The regulation of endocytic sorting and cholesterol transport by PTP1B-mediated ESCRT dephosphorylation at ER-endosome membrane contact sites

PTP1B 介导的 ER-内体膜接触位点 ESCRT 去磷酸化对内吞分选和胆固醇转运的调节

• 批准号: MR/P010091/1
• 负责人: Clare Futter
• 金额: $ 58.02万
• 依托单位: University College London
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2017
• 资助国家: 英国
• 起止时间: 2017 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=MR%2FP010091%2F1
• 关键词: regulation; endocytic; sorting; cholesterol; transport

The transport of proteins and lipids to the right cellular location is critical for the function of all cells. Endocytosis is a mechanism of uptake of extracellular molecules and cell surface membrane proteins into intracellular membrane compartments, called endosomes. Within the endosome, proteins and lipids are sorted. Unwanted cell membrane proteins and lipids can then be removed by delivering them to lysosomes where they are degraded. This degradative pathway is balanced by new synthesis of membrane proteins and lipids in the endoplasmic reticulum (ER). Overproduction or impaired degradation of membrane proteins or lipids can lead to multiple diseases, including cancer and lipid storage disorders. The degradative pathway is important for regulating signaling from growth factor receptors, such as the EGF receptor (EGFR), a cell surface signaling protein that regulates cell division. Dysregulated signaling from the EGFR occurs in about 50% of human cancers and is a target for cancer therapies. It activates signaling pathways by chemically modifying both itself and other signaling proteins. This modification, called phosphorylation, is reversible by dephosphorylation. The EGFR can be dephosphorylated by a phosphatase on the ER, PTP1B. Proteins and lipids can shuttle from cell membrane to membrane via small transport vesicles. Different cellular membranes can also form membrane contacts where they come very close together to form sites where proteins on the apposing membranes can interact and lipids can be flipped directly from one membrane to the other. We recently found that the ER and the endocytic pathway form membrane contacts and have identified molecules that tether the apposing membranes at the contact. We showed that ER:endosome membrane contacts are necessary for PTP1B to dephosphorylate endocytosed EGFR, and that PTP1B activity promotes delivery of EGFR to the lysosome for degradation. Some of the molecules that regulate lysosomal delivery of EGFR are themselves regulated by PTP1B-mediated dephosphorylation and one of those molecules, Hrs, has also been implicated in cholesterol exchange between endosomes and the ER.Cholesterol is a critical component of cell membranes obtained by endocytosis of dietary LDL, or by new synthesis in the ER. Excess endocytosed cholesterol is transferred to the ER and packaged into lipid droplets for storage. However, the endocytic pathway needs a certain amount of cholesterol to function. So when LDL-derived cholesterol is low, newly synthesized cholesterol is transferred from the ER to endosomes. We showed that ER:endosome membrane contacts regulate exchange of cholesterol between the ER and endosomes. Regulation of endosomal cholesterol is important for multiple processes. Endosomal cholesterol is required for normal function of the degradative pathway, but impaired egress of endosomal LDL-cholesterol is associated with lipid storage diseases, such as the childhood neurological disorder, Neimann Pick Type C disease. We hypothesise that PTP1B-mediated dephosphorylation of Hrs at membrane contact sites co-ordinates lysosomal targeting of EGFR and cholesterol exchange between endosomes and the ER. We will test this hypothesis using tools we've developed to manipulate membrane contacts, together with high-resolution electron microscopy (the only way to unequivocally identify membrane contacts) and newly developed super resolution microscopy techniques. Together these studies will provide a better understanding of how PTP1B activity at membrane contact sites regulates EGFR signaling and cholesterol exchange. This will determine how the relationship between PTP1B and EGFR can be exploited for the design of better combination cancer therapies and may yield novel therapeutic targets for the treatment of lipid storage diseases.

蛋白质和脂类的运输到正确的细胞位置对所有细胞的功能都是至关重要的。内吞作用是一种将细胞外分子和细胞表面膜蛋白摄取到细胞内膜隔间的机制，称为内小体。在内吞体内，蛋白质和脂类被分类。然后，不需要的细胞膜蛋白和脂类可以通过将它们输送到溶酶体来去除，在溶酶体中它们被降解。这一降解途径由内质网(ER)中膜蛋白和脂类的新合成来平衡。膜蛋白或脂类的过量生产或降解受损可导致多种疾病，包括癌症和脂类储存障碍。降解途径对于调节生长因子受体的信号是重要的，例如EGF受体(EGFR)，一种调节细胞分裂的细胞表面信号蛋白。大约50%的人类癌症中会发生EGFR信号的失调，这也是癌症治疗的靶点。它通过对自身和其他信号蛋白进行化学修饰来激活信号通路。这种修饰被称为磷酸化，通过去磷酸化是可逆的。EGFR可以被内质网上的磷酸酶PTP1B去磷酸化。蛋白质和脂类可以通过小的运输小泡从细胞膜穿梭到细胞膜。不同的细胞膜也可以形成膜接触，在那里它们非常接近，形成相对膜上的蛋白质可以相互作用的位置，脂类可以直接从一个膜翻到另一个膜上。我们最近发现，内质网和内吞途径形成了膜接触，并发现了在接触处拴住相对膜的分子。我们证明了内质网：内体膜接触是PTP1B对内吞的EGFR进行去磷酸化所必需的，并且PTP1B的活性促进了EGFR的运输到溶酶体进行降解。一些调节溶酶体转运EGFR的分子本身就受到PTP1B介导的去磷酸化的调节，其中一个分子HRs也参与了内体和内质网之间的胆固醇交换。胆固醇是细胞膜的关键成分，通过内吞饮食低密度脂蛋白或内质网新合成获得。过量的内吞胆固醇被转移到内质网，并包装成脂滴进行储存。然而，内吞途径需要一定量的胆固醇才能发挥作用。因此，当低密度脂蛋白衍生的胆固醇较低时，新合成的胆固醇就会从内质网转移到内吞体内。我们发现内质网：内吞体膜接触调节内质网和内容体之间的胆固醇交换。内体胆固醇的调节在多种过程中都很重要。内体胆固醇是降解途径的正常功能所必需的，但内体低密度脂蛋白-胆固醇的外排受损与脂类储存疾病有关，如儿童神经功能障碍，Neimann Pick C型病。我们假设，PTP1B介导的膜接触部位HRs的去磷酸化协同溶酶体靶向EGFR和内容体与内质网之间的胆固醇交换。我们将使用我们开发的操纵膜接触的工具，以及高分辨率电子显微镜(明确识别膜接触的唯一方法)和新开发的超分辨率显微镜技术来验证这一假设。总之，这些研究将更好地了解膜接触部位的PTP1B活性如何调节EGFR信号和胆固醇交换。这将决定如何利用PTP1B和EGFR之间的关系来设计更好的癌症联合疗法，并可能为治疗脂质储存疾病产生新的治疗靶点。

项目成果

REEP6 deficiency leads to retinal degeneration through disruption of ER homeostasis and protein trafficking.

• DOI: 10.1093/hmg/ddx149
• 发表时间: 2017-07-15
• 期刊: Human molecular genetics
• 影响因子: 3.5
• 作者: Agrawal SA;Burgoyne T;Eblimit A;Bellingham J;Parfitt DA;Lane A;Nichols R;Asomugha C;Hayes MJ;Munro PM;Xu M;Wang K;Futter CE;Li Y;Chen R;Cheetham ME
• 通讯作者: Cheetham ME

Endothelial MAPKs Direct ICAM-1 Signaling to Divergent Inflammatory Functions.

• DOI: 10.4049/jimmunol.1600823
• 发表时间: 2017-05-15
• 期刊: Journal of immunology (Baltimore, Md. : 1950)
• 作者: Dragoni S;Hudson N;Kenny BA;Burgoyne T;McKenzie JA;Gill Y;Blaber R;Futter CE;Adamson P;Greenwood J;Turowski P
• 通讯作者: Turowski P

Correlative light and immuno-electron microscopy of retinal tissue cryostat sections.

• DOI: 10.1371/journal.pone.0191048
• 发表时间: 2018
• 期刊: PloS one
• 影响因子: 3.7
• 作者: Burgoyne T;Lane A;Laughlin WE;Cheetham ME;Futter CE
• 通讯作者: Futter CE

Cholesterol Overload: Contact Sites to the Rescue!

• DOI: 10.1177/2515256419893507
• 发表时间: 2019-01-01
• 期刊: Contact (Thousand Oaks (Ventura County, Calif.))
• 作者: Enrich, Carlos;Rentero, Carles;Eden, Emily R
• 通讯作者: Eden, Emily R

ALIX Regulates Tumor-Mediated Immunosuppression by Controlling EGFR Activity and PD-L1 Presentation.

• DOI: 10.1016/j.celrep.2018.06.066
• 发表时间: 2018-07-17
• 期刊: Cell reports
• 影响因子: 8.8
• 作者: Monypenny J;Milewicz H;Flores-Borja F;Weitsman G;Cheung A;Chowdhury R;Burgoyne T;Arulappu A;Lawler K;Barber PR;Vicencio JM;Keppler M;Wulaningsih W;Davidson SM;Fraternali F;Woodman N;Turmaine M;Gillett C;Franz D;Quezada SA;Futter CE;Von Kriegsheim A;Kolch W;Vojnovic B;Carlton JG;Ng T
• 通讯作者: Ng T