Autophagy ( IF 13.3 ) Pub Date : 2020-12-07 , DOI: 10.1080/15548627.2020.1852725 Subhankar Dolai 1 , Toshimasa Takahashi 2 , Tairan Qin 2 , Tao Liang 2 , Li Xie 2 , Fei Kang 2 , Yi-Fan Miao 2 , Huanli Xie 2 , Youhou Kang 2 , Justin Manuel 1 , Erin Winter 1 , Paul A Roche 3 , Mark S Cattral 1 , Herbert Y Gaisano 1, 2
ABSTRACT
Intrapancreatic trypsin activation by dysregulated macroautophagy/autophagy and pathological exocytosis of zymogen granules (ZGs), along with activation of inhibitor of NFKB/NF-κB kinase (IKK) are necessary early cellular events in pancreatitis. How these three pancreatitis events are linked is unclear. We investigated how SNAP23 orchestrates these events leading to pancreatic acinar injury. SNAP23 depletion was by knockdown (SNAP23-KD) effected by adenovirus-shRNA (Ad-SNAP23-shRNA/mCherry) treatment of rodent and human pancreatic slices and in vivo by infusion into rat pancreatic duct. In vitro pancreatitis induction by supraphysiological cholecystokinin (CCK) or ethanol plus low-dose CCK were used to assess SNAP23-KD effects on exocytosis and autophagy. Pancreatitis stimuli resulted in SNAP23 translocation from its native location at the plasma membrane to autophagosomes, where SNAP23 would bind and regulate STX17 (syntaxin17) SNARE complex-mediated autophagosome-lysosome fusion. This SNAP23 relocation was attributed to IKBKB/IKKβ-mediated SNAP23 phosphorylation at Ser95 Ser120 in rat and Ser120 in human, which was blocked by IKBKB/IKKβ inhibitors, and confirmed by the inability of IKBKB/IKKβ phosphorylation-disabled SNAP23 mutant (Ser95A Ser120A) to bind STX17 SNARE complex. SNAP23-KD impaired the assembly of STX4-driven basolateral exocytotic SNARE complex and STX17-driven SNARE complex, causing respective reduction of basolateral exocytosis of ZGs and autolysosome formation, with consequent reduction in trypsinogen activation in both compartments. Consequently, pancreatic SNAP23-KD rats were protected from caerulein and alcoholic pancreatitis. This study revealed the roles of SNAP23 in mediating pathological basolateral exocytosis and IKBKB/IKKβ’s involvement in autolysosome formation, both where trypsinogen activation would occur to cause pancreatitis. SNAP23 is a strong candidate to target for pancreatitis therapy.
Abbreviations: AL: autolysosome; AP: acute pancreatitis; AV: autophagic vacuole; CCK: cholecystokinin; IKBKB/IKKβ: inhibitor of nuclear factor kappa B kinase subunit beta; SNAP23: synaptosome associated protein 23; SNARE: soluble NSF (N-ethylmaleimide-sensitive factor) attachment protein receptor; STX: syntaxin; TAP: trypsinogen activation peptide; VAMP: vesicle associated membrane protein; ZG: zymogen granule.
中文翻译:
胰腺特异性 SNAP23 消耗通过减弱病理性基底外侧胞吐作用和胰蛋白酶激活自溶酶体的形成来预防胰腺炎
摘要
通过失调的巨自噬/自噬和酶原颗粒 (ZGs) 的病理性胞吐作用激活胰腺内胰蛋白酶,以及激活 NFKB/NF-κB 激酶 (IKK) 抑制剂是胰腺炎中必要的早期细胞事件。这三种胰腺炎事件如何联系尚不清楚。我们研究了 SNAP23 如何协调这些导致胰腺腺泡损伤的事件。SNAP23 消耗是通过腺病毒-shRNA ( Ad-SNAP23-shRNA/mCherry ) 影响的敲低 (SNAP23-KD)) 治疗啮齿动物和人胰腺切片,并通过输注到大鼠胰管中进行体内治疗。超生理性胆囊收缩素 (CCK) 或乙醇加低剂量 CCK 诱导的体外胰腺炎用于评估 SNAP23-KD 对胞吐作用和自噬的影响。胰腺炎刺激导致 SNAP23 从其在质膜的天然位置易位到自噬体,其中 SNAP23 将结合并调节 STX17(syntaxin17)SNARE 复合物介导的自噬体-溶酶体融合。这种 SNAP23 重定位归因于 IKBKB/IKKβ 介导的大鼠 Ser95 Ser120 和人 Ser120 处的 SNAP23 磷酸化,这被 IKBKB/IKKβ 抑制剂阻断,并通过 IKBKB/IKKβ 磷酸化禁用的 SNAP23 突变体 (Ser95A Ser120A) 的失能得到证实结合 STX17 SNARE 复合物。SNAP23-KD 损害了 STX4 驱动的基底外侧胞吐 SNARE 复合物和 STX17 驱动的 SNARE 复合物的组装,导致 ZG 的基底外侧胞吐作用和自溶酶体形成分别减少,从而导致两个隔室中的胰蛋白酶原活化减少。因此,胰腺 SNAP23-KD 大鼠免受雨蛙素和酒精性胰腺炎的侵害。该研究揭示了 SNAP23 在介导病理性基底外侧胞吐作用和 IKBKB/IKKβ 参与自溶酶体形成中的作用,这两种情况都会发生胰蛋白酶原激活导致胰腺炎。SNAP23 是胰腺炎治疗靶向的有力候选者。结果导致两个隔室中的胰蛋白酶原活化减少。因此,胰腺 SNAP23-KD 大鼠免受雨蛙素和酒精性胰腺炎的侵害。该研究揭示了 SNAP23 在介导病理性基底外侧胞吐作用和 IKBKB/IKKβ 参与自溶酶体形成中的作用,这两种情况都会发生胰蛋白酶原激活导致胰腺炎。SNAP23 是胰腺炎治疗靶向的有力候选者。结果导致两个隔室中的胰蛋白酶原活化减少。因此,胰腺 SNAP23-KD 大鼠免受雨蛙素和酒精性胰腺炎的侵害。该研究揭示了 SNAP23 在介导病理性基底外侧胞吐作用和 IKBKB/IKKβ 参与自溶酶体形成中的作用,这两种情况都会发生胰蛋白酶原激活导致胰腺炎。SNAP23 是胰腺炎治疗靶向的有力候选者。
缩写: AL:自溶酶体;AP:急性胰腺炎;AV:自噬泡;CCK:胆囊收缩素;IKBKB/IKKβ:核因子κB激酶亚基β抑制剂;SNAP23:突触体相关蛋白 23;SNARE:可溶性NSF(N-乙基马来酰亚胺敏感因子)附着蛋白受体;STX:语法;TAP:胰蛋白酶原活化肽;VAMP:囊泡相关膜蛋白;ZG:酶原颗粒。