Ca Binding Protein Research Articles

Early cellular and synaptic changes in dopaminoceptive forebrain regions of juvenile mice following gestational exposure to valproate.

Gestational exposure of mice to valproic acid (VPA) is one currently used experimental model for the investigation of typical failure symptoms associated with autism spectrum disorder (ASD). In the present study we hypothesized that the reduction of dopaminergic source neurons of the VTA, followed by perturbed growth of the mesotelencephalic dopamine pathway (MT), should also modify pattern formation in the dopaminoceptive target regions (particularly its mesoaccumbens/mesolimbic portion). Here, we investigated VPA-evoked cellular morphological (apoptosis-frequency detected by Caspase-3, abundance of Ca-binding proteins, CaBP), as well as synaptic proteomic (western blotting) changes, in selected dopaminoceptive subpallial, as compared to pallial, regions of mice, born to mothers treated with 500 mg/kg VPA on day 13.5 of pregnancy. We observed a surge of apoptosis on VPA treatment in nearly all investigated subpallial and pallial regions; with a non-significant trend of similar increase the nucleus accumbens (NAc) at P7, the age at which the MT pathway reduction has been reported (also supplemented by current findings). Of the CaBPs, calretinin (CR) expression was decreased in pallial regions, most prominently in retrosplenial cortex, but not in the subpallium of P7 mice. Calbindin-D 28K (CB) was selectively reduced in the caudate-putamen (CPu) of VPA exposed animals at P7 but no longer at P60, pointing to a potency of repairment. The VPA-associated overall increase in apoptosis at P7 did not correlate with the abundance and distribution of CaBPs, except in CPu, in which the marked drop of CB was negatively correlated with increased apoptosis. Abundance of parvalbumin (PV) at P60 showed no significant response to VPA treatment in any of the observed regions we did not find colocalization of apoptotic (Casp3+) cells with CaBP-immunoreactive neurons. The proteomic findings suggest reduction of tyrosine hydroxylase in the crude synaptosome fraction of NAc, but not in the CPu, without simultaneous decrease of the synaptic protein, synaptophysin, indicating selective impairment of dopaminergic synapses. The morpho-functional changes found in forebrain regions of VPA-exposed mice may signify dendritic and synaptic reorganization in dopaminergic target regions, with potential translational value to similar impairments in the pathogenesis of human ASD.

Vitamin D and calcium co-therapy mitigates pre-established cadmium nephropathy by regulating renal calcium homeostatic molecules and improving anti-oxidative and anti-inflammatory activities in rat

BackgroundCadmium (Cd) is a major environmental pollutant and chronic toxicity could induce nephropathy by increasing renal oxidative stress and inflammation. Although vitamin D (VD) and calcium (Ca) prophylactic treatments attenuated Cd-induced cell injury, none of the prior studies measure their renoprotective effects against pre-established Cd-nephropathy. AimsTo measure the alleviating effects of VD and/or Ca single and dual therapies against pre-established nephrotoxicity induced by chronic Cd toxicity prior to treatment initiation. MethodsForty male adult rats were allocated into: negative controls (NC), positive controls (PC), Ca, VD and VC groups. The study lasted for eight weeks and all animals, except the NC, received CdCl2 in drinking water (44 mg/L) throughout the study. Ca (100 mg/kg) and/or VD (350 IU/kg) were given (five times/week) during the last four weeks to the designated groups. Subsequently, the expression of transforming growth factor-β (TGF-β1), inducible nitric oxide synthase (iNOS), neutrophil gelatinase-associated lipocalin (NGAL), kidney injury molecule-1 (KIM-1), VD synthesising (Cyp27b1) and catabolizing (Cyp24a1) enzymes with VD receptor (VDR) and binding protein (VDBP) was measured in renal tissues. Similarly, renal expression of Ca voltage-dependent channels (CaV1.1/CaV3.1), store-operated channels (RyR1/ITPR1), and binding proteins (CAM/CAMKIIA/S100A1/S100B) were measured. Serum markers of renal function alongside several markers of oxidative stress (MDA/H2O2/GSH/GPx/CAT) and inflammation (IL-6/TNF-α/IL-10) together with renal cell apoptosis and expression of caspase-3 were also measured. ResultsThe PC group exhibited hypovitaminosis D, hypocalcaemia, hypercalciuria, proteinuria, reduced creatinine clearance, and increased renal apoptosis/necrosis with higher caspase-3 expression. Markers of renal tissue damage (TGF-β1/iNOS/NGAL/KIM-1), oxidative stress (MDA/H2O2), and inflammation (TNF-α/IL-1β/IL-6) increased, whilst the antioxidants (GSH/GPx/CAT) and IL-10 decreased, in the PC group. The PC renal tissues also showed abnormal expression of Cyp27b1, Cyp24a1, VDR, and VDBP, alongside Ca-membranous (CaV1.1/CaV3.1) and store-operated channels (RyR1/ITPR1) and cytosolic Ca-binding proteins (CAM/CAMKIIA/S100A1/S100B). Although VD was superior to Ca monotherapy, their combination revealed the best mitigation effects by attenuating serum and renal tissue Cd concentrations, inflammation and oxidative stress, alongside modulating the expression of VD/Ca-molecules. ConclusionsThis study is the first to show improved alleviations against Cd-nephropathy by co-supplementing VD and Ca, possibly by better regulation of Ca-dependent anti-oxidative and anti-inflammatory actions.

Rabies virus infection is associated with variations in calbindin D-28K and calretinin mRNA expression levels in mouse brain tissue.

Rabies virus (RABV) infection leads to a fatal neurological outcome in humans and animals and is associated with major alterations in cellular gene expression. In this study, we describe the effects of RABV infection on the mRNA expression levels of two genes, encoding the Ca2+-binding proteins (Ca-BPs) calbindin D-28K (Calb1) and calretinin (Calb2), in the brains of BALB/c mice. Sixty 4-week-old mice were divided into two test groups and one control group. Mice were inoculated intramuscularly with either a street rabies virus (SRV) strain or a challenge virus standard (CVS-11) strain and sacrificed at 3-day intervals up to day 18 postinfection. A direct fluorescent antibody test (DFAT) was used to verify the presence of RABV antigen in brain tissues, and real-time quantitative PCR (RT-PCR) was used to assess gene expression. Infection with both RABV strains resulted in significant (p < 0.05) increases in Calb1 and Calb2 expression in the test animals when compared with the controls at various time points in the study. Correlation analysis indicated very weak insignificant (p > 0.05) negative and positive relationships, respectively, between Calb1 expression (r = -0.04) and Calb2 expression (r = 0.08) with viral load (CVS-11 strain). Insignificant (p > 0.05) relationships were also observed Calb1 expression (r = -0.28) and Calb2 expression (r = 0.06) and viral load for the SRV strain.The observed alterations in Calb1 and Calb2 expression in this study indicate possible impairments in neuronal Ca2+ buffering and Ca2+ homeostasis as a result of RABV infection and, consequently, possible involvement of calbindin-D28K and calretinin in the neuropathogenesis of rabies.

Dysregulation of metalloproteins in ischemic heart disease patients with systolic dysfunction

Ischemic heart disease (IHD) is the leading cause of mortality worldwide. Metalloproteins have been linked to human health and diseases. The molecular functions of metalloproteins in IHD is not well understood and require further exploration. The objective of this study was to find out the role of metalloproteins in the pericardial fluid of IHD patients having normal (EF > 45) and impaired (EF < 45) left ventricular ejection fraction (LVEF). IHD patients were grouped into two categories: LVEF<45 (n = 12) and LVEF >45 (n = 33). Pooled samples of pericardial fluid were fractionated by using ZOOM-isoelectric focusing (IEF) followed by further processing using one-dimensional gel electrophoresis (1D SDS-PAGE) and filter-aided sample preparation (FASP). Tryptic peptides of each fraction and differential bands were then analyzed by nano-LC-ESI-MS/MS. Protein identification was performed through a Mascot search engine using NCBI-Prot and SwissProt databases. A total of 1082 proteins including 154 metalloproteins were identified. In the differential bands, 60 metalloproteins were identified, while 115 metalloproteins were identified in all ZOOM-IEF fractions. Twelve differentially expressed metalloproteins were selected in the intense bands according to their molecular weight (MW) and isoelectric point (pI). The 12 differentially expressed metalloprotein includes ceruloplasmin, Prothrombin, Vitamin K-dependent protein, Fibulin-1, Ribosomal protein S6 kinase alpha-6, nidogen, partial, Serum albumin, Hemopexin, C-reactive protein, Serum amyloid P-component, and Intelectin-1 protein which were all up-regulated while serotransferrin is the only metalloprotein that was down-regulated in impaired (LVEF<45) group. Among the metalloproteins, Zn-binding proteins are 36.5 % followed by Ca-binging 32.2 %, and Fe-binging 12.2 %. KEGG, pathway analysis revealed the association of ceruloplasmin and serotransferrin with the ferroptosis pathway. In conclusion, 154 metalloproteins were identified of them the Zn-binding protein followed by Ca-binding and Fe-binding proteins were the most abundant metalloproteins. The two metalloproteins, the Cu-binding protein ceruloplasmin, and Fe-binding protein serotransferrin are involved in the ferroptosis pathway, an iron-dependent form of regulated cell death that has been linked to cardiac pathology, especially in IHD patients having impaired systolic (LVEF<45) dysfunction. However, further research is required to validate these findings.

Regulation of Presynaptic Calcium Channels.

Voltage-gated calcium channels (VGCCs), especially Cav2.1 and Cav2.2, are the major mediators of Ca2+ influx at the presynaptic membrane in response to neuron excitation, thereby exerting a predominant control on synaptic transmission. To guarantee the timely and precise release of neurotransmitters at synapses, the activity of presynaptic VGCCs is tightly regulated by a variety of factors, including auxiliary subunits, membrane potential, G protein-coupled receptors (GPCRs), calmodulin (CaM), Ca2+-binding proteins (CaBP), protein kinases, various interacting proteins, alternative splicing events, and genetic variations.

Impaired Dynamic Sarcoplasmic Reticulum Ca Buffering in Autosomal Dominant CPVT2.

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a potentially lethal cardiac arrhythmia syndrome triggered by catecholamines released during exercise, stress, or sudden emotion. Variants in the calsequestrin-2 gene (CASQ2), encoding the major calcium (Ca) binding protein in the sarcoplasmic reticulum (SR), are the second most common cause of CPVT. Recently, several CASQ2 gene variants, such as CASQ2-K180R, have been linked to an autosomal dominant form of Casq2-linked CPVT (CPVT2), but the underlying mechanism is not known. A K180R mouse model was generated using CRIPSR/Cas9. Heterozygous and homozygous K180R mice were studied using telemetry ECG recordings in vivo. Ventricular cardiomyocytes were isolated and studied using fluorescent Ca indicators and patch clamp. Expression levels and localization of SR Ca-handling proteins were evaluated using Western blotting and immunostaining. Intra-SR Ca kinetics were quantified using low-affinity Ca indicators. K180R mice exhibit an autosomal dominant CPVT phenotype following exercise or catecholamine stress. Upon catecholamine stress, K180R ventricular cardiomyocytes exhibit increased spontaneous SR Ca release events, triggering delayed afterdepolarizations and spontaneous beats. K180R had no effect on levels of Casq2, Casq2 polymers, or other SR Ca-handling proteins. Intra-SR Ca measurements revealed that K180R impaired dynamic intra-SR Ca buffering, resulting in a more rapid rise of free Ca in the SR during diastole. Steady-state SR Ca buffering and total SR Ca content were not changed. Consistent with the reduced dynamic intra-SR buffering, K180R causes reduced SR Ca release refractoriness. CASQ2-K180R causes CPVT2 via a heretofore unknown mechanism that differs from CASQ2 variants associated with autosomal recessive CPVT2. Unlike autosomal recessive CASQ2 variants, K180R impairs the dynamic buffering of Ca within the SR without affecting total SR Ca content or Casq2 protein levels. Our data provide insight into the molecular mechanism underlying autosomal dominant CPVT2.

Oncomodulin (OCM) uniquely regulates calcium signaling in neonatal cochlear outer hair cells

In cochlear outer hair cells (OHCs), a network of Ca2+ channels, pumps and Ca2+-binding proteins (CaBPs) regulates the localization, spread, and magnitude of free Ca2+ ions. During early postnatal development, OHCs express three prominent mobile EF-hand CaBPs: oncomodulin (OCM), α-parvalbumin (APV) and sorcin. We have previously shown that deletion of Ocm (Ocm-/-) gives rise to progressive cochlear dysfunction in young adult mice. Here, we show that changes in Ca2+ signaling begin early in postnatal development of Ocm-/- mice. While mutant OHCs exhibit normal electrophysiological profiles compared to controls, their intracellular Ca2+ signaling is altered. The onset of OCM expression at postnatal day 3 (P3) causes a developmental change in KCl-induced Ca2+ transients in OHCs and leads to slower KCl-induced Ca2+ transients than those elicited in cells from Ocm-/- littermates. We compared OCM buffering kinetics with other CaBPs in animal models and cultured cells. In a double knockout of Ocm and Apv (Ocm-/-;Apv-/-), mutant OHCs show even faster Ca2+ kinetics, suggesting that APV may also contribute to early postnatal Ca2+ signaling. In transfected HEK293T cells, OCM slows Ca2+ kinetics more so than either APV or sorcin. We conclude that OCM controls the intracellular Ca2+ environment by lowering the amount of freely available [Ca2+]i in OHCs and transfected HEK293T cells. We propose that OCM plays an important role in shaping the development of early OHC Ca2+ signals through its inimitable Ca2+ buffering capacity.

PO-675-05 AUTOSOMAL DOMINANT CPVT2 IS CAUSED BY IMPAIRED DYNAMIC CALCIUM BUFFERING IN THE SARCOPLASMIC RETICULUM

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a cardiac arrhythmia syndrome triggered by catecholamines released during exercise, stress, or sudden emotion. Variants in the calsequestrin-2 gene (CASQ2) encoding an abundant calcium (Ca) binding protein in the sarcoplasmic reticulum (SR), are the second most common cause of CPVT. Recently, several CASQ2 gene variants, such as CASQ2-K180R, have been linked to an autosomal dominant form of Casq2-linked CPVT (CPVT2) but the underlying mechanism is not known.

Identification of the interactions between specific genetic polymorphisms and nutrient intake associated with general and abdominal obesity in middle-aged adults

Comprehensive understanding of gene-diet interactions is necessary to establish proper dietary guidelines to prevent and manage general and abdominal obesity. We investigated the role of genetic variants and their interactions with general and abdominal obesity-associated nutrients using a largescale genome-wide association study of Korean adults. A total of 50,808 participants from a Korean genome and epidemiology study were included. Dietary intake was assessed using a food frequency questionnaire. Obesity was defined as a body mass index ≥25kg/m2. Abdominal obesity (AO) was defined as waist circumference ≥90cm and 80cm in males and females, respectively. Dietary nutrient intake was classified based on Korean Dietary Reference Intakes (DRIs). Odds ratios and 95% confidence intervals were calculated after adjusting for age, sex, exercise, smoking, alcohol drinking, total energy consumption, PC1, and PC2. Among the individuals consuming fat (%) above DRI, carriers of Ca binding protein 39 (CAB39)- rs6722579 minor allele (A) have a higher risk of AO than those not carrying the SNP (odds ration [OR]=3.73, p-value=2.05e-07; interaction p-value=1.80e-07). Among the individuals consuming vitamin C above DRI, carriers of carboxypeptidase Q (CPQ)- rs59465035 minor allele (T) have a lower risk of AO than those without that SNP (OR=0.89, p-value=1.44e-08; interaction p-value=9.50e-06). The genetic association with obesity was stronger among individuals with a genetic variant rs4130113 near GHR gene region in those consume folate above DRI and with a genetic variant rs5760920 near CRYBB2 gene region in those consume vitamin B2 above DRI. Our study results suggested that interactions of specific polymorphisms at loci and certain nutrients may influence obesity and abdominal obesity.

1,25-Dihydroxycholecalciferol Improved the Growth Performance and Upregulated the Calcium Transporter Gene Expression Levels in the Small Intestine of Broiler Chickens.

1,25-Dihydroxycholecalciferol (1,25-(OH)2-D3) is the final active product of vitamin D. This study aimed to investigate the effects of 1,25-(OH)2-D3 on growth performance, bone development, and calcium (Ca) transporter gene expression levels in the small intestine of broiler chickens. On the day of hatching, 140 female Ross 308 broilers were randomly allotted into two treatments with five replicates (14 birds per replicate). Two levels of 1,25-(OH)2-D3 (0 and 1.25 µg/kg) were added to the basal diet without vitamin D. Results showed that the addition of 1.25 µg/kg 1,25-(OH)2-D3 increased the average daily feed intake and the average daily gain and decreased the feed conversion ratio and mortality in 1- to 19-day-old broiler chickens compared with the basal diet without vitamin D (P<0.05). 1,25-(OH)2-D3 also enhanced the length, weight, ash weight, and the percentage contents of ash, Ca, and P in the tibia and femur of broilers (P<0.05). The mRNA expression levels of the Ca-binding protein (CaBP-D28k) in the duodenum, jejunum, and ileum of 19-day-old broilers increased to 88.1-, 109.1-, and 2.7-fold, respectively, after adding 1,25-(OH)2-D3 (P<0.05). The mRNA expression levels of the plasma membrane Ca ATPase 1b (PMCAlb) in the duodenum and the sodium (Na)/ Ca exchanger 1 (NCX1) in the duodenum and the jejunum were also enhanced to 1.57-2.86 times with the addition of 1,25-(OH)2-D3 (P<0.05). In contrast, the mRNA expression levels of PMCA1b and NCX1 in the ileum and that of vitamin D receptor (VDR) in the small intestine were not affected by 1,25-(OH)2-D3 (P>0.05). These data indicate that 1,25-(OH)2-D3 upregulated Ca transporter gene transcription and promoted Ca2+ absorption in the small intestine, especially in the proximal intestine (duodenum and jejunum), thereby improving growth performance and bone mineralization in broiler chickens.

Deletion of Oncomodulin Gives Rise to Early Progressive Cochlear Dysfunction in C57 and CBA Mice.

Ca2+ signaling is a major contributor to sensory hair cell function in the cochlea. Oncomodulin (OCM) is a Ca2+ binding protein (CaBP) preferentially expressed in outer hair cells (OHCs) of the cochlea and few other specialized cell types. Here, we expand on our previous reports and show that OCM delays hearing loss in mice of two different genetic backgrounds: CBA/CaJ and C57Bl/6J. In both backgrounds, genetic disruption of Ocm leads to early progressive hearing loss as measured by auditory brainstem response (ABR) and distortion product otoacoustic emission (DPOAE). In both strains, loss of Ocm reduced hearing across lifetime (hearing span) by more than 50% relative to wild type (WT). Even though the two WT strains have very different hearing spans, OCM plays a considerable and similar role within their genetic environment to regulate hearing function. The accelerated age-related hearing loss (ARHL) of the Ocm KO illustrates the importance of Ca2+ signaling in maintaining hearing health. Manipulation of OCM and Ca2+ signaling may reveal important clues to the systems of function/dysfunction that lead to ARHL.

Dietary calcium levels regulate calcium transporter gene expression levels in the small intestine of broiler chickens

ABSTRACT 1. This study investigated the effect of dietary calcium (Ca) levels on growth performance, bone development and Ca transporter gene expression levels in the small intestine of broiler chickens. 2. On the day of hatch, 350, Ross 308 male broilers were randomly allotted to one of five treatments with five replicate pens each and 14 birds per pen. Dietary Ca levels in feed were 5.0, 7.0, 9.0, 11.0 and 13.0 g/kg, in which 9.0 g/kg was in the control diet. All diets contained 4.5 g/kg non-phytate phosphorus (NPP). 3. The increase in dietary Ca levels from 5.0 to 13.0 g/kg did not affect the growth performance of 1- to 18-day-old broilers (P > 0.05). 4. Increasing the Ca levels linearly increased the ash weight and the contents of ash, Ca and phosphorus (P) in the tibia of broilers at 18 days of age (P < 0.05). The contents of ash, Ca and P in broilers fed with 9.0 g/kg Ca were higher than those in birds fed with 5.0 g/kg Ca (P < 0.05). 5. Increasing the Ca levels linearly decreased mRNA expression levels of the Ca-binding protein 28-kDa (CaBP-D28k), plasma membrane Ca-transporting ATPase 1b (PMCAlb), sodium (Na)/Ca exchanger 1 (NCX1), nuclear vitamin D receptor (nVDR) and membrane vitamin D receptor (mVDR) in the duodenum of broilers at 18 d of age (P < 0.05). Similar results were seen in the jejunum and ileum. Broilers fed 9.0–13.0 g/kg Ca in feed had lower mRNA expression levels of CaBP-D28k and PMCAlb in the small intestine than birds fed 5.0 g/kg Ca in feed (P < 0.05). 6. The data indicated that low levels of dietary Ca stimulated its transporter gene transcription and promoted absorption, but high levels of Ca inhibited transporter gene expression and prevented excessive absorption in the small intestine of broiler chickens.

Calcium in the Backstage of Malaria Parasite Biology.

The calcium ion (Ca2+) is a ubiquitous second messenger involved in key biological processes in prokaryotes and eukaryotes. In Plasmodium species, Ca2+ signaling plays a central role in the parasite life cycle. It has been associated with parasite development, fertilization, locomotion, and host cell infection. Despite the lack of a canonical inositol-1,4,5-triphosphate receptor gene in the Plasmodium genome, pharmacological evidence indicates that inositol-1,4,5-triphosphate triggers Ca2+ mobilization from the endoplasmic reticulum. Other structures such as acidocalcisomes, food vacuole and mitochondria are proposed to act as supplementary intracellular Ca2+ reservoirs. Several Ca2+-binding proteins (CaBPs) trigger downstream signaling. Other proteins with no EF-hand motifs, but apparently involved with CaBPs, are depicted as playing an important role in the erythrocyte invasion and egress. It is also proposed that a cross-talk among kinases, which are not members of the family of Ca2+-dependent protein kinases, such as protein kinases G, A and B, play additional roles mediated indirectly by Ca2+ regulation. This statement may be extended for proteins directly related to invasion or egress, such as SUB1, ERC, IMC1I, IMC1g, GAP45 and EBA175. In this review, we update our understanding of aspects of Ca2+-mediated signaling correlated to the developmental stages of the malaria parasite life cycle.

Structural Changes beyond the EF-Hand Contribute to Apparent Calcium Binding Affinities: Insights from Parvalbumins.

Members of the parvalbumin (PV) family of calcium (Ca2+) binding proteins (CBPs) share a relatively high level of sequence similarity. However, their Ca2+ affinities and selectivities against competing ions like Mg2+ can widely vary. We conducted molecular dynamics simulations of several α-parvalbumin (αPV) constructs with micromolar to nanomolar Ca2+ affinities to identify structural and dynamic features that contribute to their binding of ions. Specifically, we examined a D94S/G98E construct with a lower Ca2+ affinity (≈-18 kcal/mol) relative to the wild type (WT) (≈-22 kcal/mol) and an S55D/E59D variant with enhanced affinity (≈-24 kcal/mol). Additionally, we also examined the binding of Mg2+ to these isoforms, which is much weaker than Ca2+. We used mean spherical approximation (MSA) theory to evaluate ion binding thermodynamics within the proteins' EF-hand domains to account for the impact of ions' finite sizes and the surrounding electrolyte composition. While the MSA scores differentiated Mg2+ from Ca2+, they did not indicate that Ca2+ binding affinities at the binding loop differed between the PV isoforms. Instead, molecular mechanics generalized Born surface area (MM/GBSA) approximation energies, which we used to quantify the thermodynamic cost of structural rearrangement of the proteins upon binding ions, indicated that S55D/E59D αPV favored Ca2+ binding by -20 kcal/mol relative to WT versus 30 kcal/mol for D94S/G98E αPV. Meanwhile, Mg2+ binding was favored for the S55D/E59D αPV and D94S/G98E αPV variants by -18.32 and -1.65 kcal/mol, respectively. These energies implicate significant contributions to ion binding beyond oxygen coordination at the binding loop, which stemmed from changes in α-helicity, β-sheet character, and hydrogen bonding. Hence, Ca2+ affinity and selectivity against Mg2+ are emergent properties stemming from both local effects within the proteins' ion binding sites as well as non-local contributions elsewhere. Our findings broaden our understanding of the molecular bases governing αPV ion binding that are likely shared by members of the broad family of CBPs.

Assessing the Role of Calmodulin's Linker Flexibility in Target Binding.

Calmodulin (CaM) is a highly-expressed Ca binding protein known to bind hundreds of protein targets. Its binding selectivity to many of these targets is partially attributed to the protein’s flexible alpha helical linker that connects its N- and C-domains. It is not well established how its linker mediates CaM’s binding to regulatory targets yet. Insights into this would be invaluable to understanding its regulation of diverse cellular signaling pathways. Therefore, we utilized Martini coarse-grained (CG) molecular dynamics simulations to probe CaM/target assembly for a model system: CaM binding to the calcineurin (CaN) regulatory domain. The simulations were conducted assuming a ‘wild-type’ calmodulin with normal flexibility of its linker, as well as a labile, highly-flexible linker variant to emulate structural changes that could be induced, for instance, by post-translational modifications. For the wild-type model, 98% of the 600 simulations across three ionic strengths adopted a bound complex within 2 μs of simulation time; of these, 1.7% sampled the fully-bound state observed in the experimentally-determined crystallographic structure. By calculating the mean-first-passage-time for these simulations, we estimated the association rate to be 8.7 × 10 M s, which is similar to the diffusion-limited, experimentally-determined rate of 2.2 × 10 M s. Furthermore, our simulations recapitulated its well-known inverse relationship between the association rate and the solution ionic strength. In contrast, although over 97% of the labile linker simulations formed tightly-bound complexes, only 0.3% achieved the fully-bound configuration. This effect appears to stem from a difference in the ensembles of extended and collapsed states which are controlled by the linker flexibility. Therefore, our simulations suggest that variations in the CaM linker’s propensity for alpha helical secondary structure can modulate the kinetics of target binding.

Autosomal-Dominant CASQ2-K180R Causes CPVT by Altering Intra-SR Calcium Buffering without Reducing Casq2 Protein Levels

Calsequestrin-2 (Casq2) is a high capacity, low affinity calcium (Ca) binding protein located in the junctional sarcoplasmic reticulum (SR) of cardiac myocytes. Casq2 interacts with the ryanodine receptor (RyR2) to regulate the amount of Ca released during the excitation-contraction coupling cycle. Mutations in Casq2 can cause the severe genetic arrhythmia syndrome catecholaminergic polymorphic ventricular tachycardia (CPVT). Casq2-linked CPVT is usually autosomal-recessive, causing a severe reduction in Casq2 protein.

Влияние L-альфа-аминоадипиновой кислоты на нейроны и глиальные клетки стриатума мозга крыс

Introduction. Few published studies have examined the role of glia-neuron interactions in neurodegen-eration. In this regard, the search for new experimental models is important. L-alpha-aminoadipic acid (L-AA), being a structural analogue of glutamate with a selective toxic effect on astroglia, is of particular interest. However, morphological and neurochemical changes caused by L-AA still remain unclear. The aim of the study was to characterize immunomorphological changes of glia and neurons in the striata of rats after L-AA administration. Materials and methods. On days 3 and 12 after L-AA stereotaxic administration, we studied astrocytic pro-teins localization using immunofluorescence methods: GFAP, vimentin, glutamine synthetase, along with oligodendroglia (by cyclonucleotidphosphatase expression), microglia (IBA1 Ca-binding protein), neuronal nuclear protein NeuN, and the astroglia proliferative activity (based on Ki67 localization). Results. We detected astrocyte death and a decrease in glutamine synthetase immunoreactivity in the le-sioned area, but no changes in the microglia reaction and the L-AA effect on neurons and oligodendroglia. The astrocyte loss was replenished by proliferation and migration of newly formed immature astrocytes, and a glial scar formed on day 12 after the surgery. Conclusion. L-AA administration, which causes the death of striatum astrocytes in the injection area, can serve as a convenient model for studying reactive changes in astroglia and astrocytic dysfunction while revealing the pathogenetic patterns of neurodegenerative processes. Keywords: astrocytes, striatum, L-alpha-aminoadipic acid, gliosis, glial toxin

Purification, crystallization and X-ray analysis of Pf-SCP (sarcoplasmic Ca-binding protein), related to storage and transport of calcium in mantle of Pinctada fucata

Pf-SCP, a 21 kDa protein with two EF-hand motifs and a phosphorylation site, was identified from mantle tissue and binds to calcium ions and transports calcium components from cell to the shell of Pinctada fucata. To reveal the molecular basis of the calcium binding activity of Pf-SCP, we expressed the recombinant protein of full-length Pf-SCP in Escherichia coli. Recombinant Pf-SCP (rPf-SCP) purified by Ni affinity chromatography and size exclusion chromatography appeared as a single band on SDS-PAGE. The circular dichroism spectroscopy showed that the α-helix content decreased when rPf-SCP interacted with both calcium ions and calcium carbonate. Western blotting and immunostaining verified the Pf-SCP expression in the shell and localization most in the mantle epithelial cells. To further understand the structural and functional regulation of Pf-SCP by calcium ions and calcium carbonate, the crystallization experiments of rPf-SCP in the presence of calcium ions were performed. A crystal of rPf-SCP obtained in the presence of calcium ions diffracted X-rays up to a resolution of 1.8 Å. The space group of the crystal is C2 with unit cell parameters of a = 96.828 Å, b = 55.906 Å, c = 102.14 Å and β = 90.009°, indicating that three molecules of rPf-SCP are contained in an asymmetric unit as estimated at the value of the Matthews coefficient. These results suggest that Pf-SCP may play a role in calcium ions transportation and shell mineralization by concentrating calcium ions inside the mantle epithelial cells and interacting with calcium carbonate molecules.

Structural and functional diversity of Entamoeba histolytica calcium-binding proteins.

Entamoeba histolytica (E. histolytica) is an etiological agent of human amoebic colitis, and it causes a high level of morbidity and mortality worldwide, particularly in developing countries. Ca2+ plays a pivotal role in amoebic pathogenesis, and Ca2+-binding proteins (CaBPs) of E. histolytica appear to be a major determinant in this process. E. histolytica has 27-EF-hand containing CaBPs, suggesting that this organism has complex Ca2+ signaling cascade. E. histolytica CaBPs share (29-47%) sequence identity with ubiquitous Ca2+-binding protein calmodulin (CaM); however, they do not show any significant structural similarity, indicating lack of a typical CaM in this organism. Structurally, these CaBPs are very diverse among themselves, and perhaps such diversity allows them to recognize different cellular targets, thereby enabling them to perform a range of cellular functions. The presence of such varied signaling molecules helps parasites to invade host cells and advance in disease progression. In the past two decades, tremendous progress has been made in understanding the structure of E. histolytica CaBPs by using the X-ray or NMR method. To gain greater insight into the structural and functional diversity of these amoebic CaBPs, we analyzed and compiled all the available literature. Most of the CaBPs has about 150 amino acids with 4-EF hand or EF-hand-like sequences, similar to CaM. In a few cases, all the EF-hand motifs are not capable of binding Ca2+, suggesting them to be pseudo EF-hand motifs. The CaBPs perform diverse cellular signaling that includes cytoskeleton remodeling, phagocytosis, cell proliferation, migration of trophozoites, and GTPase activity. Overall, the structural and functional diversity of E. histolytica CaBPs compiled here may offer a basis to develop an efficient drug to counter its pathogenesis.

Entamoeba histolytica and pathogenesis: A calcium connection.

Calcium signaling plays a key role in many essential processes in almost all eukaryotic systems. It is believed that it may also be an important signaling system of the protist parasite Entamoeba histolytica. Motility, adhesion, cytolysis, and phagocytosis/trogocytosis are important steps in invasion and pathogenesis of E. histolytica, and Ca2+ signaling is thought to be associated with these processes leading to tissue invasion. There are a large number of Ca2+-binding proteins (CaBPs) in E. histolytica, and a number of these proteins appear to be associated with different steps in pathogenesis. The genome encodes 27 EF-hand–containing CaBPs in addition to a number of other Ca2+-binding domain/motif-containing proteins, which suggest intricate calcium signaling network in this parasite. Unlike other eukaryotes, a typical calmodulin-like protein has not been seen in E. histolytica. Though none of the CaBPs display sequence similarity with a typical calmodulin, extensive structural similarity has been seen in spite of lack of significant functional overlap with that of typical calmodulins. One of the unique features observed in E. histolytica is the identification of CaBPs (EhCaBP1, EhCaBP3) that have the ability to directly bind actin and modulate actin dynamics. Direct interaction of CaBPs with actin has not been seen in any other system. Pseudopod formation and phagocytosis are some of the processes that require actin dynamics, and some of the amoebic CaBPs (EhC2Pk, EhCaBP1, EhCaBP3, EhCaBP5) participate in this process. None of these E. histolytica CaBPs have any homolog in organisms other than different species of Entamoeba, suggesting a novel Ca2+ signaling pathway that has evolved in this genus.