Elucidating pro-metastatic collagen modifying activities of lysyl hydroxylase 2

随着肿瘤的发展，它们会积累交联型胶原纤维，从而增强基质硬度， 从而激活胶原蛋白受体，从而触发癌细胞的侵袭和扩散。我们之前 有报道称，一种名为赖氨酰羟基酶2(LH2)的胶原修饰酶在转移性肿瘤中高表达。 通过增加一种特别稳定的胶原蛋白的数量促进肺癌细胞的转移 这种交联物称为羟赖氨酸醛衍生的胶原交联物(HLCC)。虽然LH家族成员 (LH1-3)具有高度保守的促黄体生成素和葡萄糖半乳糖基转移酶(GGT)结构域，据报道LH2缺乏 GGT活性，在LHS中是独一无二的，因为它能够羟化N-和C-胶原上的赖氨酸(LYS)残基。 端粒(“端肽”)是产生HLCC所必需的。LH2的S唯一功能的结构基础 具体情况尚不清楚。这里提出的研究将解决这一关键的知识差距。在此基础上 对于我们最近解决的胶原LH域和GGT域的晶体结构，我们证明了LH2具有端肽 黄体生成素(t-黄体生成素)活性源于一个独特的碱性残基簇，它与酸性物质产生静电相互作用 胶原蛋白上的残留物。此外，通过使用我们开发的一种新的胶原GGT活性测定， 比以前报道的更敏感，我们证明了LH2由于选择性剪接而具有GGT活性 外显子13a编码的环，位于GGT活性位点的入口处。我们证明了LH2异构体 缺失(LH2a)或含有(LH2b)外显子13a在TCGA肺癌队列中差异表达，并且 LH2 b是在原位KMLC模型中表达的主要亚型，在该模型中，LH2促进转移 并导致肿瘤内免疫力的广泛变化。我们开发了定义的胶原蛋白基质， 在LH2介导的HLCC(完全的或糖化的)中不足或充足，并使用这些矩阵作为工具来显示 HLCC影响肺癌细胞的行为。根据这些初步结果，我们假设 LH2通过双重(促黄体生成素和谷氨酸转移酶介导的)胶原修饰推动肺癌转移，并将测试 这个假设是通过完成3个具体目标来实现的。目的1：论证LH2与S之间的因果关系 静电与胶原的相互作用，HLCC的形成，与肺癌转移。目标2：演示 LH2‘S外显子13a和胶原糖基化与转移的因果关系目标3：实现 证明LH2‘S双重(t-黄体生成素和谷氨酸转氨酶介导)胶原修饰与 LAIR-1介导的免疫抑制。总而言之，我们的提议的新颖性在于一个假设，即 基于对LH2的S双酶活性和我们开发的工具的独特洞察 假设(例如，晶体结构、酶分析和定义的胶原基质)。来自这些的发现 研究将为未来对我们已经确定的选择性LH2拮抗剂的测试提供基础 高通量的屏幕已经开始在这一应用范围之外进行优化。

As they progress, malignant tumors accumulate cross-linked collagen fibrils that enhance matrix stiffness, thereby activating collagen receptors that trigger cancer cell invasion and dissemination. We previously reported that a collagen modifying enzyme called lysyl hydroxylase 2 (LH2) is highly expressed in metastatic lung cancer cells and promotes metastasis by increasing the amount of a particularly stable type of collagen cross-link called hydroxylysine aldehyde-derived collagen cross-link (HLCC). Although LH family members (LH1-3) have highly conserved LH and glucosylgalactosyltransferase (GGT) domains, LH2 reportedly lacks GGT activity and is unique among LHs in its ability to hydroxylate lysine (lys) residues on collagen N- and C- termini (“telopeptides”) that are required to generate HLCCs. The structural basis for LH2's unique functional properties remains unclear. The studies proposed herein will address this crucial knowledge gap. On the basis of collagen LH and GGT domain crystal structures that we recently solved, we show that LH2 has telopeptidyl LH (t-LH) activity owing to a unique basic residue cluster that generates electrostatic interactions with acidic residues on collagen. Furthermore, by using a new collagen GGT activity assay we developed that is more sensitive than ones reported previously, we show that LH2 has GGT activity owing to an alternatively spliced exon 13a-encoded loop that resides at the entrance of the GGT active site. We show that LH2 isoforms that lack (LH2a) or contain (LH2b) exon 13a are differentially expressed in the TCGA lung cancer cohort, and that LH2b is the predominant isoform expressed in an orthotopic KMLC model in which LH2 promotes metastasis and causes widespread alterations in intra-tumoral immunity. We developed defined collagen matrices that are deficient or replete in LH2-mediated HLCCs (total or glucosylated) and used these matrices as tools to show that HLCCs influence lung cancer cell behaviors. On the basis of these preliminary results, we postulate that LH2 drives lung cancer metastasis through dual (LH- and GGT-mediated) collagen modifications and will test this hypothesis by completing 3 specific aims. Aim 1: To demonstrate a causal relationship between LH2's electrostatic interactions with collagen, HLCC formation, and lung cancer metastasis. Aim 2: To demonstrate a causal relationship between inclusion of LH2's exon 13a, collagen glucosylation, and metastasis. Aim 3: To demonstrate a causal relationship between LH2's dual (t-LH- and GGT-mediated) collagen modifications and LAIR-1-mediated immunosuppression. In summary, the novelty of our proposal rests in an hypothesis that is based on unique insight into LH2's dual enzymatic activities and the tools we developed to generate that hypothesis (e.g., crystal structures, enzymatic assays, and defined collagen matrices). Findings from these studies will provide a basis for future testing of selective LH2 antagonists that we have already identified from high-throughput screens and have begun to optimize outside the scope of this application.