MK-STYX as a regulator in stress granule clearance

MK-STYX 作为应激颗粒清除调节剂

• 批准号: 1909316
• 负责人: Shanta Hinton
• 金额: $ 90万
• 依托单位: College of William and Mary
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-08-01 至 2025-01-31
• 项目状态: 未结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1909316&HistoricalAwards=false
• 关键词: MK; STYX; regulator; stress; granule

Cellular stress may lead to harmful conditions that alter or prevent the completion of a normal routine; therefore, a dynamic response must be established to maintain balance of cellular functions under stressful conditions. This project investigates how cells adapt to a stressful environment to return to normal. The researchers involved in this project will be undergraduate and graduate students who will participate in independent projects to determine the mechanisms of how genetic information is stored and influenced by a specific protein under normal and stressful conditions. They will have the opportunity to continue their research in Canada at the Lunenfeld-Tanenbaum Research Institute (Toronto, Ontario). Their findings will be presented at local, national, and international conferences such as at William and Mary (Williamsburg), San Diego, and Greece. In addition, these students will develop and use a children friendly workshop to teach local summer camp students and their parents about the importance of cells keeping a balance, under toxic conditions. This research will also allow selected students at William and Mary and Hampton University to travel to a leading research facility, Cold Spring Harbor Laboratory (Long Island, NY), to engage and build networks with scientists highly active in various disciplines. Thus, the overall scope of this project allows undergraduates and Master's students to integrate education and research in the stress response field.Cells have protective mechanisms to safeguard stalled mRNAs from harmful conditions. They form stress granules (SG). SG are large (100-200 nm) cytoplasmic RNA-protein complexes that transiently form under stress and disassemble upon return to normal conditions. When SG coalesce for too long, they become toxic, disrupting cellular homeostasis, highlighting the importance of understanding SG clearance. Investigators in this project previously reported that SG assembly was decreased by the signaling protein MK-STYX (MAP kinase serine/threonine/tyrosine binding protein; a unique catalytically inactive phosphatase), whereas the active mutant of MK-STYX induces SG. The mechanism by which MK-STYX decreases SG remains unclear. The current project explores the molecular mechanisms by which MK-STYX regulates SG by addressing the following questions: 1) Which domain of MK-STYX elicits a decrease in SG? 2) What are the effects of MK-STYX on HDAC6 and the motor protein dynein? 3) Does MK-STYX have a role in autophagy, which clears stress granules? Investigators have identified a phenotype that will allow substantial structural and functional assays of MK-STYX, including identification of the MK-STYX domains and the important protein interactions of MK-STYX that lead to a decrease in SG. Various fluorescent protein-tagged mutants of MK-STYX, such as truncated domains and the active mutant (catalytic activity restored), will be overexpressed in HeLa and HEK/293 cells stably expressing the SG nucleator G3BP1, and observed for SG, and analyzed further through proteomics. HDAC6 is a critical component of SG; its deacetylation activity is required for SG. Colocalization, immunoblotting for acetylation and ubiquitination (important for HDAC6 function), immunoprecipitation, and deacetylation activity assays will address whether MK-STYX affects the activity of HDAC6. Dynein is required in conjunction with HDAC6 for SG. Thus, it is important to determine the effects of MK-STYX on these proteins. Since autophagy and MK-STYX each negatively affect SG formation, it is important to determine the effects of MK-STYX on autophagy. A combination of TEM, immunoblotting (LC3-autophagsomes marker, ubiquitin, etc.), radioactive pulse chase analysis, tandem mRFP/mCherry-GFP colocalization microscopy, and autophagic sequestration assays (autophagy inhibitors) will be used, resulting in the development and integration of computational analysis. The long-term objective of this research is to understand how MK-STYX regulates signaling pathways.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

细胞应激可能导致有害的情况，改变或阻止正常程序的完成；因此，必须建立动态反应，以维持细胞功能在应激条件下的平衡。这个项目研究细胞如何适应压力环境以恢复正常。参与这个项目的研究人员将是本科生和研究生，他们将参与独立的项目，以确定基因信息在正常和压力条件下如何存储和受到特定蛋白质影响的机制。他们将有机会在加拿大安大略省多伦多的鲁南菲尔德-塔南鲍姆研究所继续他们的研究。他们的发现将在地方、国家和国际会议上公布，例如威廉和玛丽(威廉斯堡)、圣地亚哥和希腊。此外，这些学生将开发和使用一个儿童友好工作坊，向当地夏令营学生和他们的父母传授细胞在有毒条件下保持平衡的重要性。这项研究还将允许威廉和玛丽大学和汉普顿大学选定的学生前往领先的研究机构--冷泉港实验室(纽约州长岛)，与在不同学科高度活跃的科学家接触并建立网络。因此，这个项目的总体范围允许本科生和硕士学生在应激反应领域整合教育和研究。细胞具有保护机制，以保护停滞的mRNA免受有害条件的影响。它们形成应力颗粒(SG)。SG是大的(100-200 nm)细胞质RNA-蛋白质复合体，在胁迫下瞬时形成，并在恢复正常条件时分解。当SG结合太久时，它们就会变得有毒，扰乱细胞动态平衡，突显了解SG清除的重要性。该项目的研究人员先前报道了SG组装被信号蛋白MK-Styx(MAP激酶丝氨酸/苏氨酸/酪氨酸结合蛋白；一种独特的催化非活性磷酸酶)减少，而MK-Styx的活性突变体诱导SG。MK-Styx降低SG的机制尚不清楚。本项目通过解决以下问题来探讨MK-Styx调节SG的分子机制：1)MK-Styx的哪个结构域导致SG减少？2)MK-Styx对HDAC6和运动蛋白dynein有什么影响？3)MK-Styx在清除应激颗粒的自噬中起作用吗？研究人员已经确定了一种表型，可以对MK-Styx进行实质性的结构和功能分析，包括鉴定MK-Styx结构域和导致SG减少的MK-Styx的重要蛋白质相互作用。MK-Styx的各种荧光蛋白标记突变体，如截短结构域和活性突变体(催化活性恢复)，将在稳定表达SG核因子G3BP1的HeLa和HEK/293细胞中过表达，并观察SG，并通过蛋白质组学进一步分析。HDAC6是SG的重要组成部分，其脱乙酰活性是SG所必需的。共定位、乙酰化和泛素化的免疫印迹(对HDAC6功能很重要)、免疫沉淀和去乙酰化活性分析将解决MK-Styx是否影响HDAC6的活性。对于SG，需要动力蛋白与HDAC6一起使用。因此，确定MK-Styx对这些蛋白质的影响是很重要的。由于自噬和MK-Styx都对SG的形成产生负面影响，因此确定MK-Styx对自噬的影响是很重要的。将结合使用透射电子显微镜、免疫印迹(LC3-自噬标记、泛素等)、放射性脉冲追逐分析、串联MRFP/mCherry-GFP共定位显微镜和自噬隔离分析(自噬抑制物)，从而导致计算分析的发展和整合。这项研究的长期目标是了解MK-Styx如何监管信号通路。这一奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。