N-ethylmaleimide Sensitive Factor Attachment Protein Research Articles

Mutations of Single Residues in the Complexin N-terminus Exhibit Distinct Phenotypes in Synaptic Vesicle Fusion.

The release of neurotransmitters (NTs) at central synapses is dependent on a cascade of protein interactions, specific to the presynaptic compartment. Among those dedicated molecules, the cytosolic complexins play an incompletely defined role as synaptic transmission regulators. Complexins are multidomain proteins that bind soluble N-ethylmaleimide sensitive factor attachment protein receptor complexes, conferring both inhibitory and stimulatory functions. Using systematic mutagenesis and comparing reconstituted in vitro membrane fusion assays with electrophysiology in cultured neurons from mice of either sex, we deciphered the function of the N-terminus of complexin (Cpx) II. The N-terminus (amino acid 1-27) starts with a region enriched in hydrophobic amino acids (1-12), which binds lipids. Mutants maintaining this hydrophobic character retained the stimulatory function of Cpx, whereas exchanges introducing charged residues perturbed both spontaneous and evoked exocytosis. Mutants in the more distal region of the N-terminal domain (amino acid 11-18) showed a spectrum of effects. On the one hand, mutation of residue A12 increased spontaneous release without affecting evoked release. On the other hand, replacing D15 with amino acids of different shapes or hydrophobic properties (but not charge) not only increased spontaneous release but also impaired evoked release. Most surprising, this substitution reduced the size of the readily releasable pool, a novel function for Cpx at mammalian synapses. Thus, the exact amino acid composition of the Cpx N-terminus fine-tunes the degree of spontaneous and evoked NT release.

Investigating the potential role of α-SNAP in preventing chemotherapy-induced ovarian dysfunction: Insights from cellular and animal models

BackgroundThe phosphoinositide 3-kinase/Akt/mammalian target of rapamycin complex 1 (PI3K/Akt/mTORC1) pathway plays a crucial role in the activation of primordial follicles. However, excessive activation and the loss of primordial follicles can lead to ovarian dysfunction. The alpha-soluble N-ethylmaleimide sensitive factor attachment protein (α-SNAP) protein has been implicated in PI3K/Akt/mTORCl signaling, suggesting its potential involvement in follicle activation. Thus, this study aimed to explore the role of α-SNAP in the activation of the PI3K/Akt/mTORC1 signaling pathway and its ability to mitigate the effects of cisplatin on ovarian function, using both in vitro and in vivo models. MethodsWe transfected KGN human ovarian granulosa cells (GCs) with small interfering RNA (siRNA) targeting α-SNAP to investigate the effects of α-SNAP inhibition on GC proliferation and apoptosis, as well as on the activity of the PI3K/Akt/mTORC1 pathway. In a mouse model, α-SNAP siRNA was delivered via an adeno-associated virus before treatment with cisplatin to assess its effects on follicle activation and ovarian function. Follicle counts at various growth stages, western blotting, and immunohistochemistry analyses were conducted to detect the expression of cleaved caspase-3, Ki67, α-SNAP, and p-mTOR. Additionally, the serum concentrations of anti-Müllerian hormone (AMH) were measured through an enzyme-linked immunosorbent assay. ResultsIn vitro, α-SNAP depletion prevented GC proliferation by inhibiting the PI3K/Akt/mTORC1 pathway, thereby indicating its role in the regulation of cell growth. In vivo, α-SNAP knockdown attenuated the cisplatin-induced overactivation of primordial follicles by suppressing the PI3K/Akt/mTORC1 signaling pathway and partially restoring AMH levels. In addition, the expression and distribution patterns of cleaved caspase-3, Ki67, α-SNAP, and p-mTOR varied across different follicular growth stages, suggesting a protective effect against chemotherapy-induced ovarian damage. ConclusionsInhibiting α-SNAP may attenuate GC proliferation by suppressing the PI3K/Akt/mTORC1 pathway, thereby mitigating the overactivation and loss of primordial follicles induced by cisplatin. Targeting α-SNAP may emerge as a novel strategy to prevent ovarian damage resulting from chemotherapy. However, these conclusions warrant repeated testing, and the mechanistic underpinnings of α-SNAP must be further elucidated in the future.

Early Endosomes Act as Local Exocytosis Hubs to Repair Endothelial Membrane Damage.

The plasma membrane of a cell is subject to stresses causing ruptures that must be repaired immediately to preserve membrane integrity and ensure cell survival. Yet, the spatio-temporal membrane dynamics at the wound site and the source of the membrane required for wound repair are poorly understood. Here, it is shown that early endosomes, previously only known to function in the uptake of extracellular material and its endocytic transport, are involved in plasma membrane repair in human endothelial cells. Using live-cell imaging and correlative light and electron microscopy, it is demonstrated that membrane injury triggers a previously unknown exocytosis of early endosomes that is induced by Ca2+ entering through the wound. This exocytosis is restricted to the vicinity of the wound site and mediated by the endosomal soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) VAMP2, which is crucial for efficient membrane repair. Thus, the newly identified Ca2+ -evoked and localized exocytosis of early endosomes supplies the membrane material required for rapid resealing of a damaged plasma membrane, thereby providing the first line of defense against damage in mechanically challenged endothelial cells.

Role of complexin-lipid interactions in controlling snare-mediated membrane fusion.

Fast and reliable neurotransmitter release is essential for signaling fidelity at neuronal synapses. Synaptic exocytosis is a tightly controlled process accomplished through Ca2+ triggered fusion of neurotransmitter-laden vesicles with the presynaptic plasma membrane. This process is mediated by SNARE (soluble N-ethylmaleimide sensitive factor attachment protein receptor) proteins that zipper into a four-helix bundle to drive membrane fusion. The neuronal SNARE complex, consisting of Syntaxin-1a, SNAP-25, and Synaptobrevin-2, is intrinsically capable of catalyzing slow and Ca2+ independent fusion in reconstituted fusion assays in vitro. A single model of how interactions with SNARE regulatory proteins impart physiological properties seen in neurons, such as Ca2+ specificity, has yet to be established. Complexin-1 (Cpx) is a small, soluble regulatory protein that binds SNAREs and lipid membranes. Cpx is proposed to facilitate fusion in the presence of Ca2+ as well as suppress fusion in the absence of Ca2+ through interactions of its central domain with SNARE complexes in their prefusion and postfusion states. Additionally, the Cpx C-terminal domain interacts with membranes, an interaction that has been proposed to facilitate function. Recent work has shown that Cpx's lipid-binding domains can differentiate between membranes of different compositions, though the functional relevance of this finding has yet to be established. Our group has previously developed a hybrid fusion assay that combines synthetic, reconstituted, and native components to reproduce key features of synaptic exocytosis. This method mimics intracellular conditions more closely than a reconstituted system with only purified components while maintaining the ability to easily alter lipid, protein, and ion concentrations. We utilize this system with wild-type and mutant Cpx, along with other protein and lipid regulators of SNAREs, to investigate how interaction with and composition of the fusing membranes controls Cpx's regulatory function.

A novel association between platelet filamin A and soluble N-ethylmaleimide sensitive factor attachment proteins regulates granule secretion

Background and ObjectiveThe molecular mechanisms that underpin platelet granule secretion remain poorly defined. Filamin A (FLNA) is an actin-crosslinking and signaling scaffold protein whose role in granule exocytosis has not been explored despite evidence that FLNA gene mutations confer platelet defects in humans. Methods and ResultsUsing platelets from platelet-specific conditional Flna-knockout mice, we showed that the loss of FLNA confers a severe defect in alpha (α)- and dense (δ)-granule exocytosis, as measured based on the release of platelet factor 4 (aka CXCL4) and adenosine triphosphate (ATP), respectively. This defect was observed following activation of both immunoreceptor tyrosine-based activation motif (ITAM) signaling by collagen-related peptide (CRP) and G protein–coupled receptor (GPCR) signaling by thrombin and the thromboxane mimetic U46619. CRP–induced spikes in intracellular calcium [Ca2+]i were impaired in FLNA-null platelets relative to controls, confirming that FLNA regulates ITAM-driven proximal signaling. In contrast, GPCR-mediated spikes in [Ca2+]i in response to thrombin and U46619 were unaffected by FLNA. Normal platelet secretion requires complexing of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins synaptosomal-associated protein 23 (SNAP23) and syntaxin-11 (STX11). We determined that FLNA coimmunoprecipitates with both SNAP23 and STX11 upon platelet stimulation. ConclusionFLNA regulates GPCR-driven platelet granule secretion and associates with SNAP23 and STX11 in an activation-dependent manner.

Epigallocatechin gallate inhibits SNARE-dependent membrane fusion by blocking trans-SNARE assembly.

Insulin secretion is a signal-triggered process that requires membrane fusion between the secretory granules and plasma membrane in pancreatic β cells. The exocytosis of insulin is mediated by target-soluble N-ethylmaleimide sensitive factor attachment protein receptors (SNAREs) on the plasma membrane and vesicle-SNAREs on the vesicles, which assemble into a quaternary trans-SNARE complex to initiate the fusion. Expression of fusion proteins is reduced in the islets of patients with type II diabetes, indicating that SNARE-mediated fusion defect is closely related to insulin-based metabolic diseases. Previous studies have suggested that epigallocatechin gallate (EGCG) has an inhibitory effect on membrane fusion. In the present study, we performed in vitro reconstitution assays to unravel the molecular mechanisms of EGCG in SNARE-mediated insulin secretory vesicle fusion. Our data show that EGCG efficiently inhibits insulin secretory SNARE-mediated membrane fusion. Mechanistic studies indicated that EGCG blocks the formation of the trans-SNARE complex. Furthermore, calcium/synaptotagmin-7-stimulated fusion kinetics were largely reduced by EGCG, confirming that it is a potential regulator of SNARE-dependent insulin secretion. Our findings suggest that the trans-SNARE complex might be a promising target for controlling SNARE-dependent vesicle fusion.

Aberrant autophagy in lysosomal storage disorders marked by a lysosomal SNARE protein shortage due to suppression of endocytosis.

Lysosomal storage disorders (LSDs) are inherited metabolic diseases caused by genetic defects in lysosomal enzymes or related factors. LSDs are associated with excessive accumulation of natural substrates in lysosomes leading to central nervous system and peripheral tissue damage. Abnormal autophagy is also involved in pathogenesis, although the underlying mechanisms remain unclear. We demonstrated that impairment of lysosome-autophagosome fusion is due to suppressed endocytosis in LSDs. The fusion was reduced in several LSD cells and the brains of LSD model mice, suggesting that the completion of autophagy is suppressed by the accumulation of substrates. In this brain, the expression of the soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) proteins, VAMP8 and Syntaxin7, was decreased on the lysosomal surface but not intracellular. This aberrant autophagy preceded the development of pathological phenotypes in LSD-model mice. Furthermore, the enzyme deficiency leading to the substrate accumulation could suppress endocytosis, and the inhibited endocytosis decreased SNARE proteins localized on lysosomes. These findings suggest that the shortage of SNARE proteins on lysosomes is one of the reasons for the impairment of lysosome-autophagosome fusion in LSD cells. Defects in lysosomal enzyme activity suppress endocytosis and decrease the supply of intracellular SNARE proteins recruited to lysosomes. This shortage of lysosomal SNARE proteins impairs lysosome-autophagosome fusion in lysosomal storage disorders.

Secretory membrane traffic in plant-microbe interactions.

Plant defense responses include the extracellular release of defense-related molecules, such as pathogenesis-related proteins and secondary metabolites, as well as cell wall materials. This primarily depends on the trafficking of secretory vesicles to the plasma membrane, where they discharge their contents into the apoplastic space via soluble N-ethylmaleimide sensitive factor attachment protein receptor-assisted exocytosis. However, some pathogenic and symbiotic microbes have developed strategies to manipulate host plant exocytic pathways. Here, we discuss the mechanisms by which plant exocytic pathways function in immunity and how microbes have evolved to manipulate those pathways.

Apicoplast biogenesis mediated by ATG8 requires the ATG12–ATG5-ATG16L and SNAP29 complexes in Toxoplasma gondii

ABSTRACT In apicomplexan parasites, the macroautophagy/autophagy machinery is repurposed to maintain the plastid-like organelle apicoplast. Previously, we showed that in Toxoplasma and Plasmodium, ATG12 interacts with ATG5 in a non-covalent manner, in contrast to the covalent interaction in most organisms. However, it remained unknown whether apicomplexan parasites have functional orthologs of ATG16L1, a protein that is essential for the function of the covalent ATG12–ATG5 complex in vivo in other organisms. Furthermore, the mechanism used by the autophagy machinery to maintain the apicoplast is unclear. We report that the ATG12–ATG5-ATG16L complex exists in Toxoplasma gondii (Tg). This complex is localized on isolated structures at the periphery of the apicoplast dependent on TgATG16L. Inducible depletion of TgATG12, TgATG5, or TgATG16L caused loss of the apicoplast and affected parasite growth. We found that a putative soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) protein, synaptosomal-associated protein 29 (TgSNAP29, Qbc SNARE), is required to maintain the apicoplast in T. gondii. TgSNAP29 depletion disrupted TgATG8 localization at the apicoplast. Additionally, we identified a putative ubiquitin-interacting motif-docking site (UDS) of TgATG8. Mutation of the UDS site abolished TgATG8 localization on the apicoplast but not lipidation. These findings suggest that the TgATG12–TgATG5-TgATG16L complex is required for biogenesis of the apicoplast, in which TgATG8 is translocated to the apicoplast via vesicles in a SNARE -dependent manner in T. gondii. Abbreviations: AID: auxin-inducible degron; CCD: coiled-coil domain; HFF: human foreskin fibroblast; IAA: indole-3-acetic acid; LAP: LC3-associated phagocytosis; NAA: 1-naphthaleneacetic acid; PtdIns3P: phosphatidylinositol-3-phosphate; SNARE: soluble N-ethylmaleimide sensitive factor attachment protein receptor; UDS: ubiquitin-interacting motif-docking site; UIM: ubiquitin-interacting motif.

AQP2 trafficking in health and diseases: an updated overview.

Renal collecting duct principal cells play a key role in controlling body water balance. Principal cells express the water channels AQP2, AQP3, and AQP4 that mediate renal water reabsorption. AQP3 and AQP4 are expressed at the basolateral membrane constitutively. Conversely, AQP2 is localized in intracellular vesicles and translocates to the plasma membrane under vasopressin action. Stimulation with vasopressin activates the cAMP/PKA signal transduction pathway that induces the redistribution of AQP2 from an intracellular pool to the apical plasma membrane. AQP2 trafficking and function depend on multiple post-translational modifications. Moreover, several proteins control different steps activated by the vasopressin stimulation that triggers the redistribution of the AQP2 vesicles. A-kinase anchoring proteins (AKAPs) together with phosphodiesterases and adenylate cyclases play crucial roles in modulating local changes of cAMP. Soluble N-ethylmaleimide sensitive fusion factor attachment protein receptors (SNARE), cytoskeletal proteins, and the small GTPases of the Rho family regulate the fusion and the endocytotic retrieval of AQP2 vesicles. Abnormal vasopressin signaling and altered AQP2 expression or trafficking can lead to disorders characterized by deregulated mechanisms controlling water homeostasis. This review provides updated data on the molecular signals regulating vasopressin-induced AQP2 trafficking in health and disease.

Exocytosis Proteins: Typical and Atypical Mechanisms of Action in Skeletal Muscle.

Insulin-stimulated glucose uptake in skeletal muscle is of fundamental importance to prevent postprandial hyperglycemia, and long-term deficits in insulin-stimulated glucose uptake underlie insulin resistance and type 2 diabetes. Skeletal muscle is responsible for ~80% of the peripheral glucose uptake from circulation via the insulin-responsive glucose transporter GLUT4. GLUT4 is mainly sequestered in intracellular GLUT4 storage vesicles in the basal state. In response to insulin, the GLUT4 storage vesicles rapidly translocate to the plasma membrane, where they undergo vesicle docking, priming, and fusion via the high-affinity interactions among the soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) exocytosis proteins and their regulators. Numerous studies have elucidated that GLUT4 translocation is defective in insulin resistance and type 2 diabetes. Emerging evidence also links defects in several SNAREs and SNARE regulatory proteins to insulin resistance and type 2 diabetes in rodents and humans. Therefore, we highlight the latest research on the role of SNAREs and their regulatory proteins in insulin-stimulated GLUT4 translocation in skeletal muscle. Subsequently, we discuss the novel emerging role of SNARE proteins as interaction partners in pathways not typically thought to involve SNAREs and how these atypical functions reveal novel therapeutic targets for combating peripheral insulin resistance and diabetes.

Neuronal SNARE complex assembly guided by Munc18-1 and Munc13-1.

Neurotransmitter release by Ca2+ -triggered synaptic vesicle exocytosis is essential for information transmission in the nervous system. The soluble N-ethylmaleimide sensitive factor attachment protein receptors (SNAREs) syntaxin-1, SNAP-25, and synaptobrevin-2 form the SNARE complex to bring synaptic vesicles and the plasma membranes together and to catalyze membrane fusion. Munc18-1 and Munc13-1 regulate synaptic vesicle priming via orchestrating neuronal SNARE complex assembly. In this review, we summarize recent advances toward the functions and molecular mechanisms of Munc18-1 and Munc13-1 in guiding neuronal SNARE complex assembly, and discuss the functional similarities and differences between Munc18-1 and Munc13-1 in neurons and their homologs in other intracellular membrane trafficking systems.

BOTULINUM

Botulinum neurotoxins (BoNT) are toxins produced by bacteria of the genus Clostridium botulinum and are considered as the most potent toxins nowadays. Firstly, known as a food poison, later explored as a biological weapon and currently considered a great therapeutic resource. Seven different BoNT serotypes (A-G) have already been identified. Among them, serotypes A and B are used in clinics, and serotype A is the most used for the highest efficacy and safety. BoNT is widely known and used in aesthetics, through intramuscular injections to attenuate dynamic wrinkles. Additionally, it has been used as therapy in the treatment of strabismus, blepharospasm, cervical dystonia, hyperhidrosis, sialorrhea, hemifacial spasm, and many other clinical conditions in areas such as urology, neurology, ophthalmology, gastroenterology and more recently evaluated in psychiatry. BoNTs are also being studied for a new therapeutic horizon: the treatment of pain. In some painful syndromes that can be treated with BoNT, the mechanism of action is related to the inhibition of muscle contraction through inhibition of acetylcholine release at the neuromuscular junction. This effect involves a selective toxin action on SNARE family proteins (soluble N-ethylmaleimide sensitive factor attachment protein receptor), which mediate the acetylcholine exocytosis process. Moreover, it shows analgesic effect in some clinical conditions that are not related to excessive muscle contraction, such as in the treatment of migraine. Therefore, it has been suggested that BoNT is capable of producing analgesia independent of muscle contraction inhibition. This evidence has stimulated the study of BoNT in other painful conditions, such as trigeminal neuralgia. One of the advantages of BoNT clinical application is its safety. Major adverse effects are application-related and include pain, erythema, pruritus, edema, bruising, hematoma, paraesthesia, and ptosis. In this review BoNT will be analyzed in the historical, pharmacological, biochemical and clinical aspects, but the focus will be on the investigation of the analgesic effect of BoNT that occurs regardless of its muscle relaxant effect.

The complexin C-terminal amphipathic helix stabilizes the fusion pore open state by sculpting membranes.

Neurotransmitter release is mediated by proteins that drive synaptic vesicle fusion with the presynaptic plasma membrane. While soluble N-ethylmaleimide sensitive factor attachment protein receptors (SNAREs) form the core of the fusion apparatus, additional proteins play key roles in the fusion pathway. Here, we report that the C-terminal amphipathic helix of the mammalian accessory protein, complexin (Cpx), exerts profound effects on membranes, including the formation of pores and the efficient budding and fission of vesicles. Using nanodisc-black lipid membrane electrophysiology, we demonstrate that the membrane remodeling activity of Cpx modulates the structure and stability of recombinant exocytic fusion pores. Cpx had particularly strong effects on pores formed by small numbers of SNAREs. Under these conditions, Cpx increased the current through individual pores 3.5-fold, and increased the open time fraction from roughly 0.1 to 1.0. We propose that the membrane sculpting activity of Cpx contributes to the phospholipid rearrangements that underlie fusion by stabilizing highly curved membrane fusion intermediates.

Epileptic Phenotypes Associated With SNAREs and Related Synaptic Vesicle Exocytosis Machinery.

SNAREs (soluble N-ethylmaleimide sensitive factor attachment protein receptor) are an heterogeneous family of proteins that, together with their key regulators, are implicated in synaptic vesicle exocytosis and synaptic transmission. SNAREs represent the core component of this protein complex. Although the specific mechanisms of the SNARE machinery is still not completely uncovered, studies in recent years have provided a clearer understanding of the interactions regulating the essential fusion machinery for neurotransmitter release. Mutations in genes encoding SNARE proteins or SNARE complex associated proteins have been associated with a variable spectrum of neurological conditions that have been recently defined as “SNAREopathies.” These include neurodevelopmental disorder, autism spectrum disorder (ASD), movement disorders, seizures and epileptiform abnormalities. The SNARE phenotypic spectrum associated with seizures ranges from simple febrile seizures and infantile spasms, to severe early-onset epileptic encephalopathies. Our study aims to review and delineate the epileptic phenotypes associated with dysregulation of synaptic vesicle exocytosis and transmission, focusing on the main proteins of the SNARE core complex (STX1B, VAMP2, SNAP25), tethering complex (STXBP1), and related downstream regulators.

The decoy SNARE Tomosyn sets tonic versus phasic release properties and is required for homeostatic synaptic plasticity.

Synaptic vesicle (SV) release probability (Pr) is a key presynaptic determinant of synaptic strength established by cell-intrinsic properties and further refined by plasticity. To characterize mechanisms that generate Pr heterogeneity between distinct neuronal populations, we examined glutamatergic tonic (Ib) and phasic (Is) motoneurons in Drosophila with stereotyped differences in Pr and synaptic plasticity. We found the decoy soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) Tomosyn is differentially expressed between these motoneuron subclasses and contributes to intrinsic differences in their synaptic output. Tomosyn expression enables tonic release in Ib motoneurons by reducing SNARE complex formation and suppressing Pr to generate decreased levels of SV fusion and enhanced resistance to synaptic fatigue. In contrast, phasic release dominates when Tomosyn expression is low, enabling high intrinsic Pr at Is terminals at the expense of sustained release and robust presynaptic potentiation. In addition, loss of Tomosyn disrupts the ability of tonic synapses to undergo presynaptic homeostatic potentiation.

SNARE Regulatory Proteins in Synaptic Vesicle Fusion and Recycling.

Membrane fusion is a universal feature of eukaryotic protein trafficking and is mediated by the soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) family. SNARE proteins embedded in opposing membranes spontaneously assemble to drive membrane fusion and cargo exchange in vitro. Evolution has generated a diverse complement of SNARE regulatory proteins (SRPs) that ensure membrane fusion occurs at the right time and place in vivo. While a core set of SNAREs and SRPs are common to all eukaryotic cells, a specialized set of SRPs within neurons confer additional regulation to synaptic vesicle (SV) fusion. Neuronal communication is characterized by precise spatial and temporal control of SNARE dynamics within presynaptic subdomains specialized for neurotransmitter release. Action potential-elicited Ca2+ influx at these release sites triggers zippering of SNAREs embedded in the SV and plasma membrane to drive bilayer fusion and release of neurotransmitters that activate downstream targets. Here we discuss current models for how SRPs regulate SNARE dynamics and presynaptic output, emphasizing invertebrate genetic findings that advanced our understanding of SRP regulation of SV cycling.

Chemical and biological properties of anti-wrinkle peptide Argireline

Argireline, a peptide with the sequence: Ac-Glu-Glu-Met-Gln-Arg-Arg-NH2, also known as Acetyl Hexapeptide-8, reduces facial lines and wrinkles by destabilization of the formation of the SNARE complex (SNAP Receptor, soluble N-ethylmaleimide sensitive factor (NSF) attachment protein receptor), thus preventing muscle contraction. It is a biosafe cosmetic alternative to the botulinum toxin. The method of choice in bioactive peptide analysis is reversed-phase high performance liquid chromatography coupled with mass spectrometry (LC-MS). The aim of this work as to present the properties of Argireline and the analysis of cosmetic products containing this peptide. Previous reports on possible Argireline transformations in cosmetic formulations have not confirmed deacetylation, whereas the oxidation of methionine residue was detected by our team. As the biological activity of the oxidized Argireline is not known, further biological studies, as well as efficient analytical procedures for transformation monitoring and quality control in cosmetic products, are necessary.

The canonical α-SNAP is essential for gametophytic development in Arabidopsis.

The development of male and female gametophytes is a pre-requisite for successful reproduction of angiosperms. Factors mediating vesicular trafficking are among the key regulators controlling gametophytic development. Fusion between vesicles and target membranes requires the assembly of a fusogenic soluble N-ethylmaleimide sensitive factor attachment protein receptors (SNAREs) complex, whose disassembly in turn ensures the recycle of individual SNARE components. The disassembly of post-fusion SNARE complexes is controlled by the AAA+ ATPase N-ethylmaleimide-sensitive factor (Sec18/NSF) and soluble NSF attachment protein (Sec17/α-SNAP) in yeast and metazoans. Although non-canonical α-SNAPs have been functionally characterized in soybeans, the biological function of canonical α-SNAPs has yet to be demonstrated in plants. We report here that the canonical α-SNAP in Arabidopsis is essential for male and female gametophytic development. Functional loss of the canonical α-SNAP in Arabidopsis results in gametophytic lethality by arresting the first mitosis during gametogenesis. We further show that Arabidopsis α-SNAP encodes two isoforms due to alternative splicing. Both isoforms interact with the Arabidopsis homolog of NSF whereas have distinct subcellular localizations. The presence of similar alternative splicing of human α-SNAP indicates that functional distinction of two α-SNAP isoforms is evolutionarily conserved.

Syntaxin of plants31 (SYP31) and SYP32 is essential for Golgi morphology maintenance and pollen development.

Pollen development is a key process for the sexual reproduction of angiosperms. The Golgi plays a critical role in pollen development via the synthesis and transport of cell wall materials. However, little is known about the molecular mechanisms underlying the maintenance of Golgi integrity in plants. In Arabidopsis thaliana, syntaxin of plants (SYP) 3 family proteins SYP31 and SYP32 are the only two Golgi-localized Qa-soluble N-ethylmaleimide sensitive factor attachment protein receptors (SNAREs) with unknown endogenous functions. Here, we demonstrate the roles of SYP31 and SYP32 in modulating Golgi morphology and pollen development. Two independent lines of syp31/+ syp32/+ double mutants were male gametophytic lethal; the zero transmission rate of syp31 syp32 mutations was restored to largely normal levels by pSYP32:SYP32 but not pSYP32:SYP31 transgenes, indicating their functional differences in pollen development. The initial arrest of syp31 syp32 pollen occurred during the transition from the microspore to the bicellular stage, where cell plate formation in pollen mitosis I (PMI) and deposition of intine were abnormal. In syp31 syp32 pollen, the number and length of Golgi cisterna were significantly reduced, accompanied by many surrounding vesicles, which could be largely attributed to defects in anterograde and retrograde trafficking routes. SYP31 and SYP32 directly interacted with COG3, a subunit of the conserved oligomeric Golgi (COG) complex and were responsible for its Golgi localization, providing an underlying mechanism for SYP31/32 function in intra-Golgi trafficking. We propose that SYP31 and SYP32 play partially redundant roles in pollen development by modulating protein trafficking and Golgi structure.