Botulinum E Toxin Light Chain Does Not Cleave SNAP-23 and Only Partially Impairs Insulin Stimulation of GLUT4 Translocation in 3T3-L1 Cells

Macaulay, S. Lance; Rea, Shane L.; Gough, K. H.; Ward, Colin W.; James, David E.

doi:10.1006/bbrc.1997.7143

Cited by 21 publications

(5 citation statements)

References 40 publications

(13 reference statements)

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“…SNAP23 is able to replace SNAP25 and rescue insulin secretion in BoNT/E-treated HIT cells (23). Murine SNAP23 is naturally toxin-resistant (24). We recently generated SNAP23 mutants displaying a decreased raft affinity compared with the wild-type protein because of the mutation of cysteines at amino acid positions 79 or 83 to phenylalanine (Fig.…”

Section: Resultsmentioning

confidence: 99%

Lipid Raft Association of SNARE Proteins Regulates Exocytosis in PC12 Cells

Salaün

Gould

Chamberlain

2005

Journal of Biological Chemistry

127

115

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SNAP25 and SNAP23 are plasma membrane SNARE proteins essential for regulated exocytosis in diverse cell types. Several recent studies have shown that these proteins are partly localized in lipid rafts, domains of the plasma membrane enriched in sphingolipids, and cholesterol. Here, we have employed cysteine mutants of SNAP25/SNAP23, which have modified affinities for raft domains, to examine whether raft association of these proteins is important for the regulation of exocytosis. PC12 cells were engineered that express the light chain of botulinum neurotoxin; in these cells all of the SNAP25 was cleaved to a lower molecular weight form, and regulated exocytosis was essentially absent. Exocytosis was rescued by expressing toxin-resistant SNAP25 or wildtype SNAP23, which is naturally toxin-resistant. Remarkably, a mutant SNAP25 protein with an increased affinity for rafts displayed a reduced ability to support exocytosis, whereas SNAP23 mutants with a decreased affinity for rafts displayed an enhancement of exocytosis when compared with wild-type SNAP23. The effects of the mutant proteins on exocytosis were dependent upon the integrity of the plasma membrane and lipid rafts. These results provide the first direct evidence that rafts regulate SNARE function and exocytosis and identify the central cysteine-rich region of SNAP25/23 as an important regulatory domain.Exocytosis, the fusion of intracellular vesicles with the plasma membrane, mediates the secretion of molecules from the cell and the insertion of proteins and lipids into the plasma membrane. This membrane fusion event needs to be tightly regulated when vesicles contain molecules such as neurotransmitters, adrenaline, or insulin. A large number of proteins have been identified that function in exocytosis (1, 2). Among these, SNARE 1 proteins have emerged as potential membrane fusion catalysts (3, 4). Membrane fusion requires the interaction of Q-SNAREs present on the plasma membrane with R-SNAREs residing on the vesicle membrane. In neuronal and neuroendocrine cells, the Q-SNAREs that function in regulated exocytosis are syntaxin 1 and SNAP25, whereas the R-SNARE is VAMP/ synaptobrevin (5).Recently, there has been significant interest in the domain distribution of Q-SNAREs present at the plasma membrane. A number of studies have suggested that Q-SNAREs are partly localized in lipid rafts, lipid microdomains in the plasma membrane enriched in sphingolipids, and cholesterol (6 -14). As rafts have been proposed to function in the regulation of numerous signal transduction (15) and membrane traffic pathways (16), these observations raise the intriguing possibility that rafts may regulate SNARE function and, hence, exocytosis. As yet, the importance of the raft association of SNARE proteins for exocytosis has not been examined.Recent work from our group has reported that raft association of SNAP25 and its ubiquitous homologue, SNAP23, is mediated by the cysteine-rich domains of these proteins (17). We identified mutations within SNAP23 that decreased its raft a...

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Section: Resultsmentioning

confidence: 99%

Lipid Raft Association of SNARE Proteins Regulates Exocytosis in PC12 Cells

Salaün

Gould

Chamberlain

2005

Journal of Biological Chemistry

127

115

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“…Synaptobrevin, SNAP-25 and syntaxin forms a 1:1:1 stoichiometric complex. Interaction between vesicle-associated SNAREs [(v-SNARE), soluble NSF (N-ethylmaleimide-sensitive factor) attachment receptor] and target-associated SNAREs (t-SNARE) forms a high affinity complex [36][37][38][39][40][41][42].…”

Section: Mechanism Of Actionmentioning

confidence: 99%

Treating Benign Prostatic Hyperplasia with Botulinum Neurotoxin

Brisinda

Vanella²,

Marniga³

et al. 2011

CMC

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Botulinum toxin (BoNT) has been increasingly used in the interventional treatment of several disorders; the use of this agent has extended to a plethora of conditions including focal dystonia, spasticity, inappropriate contraction in most gastrointestinal sphincters, eye movement disorders, hyperhidrosis, genitourinary disorders and aesthetically undesirable hyperfunctional facial lines. In addition, BoNT is being investigated for the control of pain, and for the management of tension or migraine headaches and myofascial pain syndrome. Benign prostatic hyperplasia (BPH) is a common condition in ageing men; the goal of therapy is to reduce the lower urinary tract symptoms (LUTS) associated with BPH and to improve the quality of life. However, medical treatment, including drugs that relax smooth muscle within the prostate and drugs that shrink the gland are not totally effective or without complications. The standard surgical treatment for BPH is progressively changing to minimally invasive therapies, but none of them has provided clear results. The use of BoNT-A to inhibit the autonomic efferent effects on prostate growth and contraction, and inhibit the abnormal afferent effects on prostate sensation, might be an alternative treatment for BPH. BoNT injections have several advantages over drugs and surgical therapies in the management of intractable or chronic disease; systemic pharmacologic effects are rare, permanent destruction of tissue does not occur, and graded degrees of relaxation may be achieved by varying the dose injected. In this paper, clinical experience over the last years with BoNT in BPH impaired patients will be illustrated.

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“…It has been reported that hSNAP‐23 was resistant to the action of BoNT/E (Macaulay et al, 1997). In a similar manner, mSNAP‐23 was shown to resist moderate concentrations of BoNT/A and C (Chen et al, 1997) or BoNT/E (Macaulay et al, 1997). In contrast, Washbourne et al (1997) reported that mSNAP‐23 was cleaved by BoNT/E.…”

Section: Murine and Human Snap‐25 Isoforms Display Distinct Sensitivimentioning

confidence: 99%

Proteolysis of SNAP‐25 Isoforms by Botulinum Neurotoxin Types A, C, and E

Vaidyanathan¹,

Yoshino²,

Jahnz³

et al. 1999

Journal of Neurochemistry

190

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Abstract:Tetanus toxin and the seven serologically distinct botulinal neurotoxins (BoNT/A to BoNT/G) abrogate synaptic transmission at nerve endings through the action of their light chains (L chains), which proteolytically cleave VAMP (vesicle-associated membrane protein)/ synaptobrevin, SNAP-25 (synaptosome-associated protein of 25 kDa), or syntaxin. BoNT/C was reported to proteolyze both syntaxin and SNAP-25. Clostridia produce several powerful neurotoxins; tetanus toxin and botulinal neurotoxins BoNT/A to BoNT/G cause the clinical manifestations of tetanus and botulism in a large variety of animal species and humans. The toxins are synthesized as single-chain polypeptides with molar masses of ϳ150 kDa. On lysis of the bacteria and activation by proteolytic cleavage, the light chains (L chains; 50 kDa) remain disulfide bound to the heavy chains (H chains; 100 kDa). The extreme neurotoxicity is largely ascribed to the H chains, which bind to neuronal receptors that internalize the holotoxins, and translocate the L chains into the cytosol. Here, the L chains block fusion of synaptic vesicles with the presynaptic membrane (Simpson, 1989).The genes of the eight known clostridial neurotoxins have been cloned and characterized (for review, see Niemann et al., 1994). The L chains contain a Zn 2ϩ -binding motif, His-Glu-X-X-His, also found in an increasing number of zinc-dependent metalloproteases (Jongeneel et al., 1989). Soon after demonstration of cleavage of VAMP (vesicle-associated membrane protein)/synaptobrevin (Trimble et al., 1988) by tetanus toxin and BoNT/B at the same peptide bond (Link et al., 1992;Schiavo et al., 1992), substrates and scissile bonds were identified for all other botulinum serotypes. These studies revealed that BoNT/D, BoNT/F, and BoNT/G also hydrolyze synaptobrevin, although each at a different peptide bond. BoNT/A and BoNT/E cleave SNAP-25 (synaptosome-associated protein of 25 kDa), again at distinct sites close to the C-terminus (for review, see Received March 20, 1998; revised manuscript received July 20, 1998; accepted July 23, 1998. Address correspondence and reprint requests to Dr. T. Binz at Department of Biochemistry, Medizinische Hochschule Hannover, Carl-Neuberg-Straße 1, D-30623, Hannover, Germany.The present address of Dr. V. V. Vaidyanathan is Experimental Immunology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, U.S.A.Abbreviations used: BoNT, botulinal neurotoxin; GST, glutathione S-transferase; GT, glutathione; L chain, light chain; NSF, Nethylmaleimide sensitive fusion protein; PAGE, polyacrylamide gel electrophoresis; SDS, sodium dodecyl sulfate; SNAP, soluble NSF attachment protein; SNAP-23, 23-kDa isoform of SNAP-25; SNAP-25, synaptosome-associated protein of 25 kDa; hSNAP-23 and mSNAP-23, human and murine SNAP-23, respectively; SNARE, soluble NSF attachment protein receptor; VAMP, vesicle-associated membrane protein.

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Botulinum E Toxin Light Chain Does Not Cleave SNAP-23 and Only Partially Impairs Insulin Stimulation of GLUT4 Translocation in 3T3-L1 Cells

Cited by 21 publications

References 40 publications

Lipid Raft Association of SNARE Proteins Regulates Exocytosis in PC12 Cells

Lipid Raft Association of SNARE Proteins Regulates Exocytosis in PC12 Cells

Treating Benign Prostatic Hyperplasia with Botulinum Neurotoxin

Proteolysis of SNAP‐25 Isoforms by Botulinum Neurotoxin Types A, C, and E

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