PYRIN domains 的表現，純化及三維核磁共振結構分析

Abstract

先天免疫系統招募各種穿膜及細胞質中生殖系編碼 (germline-encoded)的模式識別受體(pattern recognition receptors, PRRs) 來偵測外來病原的pathogen-associatedmolecular patterns (PAMPs)，像是脂多醣 (lipopolysaccharide, LPS), 肽聚醣(peptidoglycan, PGN),鞭毛(flagellin), 核酸(DNA) 及CpG DNA. 這些被PAMPs 刺激的受體便會啟動細胞內的訊息傳遞以達到宿主防禦的反應。 當PRRs 的leucine-rich repeat (LRR) 辨認到各種各樣的PAMPs, 細胞質中nucleotide-binding domain and leucine-rich repeat-containing receptors (NLRs)的N 端Pyrin domain (PYD) 利用它的homotypic 作用力使訊號傳到下游。PYD-containingNLRs (NLRPs)家族總共有14 個成員，而其中幾個發生突變跟人類發炎失調疾病息息相關。 人類的PYNOD, 亦稱NLRP10 是一個新穎的發炎負向調控者，其特徵是缺乏C端的LRR。通過它的PYD domain, PYNOD抑制ASC (apoptosis-associated speck-like protein containing a CARD)的聚合反應，使capase-1 的活化以及caspase-1 媒介的1L-1β 成熟化受到壓制。由於PYNOD 具有抗發炎性，從結構的角度了解它跟ASC的PYD-PYD 的結合模式是重要的。在本篇論文中，我主要研究PYNOD 的三維核磁共振結構和它的動態，結果顯示其外形是由6 個Helices 構成的桶狀構形，連接H2 和H3 有突出的loop L3。骨架動態數據發現這loop L3 及H3 比其他5 段Helices有相對性較高的彈性。

The innate immune system recruits various transmembrane and cytoplasmic germline-encoded pattern recognition receptors (PRRs) to detect pathogen-associated molecular patterns (PAMP) motifs of microbial invaders, such as lipopolysaccharides (LPS), peptidoglycan (PGN), flagellin, ds-DNA, and CpG DNA. PAMP-activated receptors then trigger intracellular signaling cascades that lead to a spectrum of host defense reactions. While diverse PAMP motifs are recognized by the leucine-rich repeat (LRR) protein modules present in PRRs, the N-terminal pyrin domain (PYD) of cytoplasmic nucleotide-binding domain and leucine-rich repeat-containing receptors (NLRs) links PAMP-recognition to downstream signaling cascades through specific homotypic interactions. There are 14 members of PYD-containing NLRs (NLRPs) and mutations in several of them have been implicated in numerous human inflammatory disorders. Human PYNOD/NLRP10 is a novel negative regulator of inflammation characterized by the lack of a C-terminal LRR domain. Via its PYD domain, PYNOD suppresses oligomerization of ASC (apoptosis-associated speck-like protein containing a CARD), a critical PYD-containing signaling adaptor protein, which leads to inhibition of caspase-1 activation and caspase-1 mediated maturation of 1L-1β. Owing to its important anti-inflammatory activity, we have sought to elucidate the structural basis of PYD-mediated interaction of PYNOD with ASC. Here I present the structure and dynamics of the human PYNOD PYD, which shows that PYNOD PYD adopts a core six-helix bundle structure with a prominent loop L3 between helices H2 and H3. Backbone 15N relaxation data revealed this L3 loop and the H3 helix display a greater degree of conformational disorder than the other five helices.