C2A Domain Research Articles

Surface display of spring viremia of carp virus glycoprotein on Lactococcus lactis and its protection efficacy in common carp (Cyprinus carpio L.)

Spring viremia of carp virus (SVCV) causes devastating disease in aquaculture, resulting in significant economic impact. To develop an effective means against SVCV infection, a Lactococcus lactis (L.lactis) based subunit vaccine (pNZ-UGA) was developed based on surface displaying of SVCV glycoprotein using anchoring motif of the cA (C terminus of the peptidoglyvsn-binding) domains of AcmA, a major autolysin from L.lactis. The surface expression of SVCV glycoprotein was verified by indirect immunofluorescence assay. The efficacy of the constructed vaccine was further evaluated in common carp. The results showed that the higher levels of specific IgM could be detected in fish vaccinated with pNZ-UGA, compared with that in PBS and L.lactis groups. Immune-related genes including TNF-α, IL-6b, IL-1β, Cxcr 1, Cxca, IFNg2b, I-IFN, and IgM expression in pNZ-UGA group were strongly up-regulated, revealing that robust innate immune response was induced. Notably, the lowest cumulative mortality (13.46%) was observed in fish vaccinated with pNZ-UGA vaccine after SVCV challenge, whereas the cumulative mortality were 100.00% and 92.31% in PBS and L.lactis groups, respectively. This study suggests the potential use of the recombinant L.lactis with surface displaying antigen proteins as effective vaccines against SVCV and other fish virus infection.

Cytotoxic Granule Trafficking and Fusion in Synaptotagmin7-Deficient Cytotoxic T Lymphocytes.

Granules of cytotoxic T lymphocytes (CTL) are derived from the lysosomal compartment. Synaptotagmin7 (Syt7) appears to be the calcium sensor triggering fusion of lysosomes in fibroblasts. Syt7 has been proposed to control cytotoxic granule (CG) fusion in lymphocytes and mice lacking Syt7 have reduced ability to clear infections. However, fusion of CG persists in the absence of Syt7. To clarify the role of Syt7 in CTL function, we have examined the fusion of cytotoxic granules of CD8+ T-lymphocytes from Syt7 knock-out mice. We have recorded granule fusion in living CTL, using total internal reflection microscopy. Since Syt7 is considered a high affinity calcium-sensor specialized for fusion under low calcium conditions, we have compared cytotoxic granule fusion under low and high calcium conditions in the same CTL. There was no difference in latencies or numbers of fusion events per CTL under low-calcium conditions, indicating that Syt7 is not required for cytotoxic granule fusion. A deficit of fusion in Syt7 KO CTL was seen when a high-calcium solution was introduced. Expressing wild type Syt7 in Syt7 KO lymphocytes reversed this deficit, confirming its Syt7-dependence. Mutations of Syt7 which disrupt calcium binding to its C2A domain reduced the efficacy of this rescue. We counted the cytotoxic granules present at the plasma membrane to determine if the lack of fusion events in the Syt7 KO CTL was due to a lack of granules. In low calcium there were no differences in fusion events per CTL, and granule numbers were similar. In high calcium, granule number was similar though wild type CTL exhibited significantly more fusion than Syt7 KO CTL. The modest differences in granule counts do not account for the lack of fusion in high calcium in Syt7 KO CTL. In Syt7 KO CTL expressing wild type Syt7, delivery of cytotoxic granules to the plasma membrane was comparable to that of wild type CTL. Syt7 KO CTL expressing Syt7 with deficient calcium binding in the C2A domain had significantly less fusion and fewer CG at the plasma membrane. These results indicate that Syt7 is involved in trafficking of CG to the plasma membrane.

The role of the C2A domain of synaptotagmin 1 in asynchronous neurotransmitter release.

Following nerve stimulation, there are two distinct phases of Ca2+-dependent neurotransmitter release: a fast, synchronous release phase, and a prolonged, asynchronous release phase. Each of these phases is tightly regulated and mediated by distinct mechanisms. Synaptotagmin 1 is the major Ca2+ sensor that triggers fast, synchronous neurotransmitter release upon Ca2+ binding by its C2A and C2B domains. It has also been implicated in the inhibition of asynchronous neurotransmitter release, as blocking Ca2+ binding by the C2A domain of synaptotagmin 1 results in increased asynchronous release. However, the mutation used to block Ca2+ binding in the previous experiments (aspartate to asparagine mutations, sytD-N) had the unintended side effect of mimicking Ca2+ binding, raising the possibility that the increase in asynchronous release was directly caused by ostensibly constitutive Ca2+ binding. Thus, rather than modulating an asynchronous sensor, sytD-N may be mimicking one. To directly test the C2A inhibition hypothesis, we utilized an alternate C2A mutation that we designed to block Ca2+ binding without mimicking it (an aspartate to glutamate mutation, sytD-E). Analysis of both the original sytD-N mutation and our alternate sytD-E mutation at the Drosophila neuromuscular junction showed differential effects on asynchronous release, as well as on synchronous release and the frequency of spontaneous release. Importantly, we found that asynchronous release is not increased in the sytD-E mutant. Thus, our work provides new mechanistic insight into synaptotagmin 1 function during Ca2+-evoked synaptic transmission and demonstrates that Ca2+ binding by the C2A domain of synaptotagmin 1 does not inhibit asynchronous neurotransmitter release in vivo.

Novel Human Polymorphisms Define a Key Role for the SLC26A6-STAS Domain in Protection From Ca2+-Oxalate Lithogenesis.

Impaired homeostasis of the carboxylic acids oxalate and citrate, dramatically increases the risk for the formation of Ca2+-oxalate kidney stones, which is the most common form of kidney stones in humans. Renal homeostasis of oxalate and citrate is controlled by complex mechanisms including epithelial transport proteins such as the oxalate transporter, SLC26A6, and the citrate transporters, the SLC13’s. These transporters interact via the SLC26A6-STAS domain in vitro, however, the role of the Sulfate Transporter and Anti-Sigma factor antagonist (STAS) domain in Ca2+-oxalate stone formation was not investigated in humans. Here, we report two novel human SLC26A6 polymorphisms identified in the STAS domain of SLC26A6 in two heterozygous carriers. Intriguingly, these individuals have low urinary citrate, but different clinical manifestations. Our in vitro experiments indicate that the homolog mutations of SLC26A6(D23H/D673N) and SLC26A6(D673N) alone abolished the expression and function of SLC26A6, and impaired the regulation of SLC13-mediated citrate transport by SLC26A6. On the other hand, the SLC26A6(R621G) variant showed reduced SLC26A6 protein expression and membrane trafficking, retained full transport activity, but impaired the regulation of the citrate transporter. Accordingly, the human SLC26A6(D23H/D673N) carrier showed a dramatic reduction in urinary citrate concentrations which resulted in Ca2+-oxalate stones formation, as opposed to the carrier of SLC26A6(R621G). Our findings indicate that the human SLC26A6-STAS domain mutations differentially impair SLC26A6 expression, function, and regulation of citrate transporters. This interferes with citrate and oxalate homeostasis thus potentially predisposes to Ca2+-oxalate kidney stones.

Deep Learning—Powered Iterative Combinatorial Auctions

In this paper, we study the design of deep learning-powered iterative combinatorial auctions (ICAs). We build on prior work where preference elicitation was done via kernelized support vector regressions (SVRs). However, the SVR-based approach has limitations because it requires solving a machine learning (ML)-based winner determination problem (WDP). With expressive kernels (like gaussians), the ML-based WDP cannot be solved for large domains. While linear or quadratic kernels have better computational scalability, these kernels have limited expressiveness. In this work, we address these shortcomings by using deep neural networks (DNNs) instead of SVRs. We first show how the DNN-based WDP can be reformulated into a mixed integer program (MIP). Second, we experimentally compare the prediction performance of DNNs against SVRs. Third, we present experimental evaluations in two medium-sized domains which show that even ICAs based on relatively small-sized DNNs lead to higher economic efficiency than ICAs based on kernelized SVRs. Finally, we show that our DNN-powered ICA also scales well to very large CA domains.

Calcium Binding Preferentially Stabilizes Apical End of Dysferlin C2A

Dysferlin is a crucial component of the eukaryotic membrane repair machinery, helping to seal microtears in the sarcolemmal membrane that arise from constant mechanical insult. Loss of this protein results in several muscle‐wasting phenotypes in both human patients and mouse models. Dysferlin contains seven C2 domains, which are well‐documented Ca2+‐dependent lipid sensors, which suggests a putative role in the calcium‐sensitive steps of membrane repair for dysferlin. A deeper understanding of the mechanisms that govern how dysferlin coordinates calcium signals with repair machinery mobilization is essential to develop therapeutic strategies. Out of the many C2 domains, the N‐terminal C2A domain is of particular interest because it has proved essential to the membrane repair process. Initial thermodynamic studies of this domain showed a curiously low energy of unfolding, suggesting that the domain is partially unfolded under resting calcium conditions. Here we use nuclear magnetic resonance spectroscopy and supporting biophysical techniques to examine the stabilizing effect of millimolar levels of calcium at the residue level and conclude that calcium binding primarily affects the apical end of the C2A domain of dysferlin. Calcium binding to C2 domains has been previously shown to induce subtle conformational shifts towards a more membrane‐competent fold, however, our studies provide the most drastic example of this phenomenon to date.

Impaired Autoinhibition of Protein Kinase Cγ in Spinocerebellar Ataxia Type 14

Spinocerebellar ataxia type 14 (SCA14) is a neurodegenerative disease caused by germline mutations in the diacylglycerol (DG)/Ca2+‐regulated protein kinase C gamma (PKCγ), leading to Purkinje cell degeneration and progressive cerebellar dysfunction. Curiously, the majority of the approximately 50 missense mutations identified in PKCγ cluster to the DG‐sensing C1A and C1B domains. Here, we use a genetically‐encoded FRET‐based C Kinase Activity Reporter (CKAR) to show that ataxia‐associated PKCγ mutants have higher basal activity in cells, and thus are less autoinhibited, than wild‐type enzyme. However, whereas reduced autoinhibition generally renders PKC sensitive to degradation, we show that mutations in the C1B domain allow translocation to membranes but protect PKCγ from phorbol ester‐induced down‐regulation. Indeed, deletion of the C1B domain prevents PKCγ down‐regulation with phorbol esters, potent ligands for the C1 domains. Strikingly, the degree of impaired autoinhibition correlates inversely with age of disease onset. Patients with the most severe mutation we examined (V138E) present with symptoms as young children, whereas symptoms in patients with the least severe mutation examined (D115Y) manifested in their 40s. To understand the structural basis of mutations outside the C1 domains, we generated a model of PKCγ using homology modeling and molecular docking. Mutations outside the C1 domains occur in regions also predicted to disrupt autoinhibition, including the pseudosubstrate, a predicted interface between the kinase and C1B domains, and the C‐terminal tail of PKCγ. Taken together, our data support a model in which SCA14 mutations enhance PKCγ activity without compromising stability. Furthermore, because many of the genetic causes of the 40+ types of SCA alter Ca2+ homeostasis, deregulated PKCγ activity may be a common cause for the disease. This raises the possibility that inhibition of PKCγ will be a potentially viable therapeutic target for SCA.Support or Funding InformationNIH T32 GM007752

Coupling of Conformational Switches in Calcium Sensor Unraveled with Local Markov Models and Transfer Entropy.

Proteins often have multiple switching domains that are coupled to each other and to the binding of ligands in order to realize signaling functions. Here we investigate the C2A domain of Synaptotagmin-1 (Syt-1), a calcium sensor in the neurotransmitter release machinery and a model system for the large family of C2 membrane binding domains. We combine extensive molecular dynamics (MD) simulations with Markov modeling in order to model conformational switching domains, their states, and their dependence on bound calcium ions. Then, we use transfer entropy to characterize how the switching domains are coupled via directed or allosteric mechanisms and give rise to the calcium sensing function of the protein. Our proposed switching mechanism contributes to the understanding of the neurotransmitter release machinery. Furthermore, the methodological approach we develop serves as a template to analyze conformational switching domains and the broad study of their coupling in macromolecular machines.

The C2A domain of synaptotagmin is an essential component of the calcium sensor for synaptic transmission.

The synaptic vesicle protein, synaptotagmin, is the principle Ca2+ sensor for synaptic transmission. Ca2+ influx into active nerve terminals is translated into neurotransmitter release by Ca2+ binding to synaptotagmin's tandem C2 domains, triggering the fast, synchronous fusion of multiple synaptic vesicles. Two hydrophobic residues, shown to mediate Ca2+-dependent membrane insertion of these C2 domains, are required for this process. Previous research suggested that one of its tandem C2 domains (C2B) is critical for fusion, while the other domain (C2A) plays only a facilitatory role. However, the function of the two hydrophobic residues in C2A have not been adequately tested in vivo. Here we show that these two hydrophobic residues are absolutely required for synaptotagmin to trigger vesicle fusion. Using in vivo electrophysiological recording at the Drosophila larval neuromuscular junction, we found that mutation of these two key C2A hydrophobic residues almost completely abolished neurotransmitter release. Significantly, mutation of both hydrophobic residues resulted in more severe deficits than those seen in synaptotagmin null mutants. Thus, we report the most severe phenotype of a C2A mutation to date, demonstrating that the C2A domain is absolutely essential for synaptotagmin's function as the electrostatic switch.

Biophysical Origins of Calcium-Inhibited Membrane Binding by the C2A Domain of Synaptotagmin-Like Protein 2

Although C2 domains canonically bind membranes in response to increased cytoplasmic calcium, there are also many examples of C2 domains known to bind membranes in the absence of calcium. Most of these are calcium-independent C2 domains that lack the conserved aspartate cluster, but the C2A domain of synapotagmin-like protein 2 (Slp-2; also called exophilin-4) has been reported to be inhibited by calcium. Here, we use a combination of fluorescence-based liposome binding and kinetic assays, mass spectrometry, and NMR spectroscopy to analyze the physical-chemical origins of this unusual property. Our data confirm that the purified recombinant C2A domain indeed binds anionic lipid membranes containing phosphatidylserine and/or phosphatidylinositol-(4,5)-bisphosphate in a manner that is inhibited by sub-millimolar concentrations of calcium. Surprisingly, however, the Slp-2 C2A domain does not show detectable binding to calcium ions in solution, despite having 3 of 5 conserved aspartate residues that form the canonical calcium binding site in C2 domains. Rather, its electrostatically driven membrane binding must be inhibited through effects of calcium on membrane lipids. Because calcium is more effective at inhibiting Slp-2 C2A membrane binding than other protein domains with comparable membrane affinities, we speculate that the inhibition may arise due to a combination of electrostatic screening and other effects, e.g. on membrane structure. Overall, this unusual C2 domain competes with calcium ions for binding to its anionic lipid targets.

Effect of Bapta and Dysferlin's C2A Domain on Recovery of Ca2D Transients After Osmotic Shock in Dysferlin-Null Myofibers

Effect of Bapta and Dysferlin's C2A Domain on Recovery of Ca2D Transients After Osmotic Shock in Dysferlin-Null Myofibers

SYTL4 downregulates microtubule stability and confers paclitaxel resistance in triple-negative breast cancer.

Background: Taxanes are frontline chemotherapeutic drugs for patients with triple-negative breast cancer (TNBC); however, chemoresistance reduces their effectiveness. We hypothesized that the molecular profiling of tumor samples before and after neoadjuvant chemotherapy (NAC) would help identify genes associated with drug resistance.Methods: We sequenced 10 samples by RNA-seq from 8 NAC patients with TNBC: 3 patients with a pathologic complete response (pCR) and the other 5 with non-pCR. Differentially expressed genes that predicted chemotherapy response were selected for in vitro functional screening via a small-scale siRNAs pool. The clinical and functional significance of the gene of interest in TNBC was further investigated in vitro and in vivo, and biochemical assays and imaging analysis were applied to study the mechanisms.Results: Synaptotagmin-like 4 (SYTL4), a Rab effector in vesicle transport, was identified as a leading functional candidate. High SYTL4 expression indicated a poor prognosis in multiple TNBC cohorts, specifically in taxane-treated TNBCs. SYTL4 was identified as a novel chemoresistant gene as validated in TNBC cells, a mouse model and patient-derived organoids. Mechanistically, downregulating SYTL4 stabilized the microtubule network and slowed down microtubule growth rate. Furthermore, SYTL4 colocalized with microtubules and interacted with microtubules through its middle region containing the linker and C2A domain. Finally, we found that SYTL4 was able to bind microtubules and inhibit the in vitro microtubule polymerization.Conclusion: SYTL4 is a novel chemoresistant gene in TNBC and its upregulation indicates poor prognosis in taxane-treated TNBC. Further, SYTL4 directly binds microtubules and decreases microtubule stability.

Structural Studies of a Novel Extended Synaptotagmin with Only Two C2 Domains from <i>Trypanosoma Brucei</i>

Extended synaptotagmins (E-Syts) are a group of evolutionarily conserved proteins localizing at membrane contact sites between the endoplasmic reticulum (ER) and the plasma membrane, where they mediate inter-membrane lipid transfer and control plasma membrane lipid homeostasis. There are three E-Syts in mammals, which all contain an N-terminal transmembrane (TM) hairpin for the association with the ER membrane, and a central synaptotagmin-like mitochondrial-lipid binding protein (SMP) domain as a carrier for phospholipids. Additionally, mammalian E-Syt1 and E-Syt2/E-Syt3 respectively have five and three C2 domains at their C-terminus, which mediate their interaction with the plasma membrane. Here we report a novel E-Syt from the protist parasite Trypanosoma brucei, TbE-Syt. Despite also having the TM hairpin and the SMP domain, TbE-Syt contains only two C2 domains, which makes it the shortest E-Syt currently identified. We determined a 1.5 A resolution crystal structure of the TbE-Syt C2B domain, which showed that it binds two Ca2+ ions. Our mutagenesis studies demonstrated that TbE-Syt-C2B binds lipids via both Ca2+- and PI(4,5)P2-dependent means. Our bioinformatics analyses showed that TbE-Syt-C2A lacks the Ca2+-binding site found in C2B but may still interact with lipids via a basic surface patch. Furthermore, in contrast to the rigid V-shaped arrangement of the C2A and C2B domains in human E-Syt2, our analysis suggests that the two C2 domains in TbE-Syt are connected by a long flexible linker. We propose a working model for how TbE-Syt might tether the ER membrane and the plasma membrane to transfer lipids between the two organelles.

Ether lipid metabolism by AADACL1 regulates platelet function and thrombosis

AbstractWe previously reported the discovery of a novel lipid deacetylase in platelets, arylacetamide deacetylase-like 1 (AADACL1/NCEH1), and that its inhibition impairs agonist-induced platelet aggregation, Rap1 GTP loading, protein kinase C (PKC) activation, and ex vivo thrombus growth. However, precise mechanisms by which AADACL1 impacts platelet signaling and function in vivo are currently unknown. Here, we demonstrate that AADACL1 regulates the accumulation of ether lipids that impact PKC signaling networks crucial for platelet activation in vitro and in vivo. Human platelets treated with the AADACL1 inhibitor JW480 or the AADACL1 substrate 1-O-hexadecyl-2-acetyl-sn-glycerol (HAG) exhibited decreased platelet aggregation, granule secretion, Ca2+ flux, and PKC phosphorylation. Decreased aggregation and secretion were rescued by exogenous adenosine 5′-diphosphate, indicating that AADACL1 likely functions to induce dense granule secretion. Experiments with P2Y12−/− and CalDAG GEFI−/− mice revealed that the P2Y12 pathway is the predominate target of HAG-mediated inhibition of platelet aggregation. HAG itself displayed weak agonist properties and likely mediates its inhibitory effects via conversion to a phosphorylated metabolite, HAGP, which directly interacted with the C1a domains of 2 distinct PKC isoforms and blocked PKC kinase activity in vitro. Finally, AADACL1 inhibition in rats reduced platelet aggregation, protected against FeCl3-induced arterial thrombosis, and delayed tail bleeding time. In summary, our data support a model whereby AADACL1 inhibition shifts the platelet ether lipidome to an inhibitory axis of HAGP accumulation that impairs PKC activation, granule secretion, and recruitment of platelets to sites of vascular damage.

Synaptotagmin 1 clamps synaptic vesicle fusion in mammalian neurons independent of complexin

Synaptic vesicle (SV) exocytosis is mediated by SNARE proteins. Reconstituted SNAREs are constitutively active, so a major focus has been to identify fusion clamps that regulate their activity in synapses: the primary candidates are synaptotagmin (syt) 1 and complexin I/II. Syt1 is a Ca2+ sensor for SV release that binds Ca2+ via tandem C2-domains, C2A and C2B. Here, we first determined whether these C2-domains execute distinct functions. Remarkably, the C2B domain profoundly clamped all forms of SV fusion, despite synchronizing residual evoked release and rescuing the readily-releasable pool. Release was strongly enhanced by an adjacent C2A domain, and by the concurrent binding of complexin to trans-SNARE complexes. Knockdown of complexin had no impact on C2B-mediated clamping of fusion. We postulate that the C2B domain of syt1, independent of complexin, is the molecular clamp that arrests SVs prior to Ca2+-triggered fusion.

Phosphatidylinositol 4,5 Bisphosphate Controls the cis and trans Interactions of Synaptotagmin 1

Synaptotagmin 1 acts as the Ca2+ sensor for synchronous neurotransmitter release; however, the mechanism by which it functions is not understood and is presently a topic of considerable interest. Here, we describe measurements on full-length membrane-reconstituted synaptotagmin 1 using site-directed spin labeling in which we characterize the linker region as well as the cis (vesicle membrane) and trans (cytoplasmic membrane) binding of its two C2 domains. In the full-length protein, the C2A domain does not undergo membrane insertion in the absence of Ca2+; however, the C2B domain will bind to and penetrate in trans to a membrane containing phosphatidylinositol 4,5 bisphosphate, even if phosphatidylserine (PS) is present in the cis membrane. In the presence of Ca2+, the Ca2+ binding loops of C2A and C2B both insert into the membrane interface; moreover, C2A preferentially inserts into PS-containing bilayers and will bind in a cis configuration to membranes containing PS even if a phosphatidylinositol 4,5 bisphosphate membrane is presented in trans. The data are consistent with a bridging activity for synaptotagmin 1 in which the two domains bind to opposing vesicle and plasma membranes. The failure of C2A to bind membranes in the absence of Ca2+ and the long unstructured segment linking C2A to the vesicle membrane indicates that synaptotagmin 1 could act to significantly shorten the vesicle-plasma membrane distance with increasing levels of Ca2+.

Structural Basis for the Distinct Membrane Binding Activity of the Homologous C2A Domains of Myoferlin and Dysferlin

Dysferlin has been implicated in acute membrane repair processes, whereas myoferlin's activity is maximal during the myoblast fusion stage of early skeletal muscle cell development. Both proteins are similar in size and domain structure; however, despite the overall similarity, myoferlin's known physiological functions do not overlap with those of dysferlin. Here we present for the first time the X‐ray crystal structure of human myoferlin C2A to 1.9 Å resolution bound to two divalent cations, and compare its three-dimensional structure and membrane binding activities to that of dysferlin C2A. We find that while dysferlin C2A binds membranes in a Ca2+-dependent manner, Ca2+ binding was the rate-limiting kinetic step for this interaction. Myoferlin C2A, on the other hand, binds two calcium ions with an affinity 3-fold lower than that of dysferlin C2A; and, surprisingly, myoferlin C2A binds only marginally to phospholipid mixtures with a high fraction of phosphatidylserine.

Evaluating the Role of Piccolo Splice Variants in Regulated Secretion of Insulin from Rat Pancreatic Cells

Secretion of insulin from pancreatic beta‐cells is required for proper glucose homeostasis; however, the molecular mechanism of insulin granule exocytosis has not been fully elucidated. In pancreatic beta‐cells, glucose‐induced insulin secretion is synergistically potentiated through an Epac2‐Rim2‐Piccolo signaling response mechanism. Both Piccolo and Rim2 were originally identified as active zone proteins in neurons and have been shown to regulate synaptic vesicle cycling and neurotransmitter signaling. A recent study showed that a number of active zone proteins, including Rim2 and Piccolo, are downregulated in human pancreatic islet cells isolated from patients with type 2 diabetes (T2D). Understanding the role of Piccolo in regulated secretion of insulin may provide valuable insights into the causes of T2D. The C‐terminal region of Piccolo contains a Ca2+‐binding C2A domain. It is likely that Piccolo may regulate secretion from insulin granule vesicles by serving as a Ca2+‐sensor. Alternative splicing of exons within the coding region of the C2A domain results in an exon‐skipping event generating either a splice variant that lacks the 27‐nucleotide exon 16 (short C2A) or a variant that retains this exon (long C2A). Exclusion of the 9 amino acid residue sequence in the short C2A domain results in enhanced Ca2+‐binding affinity. In this study, we sought to examine the differential roles of the short C2A and long C2A variants in Ca2+‐mediated insulin secretion from rat INS‐832/13 pancreatic beta‐cells. We demonstrate that INS‐832/13 cells express both long and short variants of the C2A domain and real‐time qPCR demonstrated differential expression of the variants. In addition, we examined the effects of overexpression of the short and long C2A domains on glucose‐stimulated insulin secretion from INS‐832/13 cells.Support or Funding InformationMaximizing Access to Research Careers Program at Fort Lewis College (NIH‐T34GM092711)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Insight into the mechanism of action of EP-39, a bevirimat derivative that inhibits HIV-1 maturation

Maturation of human immunodeficiency virus type 1 (HIV-1) particles is a key step for viral infectivity. This process can be blocked using maturation inhibitors (MIs) that affect the cleavage of the capsid-spacer peptide 1 (CA-SP1) junction. Here, we investigated the mechanisms underlying the activity of EP-39, a bevirimat (BVM) derivative with better hydrosolubility. To this aim, we selected in vitro EP-39- and BVM-resistant mutants. We found that EP-39-resistant viruses have four mutations within the CA domain (CA-A194T, CA-T200N, CA-V230I, and CA-V230A) and one in the first residue of SP1 (SP1-A1V). We also identified six mutations that confer BVM resistance (CA-A194T, CA-L231F, CA-L231M, SP1-A1V, SP1-S5N and SP1-V7A). To characterize the EP-39 and BVM-resistant mutants, we studied EP-39 effects on mutant virus replication and performed a biochemical analysis with both MIs. We observed common and distinct characteristics, suggesting that, although EP-39 and BVM share the same chemical skeleton, they could interact in a different way with the Gag polyprotein precursor (Pr55Gag). Using an in silico approach, we observed that EP-39 and BVM present different predicted positions on the hexameric crystal structure of the CACTD-SP1 Gag fragment. To clearly understand the relationship between assembly and maturation, we investigated the impact of all identified mutations on virus assembly by expressing Pr55Gag mutants. Finally, using NMR, we have shown that the interaction of EP-39 with a peptide carrying the SP1-A1V mutation (CA-SP1(A1V)-NC) is almost suppressed in comparison with the wild type peptide. These results suggest that EP-39 and BVM could interact differently with the Pr55Gag lattice and that the mutation of the first SP1 residue induces a loss of interaction between Pr55Gag and EP-39.

Heterodimerization of UNC-13/RIM regulates synaptic vesicle release probability but not priming in C. elegans.

UNC-13 proteins play an essential role in synaptic transmission by recruiting synaptic vesicles (SVs) to become available for release, which is termed SV priming. Here we show that the C2A domain of UNC-13L, like the corresponding domain in mammalian Munc13-1, displays two conserved binding modes: forming C2A/C2A homodimers, or forming a heterodimer with the zinc finger domain of UNC-10/RIM (C2A/RIM). Functional analysis revealed that UNC-13L's C2A promotes synaptic transmission by regulating a post-priming process. Stimulus-evoked release but not SV priming, was impaired in unc-10 mutants deficient for C2A/RIM heterodimerization, leading to decreased release probability. Disrupting C2A/C2A homodimerization in UNC-13L-rescued animals had no effect on synaptic transmission, but fully restored the evoked release and the release probability of unc-10/RIM mutants deficient for C2A/RIM heterodimerization. Thus, our results support the model that RIM binding C2A releases UNC-13L from an autoinhibitory homodimeric complex to become fusion-competent by functioning as a switch only.