The role of calmodulin in the regulation of calcium signalling proteins in health and disease

Calcium ions (Ca2+) are major secondary signalling messengers, controlling many biological processes. Signalling relies on the activity of multiple proteins to bind, sequester, transport, release and respond to Ca2+. Phospholipase C (PLC) catalyses the production of inositol 1,4,5-trisphosphate (IP3) which stimulates the intracellular release of Ca2+ via IP3 receptor (IP3R). Another primary Ca2+ release channel is the ryanodine receptor (RyR). In response to changes in Ca2+ concentration the Ca2+ sensitive protein calmodulin (CaM) binds to and releases multiple proteins, including PLC and RyR ,altering function and regulating activity. The sperm-specific PLCζ isoform stimulates oocyte activation upon fertilisation by triggering, via IP3, subsequent Ca2+ release events to produce the oscillations in the Ca2+ concentration required for embryogenesis. Dysfunctional PLCζ causes male infertility and subfertility. The structure of PLCζ and the full mechanism of action are unknown. Recently, a novel inhibitory interaction between PLCζ and CaM was observed. The cardiac-specific RyR2 isoform releases Ca2+ in cardiac myocytes during the heartbeat. Dysfunctional RyR2 activity causes life-threatening arrhythmias. CaM binding to RyR2 inhibits Ca2+ release, and dysfunctional CaM binding is arrhythmogenic. Mutations in CaM and the CaM-binding sites of RyR2 cosegregate with arrhythmias. This thesis develops the tools for subsequent investigation of the structure of PLCζ, interaction between PLCζ and CaM, altered characteristics of interaction with RyR2 by arrhythmogenic CaM mutations. Varying fusion partners and expressed amino acid coordinates improved the yield and solubility of recombinant PLCζ. Recombinant CaM protein recapitulated established parameters of CaM and Ca2+ dependent interactions between RyR2 and CaM. Arrhythmia patient mutations of CaM perturbed these divergently without altering protein secondary structure. However, the mutations altered the Ca2+ binding affinity and thermal stability of CaM. Ca2+ dependent binding between CaM and PLCζ occurred between the C-lobe of CaM with contribution from the N-lobe and no Ca2+-free binding was observed

Calmodulin Interacts and Regulates Enzyme Activity of the Mammalian Sperm Phospholipase C

Sperm-specific Phospholipase C zeta (PLCζ) is widely considered to be the sole, physiological stimulus responsible for the generation of Ca2+ oscillations that induce egg activation and early embryo development during mammalian fertilization. PLCζ, which is delivered from the fertilizing sperm into the egg cytoplasm, catalyzes the hydrolysis of its membrane-bound phospholipid substrate phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2], triggering the cytoplasmic Ca2+ oscillations through the inositol 1,4,5-trisphosphate (InsP3) signaling pathway. Despite the recent advances the detailed regulatory mechanism of PLCζ is still unclear, as binding partners of this protein within the sperm or the fertilizing egg have not yet been identified. Calmodulin (CaM) is a ubiquitous Ca2+ sensor in eukaryotic cells. A previous study has reported that CaM directly interacts and regulates the activity of PLC delta 1 protein, a somatic PLC isoform with structural similarities to sperm PLCζ. Bioinformatics analysis revealed putative CaM-binding sites on PLCζ sequence. In the present study, we have used co-immunoprecipitation analysis and we show that in the presence of Ca2+, human PLCζ directly interacts with CaM. Isothermal titration calorimetry (ITC) experiments were performed to map the interaction. Three different peptides corresponding to disparate sequences within human PLCζ were used and it was shown that PLCζ interacts with CaM via one region of the molecule. In addition, recombinant proteins corresponding to the N- and C-lobe of human CaM were used for ITC experiments, which revealed that CaM interacts with PLCζ in the presence of Ca2+, only through one of its lobe domains. In vitro PIP2 hydrolysis assays revealed that CaM alters PLCζ PIP2 hydrolytic activity at high Ca2+ concentrations and, as suggested by liposome binding assays, this appears to be due to CaM binding to PLCζ affecting proper access of the enzyme active site to its substrate PI(4,5)P2

Arrhythmogenic calmodulin E105A mutation alters cardiac RyR2 regulation leading to cardiac dysfunction in zebrafish

Calmodulin (CaM) is a universal calcium (Ca2+)‐binding messenger that regulates many vital cellular events. In cardiac muscle, CaM associates with ryanodine receptor 2 (RyR2) and regulates excitation–contraction coupling. Mutations in human genes CALM1, CALM2, and CALM3 have been associated with life‐threatening heart disorders, such as long QT syndrome (LQTS) and catecholaminergic polymorphic ventricular tachycardia. A novel de novo LQTS‐associated missense CaM mutation (E105A) was recently identified in a 6‐year‐old boy, who experienced an aborted first episode of cardiac arrest. Herein, we report the first molecular characterization of the CaM E105A mutation. Expression of the CaM E105A mutant in zebrafish embryos resulted in cardiac arrhythmia and increased heart rate, suggestive of ventricular tachycardia. In vitro biophysical and biochemical analysis revealed that E105A confers a deleterious effect on protein stability and a reduced Ca2+‐binding affinity due to loss of cooperativity. Finally, the CaM E105A mutation resulted in reduced CaM–RyR2 interaction and defective modulation of ryanodine binding. Our findings suggest that the CaM E105A mutation dysregulates normal cardiac function by a complex mechanism involving alterations in both CaM–Ca2+ and CaM–RyR2 interactions

Defective Interaction of Cam with RyR2 Cam-Binding Pocket Might Contribute to Arrhythmogenic Cardiac Disease

Ryanodine receptor 2 (RyR2) is a large transmembrane calcium (Ca2+) release channel that mediates Ca2 release from the sarcoplasmic reticulum to activate cardiac muscle contraction. Calmodulin (CaM) regulation of RyR2 is essential for normal cardiac function. A number of linear fragments of RyR2 have been reported as potential CaM-binding sequences. The sequence 3583-3603aa of human RyR2, which is highly conserved among mammalian isoforms, has been identified as a CaM-binding site in almost all relevant studies and therefore this region is considered as a well-established CaM-binding domain of RyRs. Besides 3583-3603aa region, other RyR2 regions have been also reported as potential CaM-binding sequences. Herein, we used recombinant wild-type CaMprotein and isothermal titration calorimetry (ITC) experiments to screen a number of RyR2-specific synthetic peptides corresponding to the region 4240-4277aa of RyR2, which has been previously proposed as a putative CaM-binding RyR2 region. From all the synthetic peptides screened, a peptide corresponding to 4255-4271aa region of human RyR2 was found to interact with significant affinity with RyR2, in the presence and absence of Ca2+ (Kd values 0.60 and 16.58 μM, respectively). Moreover, investigation of the interaction of four arrhythmogenic CaM mutants (N98I, D132E, D134H and Q136P) with this synthetic peptide, as well as the peptide corresponding to the well-established CaM-binding domain of RyR2 (3583-3603aa), revealed that all mutants show disparate binding properties to these two RyR2 peptides, which have been previously proposed to contribute to a putative intra-subunit CaM-binding pocket. Our findings extend our previous observations suggesting that CaM mutations may trigger arrhythmogenic cardiac disease by altering both intrinsic Ca2+-binding, as well as by dysregulating RyR2-mediated Ca2+ release via defective interaction of CaM with a distinct CaM-binding pocket that multiple RyR2 regions might contribute

Male infertility-linked point mutation disrupts the Ca2+ oscillation-inducing and PIP2 hydrolysis activity of sperm PLCζ

A male infertility-linked human PLCζ (phospholipase Cζ) mutation introduced into mouse PLCζ completely abolishes both in vitro PIP2 (phosphatidylinositol 4,5-bisphosphate) hydrolysis activity and the ability to trigger in vivo Ca2+ oscillations in mouse eggs. Wild-type PLCζ initiated a normal pattern of Ca2+ oscillations in eggs in the presence of 10-fold higher mutant PLCζ, suggesting that infertility is not mediated by a dominant-negative mechanism

Hypertrophic cardiomyopathy-linked variants of cardiac myosin binding protein C3 display altered molecular properties and actin interaction

The most common inherited cardiac disorder, hypertrophic cardiomyopathy (HCM), is characterized by thickening of heart muscle, for which genetic mutations in cardiac myosin-binding protein C3 (c-MYBPC3) gene, is the leading cause. Notably, patients with HCM display a heterogeneous clinical presentation, onset and prognosis. Thus, delineating the molecular mechanisms that explain how disparate c-MYBPC3 variants lead to HCM is essential for correlating the impact of specific genotypes on clinical severity. Herein, five c-MYBPC3 missense variants clinically associated with HCM were investigated; namely V1 (R177H), V2 (A216T), V3 (E258K), V4 (E441K) and double mutation V5 (V3 + V4), all located within the C1 and C2 domains of MyBP-C, a region known to interact with sarcomeric protein, actin. Injection of the variant complementary RNAs in zebrafish embryos was observed to recapitulate phenotypic aspects of HCM in patients. Interestingly, V3- and V5-cRNA injection produced the most severe zebrafish cardiac phenotype, exhibiting increased diastolic/systolic myocardial thickness and significantly reduced heart rate compared with control zebrafish. Molecular analysis of recombinant C0–C2 protein fragments revealed that c-MYBPC3 variants alter the C0–C2 domain secondary structure, thermodynamic stability and importantly, result in a reduced binding affinity to cardiac actin. V5 (double mutant), displayed the greatest protein instability with concomitant loss of actin-binding function. Our study provides specific mechanistic insight into how c-MYBPC3 pathogenic variants alter both functional and structural characteristics of C0–C2 domains leading to impaired actin interaction and reduced contractility, which may provide a basis for elucidating the disease mechanism in HCM patients with c-MYBPC3 mutations

Essential role of the EF-hand domain in targeting sperm phospholipase Cζ to membrane phosphatidylinositol 4,5-bisphosphate (PIP2)

Sperm-specific phospholipase C-ζ (PLCζ) is widely considered to be the physiological stimulus that triggers intracellular Ca2+ oscillations and egg activation during mammalian fertilization. Although PLCζ is structurally similar to PLCδ1, it lacks a pleckstrin homology domain, and it remains unclear how PLCζ targets its phosphatidylinositol 4,5-bisphosphate (PIP2) membrane substrate. Recently, the PLCδ1 EF-hand domain was shown to bind to anionic phospholipids through a number of cationic residues, suggesting a potential mechanism for how PLCs might interact with their target membranes. Those critical cationic EF-hand residues in PLCδ1 are notably conserved in PLCζ. We investigated the potential role of these conserved cationic residues in PLCζ by generating a series of mutants that sequentially neutralized three positively charged residues (Lys-49, Lys-53, and Arg-57) within the mouse PLCζ EF-hand domain. Microinjection of the PLCζ EF-hand mutants into mouse eggs enabled their Ca2+ oscillation inducing activities to be compared with wild-type PLCζ. Furthermore, the mutant proteins were purified, and the in vitro PIP2 hydrolysis and binding properties were monitored. Our analysis suggests that PLCζ binds significantly to PIP2, but not to phosphatidic acid or phosphatidylserine, and that sequential reduction of the net positive charge within the first EF-hand domain of PLCζ significantly alters in vivo Ca2+ oscillation inducing activity and in vitro interaction with PIP2 without affecting its Ca2+ sensitivity. Our findings are consistent with theoretical predictions provided by a mathematical model that links oocyte Ca2+ frequency and the binding ability of different PLCζ mutants to PIP2. Moreover, a PLCζ mutant with mutations in the cationic residues within the first EF-hand domain and the XY linker region dramatically reduces the binding of PLCζ to PIP2, leading to complete abolishment of its Ca2+ oscillation inducing activity

Antigen unmasking enhances visualization efficacy of the oocyte activation factor, phospholipase C zeta, in mammalian sperm

Study Question Is it possible to improve clinical visualization of phospholipase C zeta (PLCζ) as a diagnostic marker of sperm oocyte activation capacity and male fertility? Summary Answer Poor PLCζ visualization efficacy using current protocols may be due to steric or conformational occlusion of native PLCζ, hindering antibody access, and is significantly enhanced using antigen unmasking/retrieval (AUM) protocols. What is Known Already Mammalian oocyte activation is mediated via a series of intracellular calcium (Ca2+) oscillations induced by sperm-specific PLCζ. PLCζ represents not only a potential clinical therapeutic in cases of oocyte activation deficiency but also a diagnostic marker of sperm fertility. However, there are significant concerns surrounding PLCζ antibody specificity and detection protocols. Study Design, Size Duration Two PLCζ polyclonal antibodies, with confirmed PLCζ specificity, were employed in mouse, porcine and human sperm. Experiments evaluated PLCζ visualization efficacy, and whether AUM improved this. Antibodies against two sperm-specific proteins [post-acrosomal WW-binding protein (PAWP) and acrosin] were used as controls. Participants/Materials, Setting, Methods Aldehyde- and methanol-fixed sperm were subject to immunofluorescence analysis following HCl exposure (pH = 0.1–0.5), acid Tyrode's solution exposure (pH = 2.5) or heating in 10 mM sodium citrate solution (pH = 6.0). Fluorescence intensity of at least 300 cells was recorded for each treatment, with three independent repeats. Main Results and the Role of Chance Despite high specificity for native PLCζ following immunoblotting using epitope-specific polyclonal PLCζ antibodies in mouse, porcine and human sperm, immunofluorescent visualization efficacy was poor. In contrast, sperm markers PAWP and acrosin exhibited relatively impressive results. All methods of AUM on aldehyde-fixed sperm enhanced visualization efficacy for PLCζ compared to visualization efficacy before AUM (P < 0.05 for all AUM interventions), but exerted no significant change upon PAWP or acrosin immunofluorescence following AUM. All methods of AUM enhanced PLCζ visualization efficacy in mouse and human methanol-fixed sperm compared to without AUM (P < 0.05 for all AUM interventions), while no significant change was observed in methanol-fixed porcine sperm before and after. In the absence of aldehyde-induced cross-linkages, such results suggest that poor PLCζ visualization efficacy may be due to steric or conformational occlusion of native PLCζ, hindering antibody access. Importantly, examination of sperm from individual donors revealed that AUM differentially affects observable PLCζ fluorescence, and the proportion of sperm exhibiting detectable PLCζ fluorescence in sperm from different males. Limitations, Reasons for Caution Direct correlation of fertility outcomes with the level of PLCζ in the sperm samples studied was not available. Such analyses would be required in future to determine whether the improved methodology for PLCζ visualization we propose would indeed reflect fertility status. Wider Implications of the Findings We propose that AUM alters conformational interactions to enhance PLCζ epitope availability and visualization efficacy, supporting prospective application of AUM to reduce misinterpretation in clinical diagnosis of PLCζ-linked male infertility. Our current results suggest that it is perhaps prudent that previous studies investigating links between PLCζ and fertility parameters are re-examined in the context of AUM, and may pave the way for future work to answer significant questions such as how PLCζ appears to be kept in an inactive form in the sperm

Drug inhibition of redox factor-1 restores hypoxia-driven changes in tuberous sclerosis complex 2 deficient cells

Simple Summary: Tuberous sclerosis complex (TSC) is a genetic disease where patients are predisposed to tumors and neurological complications. Current therapies for this disease are not fully curative. We aimed to explore novel drug targets and therapies that could further benefit TSC patients. This work uncovered a novel pathway that drives disease in TSC cell models involving redox factor-1 (Ref-1). Ref-1 is a protein that turns on several key transcription factors that collectively promote tumor growth and survival through direct redox signaling. Processes regulated by Ref-1 include angiogenesis, inflammation, and metabolic transformation. Therefore, this work reveals a new drug target, where inhibitors of Ref-1 could have an additional benefit compared to current drug therapies. Abstract: Therapies with the mechanistic target of rapamycin complex 1 (mTORC1) inhibitors are not fully curative for tuberous sclerosis complex (TSC) patients. Here, we propose that some mTORC1-independent disease facets of TSC involve signaling through redox factor-1 (Ref-1). Ref-1 possesses a redox signaling activity that stimulates the transcriptional activity of STAT3, NF-kB, and HIF-1α, which are involved in inflammation, proliferation, angiogenesis, and hypoxia, respectively. Here, we demonstrate that redox signaling through Ref-1 contributes to metabolic transformation and tumor growth in TSC cell model systems. In TSC2-deficient cells, the clinically viable Ref-1 inhibitor APX3330 was effective at blocking the hyperactivity of STAT3, NF-kB, and HIF-1α. While Ref-1 inhibitors do not inhibit mTORC1, they potently block cell invasion and vasculature mimicry. Of interest, we show that cell invasion and vasculature mimicry linked to Ref-1 redox signaling are not blocked by mTORC1 inhibitors. Metabolic profiling revealed that Ref-1 inhibitors alter metabolites associated with the glutathione antioxidant pathway as well as metabolites that are heavily dysregulated in TSC2-deficient cells involved in redox homeostasis. Therefore, this work presents Ref-1 and associated redox-regulated transcription factors such as STAT3, NF-kB, and HIF-1α as potential therapeutic targets to treat TSC, where targeting these components would likely have additional benefits compared to using mTORC1 inhibitors alone

Drug inhibition of redox factor-1 restores hypoxic-driven changes in Tuberous Sclerosis Complex 2-deficient cells

Therapies with mechanistic target of rapamycin complex 1 (mTORC1) inhibitors are not fully curative for Tuberous Sclerosis Complex (TSC) patients. Here we propose that some mTORC1-independent disease facets of TSC involve signaling through redox factor-1 (Ref-1). Ref-1 possesses redox signaling activity that stimulates the transcriptional activity of STAT3, NF-B, and HIF-1 involved in inflammation, proliferation, angiogenesis and hypoxia, respectively. Here we demonstrate that redox signaling through Ref-1 contributes to metabolic transformation and tumor growth in TSC cell model systems. In TSC2-deficient cells, the clinically viable Ref-1 inhibitor, APX3330, was effective at blocking the hyperactivity of STAT3, NF-B, and HIF-1. While Ref-1 inhibitors do not inhibit mTORC1, they potently block cell invasion and vasculature mimicry. Of interest, we show that cell invasion and vasculature mimicry linked to Ref-1 redox signaling are not blocked by mTORC1 inhibitors. Metabolic profiling revealed that Ref-1 inhibitors alter metabolites associated with the glutathione antioxidant pathway as well as metabolites that are heavily dysregulated in TSC2-deficient cells involved in redox homeostasis. Therefore, this work presents Ref-1 and associated redox-regulated transcription factors, such as STAT3, NF-B and HIF-1, as potential therapeutic targets to treat TSC, where targeting these components would likely have additional benefits to just using mTORC1 inhibitors alone