Effect of siRNA for SNARE proteins on exocytosis from salivary glands

SNARE 蛋白的 siRNA 对唾液腺胞吐作用的影响

• 批准号: 17591945
• 负责人: TAKUMA Taishin
• 金额: $ 2.24万
• 依托单位: Health Sciences University of Hokkaido
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2005
• 资助国家: 日本
• 起止时间: 2005 至 2006
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-17591945/
• 关键词: siRNA; exocytosis; SNARE protein; salivary gland; gene knockdown; SNAP-23; dominant-negative variant

SNAP-23, a non-neuronal isoform of SNAP-25 with 2 tandem SNARE motifs (Qb and Qc), is believed to play a key role as a t-SNARE for both regulated and constitutive exocytosis in most cells that do not express SNAP-25. Here we evaluated the role of SNAP-23 in constitutive exocytotic pathways of HeLa cells. Although the level of SNAP-23 was reduced to less than 10% of the control value by gene silencing with siRNA directed against SNAP-23, exocytosis was normal when transiently expressed SEAP (secreted alkaline phosphatase) was measured as a marker of constitutive secretion. Double knockdown of SNAP-23 and syntaxin-4 also failed to inhibit the secretion. Furthermore, over-expression of δC8-SNAP-23, a dominant-negative SNAP-23, did not abrogate SEAP secretion. When the secretory pathway was visualized by the expression of GFP-tagged human growth hormone (hGH-GFP), however, the accumulation of vesicles containing hGH-GFP was observed in the peripheral plasma membrane of the cells that over-expressed δC8-SNAP-23, although the quantity of hGH-GFP release was not decreased as in the case of SEAP secretion. These results suggest that SNAP-23 is involved in constitutive exocytosis, but is not essential at least for simple exocytosis of secretory proteins.

SNAP-23是SNAP-25的非神经元异构体，具有2个串联SNARE基序（Qb和Qc），被认为在大多数不表达SNAP-25的细胞中，作为t-SNARE在调节性和构成性胞外分泌中发挥关键作用。在这里，我们评估了SNAP-23在HeLa细胞组成胞吐通路中的作用。虽然通过siRNA对SNAP-23进行基因沉默，SNAP-23的水平降低到控制值的10%以下，但当测量瞬时表达的SEAP（分泌的碱性磷酸酶）作为组成分泌的标志时，胞吐正常。SNAP-23和syntaxin-4的双敲低也未能抑制其分泌。此外，过表达δC8-SNAP-23（显性阴性SNAP-23）并没有消除SEAP的分泌。然而，当通过表达gfp标记的人生长激素（hGH-GFP）来观察分泌途径时，在过表达δC8-SNAP-23的细胞的外周质膜上观察到含有hGH-GFP的囊泡的积累，尽管hGH-GFP的释放量不像分泌SEAP的情况下减少。这些结果表明SNAP-23参与构成性胞吐，但至少对分泌蛋白的简单胞吐不是必需的。

项目成果

Role of VAMP-2, VAMP-7, and VAMP-8 in constitutive exocytosis from HSY cells.

VAMP-2、VAMP-7 和 VAMP-8 在 HSY 细胞组成型胞吐作用中的作用。

• 发表时间: 2006
• 期刊: Histochem. Cell Biol. 125
• 作者: Oishi Y;Arakawa T;Tanimura A;Itakura M;Takahashi M;Tajima Y;Mizoguchi I;Takuma T
• 通讯作者: Takuma T

Role of VAMP-2,VAMP-8 in constitutive exocytosis from HSY cells.

VAMP-2、VAMP-8 在 HSY 细胞组成型胞吐作用中的作用。

• 发表时间: 2006
• 期刊: Histochem. Cell Biol. 125
• 作者: Oishi Y;Arakawa T;Tanimura A;Itakura M;Takahashi M;Tajima Y;Mizoguchi I;Takuma T
• 通讯作者: Takuma T