Snapin Expression Research Articles

Interaction between the VP2 protein of deformed wing virus and host snapin protein and its effect on viral replication.

Deformed wing virus (DWV) is one of the causative agents of colony collapse disorder. The structural protein of DWV plays a vital role in the process of viral invasion and host infection; however, there is limited research on DWV. In this study, we screened the host protein snapin, which can interact with the VP2 protein of DWV, using the yeast two-hybrid system. Through computer simulation and GST pull-down and CO-IP assays, an interaction between snapin and VP2 was confirmed. Furthermore, immunofluorescence and co-localization experiments revealed that VP2 and snapin primarily co-localized in the cytoplasm. Consequently, RNAi was used to interfere with the expression of snapin in worker bees to examine the replication of DWV after the interference. After silencing of snapin, the replication of DWV in worker bees was significantly downregulated. Hence, we speculated that snapin was associated with DWV infection and involved in at least one stage of the viral life cycle. Finally, we used an online server to predict the interaction domains between VP2 and snapin, and the results indicate that the interaction domain of VP2 was approximately located at 56-90, 136-145, 184-190, and 239-242 aa and the snapin interaction domain was approximately located at 31-54 and 115-136 aa. This research confirmed that DWV VP2 protein could interacts with the snapin of host protein, which provides a theoretical basis for further investigation of its pathogenesis and development of targeted therapeutic drugs.

SNAPIN Regulates Cell Cycle Progression to Promote Pancreatic β Cell Growth.

In diabetes mellitus, death of β cell in the pancreas occurs throughout the development of the disease, with loss of insulin production. The maintenance of β cell number is essential to maintaining normoglycemia. SNAPIN has been found to regulate insulin secretion, but whether it induces β cell proliferation remains to be elucidated. This study aimed to explore the physiological roles of SNAPIN in β cell proliferation. SNAPIN expression increases with the age of mice and SNAPIN is down-regulated in diabetes. KEGG pathway and GO analysis showed that SNAPIN- interacting proteins were enriched in cell cycle regulation. B cell cycle was arrested in the S phase, and cell proliferation was inhibited after SNAPIN knockdown. The expression of CDK2, CDK4 and CCND1 proteins in the S phase of the cell cycle were reduced after SNAPIN knockdown, whereas they were increased after overexpression of SNAPIN. In addition, insulin protein and mRNA levels also increased or decreased after SNAPIN knockdown or overexpression, respectively. Conclusions: Our data indicate that SNAPIN mediates β cells proliferation and insulin secretion, and provide evidences that SNAPIN might be a pharmacotherapeutic target for diabetes mellitus.

Loss of Dysbindin Implicates Synaptic Vesicle Replenishment Dysregulation as a Potential Pathogenic Mechanism in Schizophrenia

The schizophrenia-susceptibility gene, dystrobrevin-binding protein 1 (DTNBP1), encodes the dysbindin protein and mediates neurotransmission and neurodevelopment in normal subjects. Functional studies show that DTNBP1 loss may cause deficient presynaptic vesicle transmission, which is related to multiple psychiatric disorders. However, the functional mechanism of dysbindin-mediated synaptic vesicle transmission has not been investigated systematically. In this study, we performed electrophysiological recordings in calyx of Held synapses. We found that excitatory postsynaptic current (EPSC) and miniature EPSC (mEPSC) amplitudes were unchanged in dysbindin-deficient synapses, but readily releasable pool (RRP) size and calcium dependent vesicle replenishment were affected during high-frequency stimulation. Moreover, dysbindin loss accompanied slightly decreases in Munc18-1 and snapin expression levels, which are associated with vesicle priming and synaptic homeostasis under high-frequency stimulation. Together, we inferred that dysbindin directly interacts with Munc18-1 and snapin to mediate calcium dependent RRP replenishment. Dysbindin loss may lead to RRP replenishment dysregulation during high-frequency stimulation, potentially causing cognitive impairment in schizophrenia.

Mitophagy regulates integrity of mitochondria at synapses and is critical for synaptic maintenance.

Synaptic mitochondria are particularly vulnerable to physiological insults, and defects in synaptic mitochondria are linked to early pathophysiology of Alzheimer's disease (AD). Mitophagy, a cargo-specific autophagy for elimination of dysfunctional mitochondria, constitutes a key quality control mechanism. However, how mitophagy ensures synaptic mitochondrial integrity remains largely unknown. Here, we reveal Rheb and Snapin as key players regulating mitochondrial homeostasis at synapses. Rheb initiates mitophagy to target damaged mitochondria for autophagy, whereas dynein-Snapin-mediated retrograde transport promotes clearance of mitophagosomes from synaptic terminals. We demonstrate that synaptic accumulation of mitophagosomes is a feature in AD-related mutant hAPP mouse brains, which is attributed to increased mitophagy initiation coupled with impaired removal of mitophagosomes from AD synapses due to defective retrograde transport. Furthermore, while deficiency in dynein-Snapin-mediated retrograde transport recapitulates synaptic mitophagy stress and induces synaptic degeneration, elevated Snapin expression attenuates mitochondrial defects and ameliorates synapse loss in AD mouse brains. Taken together, our study provides new insights into mitophagy regulation of synaptic mitochondrial integrity, establishing a foundation for mitigating AD-associated mitochondria deficits and synaptic damage through mitophagy enhancement.

Interaction of porcine reproductive and respiratory syndrome virus major envelope proteins GP5 and M with the cellular protein Snapin

BackgroundPorcine reproductive and respiratory syndrome (PRRS) is characterized by abortions in pregnant sows and respiratory disease, particularly in young pigs. The causative agent is porcine reproductive and respiratory syndrome virus (PRRSV), a member of the arterivirus family. GP5 and M are the major envelope proteins encoded by PRRSV. To further characterize these two viral proteins, a yeast two-hybrid approach was utilized to identify interacting partners of PRRSV GP5 and M proteins. MethodsInteracting partners of PRRSV GP5 and M were identified using a porcine macrophage cDNA library yeast two-hybrid screen. Subsequently, the interactions between PRRSV GP5/M and the cellular protein Snapin were mapped using truncated versions of the GP5 and M proteins in a yeast two-hybrid assay to localize the interactions. The Snapin gene from the African green monkey kidney cell line MARC-145, which is permissive to PRRSV, was cloned and sequenced, and compared to porcine Snapin. Cellular Snapin expression was reduced in PRRSV-infected cells via Snapin-specific siRNA targeting. ResultsHere we show that the cellular Snap-Associated Protein (Snapin), an accessory protein of the SNARE membrane fusion network and also a member of the BLOC-1 complex, specifically interacts with GP5 and M. Inhibition of Snapin expression via siRNA targeting of Snapin results in the reduction of PRRSV replication. ConclusionsThe PRRSV GP5 and M proteins are known to form a heterodimeric complex which is important for viral structure and infectivity, and both PRRSV proteins can interact with cellular Snapin. Snapin knock-down suggests these interactions could be important in the PRRSV lifecycle. GP5 and M proteins may interact with Snapin to exploit its roles in intracellular transport and membrane fusion.

Regulation of Synaptic Amyloid-β Generation through BACE1 Retrograde Transport in a Mouse Model of Alzheimer's Disease.

Amyloid-β (Aβ) peptides play a key role in synaptic damage and memory deficits in the early pathogenesis of Alzheimer's disease (AD). Abnormal accumulation of Aβ at nerve terminals leads to synaptic pathology and ultimately to neurodegeneration. β-site amyloid precursor protein (APP) cleaving enzyme 1 (BACE1) is the major neuronal β-secretase for Aβ generation. However, the mechanisms regulating BACE1 distribution in axons and β cleavage of APP at synapses remain largely unknown. Here, we reveal that dynein-Snapin-mediated retrograde transport regulates BACE1 trafficking in axons and APP processing at presynaptic terminals. BACE1 is predominantly accumulated within late endosomes at the synapses of AD-related mutant human APP (hAPP) transgenic (Tg) mice and patient brains. Defective retrograde transport by genetic ablation of snapin in mice recapitulates late endocytic retention of BACE1 and increased APP processing at presynaptic sites. Conversely, overexpressing Snapin facilitates BACE1 trafficking and reduces synaptic BACE1 accumulation by enhancing the removal of BACE1 from distal AD axons and presynaptic terminals. Moreover, elevated Snapin expression via stereotactic hippocampal injections of adeno-associated virus particles in mutant hAPP Tg mouse brains decreases synaptic Aβ levels and ameliorates synapse loss, thus rescuing cognitive impairments associated with hAPP mice. Altogether, our study provides new mechanistic insights into the complex regulation of BACE1 trafficking and presynaptic localization through Snapin-mediated dynein-driven retrograde axonal transport, thereby suggesting a potential approach of modulating Aβ levels and attenuating synaptic deficits in AD.SIGNIFICANCE STATEMENT β-Site amyloid precursor protein (APP) cleaving enzyme 1 (BACE1) trafficking and synaptic localization significantly influence its β secretase activity and amyloid-β (Aβ) production. In AD brains, BACE1 is accumulated within dystrophic neurites, which is thought to augment Aβ-induced synaptotoxicity by Aβ overproduction. However, it remains largely unknown whether axonal transport regulates synaptic APP processing. Here, we demonstrate that Snapin-mediated retrograde transport plays a critical role in removing BACE1 from presynaptic terminals toward the soma, thus reducing synaptic Aβ production. Adeno-associated virus-mediated Snapin overexpression in the hippocampus of mutant hAPP mice significantly decreases synaptic Aβ levels, attenuates synapse loss, and thus rescues cognitive deficits. Our study uncovers a new pathway that controls synaptic APP processing by enhancing axonal BACE1 trafficking, thereby advancing our fundamental knowledge critical for ameliorating Aβ-linked synaptic pathology.

SNAPIN is critical for lysosomal acidification and autophagosome maturation in macrophages

ABSTRACTWe previously observed that SNAPIN, which is an adaptor protein in the SNARE core complex, was highly expressed in rheumatoid arthritis synovial tissue macrophages, but its role in macrophages and autoimmunity is unknown. To identify SNAPIN's role in these cells, we employed siRNA to silence the expression of SNAPIN in primary human macrophages. Silencing SNAPIN resulted in swollen lysosomes with impaired CTSD (cathepsin D) activation, although total CTSD was not reduced. Neither endosome cargo delivery nor lysosomal fusion with endosomes or autophagosomes was inhibited following the forced silencing of SNAPIN. The acidification of lysosomes and accumulation of autolysosomes in SNAPIN-silenced cells was inhibited, resulting in incomplete lysosomal hydrolysis and impaired macroautophagy/autophagy flux. Mechanistic studies employing ratiometric color fluorescence on living cells demonstrated that the reduction of SNAPIN resulted in a modest reduction of H+ pump activity; however, the more critical mechanism was a lysosomal proton leak. Overall, our results demonstrate that SNAPIN is critical in the maintenance of healthy lysosomes and autophagy through its role in lysosome acidification and autophagosome maturation in macrophages largely through preventing proton leak. These observations suggest an important role for SNAPIN and autophagy in the homeostasis of macrophages, particularly long-lived tissue resident macrophages.

Snapin, Positive Regulator of Stimulation- Induced Ca2+ Release through RyR, Is Necessary for HIV-1 Replication in T Cells

To identify critical host factors necessary for human immunodeficiency virus 1 (HIV-1) replication, large libraries of short-peptide-aptamers were expressed retrovirally. The target of one inhibitor peptide, Pep80, identified in this screen was determined to be Snapin, a protein associated with the soluble N-ethyl maleimide sensitive factor adaptor protein receptor (SNARE) complex that is critical for calcium-dependent exocytosis during neurotransmission. Pep80 inhibited Ca2+ release from intracellular stores and blocked downstream signaling by direct interruption of the association between Snapin and an intracellular calcium release channel, the ryanodine receptor (RyR). NFAT signaling was preferentially abolished by Pep80. Expression of Snapin overcame Pep80-mediated inhibition of Ca2+/NFAT signaling and HIV-1 replication. Furthermore, Snapin induced HIV-1 replication in primary CD4+ T cells. Thus, through its interaction with RyR, Snapin is a critical regulator of Ca2+ signaling and T cell activation. Use of the genetically selected intracellular aptamer inhibitors allowed us to define unique mechanisms important to HIV-1 replication and T cell biology.

Modulation of the Cellular Distribution of Human Cytomegalovirus Helicase by Cellular Factor Snapin

Controlled regulation of genomic DNA synthesis is a universally conserved process for all herpesviruses, including human cytomegalovirus (HCMV), and plays a key role in viral pathogenesis, such as persistent infections. HCMV UL105 is believed to encode the helicase of the DNA replication machinery that needs to localize in the nuclei, the site of viral DNA synthesis. No host factors that interact with UL105 have been identified. In this study, we show that UL105 specifically interacts with Snapin, a human protein that is predominantly localized in the cytoplasm and associated with cellular vesicles. UL105 was found to interact with Snapin in both the yeast two-hybrid screen and coimmunoprecipitation experiments in HCMV-infected cells. The nuclear and cytoplasmic levels of UL105 were decreased and increased in cells overexpressing Snapin, respectively, while the levels of UL105 in the nuclei and cytoplasm were increased and decreased in cells in which the expression of Snapin was downregulated with anti-Snapin small interfering RNA (siRNA) molecules, respectively. Furthermore, viral DNA synthesis and progeny production were decreased in cells overexpressing Snapin and increased in the anti-Snapin siRNA-treated cells, respectively. Our results provide the first direct evidence to suggest that Snapin interacts with UL105 and alters its cellular distribution, leading to modulation of viral DNA synthesis and progeny production. Our study further suggests that modulation of the cellular distribution of viral helicase by Snapin may represent a possible mechanism for regulating HCMV genomic DNA synthesis, a key step during herpesvirus lytic and persistent infections.

SNAPIN: an endogenous toll-like receptor ligand in rheumatoid arthritis

ObjectiveThe mechanisms contributing to the persistent activation of macrophages in rheumatoid arthritis (RA) are not fully understood. Some studies suggest that endogenous toll-like receptor (TLR) ligands promote the chronic inflammation...

Human Cytomegalovirus Primase UL70 Specifically Interacts with Cellular Factor Snapin

Genomic DNA synthesis is a universally conserved process for all herpesviruses, including human cytomegalovirus (HCMV). HCMV UL70 is believed to encode the primase of the DNA replication machinery, a function which requires localization in the nucleus, the site of viral DNA synthesis. No host factors that interact with UL70 have been reported. In this study, we provide the first direct evidence that UL70 specifically interacts with Snapin, a human protein that is predominantly localized in the cytoplasm and is associated with cellular vesicles. The interaction between UL70 and Snapin was identified in both the two-hybrid screen in yeast and coimmunoprecipitation in human cells. The nuclear import of UL70 was decreased in cells overexpressing Snapin and increased in cells in which the expression of Snapin was downregulated with anti-Snapin small interfering RNA (siRNA) molecules, respectively. Furthermore, viral DNA synthesis and progeny production were decreased in cells overexpressing Snapin and increased in the anti-Snapin siRNA-treated cells, respectively. In contrast, no significant difference in the nuclear level of UL70, viral DNA synthesis, and progeny production was found among the parental cells and cells that either expressed a control empty vector or were treated with control siRNA molecules that did not recognize any viral or cellular transcripts. Our results suggest that Snapin may play a key role in regulating the cellular localization of UL70 in HCMV, leading to modulation of viral DNA synthesis and progeny production.

Snapin Facilitates the Synchronization of Synaptic Vesicle Fusion

Synaptic vesicle (SV) fusion is a fine-tuned process requiring a concert of fusion machineries. Using cortical neurons from snapin-deficient mice, we reveal a role for Snapin in facilitating synchronous release. In addition to reduced frequency of miniature excitatory postsynaptic currents (mini-EPSCs) and smaller release-ready vesicle pool (RRP) size, snapin deficiency results in EPSCs with multiple peaks and increased rise and decay times, reflecting "desynchronized" SV fusion. These defects impair both synaptic precision and efficacy during sustained neurotransmission. Transient expression of Snapin not only rescues the slowed kinetics of EPSCs, but also further accelerates the rate found in wild-type neurons. Furthermore, expression of Snapin-C66A, a dimerization-defective mutant with impaired interactions with SNAP-25 and Synaptotagmin, reduces the RRP size but exhibits less effect on synchronized fusion. Our studies provide mechanistic insights into a dual role of Snapin in enhancing the efficacy of SV priming and in fine-tuning synchronous SV fusion.

The SNARE-associated component SNAPIN binds PUMILIO2 and NANOS1 proteins in human male germ cells

It has been reported that a highly conserved human protein PUMILIO2 forms a complex with NANOS1 in human male germ cells, as does the Drosophila ancestor Pumilio, which binds Nanos to regulate translation of specific mRNAs. Here, we found that PUMILIO2 interacts also with SNAPIN, a modulator of SNARE complex assembly, which is involved in vesicle trafficking. We demonstrated that SNAPIN interacts additionally with NANOS1 protein. This is the first report demonstrating that the N-terminal region of NANOS1 is necessary for protein binding. In human testis, SNAPIN co-localizes with PUMILIO2 and NANOS1 in prenatal and also in spermatogenic germ cells of the adult. We describe for the first time the expression of SNAPIN in germ cells which raises possibility that SNAPIN plays an extra role in mammals which is germ cell specific. The presence of a coiled-coil domain responsible for protein-protein interaction could enable SNAPIN to be an adaptor of PUMILIO2 and NANOS1, binding other factors to regulate translation in the development of the human germ cells.

Regulation of Type VI Adenylyl Cyclase by Snapin, a SNAP25-binding Protein

In the present study, we used the N terminus (amino acids 1 approximately 160) of type VI adenylyl cyclase (ACVI) as bait to screen a mouse brain cDNA library and identified Snapin as a novel ACVI-interacting molecule. Snapin is a binding protein of SNAP25, a component of the SNARE complex. Co-immunoprecipitation analyses confirmed the interaction between Snapin and full-length ACVI. Mutational analysis revealed that the interaction domains of ACVI and Snapin were located within amino acids 1 approximately 86 of ACVI and 33-51 of Snapin, respectively. Co-localization of ACVI and Snapin was observed in primary hippocampal neurons. Moreover, expression of Snapin specifically eliminated protein kinase C (PKC)-mediated suppression of ACVI, but not that of cAMP-dependent protein kinase (PKA) or calcium. Mutation of the potential PKC and PKA phosphorylation sites of Snapin did not affect the ability of Snapin to reverse the PKC inhibitory effect on ACVI. Phosphorylation of Snapin by PKC or PKA therefore might not be crucial for Snapin action on ACVI. In contrast, Snapin(Delta33-51), which harbors an internal deletion of amino acids 33-51 did not affect PKC-mediated inhibition of ACVI, supporting that amino acids 33-51 of Snapin comprises the ACVI-interacting region. Consistently, Snapin exerted no effect on PKC-mediated inhibition of an ACVI mutant (ACVI-DeltaA87), which lacked the Snapin-interacting region (amino acids 1-86). Snapin thus reverses its action via direct interaction with the N terminus of ACVI. Collectively, we demonstrate herein that in addition to its association with the SNARE complex, Snapin also functions as a regulator of an important cAMP synthesis enzyme in the brain.