Regulation of acinar cell function

腺泡细胞功能的调节

• 批准号: RGPIN-2018-06444
• 负责人: Pin, Christopher
• 金额: $ 2.33万
• 依托单位: University of Western Ontario
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2018
• 资助国家: 加拿大
• 起止时间: 2018-01-01 至 2019-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=662206
• 关键词: Regulation; acinar; cell; function

Acinar cells produce, store and secrete enzymes through a process known as regulated exocytosis, which involves spatial and temporal Ca2+ release in response to stimulation. Control of Ca2+ concentrations ([Ca2+]) is also essential for gene transcription and protein processing. Improper [Ca2+] alters gene expression, affects protein processing and promotes apoptosis. Recently, we identified a novel isoform of Secretory Pathway Ca2+ATPase 2 (SPCA2) termed SPCA2C, which aligns with the carboxy terminus of the longer SPCA2 protein, but lacks the ATPase domain. Over-expression of SPCA2C increases ER and cytosolic [Ca2+] and affects G protein coupled receptor (GPCR) signalling and Store-Operated Ca2+ Entry (SOCE). SOCE is activated in response to low [Ca2+] in the ER, which activates STIM and ORAI channels to transport Ca2+ into the ER from outside the cell. The short term goals for the next cycle of this research program are to (1) determine mechanisms by which SPCA2C affects Ca2+ homeostasis in acinar and non-acinar cells, and (2) identify proteins that interact with SPCA2C to regulate its function.***Governing hypothesis: SPCA2C is an important regulator of Ca2+ homeostasis essential for acinar cell function.***AIMS:***(1) Identify Ca2+-signaling pathways affected by SPCA2C. Tagged SPCA2C (SPCA2CFLAG) will be expressed in HEK293 or HEK293 cells expressing YFP-tagged Orai1 (HEK293-Orai1YFP). GPCR signalling, SOCE and Store Independent Ca2+ Entry (SICE) will be examined. Fura2 imaging, co-IF, and confocal microscopy will assess cytosolic free Ca2+ concentration ([Ca2+]i) and protein localization. Pharmacological inhibitors of SOCE and GPCR signaling will determine SPCA2C's role in these pathways.***(2) Identify the role of SPCA2C on acinar cell biology. We have developed adenoviral vectors for expression of epitope-tagged SPCA2C in acinar cells cultures or in vivo through intraductal injection. We will assess acinar cell morphology, [Ca2+]i, gene expression and exocytosis ability following SPCA2C over-expression to determine its importance to these functions in acinar cells.***(3) Define the protein interactors for SPCA2C. We will perform an unbiased screen using BioID followed by mass spectrometry to identify interacting proteins for SPCA2C. We will use co-IP and Lumiere assays to validate interacting partners for SPCA2C in HEK293-Orai1YFP cells and acinar cells. Identified proteins will be characterized as described in Aims 1 and 2, These experiments will define SPCA2C interacting proteins which may also mediate [Ca2+]i.***Characterizing SPCA2C will address our Long Term Goals to (a) delineate mechanisms by which diverse Ca2+-regulating signaling pathways are coordinated to maintain acinar function and respond to environmental cues, and (b) define how changes to these pathways affect critical cell activities including transcription, protein folding and exocytosis.

腺泡细胞通过称为调节性胞吐的过程产生、储存和分泌酶，该过程涉及响应刺激的空间和时间Ca 2+释放。Ca 2+浓度（[Ca 2 +]）的控制也是基因转录和蛋白质加工的关键。不适当的[Ca 2 +]改变基因表达，影响蛋白质加工和促进细胞凋亡。最近，我们发现了一种新的分泌途径钙ATP酶2（SPCA 2）的亚型，称为SPCA 2C，它与较长的SPCA 2蛋白的羧基端对齐，但缺乏ATP酶结构域。SPCA 2C的过表达增加ER和胞质[Ca 2 +]，并影响G蛋白偶联受体（GPCR）信号传导和钙池操纵的Ca 2+内流（SOCE）。SOCE响应于ER中的低[Ca 2 +]而被激活，其激活STIM和奥赖通道以将Ca 2+从细胞外转运到ER中。本研究计划下一周期的短期目标是（1）确定SPCA 2C影响腺泡和非腺泡细胞中Ca 2+稳态的机制，以及（2）鉴定与SPCA 2C相互作用以调节其功能的蛋白质。支配假说：SPCA 2C是腺泡细胞功能所必需的Ca 2+稳态的重要调节剂。*目的：*（1）鉴定SPCA 2C影响的Ca 2+信号通路。标记的SPCA 2C（SPCA 2CFLAG）将在HEK 293或表达YFP标记的Orai 1（HEK 293-Orai 1 YFP）的HEK 293细胞中表达。将检查GPCR信号传导、SOCE和非钙库依赖性Ca 2+进入（SICE）。Fura 2成像、co-IF和共聚焦显微镜将评估胞质游离Ca 2+浓度（[Ca 2 +]i）和蛋白定位。SOCE和GPCR信号传导的药理学抑制剂将决定SPCA 2C在这些途径中的作用。(2)确定SPCA 2C对腺泡细胞生物学的作用。我们已经开发了腺病毒载体，用于在腺泡细胞培养物中或通过导管内注射在体内表达表位标记的SPCA 2C。我们将评估SPCA 2C过表达后的腺泡细胞形态、[Ca 2 +]i、基因表达和胞吐能力，以确定其对腺泡细胞中这些功能的重要性。(3)定义SPCA 2C的蛋白质相互作用物。我们将使用BioID进行无偏筛选，然后进行质谱分析，以确定SPCA 2C的相互作用蛋白。我们将使用co-IP和Lumiere检测来验证HEK 293-Orai 1 YFP细胞和腺泡细胞中SPCA 2C的相互作用伴侣。鉴定的蛋白质将如目的1和2中所述进行表征。这些实验将定义也可介导[Ca 2 +]i的SPCA 2C相互作用蛋白。表征SPCA 2C将解决我们的长期目标，以（a）描绘不同的Ca 2+调节信号通路协调以维持腺泡功能和响应环境线索的机制，（B）定义这些通路的变化如何影响关键细胞活动，包括转录，蛋白质折叠和胞吐。