Family Of Ca2 Research Articles

Contribution of Anoctamins to Cell Survival and Cell Death.

Before anoctamins (TMEM16 proteins) were identified as a family of Ca2+-activated chloride channels and phospholipid scramblases, the founding member anoctamin 1 (ANO1, TMEM16A) was known as DOG1, a marker protein for gastrointestinal stromal tumors (GIST). Meanwhile, ANO1 has been examined in more detail, and the role of ANO1 in cell proliferation and the development of different types of malignomas is now well established. While ANO5, ANO7, and ANO9 may also be relevant for growth of cancers, evidence has been provided for a role of ANO6 (TMEM16F) in regulated cell death. The cellular mechanisms by which anoctamins control cell proliferation and cell death, respectively, are just emerging; however, the pronounced effects of anoctamins on intracellular Ca2+ levels are likely to play a significant role. Recent results suggest that some anoctamins control membrane exocytosis by setting Ca2+i levels near the plasma membrane, and/or by controlling the intracellular Cl− concentration. Exocytosis and increased membrane trafficking induced by ANO1 and ANO6 may enhance membrane expression of other chloride channels, such as CFTR and volume activated chloride channels (VRAC). Notably, ANO6-induced phospholipid scrambling with exposure of phosphatidylserine is pivotal for the sheddase function of disintegrin and metalloproteinase (ADAM). This may support cell death and tumorigenic activity of IL-6 by inducing IL-6 trans-signaling. The reported anticancer effects of the anthelminthic drug niclosamide are probably related to the potent inhibitory effect on ANO1, apart from inducing cell cycle arrest through the Let-7d/CDC34 axis. On the contrary, pronounced activation of ANO6 due to a large increase in intracellular calcium, activation of phospholipase A2 or lipid peroxidation, can lead to ferroptotic death of cancer cells. It therefore appears reasonable to search for both inhibitors and potent activators of TMEM16 in order to interfere with cancer growth and metastasis.

LabCaS for Ranking Potential Calpain Substrate Cleavage Sites from Amino Acid Sequence.

Calpains are a family of Ca2+-dependent cysteine proteases involved in many important biological processes, where they selectively cleave relevant substrates at specific cleavage sites to regulate the function of the substrate proteins. Presently, our knowledge about the function of calpains and the mechanism of substrate cleavage is still limited due to the fact that the experimental determination and validation on calpain bindings are usually laborious and expensive. This chapter describes LabCaS, an algorithm that is designed for predicting the calpain substrate cleavage sites from amino acid sequences. LabCaS is built on a conditional random field (CRF) statistic model, which trains the cleavage site prediction on multiple features of amino acid residue preference, solvent accessibility information, pair-wise alignment similarity score, secondary structure propensity, and physical-chemistry properties. Large-scale benchmark tests have shown that LabCaS can achieve a reliable recognition of the cleavage sites for most calpain proteins with an average AUC score of 0.862. Due to the fast speed and convenience of use, the protocol should find its usefulness in large-scale calpain-based function annotations of the newly sequenced proteins. The online web server of LabCaS is freely available at http://www.csbio.sjtu.edu.cn/bioinf/LabCaS .

Fluorine-18 Labeling of S100 Proteins for Small Animal Positron Emission Tomography.

The interaction of S100 proteins (S100s), a multigenic family of Ca2+-binding and Ca2+-modulated proteins, with pattern recognition receptors, e.g., Toll-like receptors (TLRs), the receptor for advanced glycation end products (RAGE), or scavenger receptors (SR), is hypothesized to be of high relevance in the pathogenesis of various diseases. This includes chronic inflammatory conditions, atherosclerosis, cardiomyopathies, neurodegeneration, and progression of cancers. However, data concerning the role of circulating S100s in these pathologies are scarce. One reason for this is the shortage of suitable radiolabeling methods for direct assessment of the metabolic fate of circulating S100s in vivo. We report a radiotracer approach using radiolabeling of recombinant human S100s with the positron emitter fluorine-18 (18F) by conjugation with N-succinimidyl 4-[18F]fluorobenzoate ([18F]SFB). The methodological radiochemical part focuses on an optimized and automated synthesis of [18F]SFB comprising HPLC purification to achieve higher chemical purity. The respective radioligands, [18F]fluorobenzoylated S100s ([18F]FB-S100s), were obtained with appropriate radiochemical purities, yields, and effective molar activities. Biological applications comprise cell and tissue binding experiments in vitro, biodistribution and metabolite studies in rodents in vivo/ex vivo, and dynamic positron emission tomography studies using dedicated small animal PET systems. Radiolabeling of S100s with 18F and, particularly, the use of small animal PET provide novel probes to delineate both their metabolic fate and the functional expression of their specific receptors under normal and pathophysiological conditions in rodent models of disease.

Abstract 4667: The use of fragment-based screening approaches to improve the specificity of inhibitors targeting malignant melanoma

Abstract S100B belongs to the S100 protein family of Ca2+-binding proteins. Prior studies have shown that S100B is a biological marker for malignant melanoma. Increases in S100B levels are predictive of disease progression, recurrence, and overall low survival in the patients. It was shown previously that S100B binds to p53 and down-regulates its tumor suppression activities including growth arrest and apoptosis. Chemical biology and structure-based drug design methods are underway to inhibit S100B and restore p53 function. However, highly specific S100B inhibitors are required, so S100B can be blocked in melanoma without impairing S100A1 function in skeletal and cardiac muscle. With this goal in mind, a fragment-based screening approach using NMR and computational approaches is being applied. Results to date indicate that these methods can be used to identify specific small molecule fragments that target S100B and not S100A1. The fragments that uniquely bind S100B will then be considered with structural data of existing S100B-SBiX complexes to improve their specificity for binding S100B (i.e. versus S100A1). The inhibitor design strategy will also include improving the stability of the compounds their cell permeability, and their overall drug-like properties. The most promising of these new inhibitors will ultimately be used to target high levels of S100B found in malignant and rescue p53-dependent tumor suppression pathways in vivo with the goal of them having therapeutic value for treating malignant melanoma. Citation Format: Sreya Mukherjee, Shardell Spriggs, Paul Wilder, Wenbo Yu, Kristen Varney, Alexander D. MacKerell, David J. Weber. The use of fragment-based screening approaches to improve the specificity of inhibitors targeting malignant melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4667.

Annexins-Coordinators of Cholesterol Homeostasis in Endocytic Pathways.

The spatiotemporal regulation of calcium (Ca2+) storage in late endosomes (LE) and lysosomes (Lys) is increasingly recognized to influence a variety of membrane trafficking events, including endocytosis, exocytosis, and autophagy. Alterations in Ca2+ homeostasis within the LE/Lys compartment are implicated in human diseases, ranging from lysosomal storage diseases (LSDs) to neurodegeneration and cancer, and they correlate with changes in the membrane binding behaviour of Ca2+-binding proteins. This also includes Annexins (AnxA), which is a family of Ca2+-binding proteins participating in membrane traffic and tethering, microdomain organization, cytoskeleton interactions, Ca2+ signalling, and LE/Lys positioning. Although our knowledge regarding the way Annexins contribute to LE/Lys functions is still incomplete, recruitment of Annexins to LE/Lys is greatly influenced by the availability of Annexin bindings sites, including acidic phospholipids, such as phosphatidylserine (PS) and phosphatidic acid (PA), cholesterol, and phosphatidylinositol (4,5)-bisphosphate (PIP2). Moreover, the cytosolic portion of LE/Lys membrane proteins may also, directly or indirectly, determine the recruitment of Annexins to LE. Strikingly, within LE/Lys, AnxA1, A2, A6, and A8 differentially contribute to cholesterol transport along the endocytic route, in particular, cholesterol transfer between LE and other compartments, positioning Annexins at the centre of major pathways mediating cellular cholesterol homeostasis. Underlying mechanisms include the formation of membrane contact sites (MCS) and intraluminal vesicles (ILV), as well as the modulation of LE-cholesterol transporter activity. In this review, we will summarize the current understanding how Annexins contribute to influence LE/Lys membrane transport and associated functions.

Functions of CaBP1 and CaBP2 in the peripheral auditory system

CaBPs are a family of Ca2+ binding proteins related to calmodulin. Two CaBP family members, CaBP1 and CaBP2, are highly expressed in the cochlea. Here, we investigated the significance of CaBP1 and CaBP2 for hearing in mice lacking expression of these proteins (CaBP1 KO and CaBP2 KO) using auditory brain responses (ABRs) and distortion product otoacoustic emissions (DPOAEs). In CaBP1 KO mice, ABR wave I was larger in amplitude, and shorter in latency and faster in decay, suggestive of enhanced synchrony of auditory nerve fibers. This interpretation was supported by the greater excitability of CaBP1 KO than WT neurons in whole-cell patch clamp recordings of spiral ganglion neurons in culture, and normal presynaptic function of CaBP1 KO IHCs. DPOAEs and ABR thresholds were normal in 4-week old CaBP1 KO mice, but elevated ABR thresholds became evident at 32 kHz at 9 weeks, and at 8 and 16 kHz by 6 months of age. In contrast, CaBP2 KO mice exhibited significant ABR threshold elevations at 4 weeks of age that became more severe in the mid-frequency range by 9 weeks. Though normal at 4 weeks, DPOAEs in CaBP2 KO mice were significantly reduced in the mid-frequency range by 9 weeks. Our results reveal requirements for CaBP1 and CaBP2 in the peripheral auditory system and highlight the diverse modes by which CaBPs influence sensory processing.

Modeling of annexin A2-Membrane interactions by molecular dynamics simulations.

The annexins are a family of Ca2+-regulated phospholipid binding proteins that are involved in membrane domain organization and membrane trafficking. Although they are widely studied and crystal structures are available for several soluble annexins their mode of membrane association has never been studied at the molecular level. Here we obtained molecular information on the annexin-membrane interaction that could serve as paradigm for the peripheral membrane association of cytosolic proteins by Molecular Dynamics simulations. We analyzed systems containing the monomeric annexin A2 (AnxA2), a membrane with negatively charged phosphatidylserine (POPS) lipids as well as Ca2+ ions. On the atomic level we identify the AnxA2 orientations and the respective residues which display the strongest interaction with Ca2+ ions and the membrane. The simulation results fully agree with earlier experimental findings concerning the positioning of bound Ca2+ ions. Furthermore, we identify for the first time a significant interaction between lysine residues of the protein and POPS lipids that occurs independently of Ca2+ suggesting that AnxA2-membrane interactions can also occur in a low Ca2+ environment. Finally, by varying Ca2+ concentrations and lipid composition in our simulations we observe a calcium-induced negative curvature of the membrane as well as an AnxA2-induced lipid ordering.

Determinants and Regulation of Protein Turnover in Yeast.

Protein turnover maintains the recycling needs of theproteome, and its malfunction has been linked to aging and age-related diseases. However, not allproteins turnover equally, and the factors that contribute to accelerate or slow down turnover are mostly unknown. We measured turnover rates for 3,160 proteins in exponentially growing yeast and analyzed their dependence on physical, functional, and genetic properties. We found that functional characteristics, including protein localization, complex membership, and connectivity, have greater effect on turnover than sequence elements. We also found that protein turnover and mRNA turnover are correlated. Analysis under nutrient perturbation and osmotic stress revealed that protein turnover highly depends on cellular state and is faster when proteinsare being actively used. Finally, stress-induced changes in protein and transcript abundance correlated with changes in protein turnover. This study provides a resource of protein turnover rates and principles to understand the recycling needs of the proteome under basal conditions and perturbation.

Possible roles of transglutaminases in molecular mechanisms responsible for cancer and human neurodegenerative diseases

Transglutaminases are a family of Ca2+- dependent enzymes which catalyze posttranslational modifications of proteins. The main activity of these enzymes is the crosslinking of glutaminyl residues of a protein/peptide substrate to lysyl residues of a protein/peptide co-substrate. In addition to lysyl residues, other second nucleophilic co-substrates may include monoamines or polyamines (to form mono- or bi-substituted/ crosslinked adducts) or -OH groups (to form ester linkages). In absence of co-substrates, the nucleophile may be water, resulting in the net deamidation of the glutaminyl residue. Transglutaminase activity has been suggested to be involved in molecular mechanisms responsible for either physiological or pathological processes. In particular, transglutaminase activity has been shown to be responsible for human autoimmune diseases and Celiac Disease is just one of them. Interestingly, cancer and neurodegenerative diseases, such as Alzheimer’s Disease, Parkinson’s Disease, supranuclear palsy and Huntington’s Disease, are characterized in part by aberrant transglutaminase activity and by increased cross-linked proteins in affected tissues. This review describes the possible molecular mechanisms by which these enzymes could be responsible for such diseases and the possible use of transglutaminase inhibitors for patients with diseases characterized by aberrant transglutaminase activity.

Silence of inositol 1,4,5-trisphosphate receptor expression decreases cyantraniliprole susceptibility in Bemisia tabaci

Cyantraniliprole is the second active ingredient of anthranilic diamide insecticide, and the first to control a cross-spectrum of chewing and sucking pests such as sweetpotato whitefly, Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae). The inositol 1,4,5-trisphosphate receptor (IP3R) and ryanodine receptor (RyR) are two families of Ca2+ release channels to raise the cytoplasmic free calcium concentration when it is activated by various extracellular stimuli. Previous study proved the over-expression of ryanodine receptor (RyR) was associated with the resistance to diamide insecticides, while the roles of IP3R in diamide resistance remain unknown. In this study, a full-length cDNA sequence of IP3R was cloned from B. tabaci through RT-PCR and rapid amplification of cDNA ends (RACE). The gene (named BtIP3R) is 9922bps long, with an open reading frame (ORF) of 8202bps, encoding a predicted IP3R of 2733 amino acids. The BtIP3R shares 47–78% identity with other insect IP3Rs. Quantitative real-time PCR (qRT-PCR) analysis showed that the BtIP3R was highly expressed in larva, pseudopupa, and female adult, while lowly expressed in egg and male adult. RNA interference (RNAi) by dietary introduction of double-stranded RNA (dsRNA) of BtIP3R significantly reduced the mRNA levels of the target gene in the adult, and dramatically decreased the susceptibility of adult B. tabaci to cyantraniliprole. The results shed light on further understanding of cyantraniliprole resistance mechanisms in B. tabaci as well as in other insects.

A Gastrointestinal Calpain Complex, G-calpain, Is a Heterodimer of CAPN8 and CAPN9 Calpain Isoforms, Which Play Catalytic and Regulatory Roles, Respectively

Calpains (CAPN) are a family of Ca2+-dependent cysteine proteases that regulate various cellular functions by cleaving diverse substrates. Of the 15 mammalian calpains, CAPN8 and CAPN9 are two that are expressed predominantly in the gastrointestinal tract, where they interact to form a protease complex, termed G-calpain. However, because native G-calpain exhibits a highly restricted expression pattern, it has never been purified, and the interactions between CAPN8 and CAPN9 have not been characterized. Here, we clarified the molecular nature of G-calpain by using recombinant proteins and transgenic mice expressing FLAG-tagged CAPN8 (CAPN8-FLAG). Recombinant mouse CAPN8 and CAPN9 co-expressed in eukaryotic expression systems exhibited the same mobility as native mouse G-calpain in Blue Native-PAGE gels, and CAPN8-FLAG immunoprecipitation from stomach homogenates of the transgenic mice showed that CAPN9 was the only protein that associated with CAPN8-FLAG. These results indicated that G-calpain is a heterodimer of CAPN8 and CAPN9. In addition, active recombinant G-calpain was expressed and purified using an in vitro translation system, and the purified protease exhibited enzymatic properties that were comparable with that of calpain-2. We found that an active-site mutant of CAPN8, but not CAPN9, compromised G-calpain's substrate cleavage activity, and that the N-terminal helix region of CAPN8 and the C-terminal EF-hands of CAPN8 and CAPN9 were involved in CAPN8/9 dimerization. Furthermore, CAPN8 protein in Capn9-/- mice was almost completely lost, whereas CAPN9 was only partially lost in Capn8-/- mice. Collectively, these results demonstrated that CAPN8 and CAPN9 function as catalytic and chaperone-like subunits, respectively, in G-calpain.

Expression of anoctamins in retinal pigment epithelium (RPE).

The anoctamin (ANO, TMEM16) family of Ca2+-activated Cl- channels consists of ten members with different cellular functions (ANO1-10). ANO1 is a Ca2+-activated Cl- channel in secretory epithelial cells of exocrine pancreas, salivary glands, or enterocytes. Expression of ANO1 also promotes cell proliferation and migration of tumor cells. ANO6 is essential for Ca2+-dependent scrambling of membrane phospholipids in platelets, red blood cells, and lymphocytes. ANO10 modulates Ca2+ signals in macrophages and has a role in cerebellar ataxia and other neurological disorders. All three anoctamins have been proposed to control intracellular Ca2+ signals. Anoctamins may also form the basolateral Ca2+-activated Cl- channel in the retinal pigment epithelium (RPE). We show that native human, bovine, porcine, and mouse RPEs express ANO1, ANO6, and ANO10. Growth arrested and confluent RPR cells expressed ANO1 in the plasma membrane, whereas ANO6 and ANO10 were found in the primary cilium. Ussing chamber experiments showed that the application of ATP to the apical (retinal) side of porcine RPE induced a Ca2+-activated Cl- secretion. Activation was inhibited by basolateral (choroidal) administration of the ANO inhibitors AO1, niflumic acid (NFA), and 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS). The results suggest that ANO1 is responsible for basolateral Ca2+-dependent Cl- secretion in RPE, whereas ANO6 and ANO10 may have different functions, such as modulating Ca2+ signals.

Abstract A015: Protein arginine deiminase enzymes which citrullinate epitopes for MHC II presentation are independent predictors of survival in colorectal cancer

Abstract Citrullination of proteins is associated with a number of autoimmune diseases. Protein Arginine Deiminases (PADs) are a family of Ca2+ dependent enzymes that under cellular stress post-translationally convert arginine to citrulline within protein substrates to generate self-modified neo-antigens. It has been shown that presentation of citrullinated peptides on MHC class II stimulates CD4 T cells to mediate potent anti-tumor immunity (1). In this study we focus on the role of the PADI2 and PADI4 family members in colorectal cancer. Using a tissue microarray of colorectal cancers and compiling a comprehensive database of clinicopathological variables, the expression of PADI2 and PADI4 was assessed by immunohistochemistry in a study cohort. This data was used to correlate PADI2 and PADI4 expression with patient survival. In the study cohort 462 colorectal tumors were stained for PADI2 and PADI4. Of these 171 (37%) and 231 (50%) could not be evaluated for PADI2 and PADI4 staining respectively due to the absence of enough tissue core or no evaluable tumor cells (i.e. all stroma) in the core. Of the 291 evaluable colorectal tumors stained with a PADI2 specific antibody, only 18/291 (6.2%) tumors failed to stain. A further 153/291 (52.5%) stained weakly, 102/291 (35.1%) moderate and 18/291 (6.2%) stained strongly. Of the 231 evaluable colorectal tumors stained with a PADI4 specific antibody, no tumors failed to stain. All cases stained strongly for PADI4 expression within the nucleus. In the cytoplasm 63/231 (27.3%) stained weakly, 143/231 (61.9%) moderate and 25/231 (10.8%) stained strongly. PADI2 expression did not correlate with any clinicopathological variables whereas nuclear but not cytoplasmic PADI4 showed a strong association with histological type (p = 0.008). Kaplan-Meier analysis showed there was a correlation of PADI2 and cytoplasmic PADI4 expression with improved survival. Expression of PADI2 gave an increase in survival time from 44.8 months (95% CI 24.3 to 65.4) to 76.2 months (95% CI 69.9 to 82.4, log rank test, p = 0.012). Expression of cytoplasmic PADI4 increased survival time from 57.9 months (95% CI 43.6 to 72.3) to 77.3 months (95% CI 69.6 to 85.1, log rank test, p = 0.012). No significant correlation was observed between PADI2 and the cytoskeletal protein Vimentin or the glycolytic enzyme α-enolase both reported to be citrullinated by PAD enzymes. PADI2 expression was significantly associated with expression of the Nuclear antigen Ki67 (p = 0.046) a cellular marker for proliferation. Nuclear PADI4 significantly correlated with the cytoplasmic glycolytic enzyme α-enolase only (p = 0.001) and cytoplasmic PADI4 was highly significantly associated with α-enolase located in both the cytoplasm (p<0.001) and shorter nuclear form (p<0.001) known as MBP-1, a transcription factor known to downregulate the activity of the c-myc proto-oncogene. Cytoplasmic PADI4 expression also significantly correlated with expression of the anti-apoptotic protein Bcl2 (p = 0.028). Multivariate analysis using Cox regression showed that PADI2 (p = 0.040)/Cytoplasmic PADI4 (p = 0.036), the stress related protein MICA (p = 0.006/0.022), vascular invasion (p<0.001, 0.040) and tumor stage (p<0.001/<0.001) are all independent markers of good prognosis in colorectal cancer. These results are consistent with the hypothesis that stressed tumor cells present citrullinated epitopes allowing their growth to be controlled by T cells.

Development of a sandwich ELISA assay for quantification of human tissue transglutaminase in cell lysates and tissue homogenates.

Tissue transglutaminase (tTG) belongs to the multigene transglutaminase family of Ca2+-dependent protein cross-linking enzymes. There is a strong evidence that tTG is involved in pathology, such as neurodegenerative diseases, cancer, and celiac disease. To study physiopathological implication of tTG, a sandwich immunoassay has been developed with a new monoclonal antibody for the capture and polyclonal antibody both generated in house. Using this ready to use assay, the tTG protein level can be measured in human tissue homogenates and cells extracts easily in about 4h. The limit of detection is 1.7ng/ml; the coefficients of intra- and inter-assay variations range from 1 to 2% and from 7 to 10%, respectively. The assay is specific to tTG, and no cross reactivity with TG1, TG3, TG6, TG7, or factor XIIIa was observed. Finally, in the addition to the tTG activity assay previously developed, this assay should be a valuable tool to increase our knowledge of the tTG involvement in physiologicaland pathological states.

TrkB and the calpain dependent-Tc TrkB receptor isoforms: possible neurological therapeutic targets

Several neurological conditions share a characteristic feature: an increase in intracellular calcium levels ([Ca2+]i). It has been demonstrated that calcium influx induces changes ranging from an increase in the expression levels of several genes to the activation of proteases such as calpains. Calpains are a family of Ca2+-dependent non-lysosomal cysteine proteases, whose substrates include several proteins that play critical roles in several cellular functions including synaptic plasticity and neuronal apoptosis. TrkB is a type of tyrosine related kinase receptor that can promote neuronal survival and differentiation upon ligand binding. It has been recently shown that in several neurological diseases, the level of full-length TrkB protein decreases before the onset of neuronal death due to one of two different processes: a) a reverse regulation of TrkB isoforms mRNA, or b) calpain-mediated processing of TrkB full-length, which yields a truncated form of TrkB (Tc-TrkB). Because the most notorious feature of calpain proteolytic activity is that the products of calpain-mediated cleavage may have biological activity, here we review the hypotheses by which calpain-generated isoform Tc-TrkB may perform biological functions.

IMPS-44S100B INHIBITION WITH DULOXETINE, A SEROTONIN-NOREPINEPHRINE REUPTAKE INHIBITOR, ALTERS MACROPHAGE POLARIZATION AND ABROGATES GLIOMA GROWTH IN MICE

S100B, a member of the multigene family of Ca2 + -binding proteins, is overexpressed by most malignant gliomas but its biological role in gliomagenesis is unclear. Recently, we demonstrated that low concentrations of S100B attenuated microglia activation through the induction of STAT3. Furthermore, S100B downregulation in a murine glioma model inhibited macrophage trafficking and tumor growth. Based on these observations, we hypothesized that S100B inhibitors may have antiglioma properties through modulation of tumor microenvironment. To discover novel S100B inhibitors, we developed a high-throughput screening cell-base S100B promoter-driven luciferase reporter assay. Initial screening of 25,904 compounds in NIH and ChemBridge libraries identified 1297 hits with >85% S100B inhibitory activity. One compound, duloxetine (Dul, a serotonin-norepinephrine reuptake inhibitor) was selected for the initial proof-of-concept studies. At low concentrations (1-5 □M) Dul inhibited S100B and CCL2 production by mouse GL261 glioma cell lines, but had minimal cytotoxic activity. In vivo, Dul (30 mg/kg/14 days) inhibited S100B production, altered polarization and trafficking of macrophages and abrogated the growth of intracranial GL261 gliomas. Dul therapeutic efficacy, however, was not observed in the K-Luc glioma model that expresses low levels of S100B. These findings affirm the role of S100B in gliomagenesis and justify the development of S100B inhibitors for glioma therapy.

STIM1 is cleaved by calpain

Store-operated calcium entry (SOCE) is a pathway that moves Ca2+ across the plasma membrane and is mediated by two major proteins, STIM1 and Orai1. Here, we discovered that the cytoplasmic domain of STIM1 is a target for calpains, a family of Ca2+-activated proteases. We found that calpain cleavage of STIM1 serves to control its cellular abundance and was noticeably increased under conditions of cellular stress and apoptosis. Dysregulation of STIM1 levels has been reported to have human disease consequences and our results suggest a mechanism for controlling STIM1 abundance.

The calcium-activated chloride channel Anoctamin 1 contributes to the regulation of renal function

The role of calcium-activated chloride channels for renal function is unknown. By immunohistochemistry we demonstrate dominant expression of the recently identified calcium-activated chloride channels, Anoctamin 1 (Ano1, TMEM16A) in human and mouse proximal tubular epithelial (PTE) cells, with some expression in podocytes and other tubular segments. Ano1-null mice had proteinuria and numerous large reabsorption vesicles in PTE cells. Selective knockout of Ano1 in podocytes (Ano1-/-/Nphs2-Cre) did not impair renal function, whereas tubular knockout in Ano1-/-/Ksp-Cre mice increased urine protein excretion and decreased urine electrolyte concentrations. Purinergic stimulation activated calcium-dependent chloride currents in isolated proximal tubule epithelial cells from wild-type but not from Ano1-/-/Ksp-Cre mice. Ano1 currents were activated by acidic pH, suggesting parallel stimulation of Ano1 chloride secretion with activation of the proton-ATPase. Lack of calcium-dependent chloride secretion in cells from Ano1-/-/Ksp-Cre mice was paralleled by attenuated proton secretion and reduced endosomal acidification, which compromised proximal tubular albumin uptake. Tubular knockout of Ano1 enhanced serum renin and aldosterone concentrations, probably leading to enhanced compensatory distal tubular reabsorption, thus maintaining normal blood pressure levels. Thus, Ano1 has a role in proximal tubular proton secretion and protein reabsorption. The results correspond to regulation of the proton-ATPase by the Ano1-homolog Ist2 in yeast.

μ-Calpain as a Novel Target for Impairment of Nitric Oxide-Mediated Vascular Relaxation in Diabetes: A Mini Review.

Diabetes is one of the most prevalent metabolic disorders. In diabetes, incidence of coronary artery diseases and peripheral vascular diseases is increased 2- to 4-fold and 10-fold, respectively, compared to healthy individuals. In spite of extensive studies, the underlying mechanisms of endothelial dysfunction (ED), an early event in the development of vascular diseases, remain incompletely understood in diabetes. This mini-review discusses the role and signaling pathways of calpains - a family of Ca2+-sensitive intracellular proteases in nitric oxide (NO)-mediated ED in diabetes. We conclude that activation of calpains, especially μ-calpain, plays an important role in the pathogenesis of NO-mediated ED and inflammatory responses in diabetes which is mainly via endothelial Nitric Oxide Synthase (eNOS) inactivation/degradation in macro- and micro-vasculature. We review existing literature demonstrating that hyperhomocysteinemia, elevated plasma homocysteine level, potentiates hyperglycemia-induced ED via μ-calpain/PKCβ2 activation-induced eNOS-pThr497/495 and eNOS inactivation. μ-calpain may be a critical therapeutic target for NO-mediated ED in diabetes.

A C-type lectin (AiCTL-3) from bay scallop Argopecten irradians with mannose/galactose binding ability to bind various bacteria

C-type lectins are a family of Ca2+-dependent carbohydrate-binding proteins playing crucial roles in innate immunity of vertebrates and invertebrates. In the present study, the cDNA of a C-type lectin with one carbohydrate-recognition domain (CRD) of 127 amino acids was cloned from bay scallop Argopecten irradians (designated AiCTL-3) by rapid amplification of cDNA end (RACE) techniques based on expressed sequence tag (EST) analysis. The mRNA transcripts of AiCTL-3 could be detected in all the tested tissues including hepatopancreas, gonad, adductor muscle, heart, hemocytes, mantle and gill, with the highest expression level in hepatopancreas. After the challenges with Vibrio anguillarum and Micrococcus luteus, the mRNA expression level of AiCTL-3 was obviously up-regulated and reached the maximum level at 9h (11.87fold, P<0.01, and 20.02-fold, P<0.05, respectively). The recombinant AiCTL-3 (designated as rAiCTL-3) could bind LPS, PGN, and glucan in vitro, but could not bind mannan. And it also bound Gram-positive bacteria Staphylococcus aureus as well as Gram-negative bacteria Escherichia coli and V. anguillarum. With a Ca2+ binding site 2 EPN (Glu–Pro–Asn) motif, rAiCTL-3 could bind both mannose and galactose which was quite different from those in vertebrate. Meanwhile, it could significantly enhance the phagocytosis of scallop hemocytes in vitro. The results clearly suggested that AiCTL-3 could serve not only as a PRR participated in the immune response against various PAMPs and bacteria in non-self recognition via mannose/galactose binding specificity but an opsonin playing an important part in clearance of invaders.