Calreticulin facilitates the cell surface expression of ABCG5/G8

ATP-binding cassette (ABC) G5 (G5) and ABCG8 (G8) heterodimerize and function as sterol transporter that promote biliary excretion of neutral sterols. Both G5 and G8 interact with a lectin-like chaperone, calnexin (CNX), in the endoplasmic reticulum (ER) but the significance of this interaction remains unclear. Here, we show that not only CNX, but also its homologue calreticulin (CRT), is involved in the biosynthesis of G5/G8 sterol transporter. Both CNX and CRT interacted with immature forms of G5 and G8, and stimulated their productive folding by inhibiting their degradation. Interestingly, CRT predominantly enhanced the cell surface expression of mature G5/G8 whereas CNX did not have a similar effect. Inhibitors of N-glycan processing indicated that quality control of G5 and G8 might be differentially regulated in the ER. These findings clarify the role of CNX and CRT in the biosynthesis and quality control of G5/G8 sterol transporter.     

Identification and localization of calreticulin in plant cells

Summary In the present study we have investigated the presence and distribution of calreticulin in plant protoplasts. Calreticulin was purified from plant homogenates using a selective ammonium sulfate precipitation procedure developed for the purification of mammalian calreticulins and shown to bind calcium in⁴⁵Ca²⁺ overlay assays. The protein was localized to plant cell endoplasmic reticulum by the indirect immunofluorescence staining of protoplasts with anti-calreticulin antibodies. No calreticulin was observed within large vacuoles. We conclude that calreticulin is present in the endoplasmic reticulum of plant cells, where, by analogy to the mammalian endoplasmic reticulum, it may play a major role in Ca²⁺ binding and storage.Abbreviations ER endoplasmic reticulum - SR sarcoplasmic reticulum - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - PBS phosphate-buffered saline     

The role of MATER in endoplasmic reticulum distribution and calcium homeostasis in mouse oocytes

Ca²⁺ oscillations are a hallmark of mammalian fertilization and play a central role in the activation of development. The calcium required for these oscillations is primarily derived from the endoplasmic reticulum (ER), which accumulates in clusters at the microvillar subcortex during oocyte maturation. The migration of the ER to the cortex during maturation is thought to play an important role in rendering the ER competent to generate the calcium transients, and the redistribution of ER is believed to be primarily mediated by microtubules and microfilaments. We have previously shown that the oocyte- and early embryo-restricted maternal effect gene Mater (Nlrp5) localizes to, and is required for, formation of the oocyte cytoplasmic lattices, a tubulin-containing structure that appears to play an important role in organelle positioning and distribution during oocyte maturation. Given these observations, we hypothesized that Mater may also be required for ER redistribution and Ca²⁺ homeostasis in oocytes. To test this hypothesis, we first investigated ER localization in metaphase-II Mater^tm/tm (hypomorph) oocytes and found ER clusters to be less abundant at the microvillar cortex when compared to wild type oocytes. To examine the potential mechanisms by which MATER mediates ER redistribution, we tested whether tubulin expression levels and localization were affected in the mutant oocytes and found that the Triton-insoluble fraction of tubulin was significantly decreased in Mater^tm/tm oocytes. To identify potential functional defects associated with these ER abnormalities, we next set out to investigate if the pattern of Ca²⁺ oscillations was altered in Mater^tm/tm oocytes after fertilization in vitro. Intriguingly, Ca²⁺ oscillations in Mater^tm/tm oocytes exhibited a significantly lower first peak amplitude and a higher frequency when compared to wild type oocytes. We then found that the Ca²⁺ oscillation defect in Mater^tm/tm oocytes was likely caused by a reduced amount of Ca²⁺ in the ER stores. Taken together, these observations support the hypothesis that MATER is required for ER distribution and Ca²⁺ homeostasis in oocytes, likely due to defects in lattice-mediated ER positioning and/or redistribution.     

Fungal calcineurin complex as an antifungal target: From past to present to future

The serine/threonine phosphatase calcineurin complex has been considered a prospective target for developing novel drugs due to its importance in fungal growth, virulence, and stress responses in pathogenic fungi. Therefore, two well-known immunosuppressants, FK506 and cyclosporine A were successfully identified to inhibit calcineurin by combining with FK506-binding protein 12 and cyclophilin A, respectively. However, these drugs are immunosuppressive and may exhibit serious side effects. There is a growing number of literatures reported on further exploring functions of the calcineurin complex as promising antifungal targets. In general, the majority of the calcineurin complex structures are conserved but some functions are species-specific. Nevertheless, there still have a lot of functional motifs in the calcineurin complex that are unexplored. Therefore, further investigation and experimentation into the calcineurin complex are strongly required. This review not only has summarized previous findings but also explored bioinformatics analysis along with structural models of the calcineurin complex for finding fungal-specific regions as potential targets, laying the groundwork for future research into new therapeutics.     

巴斯德毕赤酵母Orm1蛋白在细胞生长和内质网功能维持方面的功能

【目的】鉴定巴斯德毕赤酵母ORM1基因;研究ORM1基因缺失对毕赤酵母生长、内质网压力应答、细胞钙稳态调节和活性氧水平等方面的影响。【方法】利用生物信息学软件对毕赤酵母Orm1蛋白进行序列比对和分析;利用PCR介导的同源重组法构建orm1Δ缺失菌株,将回补质粒p IB1-ORM1转入orm1Δ菌株构建回补菌株;研究ORM1基因缺失对毕赤酵母生长的影响;以Fluo-3 AM染色法测定胞质钙含量;以DCFH-DA染色法分析胞内活性氧水平;以实时荧光定量PCR技术研究ORM1基因缺失对毕赤酵母非折叠蛋白应答、钙稳态和抗氧化系统基因表达的影响;使用试剂盒分析毕赤酵母抗氧化系统过氧化氢酶(CAT)和超氧化物歧化酶(SOD)活性及谷胱甘肽(GSH)的含量。【结果】在毕赤酵母基因组数据库中比对出酿酒酵母Orm1和Orm2的同源蛋白,并将该蛋白编码基因命名为ORM1;毕赤酵母ORM1基因缺失导致细胞生长受到明显抑制,对衣霉素引起的内质网压力敏感性增强,非折叠蛋白应答激活,细胞钙稳态紊乱,活性氧积累,抗氧化系统激活。【结论】由于非折叠蛋白应答、钙稳态调节、活性氧积累等均与内质网功能息息相关,因此,巴斯德毕赤酵母ORM1基因编码的Orm1蛋白在细胞生长及内质网正常功能的维持过程中发挥重要作用。     

钙网蛋白的免疫生物学活性研究进展

钙网蛋白(calreticulin,CRT)是内质网中的Ca2+结合蛋白,对维持细胞内的Ca2+稳态具有重要作用。CRT还可作为分子伴侣,帮助新合成的内质网蛋白正确折叠。除此之外,CRT还出现在细胞膜表面及细胞外环境中,并发挥重要的免疫调节作用。膜表面的CRT参与凋亡细胞的清除和抗肿瘤免疫应答,在T细胞活化早期也发挥重要作用。可溶性CRT出现在自身免疫病患者的血清中,且其浓度与疾病活动度相关。重组可溶性CRT腹腔注射可显著影响小鼠的Th1/Th2平衡。重组可溶性CRT分子不仅具有超强免疫刺激活性,还表现出很强的佐剂效应,提示可溶性CRT可能在自身免疫性疾病的发生与发展过程中发挥一定作用。可以预见,CRT分子的免疫生物学活性将在不久的将来成为该领域研究的一个热点。     

Overexpression of calreticulin sensitizes SERCA2a to oxidative stress

Calreticulin (CRT), a Ca(2+)-binding molecular chaperone in the endoplasmic reticulum, plays a vital role in cardiac physiology and pathology. Oxidative stress is a main cause of myocardiac disorder in the ischemic heart, but the function of CRT under oxidative stress is not fully understood. In this study, the effect of overexpression of CRT on sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase (SERCA) 2a under oxidative stress was examined using myocardiac H9c2 cells transfected with the CRT gene. The in vitro activity of SERCA2a and uptake of (45)Ca(2+) into isolated microsomes were suppressed by H(2)O(2) in CRT-overexpressing cells compared with controls. Moreover, SERCA2a protein was degraded via a proteasome-dependent pathway following the formation of a complex with CRT under the stress with H(2)O(2). Thus, we conclude that overexpression of CRT enhances the inactivation and degradation of SERCA2a in the cells under oxidative stress, suggesting some pathophysiological functions of CRT in Ca(2+) homeostasis of myocardiac disease.     

Temporal Analysis of Changes in Neuronal c-fos mRNA Levels Induced by Depletion of Endoplasmic Reticulum Calcium Stores: Effect of Clamping Cytoplasmic Calcium Activity at Resting Levels

Abstract: Activation of immediate early gene expression is a key event in stress-induced neuronal cell injury. To study whether changes in cytoplasmic calcium activity are necessary to activate neuronal immediate early gene expression, endoplasmic reticulum (ER) calcium stores of primary neurons were depleted by exposing cells to thapsigargin (Tg), an irreversible inhibitor of ER Ca²⁺-ATPase. Tg-induced rise in [Ca²⁺]_i and the effect of loading neurons with the cell-permeable calcium chelator BAPTA-AM on this increase in [Ca²⁺]_i were measured in fura-2-loaded cells by fluorescence microscopy. Changes in c- fos mRNA levels were evaluated by quantitative PCR. Tg treatment of neurons produced a pronounced rise in c- fos mRNA levels (∼10-fold more than DMSO) which peaked at 1 h after exposure. The Tg-induced rise in c- fos mRNA content was unchanged (hippocampal neurons) or even increased further (cortical neurons) by preloading cells with BAPTA before incubation with Tg. It is concluded that in neuronal cells an increase in cytoplasmic calcium activity is not a prerequisite for a rise in mRNA levels of c- fos . Thus, stress-induced changes in mRNA levels of immediate early genes of neurons may also result from disturbances in ER calcium homeostasis and not necessarily by an overload of cells with calcium ions. The results of the present series of experiments cast further doubt on the widely accepted hypothesis that the stress-induced cytoplasmic overload of neurons with calcium ions is the primary event triggering cell injury.     

Widespread tissue distribution of rabbit calreticulin,a non-muscle functional analogue of calsequestrin

Summary Calreticulin was identified in a variety of rabbit tissues by Western blot analysis. Indirect immunofluorescence studies on cultured cells or frozen sections from the corresponding tissues revealed that the protein was distributed to the endoplasmic reticulum or sarcoplasmic reticulum. Calreticulin was found to be an abundant calcium-binding protein in non-muscle and smooth muscle cells and a constitutent calcium-binding protein in cardiac and skeletal muscle. From the immunoblot data, calreticulin may exist as an isoform in rabbit neural retina. The present study establishes the ubiquity of calreticulin in intracellular calcium binding.     

Interaction of Newly Synthesized Apolipoprotein B with Calnexin and Calreticulin Requires Glucose Trimming in the Endoplasmic Reticulum

Apolipoprotein B (ApoB) is the only protein component of the low density lipoproteins (LDL) in plasma. It is a glycoprotein with a molecular mass of about 550 kDa (4536 amino acids) containing 16 N-glycans. We have studied the interaction of ApoB with two lectin-like chaperones of the Endoplasmic Reticulum (ER)—Calnexin (CN) and Calreticulin (CR). Using a co-immunoprecipitation approach we observed that newly synthesized ApoB associates with CN and CR. The interaction was transient; within 30–60 min after synthesis bulk of newly formed ApoB dissociated. Using McA Rh7777 cells expressing an N-terminal fragment of ApoB we found that inhibition of glucosidases in the ER prevented the association of CN and CR to newly synthesized ApoB. The results showed that like for association with other glycoprotein substrates, trimming of glucose residues was essential for ApoB binding to CN and CR.     

Oxytocin induces intracellular Ca2+ release in cardiac fibroblasts from neonatal rats

Pituitary neuropeptide oxytocin is increasingly recognised as a cardiovascular hormone, in addition to its many regulatory roles in other organ systems. Studies in atrial and ventricular myocytes from the neonatal and adult rats have identified synthesis of oxytocin and the expression of oxytocin receptors in these cells. In cardiac fibroblasts, the most populous non-myocyte cell type in mammalian heart, the oxytocin receptors have not been described before. In the present study, we have investigated the direct effects of oxytocin on intracellular Ca²⁺ dynamics in ventricular myocytes and fibroblasts from new born rats. In myocytes, oxytocin increased the frequency of spontaneous Ca²⁺ transients and decreased their amplitude. Our data suggest that oxytocin receptors are also present and functional in the majority of cardiac fibroblasts. We used selective oxytocin receptor inhibitor L-371,257 and a number of intracellular Ca ²⁺ release blockers to investigate the mechanism of oxytocin induced Ca²⁺ signalling in cardiac fibroblasts. Our findings suggest that oxytocin induces Ca²⁺ signals in cardiac fibroblasts by triggering endoplasmic reticulum Ca²⁺ release via inositol trisphosphate activated receptors. The functional significance of the oxytocin induced Ca²⁺ signalling in cardiac fibroblasts, especially for their activation into secretory active myofibroblasts, remains to be investigated.     

Calreticulin transacylase: Genesis,mechanism of action and biological applications

Our earlier investigations have identified a unique enzyme in the endoplasmic reticulum (ER) termed Acetoxy Drug: Protein Transacetylase (TAase) catalyzing the transfer of acetyl group from polyphenolic acetates (PA) to certain receptor proteins (RP). An elegant assay procedure for TAase was developed based on the inhibition of glutathione S-transferase (GST) due to acetylation by a model acetoxycoumarin, 7, 8-Diacetoxy-4-methylcoumarin (DAMC). TAase purified from various mammalian tissue microsomes to homogeneity exhibited a molecular weight (M.wt) of 55 kDa. Further, by N-terminal sequencing TAase was identified as Calreticulin (CR), a multifunctional Ca²⁺-binding protein in ER lumen. The identity of TAase with CR was evidenced by proteomics studies such as immunoreactivity with anti-CR antibody and mass spectrometry. This function of CR was termed Calreticulin transacetylase (CRTAase). CRTAase was also found to mediate the transfer of acetyl group from DAMC to RP such as NADPH Cytochrome c Reductase (CYPR) and Nitric Oxide Synthase (NOS). The autoacetylation of purified human placental CRTAase concomitant with the acetylation of RP by DAMC was observed. CRTAase activity was found to be inhibited by Ca²⁺. Our investigations on the individual domains (N, P and C) of CR from a nematode Haemonchus contortus revealed that the P-domain alone was found to possess CRTAase activity. Based on the observation that the autoacetylated CR was a stable intermediate in the CRTAase catalyzed protein acetylation by PA, a putative mechanism was proposed. Further, CRTAase was also found capable of transferring propionyl group from a propoxy derivative of polyphenol, 7,8-Dipropoxy-4-methylcoumarin (DPMC) to RP and concomitant autopropionylation of CR was encountered. Hence, CRTAase was assigned the general term Calreticulin Transacylase. Also, CRTAase was found to act upon the biological acyl group donors, acetyl CoA and propionyl CoA. CRTAase mediated modulation of specific functional proteins by way of acylation was exploited to elicit the biological applications of PA.     

Calreticulin in the heart

Calreticulin is a Ca²⁺ binding/storage chaperone resident protein of the endoplasmic reticulum. This protein plays a key role in the calreticulin/calnexin cycle and the quality control pathways in the endoplasmic reticulum. Calreticulin deficiency is lethal due to impaired cardiac development. However, over-expression of the protein in developing and postnatal heart leads to bradycardia, complete heart block and sudden death. Ultrastructural evidence indicates that the deficiency associated with the absence of calreticulin in the heart may be due to a defect in the development of the contractile apparatus and/or a defect in development of the conductive system as well as a metabolic abnormality. Collectively, we postulate that calreticulin and endoplasmic reticulum plays an important role in cardiac development and postnatal pathologies. (Mol Cell Biochem 263: 137–142, 2004)     

Calcium microdomains and cell cycle control

The cell division cycle comprises successive rounds of genome replication and segregation that are never error-free. A complex signalling network chaperones cell cycle events to ensure that cell cycle progression does not occur until any errors detected are put right. The signalling network consists of cell cycle control proteins that are phosphorylated and dephosphorylated, synthesized and degraded interactively to generate a set of sensors and molecular switches that are thrown at appropriate times to permit or trigger cell cycle progression. In early embryos, discrete calcium signals have been shown to be a key component of the molecular switch mechanism. In somatic cells in contrast, the participation of calcium signals in cell cycle control is far from clear. Recent experiments in syncytial Drosophila embryos have shown that localised calcium signals in the nucleus and mitotic spindle can be detected. It appears that the nucleus comprises a calcium signalling microdomain bounded by endoplasmic reticulum that isolates the nucleus and spindle. These findings offer a possible explanation for the apparent absence of calcium signals in somatic cells during mitosis.     

Membrane traffic from the endoplasmic reticulum to the Golgi apparatus is disturbed by an inhibitor of the Ca2+-ATPase in the ER

Summary An electron microscopic study of cress (Lepidium sativum L.) roots treated with cyclopiazonic acid (CPA), an inhibitor of the Ca²⁺-ATPase in the endoplasmic reticulum (ER) has been carried out. Drastic changes in the endomembrane system of the secretory root cap cells were observed. After treatment with CPA dense spherical or elliptoidal aggregates of ER (diameter 2–4 m) were formed in addition to the randomly distributed ER cisternae characteristic for control cells. The formation of ER aggregates indicates that in spite of an inhibition of the Ca²⁺ -ATPase in the ER by CPA, membrane synthesis in the ER continued. The ER aggregates are interpreted as a reservoir of ER membrane material newly synthesized during the 2 h CPA-treatment. Hypertrophied Golgi cisternae and secretory vesicles, which are characteristic for secretory cells under control conditions, were completely absent. Additionally the shape of the Golgi stacks was flat and the diameter of the cisternae was shortened by about one third. These phenomena are indicative of an inactive state of the Golgi apparatus. The cellular organization of both other cell types of the root cap, meristematic cells and statocytes, was not visibly affected by CPA, both having a relatively low secretory activity. The formation of ER aggregates as well as the reduction of Golgi compartments are indications for the existence of a unidirectional transport of membrane material from the ER to the Golgi. It is suggested that the membrane traffic from the ER to the Golgi apparatus is regulated by the cytosolic and/or luminal calcium concentration in secretory cells of the root cap.Abbreviations CPA cyclopiazonic acid - ER endoplasmic reticulum