硒蛋白P通过硒代半胱氨酸基序调节动脉粥样硬化血管炎症应激与巨噬细胞功能的分子机制研究

Selenium (Se), an essential micronutrient element for mammals, plays important roles in anti-oxidative stress and inflammation immunity primarily in forms of 25 selenoproteins in human beings. Selenoprotein P (SelP) is an extracellular, most abundant and monomeric glycoprotein containing up to 10 selenocysteine (U) residues. Recently, we performed a case-control survey to explore the correlation between plasma SelP and the atherogenic index of plasma (AIP) in a population of 330 participants recruited from Meizhou regions with various Se dietary status. Preliminary data reveals that plasma SelP concentrations are inverse associations with the AIP indicating SelP close related to atherosclerosis (AS) in population. Herein, we focus on the mechanism of SelP and its U containing motifs (UM) in regulating the inflammatory signaling and macrophage activity, and also address preliminary medical transformation of SelP and UM peptides to prevent atherogenesis..Macrophages (Mø) are most numerous among leukocytes in the lesion of atherosclerosis (AS) and play a central role in the pathogenesis of plaques, but the mechanistic links between many AS associated factors and Mø mediated reversible cholesterol (CE) transportion (RCT) in atherogenesis remain unclear. To discover the posttranslational modification (PTM) modes of palmitoylation (PALM) and S-nitrosylation (SNO) on the UMs of SelP in macrophages in response to the oxidized low density lipoprotein (oxLDL) induced inflammation signals, we constructed the over expression vectors of the wild type (WT) SelP and its mutants covering all of the 10 U residues, the adjacent cysteine (C) residues and the deletions or truncates of U containing motifs expression vector for SelP and its cysteine containing (U) motif. After transfection and expression in RAW 264.7 and 293T cells, target PTMs on SelP were detected by Western Blot Kits and identified by CoIP-MS/MS hyphenated technique. To investigate the role of SelP in AS, comparative studys of pathological atherogenesis will be conducted in the mouse models of AS, which are established in ApoE-/- mice coupled with mice of whole body SelP-/-, liver or Mø conditional depletion, and transgenetic SelP mice for rescue, respectively. To further elucidate the exact mechanism in regulating Mø characteristics of RCT, CE overloading, foam cell formation, efferocytosis and ferroptosis, which dynamically sensitized and orchestrated by PALM- or/and SNO-PTMs on UMs of SelP, cell models of primary Mø (BMDM), RAW264.7 or THP-1 cell lines with SelP gene edition will also be constructed..Additionally, novel recombination and high expression system of SelP and UM peptides will be setup by screening a effective selenocysteine insertion sequence (SECIS) to solve the pre-termination and inefficiency of UGA mediated selenoprotein translation. To further medical transformation of SelP/UM peptides, the invention and design of self-assembly protein (EPN) prefabricated nanocage with Se-nano core for SelP/UM peptides delivery is also proposed in the present project. With surface modification of the innovative nano particles loading SelP/UM peptides (SePNP) to target AS vascular macrophage, its therapeutic intervention to AS should be good in prospective application..Uncovering above roles and action mechanisms of SelP through selenocysteine motifs in response to inflammatory signaling and macrophage activity are critical for understanding how impairments in low Se status and SelP expression driven the atherogenesis, and the invention of SePNP will guide the medical transformation and application of selenoproteins in the treatment of AS related diseases.

我们发现人群血浆硒蛋白P(SelP)水平与动脉粥样硬化(AS)发生风险呈负相关，人SelP含有10个硒代半胱氨酸基序(UM)，对生物应激调控起重要作用，其对AS血管炎症应激和巨噬细胞(Mø)功能的调节作用与机制亟待阐明。本项目拟利用SelP和UM突变Mø、CoIP-质谱联用分析炎症信号诱导SelP/UM棕榈酰化和亚硝基化修饰；再利用APOE与SelP基因共敲、肝脏和Mø条件敲除及SelP敲入拯救等小鼠模型，阐明SelP/UM调节AS血管炎症和Mø胆固醇转运、泡沫化、胞葬清除和铁死亡的机制，揭示SelP对AS脂质炎症信号感应的翻译后修饰方式，发现SelP对AS血管炎症信号和Mø功能调控新机制，为应用SelP和UM多肽防治AS提供理论依据；还将实现SelP和UM硒肽高效重组表达，成功制备SelP/UM硒肽，创新SelP/UM硒肽富载的纳米硒在AS血管炎症Mø靶向递送，具有防治AS转化应用前景。

硒（Se）作为人体必需的一种微量元素，主要通过25种硒蛋白发挥抗氧化、调节代谢和免疫等功能。硒蛋白P（SelP）是人体含量最丰富的一种外分泌硒蛋白成员，它含有10个独特的硒代半胱氨酸残基(U)，SelP 及其U基序（U motifs，UM)）在氧化还原胁迫、炎症应激及动脉粥样发生中的作用机制仍有待阐明。研究内容和主要结果如下：.1）人群队列分析硒营养状态、硒蛋白组和血浆SelP水平，发现血浆SelP水平与AS风险指数(AIP)呈显著负相关，高水平SelP则AS病变较轻，提示硒营养状态和循环SelP水平分析对AS预防转归具有临床参考价值。.2）构建了SelP-WT、23个突变体和2个截短型过表达质粒并在巨噬细胞和293T细胞瞬时表达，利用免疫印迹和CoIP-质谱联用分析炎症信号诱导的SelP/UM棕榈酰化和亚硝基化修饰，确认SelP的N端UXXC氧化还原基序，C端亚硝基化基序(ITUQCKE、ESCQURL)和棕榈酰化基序(KUEUPSN)。.3）利用ApoE与SelP基因敲除小鼠，发现ApoE和SelP双敲导致动脉粥样化加重，SelP敲入可较大程度拯救AS血管病变，而截短突变体的拯救效应不明显；在培养肝细胞中观察到炎症和脂质刺激诱导SelP表达和分泌，但SelP缺失巨噬细胞脂质累积和泡沫化程度均有所减轻，说明SelP在炎症和脂代谢调节中的复杂性，SelP/UM调节AS发生机制仍在研究进展中。.4）拓展研究发现人群低硒营养状态增加砷暴露介导的红细胞氧化还原失稳，进而下调CD47信号引起巨噬细胞过度红噬作用，并活化NFkB炎症信号，提示硒营养在红细胞稳态中的重要作用及分子机制；以砷暴露为例发现胁迫诱导血管内皮钙流-钙蛋白酶1响应，通过剪切内皮连接分子参与调节血管应激和内皮稳态，还发现钙蛋白酶2核转位活化，剪切释放DAMP/HMGB1炎症信号参与动脉粥样发生；首次研究证明硒代谢物甲基硒酸对血管内皮细胞铁死亡的拮抗作用，及上调Nrf2/GPX4/GSH作用机制；通过创新制备系列糖基功能化靶向纳米硒，有效调控巨噬细胞M2极化，及干预治疗AS和IBD中显示出良好的效果。这些研究具有较好的转化应用前景。