Regulation of inositol 5-phosphatase activity by the C2 domain of SHIP1 and SHIP2.

SHIP1, an inositol 5-phosphatase, plays a central role in cellular signaling. As such, it has been implicated in many conditions. Exploiting SHIP1 as a drug target will require structural knowledge and the design of selective small molecules. We have determined apo, and magnesium and phosphate-bound structures of the phosphatase and C2 domains of SHIP1. The C2 domains of SHIP1 and the related SHIP2 modulate the activity of the phosphatase domain. To understand the mechanism, we performed activity assays, hydrogen-deuterium exchange mass spectrometry, and molecular dynamics on SHIP1 and SHIP2. Our findings demonstrate that the influence of the C2 domain is more pronounced for SHIP2 than SHIP1. We determined 91 structures of SHIP1 with fragments bound, with some near the interface between the two domains. We performed a mass spectrometry screen and determined four structures with covalent fragments. These structures could act as starting points for the development of potent, selective probes.

Joubert syndrome causing mutation in C2 domain of CC2D2A affects structural integrity of cilia and cellular signaling molecules.

Cilia are organelles extend from cells to sense external signals for tuning intracellular signaling for optimal cellular functioning. They have evolved sensory and motor roles in various cells for tissue organization and homeostasis in development and post-development. More than a thousand genes are required for cilia function. Mutations in them cause multisystem disorders termed ciliopathies. The null mutations in CC2D2A result in Meckel syndrome (MKS), which is embryonic lethal, whereas patients who have missense mutations in the C2 domain of CC2D2A display Joubert syndrome (JBTS). They survive with blindness and mental retardation. How C2 domain defects cause disease conditions is not understood. To answer this question, C2 domain of Cc2d2a (mice gene) was knocked down (KD) in IMCD-3 cells by shRNA. This resulted in defective cilia morphology observed by immunofluorescence analysis. To further probe the cellular signaling alteration in affected cells, gene expression profiling was done by RNAseq and compared with the controls. Bioinformatics analysis revealed that the differentially expressed genes (DEGs) have functions in cilia. Among the 61 cilia DEGs identified, 50 genes were downregulated and 11 genes were upregulated. These cilia genes are involved in cilium assembly, protein trafficking to the cilium, intraflagellar transport (IFT), cellular signaling like polarity patterning, and Hedgehog signaling pathway. This suggests that the C2 domain of CC2D2A plays a critical role in cilia assembly and molecular signaling hosted in cilia for cellular homeostasis. Taken together, the missense mutations in the C2 domain of CC2D2A seen in JBTS might have affected cilia-mediated signaling in neurons of the retina and brain.

Hybrid human-porcine factor VIII proteins partially escape the inhibitory effects of anti-factor VIII inhibitor alloantibodies having A2 or C2 domain specificity.

INTRODUCTION: Porcine factor (pF)VIII has low cross-reactivity with anti-human (h)FVIII inhibitor alloantibodies. Clinical trials of pFVIII in congenital haemophilia A patients with inhibitor (PwHA-I) are in progress. Most polyclonal anti-hFVIII inhibitors recognize its A2 and/or C2 domain(s), and recombinant human-porcine hybrid (hp)FVIII proteins may escape neutralization by these inhibitors. AIM: To evaluate the ability of hpFVIII to limit the anti-FVIII activity of inhibitor alloantibodies. METHODS: Three hybrid proteins were created by substituting the hFVIII A2, C2 domain or both with the corresponding domains of pFVIII [termed hp(A2), hp(C2) and hp(A2/C2), respectively]. The reactivity of these hybrids was assessed by one-stage clotting assays (OSA), thrombin generation assays (TGA) and rotational thromboelastometry (ROTEM) by adding them to FVIII-deficient samples. RESULTS: OSA demonstrated that the hybrid proteins avoided neutralization by anti-FVIII A2 or C2 monoclonal antibodies (mAb) and polyclonal inhibitor-antibodies (polyAb) from PwHA-I. In TGA, thrombin generation with hp(A2) and hp(A2/C2) was not attenuated in the presence of patient IgG recognizing anti-A2 domain. In contrast, that with hFVIII and hp(C2) was suppressed by this IgG to levels equivalent to those of FVIII-deficient plasma. With anti-A2/C2 polyAb, the activity of hp(A2/C2) was unaffected. ROTEM demonstrated that the addition of hp(A2) or hp(A2/C2) to anti-A2 polyAb shortened clot times/clot formation times, whilst hFVIII or hp(C2) were ineffective. Similarly with anti-A2/C2 polyAb, hp(A2/C2) restored coagulation potential to a greater extent than hp(A2) and hp(C2). CONCLUSION: Hybrid FVIII proteins containing porcine FVIII A2 and/or C2 domain(s) could support effective therapy in PwHA-I by avoiding neutralization.

Novel anti-PTEN C2 domain monoclonal antibodies to analyse the expression and function of PTEN isoform variants.

PTEN is a major tumor suppressor gene frequently mutated in human tumors, and germline PTEN gene mutations are the molecular diagnostic of PTEN Hamartoma Tumor Syndrome (PHTS), a heterogeneous disorder that manifests with multiple hamartomas, cancer predisposition, and neurodevelopmental alterations. A diversity of translational and splicing PTEN isoforms exist, as well as PTEN C-terminal truncated variants generated by disease-associated nonsense mutations. However, most of the available anti-PTEN monoclonal antibodies (mAb) recognize epitopes at the PTEN C-terminal tail, which may introduce a bias in the analysis of the expression of PTEN isoforms and variants. We here describe the generation and precise characterization of anti-PTEN mAb recognizing the PTEN C2-domain, and their use to monitor the expression and function of PTEN isoforms and PTEN missense and nonsense mutations associated to disease. These anti-PTEN C2 domain mAb are suitable to study the pathogenicity of PTEN C-terminal truncations that retain stability and function but have lost the PTEN C-terminal epitopes. The use of well-defined anti-PTEN mAb recognizing distinct PTEN regions, as the ones here described, will help to understand the deleterious effects of specific PTEN mutations in human disease.

The Dysferlin C2A Domain Binds PI(4,5)P2 and Penetrates Membranes.

Dysferlin is a large membrane protein found most prominently in striated muscle. Loss of dysferlin activity is associated with reduced exocytosis, abnormal intracellular Ca2+ and the muscle diseases limb-girdle muscular dystrophy and Miyoshi myopathy. The cytosolic region of dysferlin consists of seven C2 domains with mutations in the C2A domain at the N-terminus resulting in pathology. Despite the importance of Ca2+ and membrane binding activities of the C2A domain for dysferlin function, the mechanism of the domain remains poorly characterized. In this study we find that the C2A domain preferentially binds membranes containing PI(4,5)P2 through an interaction mediated by residues Y23, K32, K33, and R77 on the concave face of the domain. We also found that subsequent to membrane binding, the C2A domain inserts residues on the Ca2+ binding loops into the membrane. Analysis of solution NMR measurements indicate that the domain inhabits two distinct structural states, with Ca2+ shifting the population between states towards a more rigid structure with greater affinity for PI(4,5)P2. Based on our results, we propose a mechanism where Ca2+ converts C2A from a structurally dynamic, low PI(4,5)P2 affinity state to a high affinity state that targets dysferlin to PI(4,5)P2 enriched membranes through interaction with Tyr23, K32, K33, and R77. Binding also involves changes in lipid packing and insertion by the third Ca2+ binding loop of the C2 domain into the membrane, which would contribute to dysferlin function in exocytosis and Ca2+ regulation.

A C2 domain containing plasma membrane protein of Plasmodium falciparum merozoites mediates calcium-dependent binding and invasion to host erythrocytes.

BACKGROUND: Invasion of red blood cells by Plasmodium falciparum merozoites is governed by multiple receptor-ligand interactions which are critical for bridging the two cells together. The critical function of these ligands for invasion and their direct exposure to the host immune system makes them lucrative vaccine candidates. This necessitates the discovery of new adhesins with less redundancy that mediates the binding of merozoite to the red cell, and furthermore invasion into it. Here we have identified a novel membrane associated antigen (PfC2DMA) that is conserved throughout the Plasmodium species and has a membrane targeting C2 domain at its extreme N-terminal region. METHODS: Recombinant C2dom was expressed heterologously in bacteria and purified to homogeneity. Mice antisera against C2dom was raised and used to check the expression and intraparasitic localization of the protein. RBC and Ca2+ ion binding activity of C2dom was also checked. RESULTS: C2dom exhibited specific binding to Ca2+ ions and not to Mg2+ ions. PfC2DMA localized to the surface of merozoite and recombinant C2dom bound to the surface of human RBCs. RBC receptor modification by treatment with different enzymes showed that binding of C2dom to RBC surface is neuraminidase sensitive. Mice antisera raised against C2dom of Pf C2DMA showed invasion inhibitory effects. CONCLUSION: Our findings suggest that C2dom of PfC2DMA binds to surface of red cell in a Ca2+-dependent manner, advocating a plausible role in invasion and can serve as a potential novel blood stage vaccine candidate.

An N-glycan on the C2 domain of JAGGED1 is important for Notch activation.

The canonical members of the Jagged/Serrate and Delta families of transmembrane ligands have an extracellular, amino-terminal C2 domain that binds to phospholipids and is required for optimal activation of the Notch receptor. Somatic mutations that cause amino substitutions in the C2 domain in human JAGGED1 (JAG1) have been identified in tumors. We found in reporter cell assays that mutations affecting an N-glycosylation site reduced the ligand's ability to activate Notch. This N-glycosylation site located in the C2 domain is conserved in the Jagged/Serrate family but is lacking in the Delta family. Site-specific glycan analysis of the JAG1 amino terminus demonstrated that occupancy of this site by either a complex-type or high-mannose N-glycan was required for full Notch activation in reporter cell assays. Similarly to JAG1 variants with defects in Notch binding, N-glycan removal, either by mutagenesis of the glycosylation site or by endoglycosidase treatment, reduced receptor activation. The N-glycan variants also reduced receptor activation in a Notch signaling-dependent vascular smooth muscle cell differentiation assay. Loss of the C2 N-glycan reduced JAG1 binding to liposomes to a similar extent as the loss of the entire C2 domain. Molecular dynamics simulations suggested that the presence of the N-glycan limits the orientation of JAG1 relative to the membrane, thus facilitating Notch binding. These data are consistent with a critical role for the N-glycan in promoting a lipid-binding conformation that is required to orient Jagged at the cell membrane for full Notch activation.

Down-Regulation of Double C2 Domain Alpha Promotes the Formation of Hyperplastic Nerve Fibers in Aganglionic Segments of Hirschsprung's Disease.

Hirschsprung's disease (HSCR) is a common developmental anomaly of the gastrointestinal tract in children. The most significant characteristics of aganglionic segments in HSCR are hyperplastic extrinsic nerve fibers and the absence of endogenous ganglion plexus. Double C2 domain alpha (DOC2A) is mainly located in the nucleus and is involved in Ca2+-dependent neurotransmitter release. The loss function of DOC2A influences postsynaptic protein synthesis, dendrite morphology, postsynaptic receptor density and synaptic plasticity. It is still unknown why hyperplastic extrinsic nerve fibers grow into aganglionic segments in HSCR. We detected the expression of DOC2A in HSCR aganglionic segment colons and established three DOC2A-knockdown models in the Neuro-2a cell line, neural spheres and zebrafish separately. First, we detected the protein and mRNA expression of DOC2A and found that DOC2A was negatively correlated with AChE+ grades. Second, in the Neuro-2a cell lines, we found that the amount of neurite outgrowth and mean area per cell were significantly increased, which suggested that the inhibition of DOC2A promotes nerve fiber formation and the neuron's polarity. In the neural spheres, we found that the DOC2A knockdown was manifested by a more obvious connection of nerve fibers in neural spheres. Then, we knocked down Doc2a in zebrafish and found that the down-regulation of Doc2a accelerates the formation of hyperplastic nerve fibers in aganglionic segments in zebrafish. Finally, we detected the expression of MUNC13-2 (UNC13B), which was obviously up-regulated in Grade3/4 (lower DOC2A expression) compared with Grade1/2 (higher DOC2A expression) in the circular muscle layer and longitudinal muscle layer. The expression of UNC13B was up-regulated with the knocking down of DOC2A, and there were protein interactions between DOC2A and UNC13B. The down-regulation of DOC2A may be an important factor leading to hyperplastic nerve fibers in aganglionic segments of HSCR. UNC13B seems to be a downstream molecule to DOC2A, which may participate in the spasm of aganglionic segments of HSCR patient colons.

Characterization and phylogenetic analysis of multiple C2 domain and transmembrane region proteins in maize.

BACKGROUND: Multiple C2 domain and transmembrane region proteins (MCTPs) are evolutionarily conserved and important signaling molecules. However, the MCTP gene family has not been comprehensively analyzed in maize. RESULTS: In this study, 385 MCTP genes were identified in all surveyed 38 species. Moreover, gene duplication mode exploration showed that whole genome duplication (WGD) mainly contributed to the expansion of MCTP genes in angiosperms. Phylogeny reconstruction with all surveyed species by the maximum-likelihood (ML) method showed five clades of MCTPs, Clades I to V. Each clade of MCTPs had conservative structures and motifs. Focusing on maize, 17 MCTPs were identified, and a neighborjoining (NJ) phylogenetic tree with only ZmMCTPs was also constructed. As expected, 17 MCTPs showed similar phylogenetic relationships in the neighbor-joining (NJ) tree with those in the maximum-likelihood (ML) tree and could also be divided into five subclades. Moreover, ZmMCTP members in different clades showed specific gene structure, conserved motif, and domain structure compositions. Intriguingly, most ZmMCTP genes were intronless. Analyses of isoelectric points (pIs) and grand averages of hydropathicity (GRAVYs) indicated that the N-terminus was more dispersive than the C-terminus. Further tissue-specific expression analysis indicated that duplicated ZmMCTP pairs involved in whole genome duplication (WGD) had similar expression trends. Finally, ZmMCTPs were transcriptionally altered under diverse abiotic stresses and hormone treatments. CONCLUSIONS: Our results contribute to deciphering the evolutionary history of MCTPs in maize and other plants, facilitating further functional analysis of these factors, and provide a basis for further clarification of the molecular mechanism of stress responses.

Polybasic Patches in Both C2 Domains of Synaptotagmin-1 Are Required for Evoked Neurotransmitter Release.

Synaptotagmin-1 (Syt1) is a vesicular calcium sensor required for synchronous neurotransmitter release, composed of a single-pass transmembrane domain linked to two C2 domains (C2A and C2B) that bind calcium, acidic lipids, and SNARE proteins that drive fusion of the synaptic vesicle with the plasma membrane. Despite its essential role, how Syt1 couples calcium entry to synchronous release is poorly understood. Calcium binding to C2B is critical for synchronous release, and C2B additionally binds the SNARE complex. The C2A domain is also required for Syt1 function, but it is not clear why. Here, we asked what critical feature of C2A may be responsible for its functional role and compared this to the analogous feature in C2B. We focused on highly conserved poly-lysine patches located on the sides of C2A (K189-192) and C2B (K324-327). We tested effects of charge-neutralization mutations in either region (Syt1K189-192A and Syt1K326-327A) side by side to determine their relative contributions to Syt1 function in cultured cortical neurons from mice of either sex and in single-molecule experiments. Combining electrophysiological recordings and optical tweezers measurements to probe dynamic single C2 domain-membrane interactions, we show that both C2A and C2B polybasic patches contribute to membrane binding, and both are required for evoked release. The size of the readily releasable vesicle pool and the rate of spontaneous release were unaffected, so both patches are likely required specifically for synchronization of release. We suggest these patches contribute to cooperative membrane binding, increasing the overall affinity of Syt1 for negatively charged membranes and facilitating evoked release.SIGNIFICANCE STATEMENT Synaptotagmin-1 is a vesicular calcium sensor required for synchronous neurotransmitter release. Its tandem cytosolic C2 domains (C2A and C2B) bind calcium, acidic lipids, and SNARE proteins that drive fusion of the synaptic vesicle with the plasma membrane. How calcium binding to Synaptotagmin-1 leads to release and the relative contributions of the C2 domains are unclear. Combining electrophysiological recordings from cultured neurons and optical tweezers measurements of single C2 domain-membrane interactions, we show that conserved polybasic regions in both domains contribute to membrane binding cooperatively, and both are required for evoked release, likely by increasing the overall affinity of Synaptotagmin-1 for acidic membranes.

Analysis of the MCTP Amino Acid Sequence Reveals the Conservation of Putative Calcium- and Lipid-Binding Pockets Within the C2 Domains In Silico.

MCTPs (Multiple C2 Domains and Transmembrane region Proteins) are evolutionarily and structurally related to other C2 proteins, which are central to exocytosis and membrane trafficking; however, their specific function has been little studied. MCTPs are associated with endosomes and the endoplasmic reticulum and possess three C2 domains (C2A-C2C) and two transmembrane regions (TMRs) well conserved in different species. Here, we generated structural models of the MCTP C2 domains of C. elegans and analyzed their putative function by docking, which revealed that these domains possess Ca2+- and lipid-binding pockets, suggesting that MCTPs play a significant, calcium-dependent role in membrane physiology.

Tandem C2 domains mediate dynamic organelle targeting of a DOCK family guanine nucleotide exchange factor.

Multicellular organisms use dedicator of cytokinesis (DOCK) family guanine nucleotide exchange factors (GEFs) to activate Rac/Rho-of-plants small GTPases and coordinate cell shape change. In developing tissues, DOCK signals integrate cell-cell interactions with cytoskeleton remodeling, and the GEFs cluster reversibly at specific organelle surfaces to orchestrate cytoskeletal reorganization. The domain organizations among DOCK orthologs are diverse, and the mechanisms of localization control are poorly understood. Here, we use combinations of transgene complementation and live-cell imaging assays to uncover an evolutionarily conserved and essential localization determinant in the DOCK-GEF named SPIKE1. The SPIKE1-DHR3 domain is sufficient for organelle association in vivo, and displays a complicated lipid-binding selectivity for both phospholipid head groups and fatty acid chain saturation. SPIKE1-DHR3 is predicted to adopt a C2-domain structure and functions as part of a tandem C2 array that enables reversible clustering at the cell apex. This work provides mechanistic insight into how DOCK GEFs sense compositional and biophysical membrane properties at the interface of two organelle systems.

The C2 domains of dysferlin: roles in membrane localization, Ca2+ signalling and sarcolemmal repair.

Dysferlin is an integral membrane protein of the transverse tubules of skeletal muscle that is mutated or absent in limb girdle muscular dystrophy 2B and Miyoshi myopathy. Here we examine the role of dysferlin's seven C2 domains, C2A through C2G, in membrane repair and Ca2+ release, as well as in targeting dysferlin to the transverse tubules of skeletal muscle. We report that deletion of either domain C2A or C2B inhibits membrane repair completely, whereas deletion of C2C, C2D, C2E, C2F or C2G causes partial loss of membrane repair that is exacerbated in the absence of extracellular Ca2+ . Deletion of C2C, C2D, C2E, C2F or C2G also causes significant changes in Ca2+ release, measured as the amplitude of the Ca2+ transient before or after hypo-osmotic shock and the appearance of Ca2+ waves. Most deletants accumulate in endoplasmic reticulum. Only the C2A domain can be deleted without affecting dysferlin trafficking to transverse tubules, but Dysf-ΔC2A fails to support normal Ca2+ signalling after hypo-osmotic shock. Our data suggest that (i) every C2 domain contributes to repair; (ii) all C2 domains except C2B regulate Ca2+ signalling; (iii) transverse tubule localization is insufficient for normal Ca2+ signalling; and (iv) Ca2+ dependence of repair is mediated by C2C through C2G. Thus, dysferlin's C2 domains have distinct functions in Ca2+ signalling and sarcolemmal membrane repair and may play distinct roles in skeletal muscle. KEY POINTS: Dysferlin, a transmembrane protein containing seven C2 domains, C2A through C2G, concentrates in transverse tubules of skeletal muscle, where it stabilizes voltage-induced Ca2+ transients and participates in sarcolemmal membrane repair. Each of dysferlin's C2 domains except C2B regulate Ca2+ signalling. Localization of dysferlin variants to the transverse tubules is not sufficient to support normal Ca2+ signalling or membrane repair. Each of dysferlin's C2 domains contributes to sarcolemmal membrane repair. The Ca2+ dependence of membrane repair is mediated by C2C through C2G. Dysferlin's C2 domains therefore have distinct functions in Ca2+ signalling and sarcolemmal membrane repair.

Global Identification and Characterization of C2 Domain-Containing Proteins Associated with Abiotic Stress Response in Rice (Oryza sativa L.).

C2 domain-containing proteins (C2DPs) have been identified in different genomes that contain single or multiple C2 domains in their C- or N-terminal. It possesses higher functional activity in the transmembrane regions. The identification of C2 domains were reported in a previous study, such as multiple C2 domains and transmembrane-region proteins (MCTPs) and N-terminal-TM-C2 domain proteins (NTMC2s) of rice, Arabidopsis thaliana, and cotton, whereas the C2DP gene family in rice has not been comprehensively studied, and the role of the C2DP gene in rice in response to abiotic stress is not yet fully understood. In this study, we identified 82 C2DPs in the rice genome and divided them into seven groups through phylogenetic analysis. The synteny analysis revealed that duplication events were either exhibited within the genome of rice or between the genomes of rice and other species. Through the analysis of cis-acting elements in promoters, expression profiles, and qRT-PCR results, the functions of OsC2DPs were found to be widely distributed in diverse tissues and were extensively involved in phytohormones-related and abiotic stresses response in rice. The prediction of the microRNA (miRNA) targets of OsC2DPs revealed the possibility of regulation by consistent miRNAs. Notably, OsC2DP50/51/52 as a co-tandem duplication exhibited similar expression variations and involved the coincident miRNA-regulation pathway. Moreover, the results of the genotypic variation and haplotype analysis revealed that OsC2DP17, OsC2DP29, and OsC2DP49 were associated with cold stress responses. These findings provided comprehensive insights for characterizations of OsC2DPs in rice as well as for their roles for abiotic stress.

Loss of C2 Domain Protein (C2CD5) Alters Hypothalamic Mitochondrial Trafficking, Structure, and Function.

INTRODUCTION: Mitochondria are essential organelles required for several cellular processes ranging from ATP production to cell maintenance. To provide energy, mitochondria are transported to specific cellular areas in need. Mitochondria also need to be recycled. These mechanisms rely heavily on trafficking events. While mechanisms are still unclear, hypothalamic mitochondria are linked to obesity. METHODS: We used C2 domain protein 5 (C2CD5, also called C2 domain-containing phosphoprotein [CDP138]) whole-body KO mice primary neuronal cultures and cell lines to perform electron microscopy, live cellular imaging, and oxygen consumption assay to better characterize mitochondrial alteration linked to C2CD5. RESULTS: This study identified that C2CD5 is necessary for proper mitochondrial trafficking, structure, and function in the hypothalamic neurons. We previously reported that mice lacking C2CD5 were obese and displayed reduced functional G-coupled receptor, melanocortin receptor 4 (MC4R) at the surface of hypothalamic neurons. Our data suggest that in neurons, normal MC4R endocytosis/trafficking necessities functional mitochondria. DISCUSSION: Our data show that C2CD5 is a new protein necessary for normal mitochondrial function in the hypothalamus. Its loss alters mitochondrial ultrastructure, localization, and activity within the hypothalamic neurons. C2CD5 may represent a new protein linking hypothalamic dysfunction, mitochondria, and obesity.

Molecular dynamic study on PTEN frameshift mutations in breast cancer provide c2 domain as a potential biomarker.

PTEN is a tumour suppressor gene known for regulating apoptosis, cell growth, and many other pathways. It is one of the most frequently mutated genes comprising the phosphatase domain (PD) and C terminal domain (C2). Direct therapeutic methods are not applicable for targeting PTEN because once gets mutated, it needs restoration. For mutant detection and restoration using PTEN mRNA there is a need to explore various mutations taking place in PTEN, identify their particular domains, and study their interactions within the cellular system. Here, we have tried to highlight a few such regions in the mutated PTEN of breast cancer patients. In this study, we have selected the top-most-occurring PTEN mutation in breast cancer and compared them to determine the specific properties of each mutation and its effect on functionality. Molecular dynamic simulation for 50 ns was performed on five structures to compare the structural behaviour of mutated PTEN in the system. Our finding suggests that frameshift mutations are more damaging and affect the c2 domain. Frameshift mutant fs_ACTT is the highest occurring as well as the most damaging mutation in all the compared structures. Docking study shows that substitution mutations D92H and R130Q causes loss of binding ability towards PIP2 in normal PTEN, interfering the dephosphorylation process. Overall, the C2 domain is more frequently mutated, and the amino acid residues in the C2 domain show more fluctuations compared to the other regions. Our study can provide the basis for selecting frequently mutated C2 domain as a potential therapeutic marker.Communicated by Ramaswamy H. Sarma.

Assessment of the Contribution of a Thermodynamic and Mechanical Destabilization of Myosin-Binding Protein C Domain C2 to the Pathomechanism of Hypertrophic Cardiomyopathy-Causing Double Mutation MYBPC3Δ25bp/D389V.

Mutations in the gene encoding cardiac myosin-binding protein-C (MyBPC), a thick filament assembly protein that stabilizes sarcomeric structure and regulates cardiac function, are a common cause for the development of hypertrophic cardiomyopathy. About 10% of carriers of the Δ25bp variant of MYBPC3, which is common in individuals from South Asia, are also carriers of the D389V variant on the same allele. Compared with noncarriers and those with MYBPC3Δ25bp alone, indicators for the development of hypertrophic cardiomyopathy occur with increased frequency in MYBPC3Δ25bp/D389V carriers. Residue D389 lies in the IgI-like C2 domain that is part of the N-terminal region of MyBPC. To probe the effects of mutation D389V on structure, thermostability, and protein-protein interactions, we produced and characterized wild-type and mutant constructs corresponding to the isolated 10 kDa C2 domain and a 52 kDa N-terminal fragment that includes subdomains C0 to C2. Our results show marked reductions in the melting temperatures of D389V mutant constructs. Interactions of construct C0-C2 D389V with the cardiac isoforms of myosin-2 and actin remain unchanged. Molecular dynamics simulations reveal changes in the stiffness and conformer dynamics of domain C2 caused by mutation D389V. Our results suggest a pathomechanism for the development of HCM based on the toxic buildup of misfolded protein in young MYBPC3Δ25bp/D389V carriers that is supplanted and enhanced by C-zone haploinsufficiency at older ages.

The conserved C2 phospholipid-binding domain in Delta contributes to robust Notch signalling.

Accurate Notch signalling is critical for development and homeostasis. Fine-tuning of Notch-ligand interactions has substantial impact on signalling outputs. Recent structural studies have identified a conserved N-terminal C2 domain in human Notch ligands which confers phospholipid binding in vitro. Here, we show that Drosophila ligands Delta and Serrate adopt the same C2 domain structure with analogous variations in the loop regions, including the so-called ß1-2 loop that is involved in phospholipid binding. Mutations in the ß1-2 loop of the Delta C2 domain retain Notch binding but have impaired ability to interact with phospholipids in vitro. To investigate its role in vivo, we deleted five residues within the ß1-2 loop of endogenous Delta. Strikingly, this change compromises ligand function. The modified Delta enhances phenotypes produced by Delta loss-of-function alleles and suppresses that of Notch alleles. As the modified protein is present on the cell surface in normal amounts, these results argue that C2 domain phospholipid binding is necessary for robust signalling in vivo fine-tuning the balance of trans and cis ligand-receptor interactions.

The C2 domain of calpain 5 contributes to enzyme activation and membrane localization.

The enzymatic characteristics of the ubiquitous calpain 5 (CAPN5) remain undescribed despite its high expression in the central nervous system and links to eye development and disease. CAPN5 contains the typical protease core domains but lacks the C terminal penta-EF hand domain of classical calpains, and instead contains a putative C2 domain. This study used the SH-SY5Y neuroblastoma cell line stably transfected with CAPN5-3xFLAG variants to assess the potential roles of the CAPN5 C2 domain in Ca2+ regulated enzyme activity and intracellular localization. Calcium dependent autoproteolysis of CAPN5 was documented and characterized. Mutation of the catalytic Cys81 to Ala or addition of EGTA prevented autolysis. Eighty µM Ca2+ was sufficient to stimulate half-maximal CAPN5 autolysis in cellular lysates. CAPN5 autolysis was inhibited by tri-leucine peptidyl aldehydes, but less effectively by di-Leu aldehydes, consistent with a more open conformation of the protease core relative to classical calpains. In silico modeling revealed a type II topology C2 domain including loops with the potential to bind calcium. Mutation of the acidic amino acid residues predicted to participate in Ca2+ binding, particularly Asp531 and Asp589, resulted in a decrease of CAPN5 membrane association. These residues were also found to be invariant in several genomes. The autolytic fragment of CAPN5 was prevalent in membrane-enriched fractions, but not in cytosolic fractions, suggesting that membrane association facilitates the autoproteolytic activity of CAPN5. Together, these results demonstrate that CAPN5 undergoes Ca2+-activated autoproteolytic processing and suggest that CAPN5 association with membranes enhances CAPN5 autolysis.

Multiple C2 domain-containing transmembrane proteins promote lipid droplet biogenesis and growth at specialized endoplasmic reticulum subdomains.

Lipid droplets (LDs) are neutral lipid-containing organelles enclosed in a single monolayer of phospholipids. LD formation begins with the accumulation of neutral lipids within the bilayer of the endoplasmic reticulum (ER) membrane. It is not known how the sites of formation of nascent LDs in the ER membrane are determined. Here we show that multiple C2 domain-containing transmembrane proteins, MCTP1 and MCTP2, are at sites of LD formation in specialized ER subdomains. We show that the transmembrane domain (TMD) of these proteins is similar to a reticulon homology domain. Like reticulons, these proteins tubulate the ER membrane and favor highly curved regions of the ER. Our data indicate that the MCTP TMDs promote LD biogenesis, increasing LD number. MCTPs colocalize with seipin, a protein involved in LD biogenesis, but form more stable microdomains in the ER. The MCTP C2 domains bind charged lipids and regulate LD size, likely by mediating ER-LD contact sites. Together, our data indicate that MCTPs form microdomains within ER tubules that regulate LD biogenesis, size, and ER-LD contacts. Interestingly, MCTP punctae colocalized with other organelles as well, suggesting that these proteins may play a general role in linking tubular ER to organelle contact sites.