白念珠菌内质网-质膜连接蛋白Ist2对内质网压力应答的调控作用
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摘要
内质网-质膜连接是真核细胞一种新型的膜连接体系,介导内质网与质膜之间紧密的物质与信息交换.在条件致病真菌白念珠菌(Candida albicans)中,尚未明确内质网-质膜连接蛋白的种类与功能.本研究通过荧光定位观察发现, Ist2能够与白念珠菌内质网-质膜连接共定位,其缺失导致内质网-质膜连接程度明显减弱,表明该蛋白是白念珠菌一种关键的内质网-质膜连接蛋白.通过生长曲线测定、非折叠蛋白应答(unfolded protein response,UPR)报告体系荧光检测以及胞质钙含量测定发现,在衣霉素(tunicamycin, TN)造成的内质网压力条件下, IST2缺失菌株ist2Δ/Δ生长量明显增加, UPR报告基因PRB1的表达量下降,胞质钙含量降低.进一步采用钙离子螯合剂乙二醇双(2-氨基乙基醚)四乙酸(EGTA)与TN共处理发现, EGTA能够消除ist2Δ/Δ与野生型菌株之间内质网压力耐受性的差异.因此, Ist2作为白念珠菌的内质网-质膜连接蛋白,介导内质网压力条件下胞质钙浓度的增加,造成细胞内质网压力耐受性降低,是白念珠菌内质网压力应答的负调控因子.本研究丰富了对真菌内质网-质膜连接结构与功能的认识,为新型抗真菌药物靶点的开发提供了重要依据.
The endoplasmic reticulum-plasma membrane contact(ER-PM contact) is a novel membrane contact system in eukaryotic cells that mediates the close exchange of materials and messengers between ER and PM. However, the types and function of the ER-PM tethering proteins of the opportunistic pathogenic fungus Candida albicans remain poorly understood. In the present study, we found that Ist2 co-localizes with the ER-PM contact sites of C. albicans and that its deletion causes a marked decrease in the ER-PM contact sites as revealed by fluorescence observation. This indicates that Ist2 is a key ER-PM tethering protein of C. albicans. The measurement of growth curves, fluorescence detection of the unfolded protein response(UPR) reporting system, and the measurement of the cytosolic Ca~(2+)level revealed that the IST2 deletion strain ist2Δ/Δ considerably increased in biomass, the UPR reporting gene PRB1 expression was attenuated, and the cytoplasmic Ca~(2+)levels under ER stress caused by tunicamycin decreased. In addition, the Ca~(2+)chelating agent EGTA could eliminate the difference between ist2Δ/Δ and WT in ER stress tolerance. Therefore,Ist2 is an important ER-PM tethering protein in C. albicans and a negative regulator of ER stress responses, which mediates an increase in cytosolic Ca~(2+)level under ER stress, resulting in attenuated ER stress tolerance. The results of the present study facilitate our understanding of the structure and function of the fungal ER-PM contact and provide an important basis for the development of novel antifungal targets.
The endoplasmic reticulum-plasma membrane contact(ER-PM contact) is a novel membrane contact system in eukaryotic cells that mediates the close exchange of materials and messengers between ER and PM. However, the types and function of the ER-PM tethering proteins of the opportunistic pathogenic fungus Candida albicans remain poorly understood. In the present study, we found that Ist2 co-localizes with the ER-PM contact sites of C. albicans and that its deletion causes a marked decrease in the ER-PM contact sites as revealed by fluorescence observation. This indicates that Ist2 is a key ER-PM tethering protein of C. albicans. The measurement of growth curves, fluorescence detection of the unfolded protein response(UPR) reporting system, and the measurement of the cytosolic Ca~(2+)level revealed that the IST2 deletion strain ist2Δ/Δ considerably increased in biomass, the UPR reporting gene PRB1 expression was attenuated, and the cytoplasmic Ca~(2+)levels under ER stress caused by tunicamycin decreased. In addition, the Ca~(2+)chelating agent EGTA could eliminate the difference between ist2Δ/Δ and WT in ER stress tolerance. Therefore,Ist2 is an important ER-PM tethering protein in C. albicans and a negative regulator of ER stress responses, which mediates an increase in cytosolic Ca~(2+)level under ER stress, resulting in attenuated ER stress tolerance. The results of the present study facilitate our understanding of the structure and function of the fungal ER-PM contact and provide an important basis for the development of novel antifungal targets.
引文
1 Liu L,Li J.Communications between the endoplasmic reticulum and other organelles during abiotic stress response in plants.Front Plant Sci,2019,10:749
2 Quon E,Sere Y Y,Chauhan N,et al.Endoplasmic reticulum-plasma membrane contact sites integrate sterol and phospholipid regulation.PLoSBiol,2018,16:e2003864
3 Friedman J R,Voeltz G K.The ER in 3D:A multifunctional dynamic membrane network.Trends Cell Biol,2011,21:709-717
4 Stefan C J,Manford A G,Emr S D.ER-PM connections:Sites of information transfer and inter-organelle communication.Curr Opin Cell Biol,2013,25:434-442
5 Chang C L,Chen Y J,Liou J.ER-plasma membrane junctions:Why and how do we study them?Biochim Biophys Acta(BBA)-Mol Cell Res,2017,1864:1494-1506
6 Stefano G,Brandizzi F.Advances in plant ER architecture and dynamics.Plant Physiol,2018,176:178-186
7 Naglik J R,Richardson J P,Moyes D L,et al.Candida albicans pathogenicity and epithelial immunity.PLoS Pathog,2014,10:e1004257
8 Bhosale G,Sharpe J A,Sundier S Y,et al.Calcium signaling as a mediator of cell energy demand and a trigger to cell death.Ann NY Acad Sci,2015,1350:107-116
9 Yu Q L,Ding X H,Zhang B,et al.The P-type ATPase Spf1 is required for endoplasmic reticulum functions and cell wall integrity in Candida albicans.Int J Med Microbiol,2013,303:257-266
10 Tavassoli S,Chao J T,Young B P,et al.Plasma membrane-endoplasmic reticulum contact sites regulate phosphatidylcholine synthesis.EMBORep,2013,14:434-440
11 Yu Q L.Role of the calcium homeostasis system in morphogenesis and stress response in Candida albicans(in Chinese).Dissertation for Doctoral Degree.Tianjin:Nankai University,2013[喻其林.钙稳态系统在白念珠菌形态发生及压力应答中的功能研究.博士学位论文.天津:南开大学,2013]
12 Manford A G,Stefan C J,Yuan H L,et al.ER-to-plasma membrane tethering proteins regulate cell signaling and ER morphology.Dev Cell,2012,23:1129-1140
13 Zhang D,Vjestica A,Oliferenko S.Plasma membrane tethering of the cortical ER necessitates its finely reticulated architecture.Curr Biol,2012,22:2048-2052
14 Thibault G,Ismail N,Ng D T W.The unfolded protein response supports cellular robustness as a broad-spectrum compensatory pathway.Proc Natl Acad Sci USA,2011,108:20597-20602
15 Chang C L,Hsieh T S,Yang T T,et al.Feedback regulation of receptor-induced Ca2+signaling mediated by E-Syt1 and Nir2 at endoplasmic reticulum-plasma membrane junctions.Cell Rep,2013,5:813-825
16 Jha A,Chung W Y,Vachel L,et al.Anoctamin 8 tethers endoplasmic reticulum and plasma membrane for assembly of Ca2+signaling complexes at the ER/PM compartment.EMBO J,2019,38:e101452
17 Berridge M J,Lipp P,Bootman M D.The versatility and universality of calcium signalling.Nat Rev Mol Cell Biol,2000,1:11-21
18 Papouskova K,Andrsova M,Sychrova H.Lack of cortical endoplasmic reticulum protein Ist2 alters sodium accumulation in Saccharomyces cerevisiae cells.FEMS Yeast Res,2017,17:fox011
2 Quon E,Sere Y Y,Chauhan N,et al.Endoplasmic reticulum-plasma membrane contact sites integrate sterol and phospholipid regulation.PLoSBiol,2018,16:e2003864
3 Friedman J R,Voeltz G K.The ER in 3D:A multifunctional dynamic membrane network.Trends Cell Biol,2011,21:709-717
4 Stefan C J,Manford A G,Emr S D.ER-PM connections:Sites of information transfer and inter-organelle communication.Curr Opin Cell Biol,2013,25:434-442
5 Chang C L,Chen Y J,Liou J.ER-plasma membrane junctions:Why and how do we study them?Biochim Biophys Acta(BBA)-Mol Cell Res,2017,1864:1494-1506
6 Stefano G,Brandizzi F.Advances in plant ER architecture and dynamics.Plant Physiol,2018,176:178-186
7 Naglik J R,Richardson J P,Moyes D L,et al.Candida albicans pathogenicity and epithelial immunity.PLoS Pathog,2014,10:e1004257
8 Bhosale G,Sharpe J A,Sundier S Y,et al.Calcium signaling as a mediator of cell energy demand and a trigger to cell death.Ann NY Acad Sci,2015,1350:107-116
9 Yu Q L,Ding X H,Zhang B,et al.The P-type ATPase Spf1 is required for endoplasmic reticulum functions and cell wall integrity in Candida albicans.Int J Med Microbiol,2013,303:257-266
10 Tavassoli S,Chao J T,Young B P,et al.Plasma membrane-endoplasmic reticulum contact sites regulate phosphatidylcholine synthesis.EMBORep,2013,14:434-440
11 Yu Q L.Role of the calcium homeostasis system in morphogenesis and stress response in Candida albicans(in Chinese).Dissertation for Doctoral Degree.Tianjin:Nankai University,2013[喻其林.钙稳态系统在白念珠菌形态发生及压力应答中的功能研究.博士学位论文.天津:南开大学,2013]
12 Manford A G,Stefan C J,Yuan H L,et al.ER-to-plasma membrane tethering proteins regulate cell signaling and ER morphology.Dev Cell,2012,23:1129-1140
13 Zhang D,Vjestica A,Oliferenko S.Plasma membrane tethering of the cortical ER necessitates its finely reticulated architecture.Curr Biol,2012,22:2048-2052
14 Thibault G,Ismail N,Ng D T W.The unfolded protein response supports cellular robustness as a broad-spectrum compensatory pathway.Proc Natl Acad Sci USA,2011,108:20597-20602
15 Chang C L,Hsieh T S,Yang T T,et al.Feedback regulation of receptor-induced Ca2+signaling mediated by E-Syt1 and Nir2 at endoplasmic reticulum-plasma membrane junctions.Cell Rep,2013,5:813-825
16 Jha A,Chung W Y,Vachel L,et al.Anoctamin 8 tethers endoplasmic reticulum and plasma membrane for assembly of Ca2+signaling complexes at the ER/PM compartment.EMBO J,2019,38:e101452
17 Berridge M J,Lipp P,Bootman M D.The versatility and universality of calcium signalling.Nat Rev Mol Cell Biol,2000,1:11-21
18 Papouskova K,Andrsova M,Sychrova H.Lack of cortical endoplasmic reticulum protein Ist2 alters sodium accumulation in Saccharomyces cerevisiae cells.FEMS Yeast Res,2017,17:fox011