiencies in the C4 component isotypes.     

TMEM184B promotes proliferation,migration and invasion,and inhibits apoptosis in hypopharyngeal squamous cell carcinoma

Several members of the transmembrane protein family are associated with the biological processes of human malignancies; however, the expression pattern and biological function of one family member, TMEM184B, in hypopharyngeal squamous cell carcinoma (HPSCC) are not fully understood. The expression between HPSCC tumours and adjacent normal tissues was determined by the Immunohistochemistry (IHC). A bioinformatics analysis was performed to verify the expression pattern of TMEM184B in The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases. Furthermore, in vitro assays on cell proliferation, invasion, migration and in vivo experiments on tumour growth and apoptosis of TMEM184B in HPSCC were performed. We found that the HPSCC tissues had a significantly higher expression of TMEM184B than the adjacent normal tissues. Bioinformatics analysis confirmed the different expression of TMEM184B expression in HPSCC. Furthermore, in vitro and in vivo experiments demonstrated that TMEM184B promotes HPSCC cell growth, cell invasion and migration in FaDu cells, whereas flow cytometry assay showed that TMEM184B inhibited cell apoptosis. Our study revealed for the first time that TMEM184B might serve an oncogenic function in HPSCC and could be a potential diagnostic biomarker and therapeutic target for HPSCC.     

Identification and functional characterization of TMEM16A, a Ca2+-activated Cl- channel activated by extracellular nucleotides, in biliary epithelium

Cl(-) channels in the apical membrane of biliary epithelial cells (BECs) provide the driving force for ductular bile formation. Although a cystic fibrosis transmembrane conductance regulator has been identified in BECs and contributes to secretion via secretin binding basolateral receptors and increasing [cAMP](i), an alternate Cl(-) secretory pathway has been identified that is activated via nucleotides (ATP, UTP) binding apical P2 receptors and increasing [Ca(2+)](i). The molecular identity of this Ca(2+)-activated Cl(-) channel is unknown. The present studies in human, mouse, and rat BECs provide evidence that TMEM16A is the operative channel and contributes to Ca(2+)-activated Cl(-) secretion in response to extracellular nucleotides. Furthermore, Cl(-) currents measured from BECs isolated from distinct areas of intrahepatic bile ducts revealed important functional differences. Large BECs, but not small BECs, exhibit cAMP-stimulated Cl(-) currents. However, both large and small BECs express TMEM16A and exhibit Ca(2+)-activated Cl(-) efflux in response to extracellular nucleotides. Incubation of polarized BEC monolayers with IL-4 increased TMEM16A protein expression, membrane localization, and transepithelial secretion (I(sc)). These studies represent the first molecular identification of an alternate, noncystic fibrosis transmembrane conductance regulator, Cl(-) channel in BECs and suggest that TMEM16A may be a potential target to modulate bile formation in the treatment of cholestatic liver disorders.     

Activation and translocation of PKCdelta is necessary for VEGF-induced ERK activation through KDR in HEK293T cells

VEGF-KDR/Flk-1 signal utilizes the phospholipase C-gamma-protein kinase C (PKC)-Raf-MEK-ERK pathway as the major signaling pathway to induce gene expression and cPLA2 phosphorylation. However, the spatio-temporal activation of a specific PKC isoform induced by VEGF-KDR signal has not been clarified. We used HEK293T (human embryonic kidney) cells expressing transiently KDR to examine the activation mechanism of PKC. PKC specific inhibitors and human PKCdelta knock-down using siRNA method showed that PKCdelta played an important role in VEGF-KDR-induced ERK activation. Myristoylated alanine-rich C-kinase substrate (MARCKS) translocates from the plasma membrane to the cytoplasm depending upon phosphorylation by PKC. Translocation of MARCKS-GFP induced by VEGF-KDR stimulus was blocked by rottlerin, a PKCdelta specific inhibitor, or human PKCdelta siRNA. VEGF-KDR stimulation did not induce ERK phosphorylation in human PKCdelta-knockdown HEK293T cells, but co-expression of rat PKCdelta-GFP recovered the ERK phosphorylation. Y311/332F mutant of rat PKCdelta-GFP which cannot be activated by tyrosine-phosphorylation but activated by DAG recovered the ERK phosphorylation, while C1B-deletion mutant of rat PKCdelta-GFP, which can be activated by tyrosine-phosphorylation but not by DAG, failed to recover the ERK phosphorylation in human PKCdelta-knockdown HEK293T cell. These results indicate that PKCdelta is involved in VEGF-KDR-induced ERK activation via C1B domain.     

四种旱生藓类植物的比较结构学观察

对新疆产的4种藓类植物茎、叶的表面及内部结构进行了观察,结果表明：尖叶大帽藓(Encalypta rhabdocarpa Schwaegr.)茎的中部结构类似于种子植物(单子叶)根的内皮层,其茎表皮也有类似于种子植物表皮毛(腺毛)的腺体。在阔叶紫萼藓(Grimmia laevigata (Brid.) Brid.)茎的中轴部,厚角组织发达,数层皮部厚壁组织也很发达。小石藓(Weisia controversa Hedw.)叶中肋之中有小形厚壁细胞。弯叶墙藓(Tortula reflexa Li)的茎呈多棱形,表皮层具很厚的“树皮状”老皮,其叶表皮细胞密被马蹄形及叉状粗疣。这些特征反映了该类群植物由低等向高等演化的趋势,并可作为分类学依据之一。     

慈溪麦冬甙A和B的结构

从浙江慈溪产中药麦冬（Ｏｐｈｉｏｐｏｇｏｎ ｊａｐｏｎｉｃｕｍ）中分离得到２个新的Ｃ２７甾体甙－－慈溪麦冬甙Ａ（２）和Ｂ（３）以及已知甙ｏｐｈｉｏｇｅｎｉｎ３－氧－α－Ｌ－鼠李糖吡喃基（１→２）「β－Ｄ－木糖吡喃基（１→３）」－β－Ｄ－葡萄糖吡喃甙（２）和ｏｐｈｉｏｇｅｎｉｎ ３－氧－α－Ｌ－鼠李糖吡喃基（１→２）「β－Ｄ－木糖吡喃基（１→３）」「β－Ｄ－葡萄糖吡喃基（１→４）」－β－Ｄ－葡萄糖吡     

The effect of Bcl‐2, YAMA,and XIAP over‐expression on apoptosis and adenovirus production in HEK293 cell line

Many viruses induce cell death and lysis as part of their replication and dissemination strategy, and in many cases features of apoptosis are observed. Attempts have been made to further increase productivity by prolonging cell survival via the over‐expression of anti‐apoptotic genes. Here, we extend the study to investigate the association between virus replication and apoptosis, pertinent to large‐scale vector production for gene therapy. Infection of an HEK293 cell line with a replication defective type‐5‐adenovirus expressing a GFP reporter (Ad5GFP) resulted in rapid decline in viability associated with increased virus titer. The over‐expression of bcl‐2 resulted in improved cell resistance to apoptosis and prolonged culture duration, but reduced virus specific and total productivity. In contrast, the over‐expression of pro‐caspase‐3 (Yama/CPP32/apopain) resulted in reduced cell survival but increased virus productivity. The treatment of infected cells with caspase inhibitors support the preposition that caspase‐3 dependent apoptosis, and to a lesser degree caspase‐9 dependent apoptosis, represent important steps in virus production, thus implicating the intrinsic apoptosis pathway in the production of adenovirus from HEK293 cells. The suppression of apoptosis by the over‐expression of XIAP (inhibitors of caspase family cell death proteases) further shows that caspase‐mediated activation plays an important role in virus infection and maturation. Biotechnol. Bioeng. 2009; 104: 752–765 © 2009 Wiley Periodicals, Inc.     

姜黄素诱导人成骨肉瘤MG-63细胞凋亡过程中hnRNP A2/B1表达与定位

姜黄素(curcumin)诱导处理的人成骨肉瘤MG-63细胞,在光镜和电镜观察细胞凋亡的基础上,对hnRNP A2/B1在核基质中存在、分布及其与凋亡相关基因产物在MG-63细胞中的共定位关系进行了研究.经姜黄素处理后,细胞出现染色质凝聚、细胞核固缩、凋亡小体等典型的细胞凋亡形态特征;双向凝胶电泳和质谱鉴定结果显示,hnRNP A2/B1存在于MG-63细胞核基质蛋白组分中,在姜黄素处理后细胞核基质蛋白中表达下调.蛋白质印迹杂交结果,证实hnRNP A2/B1在姜黄素处理前后的MG-63细胞核基质蛋白中的存在及其表达下调变化.免疫荧光显微镜观察显示,hnRNP A2/B1定位于MG-63细胞核基质纤维上,经姜黄素处理后出现分布位置与表达水平变化.激光扫描共聚焦显微镜的观察结果显示,hnRNP A2/B1在MG-63细胞凋亡过程中与Bax、Bcl-2、Fas和p53等基因产物具有共定位关系,且其共定位区域发生了变化.研究结果证实了hnRNP A2/B1定位于核基质纤维上,是一种核基质蛋白,在姜黄素诱导人成骨肉瘤MG-63凋亡过程中的表达与分布变化及其与凋亡相关基因的关系显然对MG-63细胞凋亡具有重要影响,这为深入揭示肿瘤细胞凋亡的机制提供了重要科学依据和深入探索的新方向.     

Isotyping of component C4 of human complement using differences in the functional activity of isotypes C4A and C4B

The difference in the functional activity of the isotypes A and B of component C4 of human complement was used to determine their ratio and to detect the inherited deficiency of the isotypes. ELISA methods were developed for the quantitative assay of component C4 (conventional sandwich method) and its functional activity. When determining the functional activity, the classic pathway of the complement and therefore of component C4 was activated on activators sorbed on ELISA microplates: immunoglobulin IgG3 or liposaccharide of theShigella sonnei cell walls, which activates the complement by binding component C1. The nascent fragment C4b is covalently bound to the target activator; C4Ab binds better to the target protein (immunoglobulin), and C4Bb to the target carbohydrate (liposaccharide). Therefore, when immunoglobulin is a target activator, isotype C4A is bound and determined; and when the complement is activated with liposaccharide, isotype C4B is determined. The radio of the activities determined by the two methods indicates the deficiency in the individual isotypes of component C4 or its absence. The rabbit polyclonal monospecific antibodies against the human component C4 and the conjugates of these antibodies with horseradish peroxidase were used in the methods described.