骨骼肌内质网Ca2+泵转运Ca2+的结构基础

Ca²⁺泵(Ca²⁺-ATPase)是调节细胞内Ca²⁺浓度的重要蛋白质之一. Ca²⁺泵在转运Ca²⁺的过程中经历一系列构象变化. 其中,E1状态为外向的Ca²⁺高亲和状态,E2状态则为内向的Ca²⁺低亲和状态. 目前,骨骼肌内质网Ca²⁺泵转运Ca²⁺过程中的几个中间状态,包括E1-2Ca²⁺,E1-ATP,E1-P-ADP,E2-Pi和E2状态的三维晶体结构已经解析. 介绍这几种状态的晶体结构,并分析Ca²⁺泵在执行功能过程中结构与功能的关系.     

钙调磷酸酶信号调控真菌生长代谢、毒力及抗逆性能

钙调磷酸酶是一种丝氨酸/苏氨酸（Ser/Thr）蛋白磷酸酶,在真菌中普遍保守,上游信号途径由Ca²⁺通道（Cch1）、转运蛋白（Mid1）、钙离子感应蛋白（CaM）、钙调蛋白依赖性磷酸酶等组成。钙调磷酸酶受钙离子和钙调蛋白调节,在调控真菌Ca²⁺稳态的钙信号级联途径中发挥着中心作用,通过钙信号级联途径参与生物学过程,调控真菌生长、发育和毒力形成来响应外界环境因素的变化,使真菌能够适应不同环境,维持正常的生命活动。本文综述了真菌钙调磷酸酶信号的组成和上下游信号转导途径、调控细胞生长代谢、毒力形成以及抗逆性能调控的研究进展;结合对真菌代谢产物合成的调控作用,对钙调磷酸酶信号作为重要合成生物学元件及调控开关进行了展望。     

钙调蛋白研究的新进展

最近对钙调蛋白(CaM)的研究,揭示了它的三维结构及其两个结构域的功能。肯定了CaM的Ⅲ、Ⅳ位是Ca²⁺结合的高亲和位,并据此提出了CaM活化靶酶的新模型。发现神经钙蛋白(CaN)为一种依赖CaM的磷酸酶和两种最强的CaM桔抗剂多肽Mastoparan和药物EBB。证明一些疾病同Ca²⁺、CaM有关。     

细胞内信号分子传导的研究进展

近年来有关细胞内信号传导的研究,着重体现在Ca²⁺信号传导途径及相应的蛋白质分子如蛋白激酶C(PKC)、钙调素(CaM)、钙调素激酶Ⅱ(CaMKⅡ),同时也对Ras途径中出现的Vav、Rap、Crk、C3G等蛋白质分子以及cAMP和NF-κB途径作了有益的补充与修改.细胞外信号分子通过以上4种途径及其相互通讯(cross-talk),激活了某些蛋白激酶,调控了基因转录及其他相关功能,其中磷酸化对蛋白激酶及转录因子活性的调节起到了非常重要的作用.     

钙的光释放技术及其在细胞研究中的应用

Ca²⁺的光释放技术通过光解作用使预先引入细胞内的光敏感性螯合剂对Ca²⁺的亲和性改变,从而实现对细胞内游离钙离子浓度的调控,有助于阐明Ca²⁺作为第二信使对电兴奋性、肌肉收缩、神经分泌等细胞功能的调制作用.     

Ca2+在粟酒裂殖酵母细胞周期时相中的作用*

以粟酒裂殖酵母(Schizosaccharomyces pombe)为研究材料,研究了Ca²⁺在细胞周期时相中的作用。当外源Ca²⁺浓度在0.5-20 mmol/L范围内,随Ca²⁺浓度增加,细胞增殖速度加快,延滞期逐渐缩短。但SD-Ca（CaCl₂省略）并不能终止Sch. Pombe的细胞周期。采用缺氮对群体细胞进行同步化,并以EGTA 螯合培养介质中低浓度的Ca²⁺,Sch. Pombe 细胞增殖被完全抑制,细胞流式法测定结果表明：细胞周期被终止在G1期。分析认为Ca²⁺ 对Sch. Pombe 细胞增殖是必不可少的,外源Ca²⁺在G1期向S期转化过程中起着关键性的作用。     

单细胞内Ca2+时空变化的激光共聚焦显微测定

应用激光扫描共聚焦显微系统(LSCM)和Fluo-3/AM荧光探剂标记技术, 测定了单个活细胞胞内游离Ca²⁺的动态变化与立体分布影像. 结果显示, 在37℃, Fluo-3/AM终浓度为6μmol/L的条件下, C57BL/6J小鼠巨噬细胞负载1h左右即可获得良好的标记效果. 相反, 若探剂浓度太高或负载时间太长, 胞内荧光强度太强, 影响在共聚焦显微镜镜下分辨细胞内结构. 因此用LSCM研究细胞内游离Ca²⁺变化时, 荧光探剂的负载应以获得最适荧光信号而不是以最大荧光强度为标准. 上述方法在其他如平滑肌细胞、卵母细胞中的测定亦获得满意的结果, 这对进一步研究各种生理和病理条件下细胞内Ca²⁺信号的动态变化、与跨膜Ca²⁺梯差的关系及对活细胞功能活动的调节提供了一种可行的、直观的研究手段.     

脉冲电场对粒细胞内Ca2+浓度影响的动态变化

利用激光共聚焦扫描显微镜和装有CCD系统的荧光显微镜 ,研究在单脉冲电场作用下经fluo 3/AM标记的鸡胚小脑粒细胞内自由Ca²⁺浓度 ( [Ca ²⁺]_i)的动态变化过程 .结果表明 :在单个电脉冲作用下 ,细胞内Ca²⁺浓度立刻升高并达到其最大值 .Ca²⁺浓度升高的幅度以及升高的速率具有电场强度的依赖性 .当细胞外Ca²⁺被过量的EGTA络合或细胞膜上的Ca²⁺通道被La ³⁺堵塞后 ,细胞内的Ca²⁺浓度仍然升高 .细胞内不同区域的Ca²⁺浓度同时升高 ;两极内的Ca²⁺浓度早于胞体的Ca²⁺浓度达到最大值并迅速恢复 .     

PK-C的研究进展

蛋白激酶C(PK-C),一种独特的磷脂敏感性,Ca²⁺依赖性蛋白激酶。它广泛传递多种细胞外信息通过细胞膜,调节细胞内许多依赖于Ca²⁺的代谢过程。无论是在细胞释放、溶解、膜运输、受体的增效与去敏、平滑肌的收缩,还是在调控细胞分化、增殖、癌变的过程中都起着关键性的作用。     

酿酒酵母细胞增殖对Ca2+需求的新证据*

本文研究了酿酒酵母细胞增殖对Ca²⁺需求的证据。结果表明:SD-Ca培养基中外加1mmol/L的Ca²⁺明显促进酿酒酵母细胞增殖,外源Ca²⁺浓度在0~20mmol/L范围内变动时,随Ca²⁺浓度增加,细胞生长到达稳定期的终浓度也越大;5、10mmol/L的EGTA可明显延缓细胞生长的延滞期,但是最终不能抑制细胞增殖;酿酒酵母在SD-Ca培养基中继代培养4次,随增殖代数增加,细胞总钙含量没有明显变化,说明酵母能够在低钙介质中生长可能是因为具有捕捉和富集钙的功能;以Fluo-3作为胞质Ca²⁺指示剂,通过激光扫描共聚焦显微镜观察,发现随胞外Ca²⁺浓度增加,胞质中游离Ca²⁺浓度也相应增加。这些证据都揭示了Ca²⁺在酿酒酵母细胞增殖过程中是必需的。     

La3+ stimulate the activity of calcineurin in two different ways

It is well known that the activity of calcineurin (CaN) could be modulated by several transitional metal ions. In the present work, the effects of a calcium analog, lanthanum ion (La³⁺), on the activity of CaN were studied. It was found that La³⁺ exerted multiple effects on CaN activity. La³⁺ could stimulate CaN in the absence of calmodulin (CaM); whereas at low concentrations of La³⁺, there was a slight inhibition of activation of CaN in the presence of CaM. Competitive experiments and limited trypsin proteolysis confirmed that La³⁺ did not act on the catalytic core of CaN, but exerted its effect through direct action on the CaN regulatory domain similar to Mg²⁺. In activity titration and spot blotting studies, La³⁺-containing CaM complexes were less effective in stimulating CaN than Ca²⁺ or Mn²⁺-containing CaM; however, the binding affinity of these metal–CaM complexes to CaN was similar. These effects of La³⁺ on CaN activity are unique among metal ions and may provide clues to understand the biological effects of La³⁺.     

Role of Calcineurin-Mediated Dephosphorylation in Modulation of an Inwardly Rectifying K+ Channel in Human Proximal Tubule Cells

Activity of an inwardly rectifying K⁺ channel with inward conductance of about 40 pS in cultured human renal proximal tubule epithelial cells (RPTECs) is regulated at least in part by protein phosphorylation and dephosphorylation. In this study, we examined involvement of calcineurin (CaN), a Ca²⁺/calmodulin (CaM)–dependent phosphatase, in modulating K⁺ channel activity. In cell-attached mode of the patch-clamp technique, application of a CaN inhibitor, cyclosporin A (CsA, 5 μM) or FK520 (5 μM), significantly suppressed channel activity. Intracellular Ca²⁺ concentration ([Ca²⁺] _i ) estimated by fura-2 imaging was elevated by these inhibitors. Since inhibition of CaN attenuates some dephosphorylation with increase in [Ca²⁺] _i , we speculated that inhibiting CaN enhances Ca²⁺-dependent phosphorylation, which might result in channel suppression. To verify this hypothesis, we examined effects of inhibitors of PKC and Ca²⁺/CaM-dependent protein kinase-II (CaMKII) on CsA-induced channel suppression. Although the PKC inhibitor GF109203X (500 nM) did not influence the CsA-induced channel suppression, the CaMKII inhibitor KN62 (20 μM) prevented channel suppression, suggesting that the channel suppression resulted from CaMKII-dependent processes. Indeed, Western blot analysis showed that CsA increased phospho-CaMKII (Thr286), an activated CaMKII in inside–out patches, application of CaM (0.6 μM) and CaMKII (0.15 U/ml) to the bath at 10^?6 M Ca²⁺ significantly suppressed channel activity, which was reactivated by subsequent application of CaN (800 U/ml). These results suggest that CaN plays an important role in supporting K⁺ channel activity in RPTECs by preventing CaMKII-dependent phosphorylation.     

Computational design of calmodulin mutants with up to 900-fold increase in binding specificity

Calmodulin (CaM) is a ubiquitous second messenger protein that regulates a variety of structurally and functionally diverse targets in response to changes in Ca²⁺ concentration. CaM-dependent protein kinase II (CaMKII) and calcineurin (CaN) are the prominent CaM targets that play an opposing role in many cellular functions including synaptic regulation. Since CaMKII and CaN compete for the available Ca²⁺/CaM, the differential affinity of these enzymes for CaM is crucial for achieving a balance in Ca²⁺ signaling. We used the computational protein design approach to modify CaM binding specificity for these two targets. Starting from the X-ray structure of CaM in complex with the CaM-binding domain of CaMKII, we optimized CaM interactions with CaMKII by introducing mutations into the CaM sequence. CaM optimization was performed with a protein design program, ORBIT, using a modified energy function that emphasized intermolecular interactions in the sequence selection procedure. Several CaM variants were experimentally constructed and tested for binding to the CaMKII and CaN peptides using the surface plasmon resonance technique. Most of our CaM mutants demonstrated small increase in affinity for the CaMKII peptide and substantial decrease in affinity for the CaN peptide compared to that of wild-type CaM. Our best CaM design exhibited an about 900-fold increase in binding specificity towards the CaMKII peptide, becoming the highest specificity switch achieved in any protein-protein interface through the computational protein design approach. Our results show that computational redesign of protein-protein interfaces becomes a reliable method for altering protein binding affinity and specificity.     

A Highlights from MBoC Selection: Dendritic and axonal mechanisms of Ca2+ elevation impair BDNF transport in Aβ oligomer–treated hippocampal neurons

Disruption of fast axonal transport (FAT) and intracellular Ca²⁺ dysregulation are early pathological events in Alzheimer''s disease (AD). Amyloid-β oligomers (AβOs), a causative agent of AD, impair transport of BDNF independent of tau by nonexcitotoxic activation of calcineurin (CaN). Ca²⁺-dependent mechanisms that regulate the onset, severity, and spatiotemporal progression of BDNF transport defects from dendritic and axonal AβO binding sites are unknown. Here we show that BDNF transport defects in dendrites and axons are induced simultaneously but exhibit different rates of decline. The spatiotemporal progression of FAT impairment correlates with Ca²⁺ elevation and CaN activation first in dendrites and subsequently in axons. Although many axonal pathologies have been described in AD, studies have primarily focused only on the dendritic effects of AβOs despite compelling reports of presynaptic AβOs in AD models and patients. Indeed, we observe that dendritic CaN activation converges on Ca²⁺ influx through axonal voltage-gated Ca²⁺ channels to impair FAT. Finally, FAT defects are prevented by dantrolene, a clinical compound that reduces Ca²⁺ release from the ER. This work establishes a novel role for Ca²⁺ dysregulation in BDNF transport disruption and tau-independent Aβ toxicity in early AD.     

Blocking gastrin and CCK-B autocrine loop affects cell proliferation and apoptosis in vitro

Since its initial discovery as Ca²⁺/calmodulin (CaM)-dependent serine/threonine protein phosphatase, calcineurin (CaN) has been extensively studied in many mammalian tissues. CaN has been shown to be involved in various biological and Ca²⁺-dependent signal transduction pathways. Over the last decade, our laboratory has been interested and has carried out numerous experiments on this specific protein phosphatase. While, a lot of research has been performed studying CaN’s involvement in ischemia, the immune system, and various mammalian tissues, not much is known about the potential role of CaN in various eye diseases. This review focuses on the studies that have been carried out in our laboratory on CaN, and specifically CaN’s involvement in the eye. We demonstrated that CaN is localized in various eye tissues (cornea, iris, ciliary body, vitreous body, retina, choroid, sclera, and optic nerve) and that both its protein expression and activity were observed in high amounts in the retina, optic nerve and cornea. Recently, we have cloned and characterized the CaN A and B subunits in the bovine retina. These initial findings suggest that CaN may play a potential role in visual transduction and various ocular diseases, including cancer.     

Autophagy genes mediate the effect of calcineurin on life span in C. elegans

Calcineurin (CaN) is a serine/threonine phosphatase, activated by Ca²⁺/calmodulin (Ca²⁺/CaM). CaN is known to regulate various cellular responses in different organisms. A recent study showed an extended life span in the calcineurin mutants of C. elegans. In this study, we report that calcineurin defective strains exhibit enhanced autophagy. In addition, we found two essential autophagy genes (bec-1 and atg-7) are required for the life-span extension in calcineurin null mutants [cnb-1(jh103)]. Thus, for the first time we suggest that autophagy genes are required for the life-span regulation in calcineurin defective C. elegans strains.     

Involvement of calcium-sensing receptor in cardiac hypertrophy-induced by angiotensinII through calcineurin pathway in cultured neonatal rat cardiomyocytes

Cardiac hypertrophy is a common pathological change accompanying cardiovascular disease. Recently, some evidence indicated that calcium-sensing receptor (CaSR) expressed in the cardiovascular tissue. However, the functional involvement of CaSR in cardiac hypertrophy remains unclear. Previous studies have shown that CaSR caused accumulation of inositol phosphate to increase the release of intracellular calcium. Moreover, Ca²⁺-dependent phosphatase calcineurin (CaN) played a vital role in the development of cardiac hypertrophy. Therefore, we investigated the expression of CaSR in cardiac hypertrophy-induced by angiotensin II (AngII) and the effects of CaSR activated by GdCl₃ on the related signaling transduction pathways. The results showed that AngII induced cardiac hypertrophy and up-regulated the expression of CaSR, meanwhile increased the intracellular calcium concentration ([Ca²⁺]_i) and activated CaN hypertrophic signaling pathway. Compared with AngII alone, the above changes were further obvious when adding GdCl₃. But the effects of GdCl₃ on the cardiac hypertrophy were attenuated by CsA, a specific inhibitor of CaN. In conclusion, these results suggest that CaSR is involved in cardiac hypertrophy-induced by AngII through CaN pathway in cultured neonatal rat cardiomyocytes.     

Molecular cloning and biochemical characterization of bovine retina calcineurin

Calcineurin (CaN) is a member of ser/thr protein phosphatase family. Earlier, we have reported that CaN is present in all eye tissues, although the activity and protein expression varied (Seitz et al., Invest Opthalmol Vis Sci, 43:15–21, 2002). We have isolated a full-length cDNA encoding bovine retina CaN. The CaN A subunit consists of 511 amino acid residues. A 10 amino acid (ATVEAIEADE) deletion before the autoinhibitory domain was observed in bovine retina CaN A compared to bovine brain CaN A. The study on CaN activity and regulation demonstrated that different metal ions have different effects on its phosphatase activity. Ni²⁺ was found to be the strongest stimulator, while Zn²⁺ was found to inhibit CaN phosphatase activity. Mn²⁺ was a relatively less effective stimulator compared to Ni²⁺. Fe²⁺ was also able to stimulate CaN phosphatase activity; in contrast, a previous study found Fe²⁺ slightly inhibited CaN activity from bovine brain. The residues at 97–201 were found to be essential for bovine retina CaN A phosphatase activity. The residues at 407–456 also had an inhibitory effect on CaN A phosphatase activity in addition to the previously known autoinhibitory domain at 457–480. These observations suggest that bovine retina CaN A might possess some distinct structural characteristics.     

The Effect of SERCA1b Silencing on the Differentiation and Calcium Homeostasis of C2C12 Skeletal Muscle Cells

The sarcoplasmic/endoplasmic reticulum Ca²⁺ATPases (SERCAs) are the main Ca²⁺ pumps which decrease the intracellular Ca²⁺ level by reaccumulating Ca²⁺ into the sarcoplasmic reticulum. The neonatal SERCA1b is the major Ca²⁺ pump in myotubes and young muscle fibers. To understand its role during skeletal muscle differentiation its synthesis has been interfered with specific shRNA sequence. Stably transfected clones showing significantly decreased SERCA1b expression (cloneC1) were selected for experiments. The expression of the regulatory proteins of skeletal muscle differentiation was examined either by Western-blot at the protein level for MyoD, STIM1, calsequestrin (CSQ), and calcineurin (CaN) or by RT-PCR for myostatin and MCIP1.4. Quantitative analysis revealed significant alterations in CSQ, STIM1, and CaN expression in cloneC1 as compared to control cells. To examine the functional consequences of the decreased expression of SERCA1b, repeated Ca²⁺-transients were evoked by applications of 120 mM KCl. The significantly higher [Ca²⁺]i measured at the 20^th and 40^th seconds after the beginning of KCl application (112±3 and 110±3 nM vs. 150±7 and 135±5 nM, in control and in cloneC1 cells, respectively) indicated a decreased Ca²⁺-uptake capability which was quantified by extracting the maximal pump rate (454±41 μM/s vs. 144±24 μM/s, in control and in cloneC1 cells). Furthermore, the rate of calcium release from the SR (610±60 vs. 377±64 μM/s) and the amount of calcium released (843±75 μM vs. 576±80 μM) were also significantly suppressed. These changes were also accompanied by a reduced activity of CaN in cells with decreased SERCA1b. In parallel, cloneC1 cells showed inhibited cell proliferation and decreased myotube nuclear numbers. Moreover, while cyclosporineA treatment suppressed the proliferation of parental cultures it had no effect on cloneC1 cells. SERCA1b is thus considered to play an essential role in the regulation of [Ca²⁺]i and its ab ovo gene silencing results in decreased skeletal muscle differentiation.