STXBP2 mutations in children with familial haemophagocytic lymphohistiocytosis type 5

The function of the Munc18-1 protein hydrophobic pocket, which interacts with the syntaxin-1 N-terminal peptide, has been highly controversial in neurosecretion. Recent analysis of patients with familial hemophagocytic lymphohistiocytosis type 5 has identified the E132A mutation in the hydrophobic pocket of Munc18-2, prompting us to examine the role of this region in the context of immune cell secretion. Double knockdown of Munc18-1 and Munc18-2 in RBL-2H3 mast cells eliminates both IgE-dependent and ionomycin-induced degranulation and causes a significant reduction in syntaxin-11 without altering expressions of the other syntaxin isoforms examined. These phenotypes were effectively rescued on reexpression of wild-type Munc18-1 or Munc18-2 but not the mutants (F115E, E132A, and F115E/E132A) in the hydrophobic pocket of Munc18. In addition, these mutants show that they are unable to directly interact with syntaxin-11, as tested through protein interaction experiments. Our results demonstrate the crucial roles of the hydrophobic pocket of Munc18 in mast cell degranulation, which include the regulation of syntaxin-11. We also suggest that the functional importance of this region is significantly different between neuronal and immune cell exocytosis.membrane fusion | FHL5

Section: Munc18-1 and Munc18-2 Can Effectively Support Mast Cell Degrsupporting

confidence: 83%

“…Analysis of Munc18-2 in patients with FHL5 has identified an E132A mutation within the hydrophobic pocket region of Munc18-2 (33). We hypothesize that the hydrophobic pocket of Munc18-2 is indeed crucial for immune cell secretion and tested this hypothesis through analyses of the stable KD and rescue of Munc18 in mast cell model RBL-2H3 cells.…”

mentioning

confidence: 99%

Crucial role of the hydrophobic pocket region of Munc18 protein in mast cell degranulation

Bin

Jung

Piggott

et al. 2013

“…SM proteins exhibit a similar loss-of-function phenotype as that of SNAREs (i.e., abrogation of fusion) and are essential for every pathway of intracellular vesicle fusion (14)(15)(16). Mutations of SM proteins give rise to a number of human diseases, including epilepsy and inflammatory disorders, as well as arthrogryposis, renal dysfunction, and cholestasis (ARC) syndrome (17)(18)(19)(20)(21). Although the mechanism of SNAREs is well established, we are only beginning to understand how SM proteins regulate vesicle fusion.…”

mentioning

confidence: 99%

Comparative studies of Munc18c and Munc18-1 reveal conserved and divergent mechanisms of Sec1/Munc18 proteins

Rathore

Lopez

et al. 2013

149

Sec1/Munc18 (SM) family proteins are essential for every vesicle fusion pathway. The best-characterized SM protein is the synaptic factor Munc18-1, but it remains unclear whether its functions represent conserved mechanisms of SM proteins or specialized activities in neurotransmitter release. To address this question, we dissected Munc18c, a functionally distinct SM protein involved in nonsynaptic exocytic pathways. We discovered that Munc18c binds to the trans-SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complex and strongly accelerates the fusion rate. Further analysis suggests that Munc18c recognizes both vesicle-rooted SNARE and target membrane-associated SNAREs, and promotes trans-SNARE zippering at the postdocking stage of the fusion reaction. The stimulation of fusion by Munc18c is specific to its cognate SNARE isoforms. Because Munc18-1 regulates fusion in a similar manner, we conclude that one conserved function of SM proteins is to bind their cognate trans-SNARE complexes and accelerate fusion kinetics. Munc18c also binds syntaxin-4 monomer but does not block target membrane-associated SNARE assembly, in agreement with our observation that six-to eightfold increases in Munc18c expression do not inhibit insulinstimulated glucose uptake in adipocytes. Thus, the inhibitory "closed" syntaxin binding mode demonstrated for Munc18-1 is not conserved in Munc18c. Unexpectedly, we found that Munc18c recognizes the N-terminal region of the vesicle-rooted SNARE, whereas Munc18-1 requires the C-terminal sequences, suggesting that the architecture of the SNARE/SM complex likely differs across fusion pathways. Together, these comparative studies of two distinct SM proteins reveal conserved as well as divergent mechanisms of SM family proteins in intracellular vesicle fusion. membrane fusion | vesicle transport | exocytosis T he fusion of intracellular vesicles with target membranes requires two classes of conserved proteins: SNAREs and SM (Sec1/Munc18) proteins (1, 2). SNAREs are membrane-associated proteins that contain characteristic stretches of 60-70 amino acids known as core domains or SNARE motifs. Fusion is initiated when the core domains of the vesicle-rooted SNARE (v-SNARE) and the target membrane-associated SNAREs (t-SNAREs) zipper into a four-helix trans-SNARE complex between two apposed bilayers (2-5). N-to C-terminal zippering of the trans-SNARE complex brings the two membranes into close apposition to fuse (6-8).First isolated in genetic screens in yeast and nematodes (9, 10), SM proteins are hydrophilic factors of 60-70 kDa that regulate membrane fusion through binding to their cognate SNAREs (11-13). SM proteins exhibit a similar loss-of-function phenotype as that of SNAREs (i.e., abrogation of fusion) and are essential for every pathway of intracellular vesicle fusion (14-16). Mutations of SM proteins give rise to a number of human diseases, including epilepsy and inflammatory disorders, as well as arthrogryposis, renal dysfunction, and cholestasis (ARC) syndrome (17-...

“…4A and previous work suggested that E132 forms a salt bridge to R4 of the bound syntaxin. We decided to concentrate on E132A, as the mutation was identified in a homozygous patient with "full-blown" hemophagocytic lymphohistiocytosis (43), and tested if, and to what extent, mutation E132A would affect syntaxin binding.…”

mentioning

confidence: 99%

Syntaxin binding mechanism and disease-causing mutations in Munc18-2

Hackmann

Graham

Ehl³

et al. 2013

Significance Understanding the molecular mechanisms that control secretion from cytotoxic T lymphocytes (CTL) and natural killer (NK) cells is the key for understanding how these cells destroy virally infected and tumourigenic cells. Precisely how mutations in Munc18-2 and syntaxin 11 (Stx11) give rise to loss of CTL and NK function and severe immunodeficiency is poorly understood. In this study we present a crystal structure of human Munc18-2 and analyze the disease-causing mutations. Our findings reveal a mechanism that allows Munc18-2 to selectively bind Stx11 and identify potential surrogate binding partners, which could restore Munc18-Stx function upon IL-2 activation.