SNAP‐23 is not essential for constitutive exocytosis in HeLa cells

Okayama, Miki; Arakawa, Toshiya; Mizoguchi, Itaru; Tajima, Yoshifumi; Teratani, Takuma

doi:10.1016/j.febslet.2007.08.046

Cited by 18 publications

(12 citation statements)

References 29 publications

(68 reference statements)

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“…Furthermore, MDA-MB-436 with high AnxA6 levels showed much less LPS-inducible TNF-α secretion compared with MDA-MB-468 cells (Figure 10D). It should be noted that Okayama and coworkers determined exocytosis of secreted alkaline phosphatase (SEAP) to remain unchanged upon RNA interference knockdown of SNAP23 and syntaxin-4 (Okayama et al , 2007). Incomplete gene silencing; compensation through other SNAREs; different combinations of SNAREs being required for SEAP and FN/cytokine release, possibly along multiple and different intracellular routes; and AnxA6-induced changes that go beyond alterations in cholesterol homeostasis could explain these differences.…”

Section: Resultsmentioning

confidence: 99%

Cholesterol transport from late endosomes to the Golgi regulates t-SNARE trafficking, assembly, and function

Reverter¹,

Rentero²,

Muga³

et al. 2011

Molecular Biology of the Cell

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This study shows that impaired cholesterol egress from late endosomes in cells with high annexin A6 levels is associated with altered soluble N-ethylmaleimide–sensitive fusion protein 23 (SNAP23) and syntaxin-4 cellular distribution and assembly and accumulation in Golgi membranes. This correlates with reduced secretion of cargo along the constitutive and SNAP23/syntaxin-4–dependent secretory pathway.

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Section: Resultsmentioning

confidence: 99%

Cholesterol transport from late endosomes to the Golgi regulates t-SNARE trafficking, assembly, and function

Reverter¹,

Rentero²,

Muga³

et al. 2011

Molecular Biology of the Cell

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“…Whereas a role for SNAP23 in constitutive exocytosis is not apparent (25), SNAP23 contributes to regulated exocytosis (26). Transfection of approximately 60% of a HeLa cell population with LC/E(K 224 D) resulted in the cleavage of approximately 45% of the SNAP23, whereas SNAP23 cleavage was not detected when HeLa cells were transfected with Wt-LC/E or a no plasmid control (Fig.…”

Section: Resultsmentioning

confidence: 99%

Engineering botulinum neurotoxin to extend therapeutic intervention

Chen

Barbieri

2009

Proc. Natl. Acad. Sci. U.S.A.

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Clostridium botulinum neurotoxins (BoNTs) are effective therapeutics for a variety of neurological disorders, such as strabismus, blepharospam, hemificial spasm, and cervical dystonia, because of the toxin's tropism for neurons and specific cleavage of neuronal soluble N-ethylmaleimide-sensitive fusion protein-attachment protein receptors (SNARE) proteins. Modifying BoNT to bind nonneuronal cells has been attempted to extend therapeutic applications. However, prerequisite to develop nonneuronal therapies requires the retargeting the catalytic activity of BoNTs to nonneuronal SNARE isoforms. Here, we reported the engineering of a BoNT derivative that cleaves SNAP23, a nonneuronal SNARE protein. SNAP23 mediates vesicle-plasma membrane fusion processes, including secretion of airway mucus, antibody, insulin, gastric acids, and ions. This mutated BoNT/E light chain LC/E(K 224 D) showed extended substrate specificity to cleave SNAP23, and the natural substrate, SNAP25, but not SNAP29 or SNAP47. Upon direct protein delivery into cultured human epithelial cells, LC/E(K 224 D) cleaved endogenous SNAP23, which inhibited secretion of mucin and IL-8. These studies show the feasibility of genetically modifying LCs to target a nonneuronal SNARE protein that extends therapeutic potential for treatment of human hypersecretion diseases.Clostridium botulinum neurotoxins (BoNTs) are the most potent protein toxins for humans (1). BoNTs elicit neuronal-specific flaccid paralysis by targeting neurons and cleaving neuronspecific soluble N-ethylmaleimide-sensitive fusion proteinattachment protein receptors (SNARE) proteins. BoNTs are organized into 3 functional domains: an N-terminal zinc-metalloprotease light chain (LC), a translocation domain (HCT), and a C-terminal receptor binding domain (HCR) (1, 2). BoNTs bind luminal domains of synaptic vesicle proteins, upon the fusion of synaptic vesicles with the plasma membrane (3-5). BoNTs are internalized into endosomes and upon acidification, the LC is translocated into the cytoplasm, where SNARE proteins are cleaved (1, 2).Mammalian neuronal exocytosis is driven by the formation of protein complexes between the vesicle SNARE, VAMP2, and the plasma membrane SNAREs, SNAP25 and syntaxin 1a (6). There are 7 serotypes of BoNTs (termed A-G) that cleave specific residues on 1 of 3 SNARE proteins: serotypes B, D, F, and G cleave VAMP-2, serotypes A and E cleave SNAP25, and serotype C cleaves SNAP25 and syntaxin 1a (1). Thus, neuronal specificity is based upon BoNT binding to neurons and cleaving neuronal isoforms of the SNARE proteins. For example, BoNT/A cleaves human SNAP25, but not the human nonneuronal isoform SNAP23 (7,8). The nonneuronal SNARE isoforms are involved in divergent cellular processes, including fusion reactions in cell growth, membrane repair, cytokinesis, and synaptic transmission (reviewed in 9).The reversible nature of muscle function after BoNT intoxication that replace toxin-affected nerves with new nerves (10) has turned the BoNT from a deadly agent to therapies for neu...

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“…MRC5 fibroblasts were transfected with siRNA (10 nM) directed against SNAP-23 (5′CCAACAGAGAUCGUAUUGATT3′) (30), an siRNA specific for IE-2 (5′AAACGCAUCUCCGAGUUGGACTT3′) (58), or the Mission siRNA Universal Negative Control #1 (Sigma-Aldrich) by using Oligofectamine (Invitrogen) in Optimem (Gibco). After 6 h, transfection reagent was removed and replaced with medium containing 10% FBS.…”

Section: Methodsmentioning

confidence: 99%

“…The functional significance of the relocalization was probed by performing a knockdown experiment with siRNA against SNAP- 23 (30). A Western blot showed a substantial decrease in SNAP-23 levels relative to the nontargeting control using β-actin for normalization, and the cells remained competent to express the IE1 protein after infection with HCMV (Fig.…”

Section: Virion Glycerophospholipid Composition Is Different From Thamentioning

confidence: 99%

Synaptic vesicle-like lipidome of human cytomegalovirus virions reveals a role for SNARE machinery in virion egress

Liu¹,

Sharon-Friling²,

Ivanova

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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Human cytomegalovirus induces and requires fatty acid synthesis. This suggests an essential role for lipidome remodeling in viral replication. We used mass spectrometry to quantify glycerophospholipids in mock-infected and virus-infected fibroblasts, as well as in virions. Although the lipid composition of mock-infected and virus-infected fibroblasts was similar, virions were markedly different. The virion envelope contained twofold more phosphatidylethanolamines and threefold less phosphatidylserines than the host cell. This indicates that the virus buds from a membrane with a different lipid composition from the host cell as a whole. Compared with published datasets, the virion envelope showed the greatest similarity to the synaptic vesicle lipidome. Synaptosomeassociated protein of 25 kDa (SNAP-25) is a component of the complex that mediates exocytosis of synaptic vesicles in neurons; and its homolog, SNAP-23, functions in exocytosis in many other cell types. Infection induced the relocation of SNAP-23 to the cytoplasmic viral assembly zone, and knockdown of SNAP-23 inhibited the production of virus. We propose that cytomegalovirus capsids acquire their envelope by budding into vesicles with a lipid composition similar to that of synaptic vesicles, which subsequently fuse with the plasma membrane to release virions from the cell.H uman cytomegalovirus (HCMV) is a member of the enveloped herpesvirus family. It is a widespread pathogen that can cause disease in immunologically immature or compromised individuals (1). HCMV infection substantially alters the metabolism of cultured fibroblasts (2), inducing a marked increase in the flux of carbon from glucose into fatty acids (3). Consistent with the enhanced flux, inhibition of acetyl-CoA carboxylase, the first committed enzyme in the fatty acid biosynthetic pathway, reduces the yield of infectious virus. Thus, lipid synthesis is required during the HCMV replication cycle. Newly synthesized membranes likely accumulate in the assembly compartment (4-8), a virus-induced organelle with virus-coded proteins and cellular proteins that include markers of the exocytic and endocytic networks. This is the site at which viral capsids acquire an envelope (9-12).Glycerophospholipids are major components of cellular membranes. This family of lipids consists of a glycerol backbone, a functional head group, and two fatty acids of varying length and unsaturation. Phosphatidic acid (PA) serves as the basic glycerophospholipid building block, with its phosphate group esterified to a head group to produce phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidylserine (PS), phosphatidylglycerol (PG), or diphosphatidylglycerol. By using mass spectrometry, it is possible to identify and quantify numerous glycerolphospholipid species, differing in head group, acyl chain length, and degree of unsaturation (13-16). This quantitative lipidomics approach has been used to generate detailed descriptions of several viral envelopes, including those acqui...

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SNAP‐23 is not essential for constitutive exocytosis in HeLa cells

Cited by 18 publications

References 29 publications

Cholesterol transport from late endosomes to the Golgi regulates t-SNARE trafficking, assembly, and function

Cholesterol transport from late endosomes to the Golgi regulates t-SNARE trafficking, assembly, and function

Engineering botulinum neurotoxin to extend therapeutic intervention

Synaptic vesicle-like lipidome of human cytomegalovirus virions reveals a role for SNARE machinery in virion egress

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