SM protein Munc18-2 facilitates transition of Syntaxin 11-mediated lipid mixing to complete fusion for T-lymphocyte cytotoxicity

Abstract

The atypical lipid-anchored Syntaxin 11 (STX11) and its binding partner, the Sec/Munc (SM) protein Munc18-2, facilitate cytolytic granule release by cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells. Patients carrying mutations in these genes develop familial hemophagocytic lymphohistiocytosis, a primary immunodeficiency characterized by impaired lytic granule exocytosis. However, whether a SNARE such as STX11, which lacks a transmembrane domain, can support membrane fusion in vivo is uncertain, as is the precise role of Munc18-2 during lytic granule exocytosis. Here, using a reconstituted "flipped" cell-cell fusion assay, we show that lipidanchored STX11 and its cognate SNARE proteins mainly support exchange of lipids but not cytoplasmic content between cells, resembling hemifusion. Strikingly, complete fusion is stimulated by addition of wild-type Munc18-2 to the assay, but not of Munc18-2 mutants with abnormal STX11 binding. Our data reveal that Munc18-2 is not just a chaperone of STX11 but also directly contributes to complete membrane merging by promoting SNARE complex assembly. These results further support the concept that SM proteins in general are part of the core fusion machinery. This fusion mechanism likely contributes to other cell-type-specific exocytic processes such as platelet secretion.SNARE | Syntaxin 11 | Munc18-2 | CTL | SM protein

Syntaxin 4 mediates endosome recycling for lytic granule exocytosis in cytotoxic T‐lymphocytes

“…STX4 siRNA‐treated cells exhibited over 60% reduction in target cell‐killing activity compared with those treated with NT‐siRNAs (Figure C). The reduction in cytotoxic activity of STX4 knockdown cells was comparable to the STX11 siRNA‐treated cells, which were previously shown to have reduced killing activity because of impairment in LG exocytosis (Figure C). On the contrary, STX3 siRNA‐treated cells, which also exhibited almost a 70% reduction in protein expression levels (Figure D), showed a slight increase in cytotoxic activity compared with NT siRNA‐treated cells (Figure C).…”

“…CTLs express several syntaxins that belong to the PM Qa‐SNAREs sub‐group—STX3, STX4 and STX11 . Previous studies have shown that STX11, but not STX3 is required for CTL‐mediated cytotoxicity. However, it has not been investigated yet whether STX4 is also implicated in this pathway.…”

“…Subjects harboring germline‐mutations in these genes manifest with familial hemophagocytic lymphohisticytosis (F‐HLH‐4 and F‐HLH‐5, respectively), a life‐threatening primary immunodeficiency that results in hyperinflammation because of an inability of the immune system to clear out viral infections or tumor cells . Current evidences suggest that STX11 and Munc18‐2 physically interact in multiple ways to drive the final fusion of the LG at the IS . On the other hand, recent studies propose that VAMP8‐mediated fusion of Rab11a‐positive recycling endosomes (Rab11a‐RE) at the IS is essential for CTL killing activity .…”

The road to lysosome‐related organelles: Insights from Hermansky‐Pudlak syndrome and other rare diseases

“…Whereas the gene underlying FHL1 has not been identified, and mutations in the gene encoding the cytolytic effector perforin underlie FHL2, FHL3‐5 are due to mutations in genes encoding the machinery required for secretion of lytic granules, platelet alpha and dense granules and other regulated secretory organelles—specifically UNC13D (encoding munc13‐4, mutated in FHL3), STX11 (encoding syntaxin‐11, mutated in FHL4) and STXBP2 (encoding munc18‐2, mutated in FHL5). STX11 is a Qa SNARE that is part of the tSNARE complex at the plasma membrane required for cytolytic granule and platelet granule secretion . Munc18‐2 is a SM family member that specifically stabilizes STX11 in platelets and CTLs and is required for efficient STX11 expression at the plasma membrane and for granule fusion .…”

“…Munc18‐2 is a SM family member that specifically stabilizes STX11 in platelets and CTLs and is required for efficient STX11 expression at the plasma membrane and for granule fusion . It is particularly critical to stabilize full fusion intermediates mediated by the lipid‐anchored STX11 . Consistent with its function, a number of FHL5 mutations disrupt the binding sites on munc18‐2 for STX11 or αSNAP or impede release of the N‐terminal STX11 peptide and thus block subsequent munc18‐2 function in facilitating SNARE complex assembly .…”