Synaptosomal-associated Protein 29 Research Articles

Porcine Reproductive and Respiratory Syndrome Virus nsp5 Induces Incomplete Autophagy by Impairing the Interaction of STX17 and SNAP29.

Porcine reproductive and respiratory syndrome virus (PRRSV) is an economically important pathogen that has devastated the worldwide swine industry for over 30 years. Autophagy is an evolutionarily conserved intracellular lysosomal degradation pathway, and previous studies have documented that PRRSV infection prompts autophagosome accumulation. However, whether PRRSV induces complete or incomplete autophagy remains controversial. Here, we demonstrated that overexpression of PRRSV nonstructural protein 5 (nsp5) induced the accumulation of autophagosomes, and a similar scenario was observed in PRRSV-infected cells. Moreover, both PRRSV infection and nsp5 overexpression activated incomplete autophagy, as evidenced by the blockage of autophagosome-lysosome fusion. Mechanistically, nsp5 overexpression, as well as PRRSV infection, inhibited the interaction of syntaxin 17 (STX17) with synaptosomal-associated protein 29 (SNAP29), two SNARE proteins that mediate autophagosome fusion with lysosomes, to impair the formation of autolysosomes. We further confirmed that nsp5 interacted with STX17, rather than SANP29, and the interacting domains of STX17 were the N-terminal motif and SNARE motif. Taken together, the findings of our study suggest a mechanism by which PRRSV induces incomplete autophagy by blocking autophagosome degradation and provide insights into the development of new therapeutics to combat PRRSV infection. IMPORTANCE A substantial number of viruses have been demonstrated to utilize or hijack autophagy to benefit their replication. In the case of porcine reproductive and respiratory syndrome virus (PRRSV), previous studies have demonstrated the proviral effects of autophagy on PRRSV proliferation. Thus, an investigation of the mechanism by which PRRSV regulates the autophagy processes can provide new insight into viral pathogenesis. Autophagic flux is a dynamic process that consists of autophagosome formation and subsequent lysosomal degradation. However, the exact effect of PRRSV infection on the autophagic flux remains disputed. In this study, we demonstrated that PRRSV infection, as well as PRRSV nsp5 overexpression, inhibited the interaction of STX17 with SNAP29 to impair the fusion of autophagosomes with lysosomes, thereby blocking autophagic flux. This information will help us to understand PRRSV-host interactions and unravel new targets for PRRS prevention and control.

Abstract 105: Novel Functions For Synaptosomal-associated Protein 29 (snap29) In Cardiac Arrhythmias

Human genetic studies and mouse models have classically linked mutations/deficiencies in components of the desmosomal cell-cell junction to arrhythmogenic right ventricular cardiomyopathy (ARVC). However, growing evidence points to the importance of the desmosome in cardiac diseases beyond ARVC, which include electrical diseases that have no impact on cardiac structure/morphology. The mechanisms of how defects in cardiac desmosomal protein homeostasis drive these distinct forms of cardiac disease remain elusive to the field. To uncover mechanisms that underlie the distinct pathophysiology (structural versus non-structural) encompassed by desmosomal mutations/loss, we performed an unbiased yeast-two-hybrid screen using an adult human heart cDNA library and the desmosomal protein, desmoplakin (DSP) to uncover new regulators of cardiac desmosomal protein homeostasis. We identified synaptosomal-associated protein 29 (SNAP29), as a novel DSP-interacting protein in the adult human heart. Traditional functions of SNAP29 are to regulate membrane fusion and play a role in autophagy; however, its role at the desmosome and in the heart is undefined. We show that SNAP29 is a subcomponent of the cardiac desmosome, as it co-localizes with DSP in the adult heart and DSP-deficient hearts harbor loss of SNAP29. Cardiomyocyte-specific SNAP29 knockout (SNAP29-cKO) mice displayed baseline and pacing-induced ventricular arrhythmias in an age-dependent manner in the absence of cardiac structural and functional deficits. We show that a loss of a subset of desmosomal proteins and connexin43 as well as upregulation of selective autophagy-mediated degradation underlie SNAP29 deficient cardiomyocyte arrhythmias. In line with this, acute blockade of autophagy was sufficient to rescue desmosomal and connexin43 protein levels as well as arrhythmias in SNAP29 deficient cardiomyocytes. In conclusion, SNAP29 insulates a subset of desmosomal and gap junction proteins from selective autophagy-mediated degradation to restrict cardiac arrhythmias. Thus, loss of SNAP29-desmosome-gap junction interactome may predispose the heart to desmosomal based diseases of an electrical nature.

Generation and Characterization of a CRISPR/Cas9-Mediated SNAP29 Knockout in Human Fibroblasts.

Loss-of-function mutations in the synaptosomal-associated protein 29 (SNAP29) lead to the rare autosomal recessive neurocutaneous cerebral dysgenesis, neuropathy, ichthyosis, and keratoderma (CEDNIK) syndrome. SNAP29 is a soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein. So far, it has been shown to be involved in membrane fusion, epidermal differentiation, formation of primary cilia, and autophagy. Recently, we reported the successful generation of two mouse models for the human CEDNIK syndrome. The aim of this investigation was the generation of a CRISPR/Cas9-mediated SNAP29 knockout (KO) in an immortalized human cell line to further investigate the role of SNAP29 in cellular homeostasis and signaling in humans independently of animal models. Comparison of different methods of delivery for CRISPR/Cas9 plasmids into the cell revealed that lentiviral transduction is more efficient than transfection methods. Here, we reported to the best of our knowledge the first successful generation of a CRISPR/Cas9-mediated SNAP29 KO in immortalized human MRC5Vi fibroblasts (c.169_196delinsTTCGT) via lentiviral transduction.

SNAP29 Restricts Cardiac Arrhythmias by Insulating A Subset of Desmosomal Proteins and Connexin43 from Autophagic Degradation

Desmosomes are strong cell-cell adhesion junctions that connect the cytoskeleton of one cell to another (via intermediate filaments) to maintain the mechanical integrity of tissues under constant stress, such as the heart. Classically, human genetic studies and mouse models have linked components of the desmosome mutations/deficiencies to arrhythmogenic right ventricular cardiomyopathy (ARVC), an inherited cardiac disease of the desmosome that impacts cardiac structure/morphology. However, growing evidence points to a role for loss of desmosomal integrity in cardiac electrical diseases (eg., Brugada syndrome, long QT, sinus node arrhythmias) that have no impact on cardiac structure/morphology; however, how desmosomal protein loss results in these distinct forms of cardiac disease remains unclear. We exploited an unbiased yeast-two-hybrid screen using the desmosomal protein, desmoplakin (DSP) as a bait, to uncover novel desmosomal protein-protein interactions that may help further explain the pathobiology (structural versus non-structural) encompassed by desmosomal mutations/loss. Through this screen, we identified the SNARE protein, synaptosomal-associated protein 29 (SNAP29), as a novel DSP-interacting protein. Traditional functions of SNAP29 are to regulate membrane fusion and play a role in autophagy; however, its role at the desmosome in the heart is undefined. We hypothesized that SNAP29 is a new subcomponent of the cardiac desmosome that regulates desmosomal protein degradation and thus, may have important roles in desmosomal based diseases. We show that SNAP29 co-localizes with DSP in adult mouse heart and that cardiac-specific DSP deficient mice hearts harbor loss of SNAP29, validating SNAP29 as a subcomponent of the cardiac desmosome. By generating novel cardiomyocyte-specific SNAP29 knockout (SNAP29-cKO) mice (using Xenopus myosin light chain 2-Cre), we show that SNAP29-cKO mouse hearts displayed baseline and pacing-induced ventricular arrhythmias in an age-dependent manner in the absence of cardiac structural and functional deficits. Molecular analyses further revealed that adult SNAP29-cKO hearts displayed a loss of a subset of desmosomal (DSP, plakophilin2) as well as gap junction (connexin43) proteins when compared to controls. These cell-cell junction defects were accompanied by an accumulation of autophagic markers (p62 and LC3BII), as well as machinery (autophagic vesicles), which functionally resulted in cardiac arrhythmias. We show that acute blockade of autophagy was sufficient to rescue levels of desmosomal and gap junction proteins as well as arrhythmias in SNAP29 deficient cardiomyocytes. In summary, our data suggest that SNAP29 insulates a subset of desmosomal and gap junction proteins from selective autophagy-mediated degradation to restrict cardiac arrhythmias and that loss of SNAP29-desmosome-gap junction interactome may predispose the heart to desmosomal based diseases of an electrical (non-structural) nature. Collectively, our results identify defects in cardiac autophagy in desmosomal disease pathogenesis, while providing a novel role for SNARE proteins in cardiac arrhythmias.

New Cohort of Patients With CEDNIK Syndrome Expands the Phenotypic and Genotypic Spectra.

ObjectiveTo report 6 new patients with cerebral dysgenesis, neuropathy, ichthyosis, and keratoderma (CEDNIK) syndrome.MethodsClinical exome or targeted sequencing were performed to elucidate the molecular genetic cause in patients with neurocognitive abnormalities and brain imaging findings.ResultsCEDNIK syndrome is a rare genetic condition caused by biallelic pathogenic loss-of-function variants in synaptosomal-associated protein 29 (SNAP29), which encodes a vesicular membrane fusion protein. Clinical manifestations include significant developmental delay/intellectual disability (DD/ID), brain abnormalities, failure to thrive, and skin abnormalities. To date, 19 patients from 10 unrelated families with CEDNIK syndrome have been reported. We report 5 additional patients with homozygous predicted loss-of-function variants in SNAP29 and one with compound heterozygous variants: a frameshift SNAP29 variant and a 370 kb deletion on 22q11.2. All patients exhibit DD/ID, ichthyosis and/or palmoplantar keratoderma, and hypotonia. Four of 6 subjects had hypomyelinated white matter on MRI, 2 of 6 had early puberty, and 4 of 6 had strabismus, which were previously rarely reported. Other phenotypes were variably present, including dysmorphic features, feeding difficulties, and recurrent respiratory infections. The cohort includes 2 siblings with a c.2T>C variant who have a relatively milder phenotype, a patient with the most C-terminal variant yet described (c.622G>T), and 3 patients with previously described variants (c.354dupG, c.487dupA).ConclusionsThis cohort of 6 additional patients expands the genotypic and phenotypic spectrum of CEDNIK syndrome, highlighting previously under-recognized features such as hypomyelination, seizures, and early puberty. Owing to reduced penetrance of the skin phenotype, cerebral dysgenesis, and neuropathy, we propose renaming this syndrome SNAP29-related disorder.

Cardiomyocyte SNAP29 Insulates Desmosomes from Targeted Protein Degradation by Selective Autophagy to Restrict Arrhythmias

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a fatal and untreatable inherited cardiomyopathy associated with ventricular dysfunction and cardiac arrhythmias leading to sudden cardiac death in the young. Early detection is hindered as people present with complex disease patterns and harbors a “concealed phase”, where lethal arrhythmias are observed in the absence of overt structural changes to the heart. Mutations and loss of components of the desmosome, a cardiac mechanical cell‐cell junction, have been linked to the development of ARVC through human and mouse genetic studies. However, limited information exists on how desmosomal protein dysregulation/loss can trigger the complex features of ARVC, especially the asymptomatic “concealed phase”. Using a yeast two‐hybrid screen we identified Synaptoso‐malassociated protein 29 (SNAP29) as a novel desmosomal (desmoplakin, DSP)‐interacting protein. Traditional functions of SNAP29 are to regulate membrane fusion and play a role in autophagy in non‐cardiomyocytes; however, its role at the desmosome and/or heart is undefined. We hypothesize that SNAP29 may play a critical role in maintaining desmosomal protein homeostasis and have direct relevance to ARVC disease features, as it is a disease associated with desmosomal protein dissolution. We show that SNAP29 co‐localizes with DSP in both the adult mouse and human heart. Cardiac‐specific SNAP29‐deficient mice (SNAP29‐cKO) displayed baseline and pacing‐ induced ventricular arrhythmias in an age‐dependent manner in the absence of cardiac structural and functional deficits. Furthermore, molecular analyses revealed that the arrhythmias found in SNAP29‐cKO hearts and cardiomyocytes were associated with a molecular loss of a subset of desmosomal proteins (DSP and plakophilin‐2) and the gap junction protein, connexin43. Furthermore, an accumulation of autophagic markers (p62 and LC3BII), as well as machinery, was observed at the cardiac cell‐cell junction in SNAP29 deficient cardiomyocytes. Acute blockade of autophagy using specific inhibitors (chloroquine and bafilomycin A1) was sufficient to rescue levels of desmosomal and gap junction proteins as well as arrhythmias in SNAP29 deficient cardiomyocytes. Our data suggest that SNAP29 insulates a subset of desmosomal proteins from selective autophagy‐mediated degradation to restrict cardiac arrhythmias. Thus, SNAP29 loss may directly contribute to the “concealed phase” of ARVC. Our results further identify autophagy defects as an underlying mechanism in desmosomal disease pathogenesis and highlight new potential therapeutic targets for ARVC.Support or Funding InformationNIH/NHLBI (HL09780)

Silencing DAPK3 blocks the autophagosome-lysosome fusion by mediating SNAP29 in trophoblast cells under high glucose treatment

Autophagy plays an essential role in gestational diabetes mellitus (GDM). Death-associated protein kinase-3 (DAPK3) regulates a variety of cellular functions; however, the relationship between DAPK3 and autophagy is unknown. In this study, we aim to investigate whether DAPK3 is associated with autophagy in GDM, and we found that DAPK3 was upregulated in the placenta of GDM patients and extravillous trophoblast cells under high-glucose conditions. Silencing DAPK3 decreased the assembly of the STX17-SNAP29-VAMP8 complex, leading to the blockade of autophagosome-lysosome fusion by mediating synaptosomal-associated protein 29 (SNAP29). Moreover, knockdown of DAPK3 ameliorates cell invasion and mediates autophagy in high glucose, and does not alter the expression of autophagy-related genes in normal glucose. Our study demonstrates the significance of DAPK3 in autophagy and GDM, which may provide new insights into the molecular mechanisms regulating trophoblast invasion.

Snap29 mutant mice recapitulate neurological and ophthalmological abnormalities associated with 22q11 and CEDNIK syndrome

Synaptosomal-associated protein 29 (SNAP29) encodes a member of the SNARE family of proteins implicated in numerous intracellular protein trafficking pathways. SNAP29 maps to the 22q11.2 region and is deleted in 90% of patients with 22q11.2 deletion syndrome (22q11.2DS). Moreover, bi-allelic SNAP29 mutations in patients are responsible for CEDNIK (cerebral dysgenesis, neuropathy, ichthyosis, and keratoderma) syndrome. A mouse model that recapitulates abnormalities found in these syndromes is essential for uncovering the cellular basis of these disorders. In this study, we report that mice with a loss of function mutation of Snap29 on a mixed CD1;FvB genetic background recapitulate skin abnormalities associated with CEDNIK, and also phenocopy neurological and ophthalmological abnormalities found in CEDNIK and a subset of 22q11.2DS patients. Our work also reveals an unanticipated requirement for Snap29 in male fertility and supports contribution of hemizygosity for SNAP29 to the phenotypic spectrum of abnormalities found in 22q11.2DS patients.

Regulation of Autophagic Flux by the 20S Proteasome

The proteolytic arm of the protein homeostasis network is maintained by both the ubiquitin-proteasome system (UPS) and autophagy. A well-balanced crosstalk between the two catabolic pathways ensures energy-efficient maintenance of cellular function. Our current understanding of the crosstalk between the UPS and autophagy is centered around substrate ubiquitination. Herein we report an additional method of crosstalk involving ubiquitin-independent 20S proteasome regulation of autophagosome-lysosome fusion. We found that enhancement of 20S proteasome activity increased the degradation of the disordered soluble N-ethylmaleimide-sensitive factor activating protein receptor proteins, synaptosomal-associated protein 29 (SNAP29), and syntaxin 17 (STX17), but not vesicle-associated membrane protein 8. This resulted in a reduction of autophagosome-lysosome fusion, which was ameliorated upon overexpression of both SNAP29 and STX17. In all, we herein present a mechanism of crosstalk between the proteasome and autophagy pathway that is regulated by ubiquitin-independent 20S proteasome-mediated degradation of SNAP29 and STX17.

Synaptosomal‐associated protein 29 is required for the autophagic degradation of hepatitis B virus

Hepatitis B virus (HBV) replication and envelopment is dependent on cellular autophagy. Previously, we have provided evidence for the extensive lysosomal degradation of HBV virions and the hepatitis B surface antigen (HBsAg), which is likely controlled by autophagosome-lysosome fusion. Synaptosomal-associated protein 29 (SNAP29) has been identified as a protein specifically mediating autophagosome-lysosome fusion. Thus, in the present study, we addressed the hypothesis that SNAP29 is required for the autophagic degradation of HBV virions and HBsAg. We found that silencing SNAP29 significantly increased the number of autophagosomes and concomitantly promoted HBV replication and HBsAg production. Conversely, SNAP29 overexpression decreased HBV production. Consistent with this, SNAP29 modulated HBV production by interacting with vesicle-associated membrane protein 8 (VAMP8) and synergistically regulated HBV replication with Rab7 complexes. Moreover, the production and release of the small HBsAg is strongly regulated by SNAP29 expression, suggesting that its export occurs partly through the autophagic pathway. Our findings provide new evidence, strongly suggesting that autophagic degradation critically determines the production of HBV virions and HBsAg and that this is controlled by the SNAP29-VAMP8 interaction.-Lin, Y., Wu, C., Wang, X., Liu, S., Kemper, T., Li, F., Squire, A., Zhu, Y., Zhang, J., Chen, X., Lu, M. Synaptosomal-associated protein 29 is required for the autophagic degradation of hepatitis B virus.

O-GlcNAc-modified SNAP29 inhibits autophagy-mediated degradation via the disturbed SNAP29-STX17-VAMP8 complex and exacerbates myocardial injury in type I diabetic rats.

The O-linked β-N-acetylglucosamine (O-GlcNAc) modification and autophagy are associated with diabetic myocardial injury, however, the molecular mechanisms between the two processes remain to be fully elucidated. The purpose of the present study was to elucidate the molecular regulation of autophagy by O-GlcNAc-modified synaptosomal-associated protein 29 (SNAP29) in diabetic myocardial injury. A rat model of type I diabetes was established via intraperitoneal injection of streptozotocin (STZ; 55 mg/kg). Significant increases in the O-GlcNAc modification and accumulation of the autophagy markers microtubule-associated protein 1 light chain 3α II/I and P62, which suggest that autophagic flux is inhibited, were observed in rats 8 weeks following STZ induction. Subsequently, the selective O-GlcNAcase inhibitor, thiamet G, increased the level of O-GlcNAc modification, which further disrupted autophagic flux; deteriorated cardiac diastolic function, as indicated by an increased left ventricular filling peak velocity/atrial contraction flow peak velocity ratio shown by echocardiography; and exacerbated myocardial abnormalities, as characterized by cardiomyocyte disorganization and fat and interstitial fibrosis accumulation. By contrast, 6-diazo-5-oxo-L-norleucine, an inhibitor of glucosamine fructose-6-phosphate aminotransferase isomerizing 1, acted as an O-GlcNAc antagonist and reduced the level of O-GlcNAc modification, which maintained autophagic flux and improved cardiac diastolic function. In vitro, high glucose (25 mM) was used to stimulate primary neonatal rat cardiomyocytes (NRCMs). Consistent with the myocardium of diabetic rats, it was also shown in the NRCMs that O-GlcNAc modification of SNAP29 negatively regulated autophagic flux. The application of the short hairpin RNA interference lysosome-associated membrane protein (LAMP2) and the autophagy inhibitor 3-methyladenine demonstrated that high glucose inhibited autophagy-mediated degradation rather than affected the initial stage of autophagy. Finally, co-immunoprecipitation was used to determine the role of the O-GlcNAc-modified substrate protein SNAP29, which acted as an SNAP29-syntaxin-17 (STX17)-vesicle-associated membrane protein 8 (VAMP8) complex during disease progression. The present study is the first, to the best of our knowledge, to demonstrate that SNAP29 is an O-GlcNAc substrate and that an increase in O-GlcNAc-modified SNAP29 inhibits SNAP29-STX17-VAMP8 complex formation, thereby inhibiting the degradation of autophagy and exacerbating myocardial injury in type I diabetic rats.

Phosphorylation of ULK1 affects autophagosome fusion and links chaperone-mediated autophagy to macroautophagy

The Unc-51 like autophagy activating kinase 1 (ULK1) complex plays a central role in the initiation stage of autophagy. However, the function of ULK1 in the late stage of autophagy is unknown. Here, we report that ULK1, a central kinase of the ULK1 complex involved in autophagy initiation, promotes autophagosome–lysosome fusion. PKCα phosphorylates ULK1 and prevents autolysosome formation. PKCα phosphorylation of ULK1 does not change its kinase activity; however, it decreases autophagosome–lysosome fusion by reducing the affinity of ULK1 for syntaxin 17 (STX17). Unphosphorylated ULK1 recruited STX17 and increased STX17′s affinity towards synaptosomal-associated protein 29 (SNAP29). Additionally, phosphorylation of ULK1 enhances its interaction with heat shock cognate 70 kDa protein (HSC70) and increases its degradation through chaperone-mediated autophagy (CMA). Our study unearths a key mechanism underlying autolysosome formation, a process in which the kinase activity of PKCα plays an instrumental role, and reveals the significance of the mutual regulation of macroautophagy and CMA in maintaining the balance of autophagy.

NEK3-mediated SNAP29 phosphorylation modulates its membrane association and SNARE fusion dependent processes

Intracellular membrane fusion depends on the presence of specific mediators, the vesicle (v-) and the target (t-) SNAREs (Soluble N-ethylmaleimide-sensitive factor, NSF, attachment protein SNAP receptors), whose interaction brings apposing membranes to close proximity and initiates their fusion. SNAP29 (synaptosomal-associated protein 29), a t-SNARE protein, is involved in multiple fusion events during intracellular transport and affects structure of organelles such as the Golgi apparatus and focal adhesions. Mutations in SNAP29 gene result in CEDNIK (Cerebral dysgenesis, neuropathy, ichthyosis and palmoplantar keratoderma) syndrome. In the present study, we show that NEK3 (NIMA-never in mitosis gene A-related kinase 3)-mediated serine 105 (S105) phosphorylation of SNAP29 directs its membrane association, without which cells present defective focal adhesion formation, impaired Golgi structure and attenuated cellular recycling. In contrast to a phosphorylation-defective serine 105 to alanine (S105A) mutant, wildtype SNAP29, partially rescued the abnormal morphology of a CEDNIK patient derived fibroblasts. Our results highlight the importance of NEK3-mediated S105 phosphorylation of SNAP29 for its membrane localization and for membrane fusion dependent processes.

CEDNIK: Phenotypic and Molecular Characterization of an Additional Patient and Review of the Literature.

Synaptosomal-associated protein 29 (SNAP29) is a t-SNARE protein that is implicated in intracellular vesicle fusion. Mutations in the SNAP29 gene have been associated with cerebral dysgenesis, neuropathy, ichthyosis, and keratoderma syndrome (CEDNIK). In patients with 22q11.2 deletion syndrome, mutations in SNAP29 on the nondeleted chromosome are linked to similar ichthyotic and neurological phenotypes. Here, the authors report a patient with cerebral dysgenesis, neuropathy, ichthyosis, and keratoderma syndrome who presented with global developmental delay, polymicrogyria, dysgenesis of the corpus callosum, optic nerve dysplasia, gaze apraxia, and dysmorphic features. He has developed ichthyosis and palmoplantar keratoderma as he has grown. Exome sequencing identified a homozygous nonsense mutation in SNAP29 gene designated as c.85C>T (p.Arg29X). The authors compare the findings in the proband with previously reported cases. The previously unreported mutation in this patient and his phenotype add to the characterization of cerebral dysgenesis, neuropathy, ichthyosis, and keratoderma syndrome and the accumulating scientific evidence that implicates synaptic protein dysfunction in various neuroectodermal conditions.

Establishment of Two Mouse Models for CEDNIK Syndrome Reveals the Pivotal Role of SNAP29 in Epidermal Differentiation

Loss-of-function mutations in the synaptosomal-associated protein 29 (SNAP29) gene cause the cerebral dysgenesis, neuropathy, ichthyosis, and keratoderma syndrome. In this study, we created total (Snap29(-/-)) as well as keratinocyte-specific (Snap29(fl/fl)/K14-Cre) Snap29 knockout mice. Both mutant mice exhibited a congenital distinct ichthyotic phenotype resulting in neonatal lethality. Mutant mice revealed acanthosis and hyperkeratosis as well as abnormal keratinocyte differentiation and increased proliferation. In addition, theepidermal barrier was severely impaired. These results indicate an essential role of SNAP29 in epidermal differentiation and barrier formation. Markedly decreased deposition of lamellar body contents in mutant mice epidermis and the observation of malformed lamellar bodies indicate severe impairments in lamellar body function due to the Snap29 knockout. We also found increased microtubule associated protein-1 light chain 3, isoform B-II levels, unchanged p62/SQSTM1 protein amounts, and strong induction of the endoplasmic reticulum stress marker C/EBP homologous protein in mutant mice. This emphasizes a role of SNAP29 in autophagy and endoplasmic reticulum stress. Our murine models serve as powerful tools for investigating keratinocyte differentiation processes and provide insights into the essential contribution of SNAP29 to epidermal differentiation.

Caenorhabditis elegansSNAP-29 is required for organellar integrity of the endomembrane system and general exocytosis in intestinal epithelial cells

It is generally accepted that soluble N-ethylmaleimide-sensitive factor attachment protein receptors mediate the docking and fusion of transport intermediates with target membranes. Our research identifies Caenorhabditis elegans homologue of synaptosomal-associated protein 29 (SNAP-29) as an essential regulator of membrane trafficking in polarized intestinal cells of living animals. We show that a depletion of SNAP-29 blocks yolk secretion and targeting of apical and basolateral plasma membrane proteins in the intestinal cells and results in a strong accumulation of small cargo-containing vesicles. The loss of SNAP-29 also blocks the transport of yolk receptor RME-2 to the plasma membrane in nonpolarized oocytes, indicating that its function is required in various cell types. SNAP-29 is essential for embryogenesis, animal growth, and viability. Functional fluorescent protein-tagged SNAP-29 mainly localizes to the plasma membrane and the late Golgi, although it also partially colocalizes with endosomal proteins. The loss of SNAP-29 leads to the vesiculation/fragmentation of the Golgi and endosomes, suggesting that SNAP-29 is involved in multiple transport pathways between the exocytic and endocytic organelles. These observations also suggest that organelles comprising the endomembrane system are highly dynamic structures based on the balance between membrane budding and fusion and that SNAP-29-mediated fusion is required to maintain proper organellar morphology and functions.