C2 Domain Research Articles

Fundamental solutions and critical Lane-Emden exponents for nonlinear integral operators in cones

In this article we study the fundamental solutions or “α-harmonic functions” for some nonlinear positive homogeneous nonlocal elliptic problems in conical domains, such asF(u)=0inCω,u=0inRN∖Cω, where ω is a proper C2 domain in SN−1 for N≥2, Cω:={x:x≠0,|x|−1x∈ω} is the cone-like domain related to ω, and F is an extremal fully nonlinear integral operator. We prove the existence of two fundamental solutions that are homogeneous and do not change signs in the cone; one is bounded at the origin and the other at infinity.As an application, we use the fundamental solutions obtained to prove Liouville type theorems in cones for supersolutions of the Lane-Emden-Fowler equation in the formF(u)+up=0inCω,u=0inRN∖Cω. We also prove a generalized Hopf type lemma in domains with corners. Most of our results are new even when F is the fractional Laplacian operator.

CircST6GAL1 knockdown alleviates pulmonary arterial hypertension by regulating miR-509-5p/multiple C2 and transmembrane domain containing 2 axis.

Hypertension is a main contributing factor of cardiovascular diseases; deregulated circular RNAs are involved in the pathogenesis of pulmonary arterial hypertension (PAH). Herein, we evaluated the function and mechanism of circST6GAL1 in PAH process. Human pulmonary artery smooth muscle cells (HPASMCs) were cultured in hypoxic environment for functional analysis. The cell counting kit-8, 5-ethynyl-2'-deoxyuridine, wound healing, and flow cytometry assays were used to investigate cell proliferation, migration, and apoptosis. qRT-PCR and Western blotting analyses were used for level measurement of genes and proteins. The binding between miR-509-5p and circST6GAL1 or multiple C2 and transmembrane domain containing 2 (MCTP2) was analyzed by dual-luciferase reporter, RNA immunoprecipitation, and pull-down assays. The monocrotaline (MCT)-induced PAH mouse models were established for in vivo assay. CircST6GAL1 was highly expressed in PAH patients and hypoxia-induced HPASMCs. Functionally, circST6GAL1 deficiency reversed hypoxia-induced proliferation and migration, as well as apoptosis arrest in HPASMCs. Mechanistically, circST6GAL1 directly targeted miR-509-5p, and MCTP2 was a target of miR-509-5p. Rescue assays showed that the regulatory effects of circST6GAL1 deficiency on hypoxia-induced HPASMCs were abolished. Moreover, forced expression of miR-509-5p suppressed HPASMC proliferation and migration and induced cell apoptosis under hypoxia stimulation, while these effects were abolished by MCTP2 overexpression. Moreover, circST6GAL1 silencing improved MCT-induced pulmonary vascular remodeling and PAH. CircST6GAL1 deficiency reversed hypoxia-induced proliferation and migration, as well as apoptosis arrest in HPASMCs, and alleviated pulmonary vascular remodeling in MCT-induced PAH mouse models through the miR-509-5p/MCTP2 axis, indicating a potential therapeutic target for PAH.

Silencing Ditylenchus destructor cathepsin L-like cysteine protease has negative pleiotropic effect on nematode ontogenesis

Ditylenchus destructor is a migratory plant-parasitic nematode that severely harms many agriculturally important crops. The control of this pest is difficult, thus efficient strategies for its management in agricultural production are urgently required. Cathepsin L-like cysteine protease (CPL) is one important protease that has been shown to participate in various physiological and pathological processes. Here we decided to characterize the CPL gene (Dd-cpl-1) from D. destructor. Analysis of Dd-cpl-1 gene showed that Dd-cpl-1 gene contains a signal peptide, an I29 inhibitor domain with ERFNIN and GNFD motifs, and a peptidase C1 domain with four conserved active residues, showing evolutionary conservation with other nematode CPLs. RT-qPCR revealed that Dd-cpl-1 gene displayed high expression in third-stage juveniles (J3s) and female adults. In situ hybridization analysis demonstrated that Dd-cpl-1 was expressed in the digestive system and reproductive organs. Silencing Dd-cpl-1 in 1-cell stage eggs of D. destructor by RNAi resulted in a severely delay in development or even in abortive morphogenesis during embryogenesis. The RNAi-mediated silencing of Dd-cpl-1 in J2s and J3s resulted in a developmental arrest phenotype in J3 stage. In addition, silencing Dd-cpl-1 gene expression in female adults led to a 57.43% decrease in egg production. Finally, Dd-cpl-1 RNAi-treated nematodes showed a significant reduction in host colonization and infection. Overall, our results indicate that Dd-CPL-1 plays multiple roles in D. destructor ontogenesis and could serve as a new potential target for controlling D. destructor.

Nanodysferlins support membrane repair and binding to TRIM72/MG53 but do not localize to t-tubules or stabilize Ca2+ signaling

Mutations in the DYSF gene, encoding the protein dysferlin, lead to several forms of muscular dystrophy. In healthy skeletal muscle, dysferlin concentrates in the transverse tubules and is involved in repairing the sarcolemma and stabilizing Ca2+ signaling after membrane disruption. The DYSF gene encodes 7-8 C2 domains, several Fer and Dysf domains, and a C-terminal transmembrane sequence. Because its coding sequence is too large to package in adeno-associated virus, the full-length sequence is not amendable to current gene delivery methods. Thus, we have examined smaller versions of dysferlin, termed “nanodysferlins”, designed to eliminate several C2 domains, specifically C2 domains D, E and F; B, D and E; and B, D, E and F. We also generated a variant by replacing 8 amino acids in C2G in the nanodysferlin missing domains D through F. We electroporated dysferlin-null A/J mouse myofibers with Venus fusion constructs of these variants, or as untagged nanodysferlins together with GFP, to mark transfected fibers We found that, although these nanodysferlins failed to concentrate in transverse tubules, 3 of them supported membrane repair after laser wounding while all 4 bound the membrane repair protein, TRIM72/MG53, similar to wild type dysferlin. By contrast, they failed to suppress Ca2+ waves after myofibers were injured by mild hypoosmotic shock. Our results suggest that the internal C2 domains of dysferlin are required for normal t-tubule localization and Ca2+ signaling and that membrane repair does not require these C2 domains.

The First Defined Null Allele of the Notch Regulator, a Suppressor of Deltex: Uncovering Its Novel Roles in Drosophila melanogaster Oogenesis.

Suppressor of deltex (Su(dx)) is a Drosophila melanogaster member of the NEDD4 family of the HECT domain E3 ubiquitin ligases. Su(dx) acts as a regulator of Notch endocytic trafficking, promoting Notch lysosomal degradation and the down-regulation of both ligand-dependent and ligand-independent signalling, the latter involving trafficking through the endocytic pathway and activation of the endo/lysosomal membrane. Mutations of Su(dx) result in developmental phenotypes in the Drosophila wing that reflect increased Notch signalling, leading to gaps in the specification of the wing veins, and Su(dx) functions to provide the developmental robustness of Notch activity to environmental temperature shifts. The full developmental functions of Su(dx) are unclear; however, this is due to a lack of a clearly defined null allele. Here we report the first defined null mutation of Su(dx), generated by P-element excision, which removes the complete open reading frame. We show that the mutation is recessive-viable, with the Notch gain of function phenotypes affecting wing vein and leg development. We further uncover new roles for Su(dx) in Drosophila oogenesis, where it regulates interfollicular stalk formation, egg chamber separation and germline cyst enwrapment by the follicle stem cells. Interestingly, while the null allele exhibited a gain in Notch activity during oogenesis, the previously described Su(dx)SP allele, which carries a seven amino acid in-frame deletion, displayed a Notch loss of function phenotypes and an increase in follicle stem cell turnover. This is despite both alleles displaying similar Notch gain of function in wing development. We attribute this unexpected context-dependent outcome of Su(dx)sp being due to the partial retention of function by the intact C2 and WW domain regions of the protein. Our results extend our understanding of the developmental role of Su(dx) in the tissue renewal and homeostasis of the Drosophila ovary and illustrate the importance of examining an allelic series of mutations to fully understand developmental functions.

A novel framework for functional annotation of variants of uncertain significance in ID/ASD risk gene CC2D1A.

Intellectual disability (ID) and autism spectrum disorder (ASD) are genetically heterogeneous with hundreds of identified risk genes, most affecting only a few patients. Novel missense variants in these genes are being discovered as clinical exome sequencing is now routinely integrated into diagnosis, yet most of them are annotated as variants of uncertain significance (VUS). VUSs are a major roadblock in using patient genetics to inform clinical action. We developed a framework to characterize VUSs in Coiled-coil and C2 domain containing 1A (CC2D1A), a gene causing autosomal recessive ID with comorbid ASD in 40% of cases. We analyzed seven VUSs (p.Pro319Leu, p.Ser327Leu, p.Gly441Val, p.Val449Met, p.Thr580Ile, p.Arg886His and p.Glu910Lys) from four cases of individuals with ID and ASD. Variants were cloned and overexpressed in HEK293 individually and in their respective heterozygous combination. CC2D1A is a signaling scaffold that positively regulates PKA-CREB signaling by repressing phosphodiesterase 4D (PDE4D) to prevent cAMP degradation. After testing multiple parameters including direct interaction between PDE4D and CC2D1A, cAMP levels and CREB activation, we found that the most sensitive readout was CREB transcriptional activity using a luciferase assay. Compared to WT CC2D1A, five VUSs (p.Pro319Leu, p.Gly441Val, p.Val449Met, p.Thr580Ile, and p.Arg886His) led to significantly blunted response to forskolin induced CREB activation. This luciferase assay approach can be scaled up to annotate ~150 CC2D1A VUSs that are currently listed in ClinVar. Since CREB activation is a common denominator for multiple ASD/ID genes, our paradigm can also be adapted for their VUSs.

Identification of tomato C2 gene family and functional analysis of the inflorescence branching regulatory gene SlC2–25

The C2 domain plays an important role in plant growth and development. We performed genome-wide identification of SlC2 gene family members by bioinformatics, and predictive analysis of chromosome distribution, evolutionary relationships, gene structure and colinearity of their family members. The results showed that a total of 103 SlC2 genes were screened, according to the order of their physical coordinates, 103 SlC2 genes were named from SlC2–1 to SlC2–103. The amino acid lengths ranged from 107 to 2133aa, the molecular weights ranged from 12,416.96 to 229,427.7 Da, and the isoelectric points ranged from 4.18 to 9.94. The cluster analysis was divided into six subgroups, and the gene sequences were highly conserved. Based on the pre-laboratory transcriptome data, we screened the SlC2–25 gene, analyzed the expression pattern of SlC2–25 in two kinds of tomato inflorescences by RT-qPCR, and preliminarily explored the function of SlC2–25 in the regulation of tomato inflorescence meristem by using gene editing technology, and found that SlC2–25 was a negative regulator of tomato inflorescence branching. In addition, the endogenous hormone content of tomato inflorescences was determined, and it was hypothesized that the SlC2s genes might be involved in the regulatory process of tomato inflorescence branching by altering the content of endogenous hormones in the plant or by interacting with a variety of proteins.

The circMYBL2-Encoded p185 Protein Suppresses Colorectal Cancer Progression by Inhibiting Serine Biosynthesis.

Circular RNAs are a class of covalently closed single-stranded loop RNAs that have been implicated to play a functional role in almost all types of cancers. Previous studies have revealed that circMYBL2 acts as a tumor-promoting circular RNA. In this study, we found that circMYBL2 in colorectal cancer encodes a 185-amino acid protein, p185. Functionally, circMYBL2-encoded p185 suppressed the growth and aggressiveness of colorectal cancer cells in vitro and in vivo. Mechanistically, p185 counteracted ubiquitin C-terminal hydrolase L3 (UCHL3)-mediated deubiquitination of phosphoglycerate dehydrogenase (PHGDH) by competitively binding to the C1 domain of UCHL3, resulting in PHGDH degradation and a subsequent reduction in serine and glycine biosynthesis. These data revealed that the circMYBL2-encoded p185 isoform serves as a tumor suppressor to inhibit the progression of colorectal cancer by reducing serine biosynthesis. Significance: A p185 protein encoded by circMYBL2 functions as a tumor suppressor that inhibits the progression of colorectal cancer by increasing the degradation of PHGDH to reduce serine biosynthesis.

On the role of dysferlin in striated muscle: membrane repair, t-tubules and Ca2+ handling.

Dysferlin is a 237kDa membrane-associated protein characterised by multiple C2 domains with a diverse role in skeletal and cardiac muscle physiology. Mutations in DYSF are known to cause various types of human muscular dystrophies, known collectively as dysferlinopathies, with some patients developing cardiomyopathy. A myriad of in vitro membrane repair studies suggest that dysferlin plays an integral role in the membrane repair complex in skeletal muscle. In comparison, less is known about dysferlin in the heart, but mounting evidence suggests that dysferlin's role is similar in both muscle types. Recent findings have shown that dysferlin regulates Ca2+ handling in striated muscle via multiple mechanisms and that this becomes more important in conditions of stress. Maintenance of the transverse (t)-tubule network and the tight coordination of excitation-contraction coupling are essential for muscle contractility. Dysferlin regulates the maintenance and repair of t-tubules, and it is suspected that dysferlin regulates t-tubules and sarcolemmal repair through a similar mechanism. This review focuses on the emerging complexity of dysferlin's activity in striated muscle. Such insights will progress our understanding of the proteins and pathways that regulate basic heart and skeletal muscle function and help guide research into striated muscle pathology, especially that which arises due to dysferlin dysfunction.

Massive ER protein disposal by reticulophagy receptors and selective disposal by TOLLIP

ABSTRACT Proteostasis of the endoplasmic reticulum (ER) is maintained by coordinated action of two major catabolic pathways: proteasome-dependent ER-associated degradation (ERAD) and less characterized lysosomal pathways. Recent studies on ER-specific autophagy (termed “reticulophagy”) have highlighted the importance of lysosomes for ER proteostasis. Key to this process are proteins termed reticulophagy receptors that connect ER fragments and Atg8-family proteins, facilitating the lysosomal degradation of both native and aberrant ER proteins in a relatively nonselective manner. In contrast, our recent work identified TOLLIP as a novel type of cargo receptor specifically dedicated to the lysosomal degradation of aberrant ER membrane proteins. The clients of TOLLIP include an engineered model substrate, which mimics an ER-retained aberrant membrane protein, and motor neuron disease-linked misfolded mutants of VAPB and BSCL2/Seipin. TOLLIP acts as a receptor to connect these aberrant ER membrane proteins and phosphatidylinositol-3-phosphate (PtdIns3P) by recognizing the former through its misfolding-sensing intrinsically disordered region (IDR) and ubiquitin-binding CUE domain, and the latter through its C2 domain. These interactions enable PtdIns3P-dependent vesicular trafficking of aberrant membrane proteins to lysosomes without promoting reticulophagic turnover of bulk ER.

Functional characterization of sugarcane ScFTIP1 reveals its role in Arabidopsis flowering

The timing of floral transition is essential for reproductive success in flowering plants. In sugarcane, flowering time affects the production of sugar and biomass. Although the function of the crucial floral pathway integrators, FLOWERING LOCUS T (FT), in sugarcane, has been uncovered, the proteins responsible for FT export and the underlying mechanism remain unexplored. In this study, we identified a member of the multiple C2 domain and transmembrane region proteins (MCTPs) family in sugarcane, FT-interacting protein 1 (ScFTIP1), which was localized to the endoplasmic reticulum. Ectopic expression of ScFTIP1 in the Arabidopsis mutant ftip1-1 rescued the late-flowering phenotype. ScFTIP1 interacted with AtFT in vitro and in vivo assays. Additionally, ScFTIP1 interacted with ScFT1 and the floral inducer ScFT3. Furthermore, we found that the NAC member, ScNAC23, could directly bind to the ScFTIP1 promoter and negatively regulate its transcription. Overall, our findings revealed the function of ScFTIP1 and proposed a potential mechanism underlying flowering regulation in sugarcane.

Intraphagosomal Free Ca2+ Changes during Phagocytosis.

Phagocytosis (and endocytosis) is an unusual cellular process that results in the formation of a novel subcellular organelle, the phagosome. This phagosome contains not only the internalised target of phagocytosis but also the external medium, creating a new border between extracellular and intracellular environments. The boundary at the plasma membrane is, of course, tightly controlled and exploited in ionic cell signalling events. Although there has been much work on the control of phagocytosis by ions, notably, Ca2+ ions influxing across the plasma membrane, increasing our understanding of the mechanism enormously, very little work has been done exploring the phagosome/cytosol boundary. In this paper, we explored the changes in the intra-phagosomal Ca2+ ion content that occur during phagocytosis and phagosome formation in human neutrophils. Measuring Ca2+ ion concentration in the phagosome is potentially prone to artefacts as the intra-phagosomal environment experiences changes in pH and oxidation. However, by excluding such artefacts, we conclude that there are open Ca2+ channels on the phagosome that allow Ca2+ ions to "drain" into the surrounding cytosol. This conclusion was confirmed by monitoring the translocation of the intracellularly expressed YFP-tagged C2 domain of PKC-γ. This approach marked regions of membrane at which Ca2+ influx occurred, the earliest being the phagocytic cup, and then the whole cell. This paper therefore presents data that have novel implications for understanding phagocytic Ca2+ signalling events, such as peri-phagosomal Ca2+ hotspots, and other phenomena.

Tigilanol tiglate is an oncolytic small molecule that induces immunogenic cell death and enhances the response of both target and non-injected tumors to immune checkpoint blockade

BackgroundTigilanol tiglate (TT) is a protein kinase C (PKC)/C1 domain activator currently being developed as an intralesional agent for the treatment of various (sub)cutaneous malignancies. Previous work has shown that...

Determining the Algorithm of Rehabilitation Procedures in Patients with Brachial Plexus Injuries Based on the Prospective Single-Centre Clinical Neurophysiology Studies: Preliminary Results

The clinical neurophysiological tests allow us to determine the type, extent, and nature of brachial plexus damage. They are crucial in decision making regarding surgical procedures or conservative treatment. This report aimed to present an algorithm for rehabilitation procedures in patients with brachial plexus injury of various origins based on the results of neurophysiology findings for the selection of procedures supporting the process of nerve and muscle regeneration. The research group consisted of patients whose medical documentation was analysed concerning the reason, level, and localization of damage to the brachial plexus structures, surgical and or rehabilitative treatment, as well as the MRI results. Among the group of fourteen patients, the clinical studies showed the greatest incidence of brachial plexus injuries of the mixed (both pre- and postganglionic), all trunks, and cervical root injuries, respectively. Results of the motor evoked potentials (MEP) and electroneurography (ENG) recordings induced at levels of spinal roots and Erb’s point showed a decrease of more than 40% in amplitudes on the symptomatic side in comparison to the asymptomatic side. This diffeence was recorded for the axillary and radial innervation and the C5, C6, and C7 root domains, ranging from 57% to 66%; the lowest decrease was recorded following electrical stimulation at Erb’s point for the ulnar nerve (34%). The latency prolongation on the symptomatic side in CMAP and MEP tests ranged from 0.2 to 1.7 ms, with the most following magnetic stimulation of the C5 cervical root for the axillary innervation. Most of the results indicated the axonotmesis and neuropraxia type of injury in motor fibers (40%) confirmed by EMG results. The sensory conduction studies (SNCS) in distal nerve branches did not confirm the severe advancement of the brachial plexus injury (63%). The proposed algorithm of the physiotherapeutic procedures should be mainly targeted for recovery of motor dysfunction as the consequence of brachial plexus injury. Rehabilitation should incorporate the treatment supporting nerve regeneration, muscle strengthening, and maintaining functional ranges of motion of the injured extremities. The rehabilitation treatment for patients with brachial plexus injuries is an individualised process, and the selection of procedures and the effectiveness of the treatment undertaken should be confronted with results of neurophysiological tests verifying the motor neural transmission from the level of the cervical motor centre to the effector, peripheral nerve function, and muscle’s motor unit activity.

The self-reactive FVIII T cell repertoire in healthy individuals relies on a short set of epitopes and public clonotypes.

Non-mutated FVIII-specific CD4 T cell epitopes have been recently found to contribute to the development of inhibitors in patients with hemophilia A (HA), while auto-reactive CD4 T cells specific to FVIII circulate in the blood of healthy individuals at a frequency close to the foreign protein ovalbumin. Thus, although FVIII is a self-protein, the central tolerance raised against FVIII appears to be low. In this study, we conducted a comprehensive analysis of the FVIII CD4 T cell repertoire in 29 healthy donors. Sequencing of the CDR3β TCR region from isolated FVIII-specific CD4 T cells revealed a limited usage and pairing of TRBV and TRBJ genes as well as a mostly hydrophobic composition of the CDR3β region according to their auto-reactivity. The FVIII repertoire is dominated by a few clonotypes, with only 13 clonotypes accounting for half of the FVIII response. Through a large-scale epitope mapping of the full-length FVIII sequence, we identified 18 immunodominant epitopes located in the A1, A3, C1, and C2 domains and covering half of the T cell response. These epitopes exhibited a broad specificity for HLA-DR or DP molecules or both. T cell priming with this reduced set of peptides revealed that highly expanded clonotypes specific to these epitopes were responsible individually for up to 32% of the total FVIII repertoire. These FVIII T cell epitopes and clonotypes were shared among HLA-unrelated donors tested and previously reported HA patients. Our study highlights the role of the auto-reactive T cell response against FVIII in HA and its similarity to the response observed in healthy individuals. Thus, it provides valuable insights for the development of new tolerance induction and deimmunization strategies.

Let it stick: Strategies and applications for intracellular plasma membrane targeting of proteins in Saccharomyces cerevisiae.

Lipid binding domains and protein lipidations are essential features to recruit proteins to intracellular membranes, enabling them to function at specific sites within the cell. Membrane association can also be exploited to answer fundamental and applied research questions, from obtaining insights into the understanding of lipid metabolism to employing them for metabolic engineering to redirect fluxes. This review presents a broad catalog of membrane binding strategies focusing on the plasma membrane of Saccharomyces cerevisiae. Both lipid binding domains (pleckstrin homology, discoidin-type C2, kinase associated-1, basic-rich and bacterial phosphoinositide-binding domains) and co- and post-translational lipidations (prenylation, myristoylation and palmitoylation) are introduced as tools to target the plasma membrane. To provide a toolset of membrane targeting modules, respective candidates that facilitate plasma membrane targeting are showcased including their in vitro and in vivo properties. The relevance and versatility of plasma membrane targeting modules are further highlighted by presenting a selected set of use cases.

Evolutionary and Structural Analysis of PP16 in Viridiplantae.

Members of the phloem protein 16 (PP16) gene family are induced by elicitors in rice and the corresponding proteins from cucurbits, which display RNA binding and intercellular transport activities, are accumulated in phloem sap. These proteins facilitate the movement of protein complexes through the phloem translocation flow and may be involved in the response to water deficit, among other functions. However, there is scant information regarding their function in other plants, including the identification of paralog genes in non-vascular plants and chlorophytes. In the present work, an evolutionary and structural analysis of the PP16 family in green plants (Viridiplantae) was carried out. Data mining in different databases indicated that PP16 likely originated from a larger gene present in an ancestral lineage that gave rise to chlorophytes and multicellular plants. This gene encodes a protein related to synaptotagmin, which is involved in vesicular transport in animal systems, although other members of this family play a role in lipid turnover in endomembranes and organelles. These proteins contain a membrane-binding C2 domain shared with PP16 proteins in vascular plants. In silico analysis of the predicted structure of the PP16 protein family identified several β-sheets, one α-helix, and intrinsically disordered regions. PP16 may have been originally involved in vesicular trafficking and/or membrane maintenance but specialized in long-distance signaling during the emergence of the plant vascular system.

PTEN hamartoma tumor syndrome: Clinical and genetic characterization in pediatric patients.

The aim of this study was to provide a full characterization of a cohort of 11 pediatric patients diagnosed with PTEN hamartoma tumor syndrome (PHTS). Eleven patients with genetic diagnostic of PHTS were recruited between February 2019 and April 2023. Clinical, imaging, demographic, and genetic data were retrospectively collected from their hospital medical history. Regarding clinical manifestations, macrocephaly was the leading sign, present in all patients. Frontal bossing was the most frequent dysmorphism. Neurological issues were present in most patients. Dental malformations were described for the first time, being present in 27% of the patients. Brain MRI showed anomalies in 57% of the patients. No tumoral lesions were present at the time of the study. Regarding genetics, 72% of the alterations were in the tensin-type C2 domain of PTEN protein. We identified four PTEN genetic alterations for the first time. PTEN mutations appear with a wide variety of clinical signs and symptoms, sometimes associated with phenotypes which do not fit classical clinical diagnostic criteria for PHTS. We recommend carrying out a genetic study to establish an early diagnosis in children with significant macrocephaly. This facilitates personalized monitoring and enables anticipation of potential PHTS-related complications.

Copines, a Family of Calcium Sensor Proteins and Their Role in Brain Function.

The Copines are a family of evolutionary conserved calcium-binding proteins found in most eukaryotic organisms from protists to humans. They share a unique architecture and contain tandem C2 domains and a Von Willebrand factor type A (VWA) domain. C2 domains in Copines bind calcium, phospholipids, and other proteins and mediate the transient association of these proteins with biological membranes at elevated calcium levels. The VWA domain also binds calcium and is involved in protein-protein interactions. Here, we provide a comprehensive review of the sequences, structures, expression, targeting, and function of the entire family of known Copine proteins (Copine 1-9 in mammals) with a particular emphasis on their functional roles in the mammalian brain. Neuronal Copines are implicated in a wide array of processes from cell differentiation to synaptic transmission and plasticity and are also linked to several pathological conditions from cancers to brain diseases. This review provides the most up-to-date insights into the structure and function of Copines, with an emphasis on their role in brain function.

Troponin Structural Dynamics in the Native Cardiac Thin Filament Revealed by Cryo Electron Microscopy

Cardiac muscle contraction occurs due to repetitive interactions between myosin thick and actin thin filaments (TF) regulated by Ca2+ levels, active cross-bridges, and cardiac myosin-binding protein C (cMyBP-C). The cardiac TF (cTF) has two nonequivalent strands, each comprised of actin, tropomyosin (Tm), and troponin (Tn). Tn shifts Tm away from myosin-binding sites on actin at elevated Ca2+ levels to allow formation of force-producing actomyosin cross-bridges. The Tn complex is comprised of three distinct polypeptides – Ca2+-binding TnC, inhibitory TnI, and Tm-binding TnT. The molecular mechanism of their collective action is unresolved due to lack of comprehensive structural information on Tn region of cTF. C1 domain of cMyBP-C activates cTF in the absence of Ca2+ to the same extent as rigor myosin. Here we used cryo-EM of native cTFs to show that cTF Tn core adopts multiple structural conformations at high and low Ca2+ levels and that the two strands are structurally distinct. At high Ca2+ levels, cTF is not entirely activated by Ca2+ but exists in either partially or fully activated state. Complete dissociation of TnI C-terminus is required for full activation. In presence of cMyBP-C C1 domain, Tn core adopts a fully activated conformation, even in absence of Ca2+. Our data provide a structural description for the requirement of myosin to fully activate cTFs and explain increased affinity of TnC to Ca2+ in presence of active cross-bridges. We suggest that allosteric coupling between Tn subunits and Tm is required to control actomyosin interactions.