Molecular Mechanisms of Wnt Transport

Wnt 转运的分子机制

• 批准号: 10753139
• 负责人: Filippo Mancia
• 金额: $ 48.01万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-18 至 2028-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10753139
• 关键词: Adult; Affect; Autocrine Communication; Binding; Biochemical; Biological Assay; Carrier Proteins; Cell membrane; Cell surface; Cells; Chimera organism; Complex; Coupled; Cryoelectron Microscopy; Cysteine-Rich Domain; Data; Dedications; Destinations; Development; Diffusion; Dissociation; Endoplasmic Reticulum; Fab Immunoglobulins; Family; G-Protein-Coupled Receptors; Heterodimerization; Histidine; Homeostasis; Human; Hydrophobicity; In Vitro; Intestinal Cancer; Intestines; Kidney; Lateral; Ligands; Lipid Bilayers; Lipids; Location; Membrane; Membrane Proteins; Membrane Transport Proteins; Modeling; Modification; Molecular; Molecular Conformation; Morphogenesis; Movement; Mutagenesis; Pattern; Polysaccharides; Proteins; Proteoglycan; Publishing; Resolution; Route; Signal Transduction; Site; Structure; Surface; System; Technology; Testing; Tissues; Transferase; Transmembrane Domain; exosome; experience; experimental study; glycosylation; inhibitor; insight; intestinal homeostasis; migration; molecular dynamics; palmitoleate; particle; prevent; protein protein interaction; receptor; stem cell division; stem cell niche

ABSTRACT Wnts are a family of evolutionary conserved, secreted ligands that act at short range to coordinate morphogenetic movements and cell fate decisions. Secretion of Wnt and subsequent recognition by its cognate receptor require its O-palmitoleation in the endoplasmic reticulum. This modification is carried out by PORCN, that then transfers Wnt to WLS, its dedicated integral membrane transporter. How WLS associates with Wnt in the ER, and subsequently delivers it to the receptor Frizzled on the receiving cell remains largely unknown, and is the focus of this Application. Currently, there is a limited understanding of how WLS binds Wnts and transport them to the cell membrane. Here we present as recently published data the structure of human WLS, in a complex with WNT8A, determined using single-particle cryo-electron microscopy to 3.2 Å resolution. The WLS membrane domain resembles a G protein-coupled receptor (GPCR), albeit with one additional transmembrane helix. The O-palmitoleated Wnt hairpin loop 2 inserts into a conserved central cavity of the GPCR-related domain, with the attached palmitoleate protruding out into the lipid bilayer. Highly conserved patches on the outward-facing surfaces of the transmembrane domain of WLS, adjacent to a hydrophobic gateway, suggest potential mechanisms for Wnt transfer from PORCN and to Frizzled. We propose to further study the interaction between WLS and Wnt, using the available structural information as starting point. First, we will investigate how Wnt associate with WLS. We propose to determine the structure of WLS in its apo – absent Wnt – state, as well as probe the putative site of entrance of Wnt into WLS by structure- guided mutagenesis and biochemical assays. Our preliminary results suggest a direct interaction between WLS and PORCN. Second, we will test how changes in sequence and glycosylation pattern can affect Wnt secretion and destination, as well as the effect of pH on the interaction between the two proteins, using structure-guided mutagenesis as well as Wnt transport and delivery assays in primary intestinal cells. Third, we propose to study the release of Wnt to subsequent receptors. Our preliminary results suggest a direct interaction between WLS and the Frizzled receptor, and we will test this hypothesis using detailed structure function analyses.

摘要 WNTs是一类进化上保守的、分泌的配体，它们在短距离内协调形态发生 运动和细胞命运的决定。Wnt的分泌及其同源受体的后续识别需要 它在内质网上的O-棕榈油化。此修改由PORCN执行，然后将 WNT至WLS，其专用的整体膜转运器。WLS如何在急诊室与WNT关联，以及 随后将其递送到受体Frizzled上的接收细胞在很大程度上仍然未知，并是焦点 此应用程序的。 目前，对于WLS如何与WNTs结合并将其转运到细胞膜上的了解有限。 在这里，我们展示了最近发表的数据，人类WLS的结构，在与WNT8A的复合体中，确定 使用单粒子低温电子显微镜，分辨率为3.2？WLS膜结构域类似于G 蛋白偶联受体(GPCR)，尽管有一个额外的跨膜螺旋。O-棕榈油化的WNT 发夹环2插入GPCR相关结构域的保守中央空腔，并连接棕榈酸酯 突出到脂质双层中。在向外的表面上高度保守的补丁 WLS的跨膜结构域与疏水通道相邻，提示WNT的潜在机制 从PORCN转机到弗里兹莱德。 我们建议利用现有的结构信息来进一步研究WLS和WNT之间的相互作用 起点。首先，我们将调查WNT是如何与WLS相关联的。我们建议确定 WLS处于缺失的WNT状态，以及通过构造探测WNT进入WLS的假定位置-- 引导性诱变和生化分析。我们的初步结果表明WLS之间存在直接的相互作用 和PORCN。其次，我们将测试序列和糖基化模式的变化如何影响Wnt的分泌 和目的地，以及pH对两种蛋白质之间相互作用的影响 在原代肠道细胞中的诱变以及Wnt的运输和传递试验。第三，我们建议研究 将Wnt释放给后续受体。我们的初步结果表明WLS之间存在直接的相互作用 和Frizzled型受体，我们将使用详细的结构功能分析来检验这一假说。