Elucidating pro-metastatic collagen modifying activities of lysyl hydroxylase 2

随着恶性肿瘤的进展，它们积累交联的胶原纤维，增强基质硬度， 从而激活引发癌细胞侵入和扩散的胶原受体。我们之前 报道称，一种称为赖氨酰羟化酶2（LH 2）的胶原修饰酶在转移性乳腺癌中高度表达。 肺癌细胞，并通过增加一种特别稳定的胶原蛋白的量来促进转移。 交联称为羟基赖氨酸衍生胶原交联（HLCC）。LH家族成员 （LH 1 -3）具有高度保守的LH和葡糖基半乳糖基转移酶（GGT）结构域，据报道LH 2缺乏 GGT活性，并且在LH中是独特的，其能够羟基化胶原蛋白N-和C-上的赖氨酸（lys）残基。 末端（“端肽”）。LH 2独特功能的结构基础 属性尚不清楚。本文提出的研究将解决这一关键的知识差距。根据 胶原蛋白LH和GGT结构域的晶体结构，我们最近解决，我们表明，LH 2具有端肽基 LH（t-LH）活性，由于独特的碱性残基簇与酸性氨基酸产生静电相互作用 胶原蛋白上的残留物。此外，通过使用我们开发的新的胶原蛋白GGT活性测定， 比以前报道的敏感，我们表明，LH 2具有GGT活性，由于选择性剪接， 外显子13 a编码的环，其位于GGT活性位点的入口处。我们发现LH 2亚型， 缺失（LH 2a）或含有（LH 2b）外显子13 a在TCGA肺癌组群中差异表达， LH 2b是原位KMLC模型中表达的主要同种型，其中LH 2促进转移 并引起肿瘤内免疫的广泛改变。我们开发了定义的胶原蛋白基质， LH 2介导的HLCC（总或葡萄糖基化）缺乏或充满，并使用这些基质作为工具来显示 HLCCs影响肺癌细胞的行为。根据这些初步结果，我们假设， LH 2通过双重（LH和GGT介导的）胶原修饰驱动肺癌转移，并将测试 通过完成3个具体目标来实现这一假设。目的1：证明LH 2与 与胶原的静电相互作用、HLCC形成和肺癌转移。目标2：展示 LH 2外显子13 a、胶原糖基化和转移之间的因果关系。目标3： 证明LH 2的双重（t-LH和GGT介导的）胶原蛋白修饰之间的因果关系， LAIR-1介导的免疫抑制。总之，我们的建议的新奇在于一个假设， 基于对LH 2的双重酶活性的独特见解以及我们开发的产生这种酶活性的工具， 假设（例如，晶体结构、酶测定和确定的胶原基质）。这些调查结果 这些研究将为将来测试我们已经从LH 2中鉴定出的选择性LH 2拮抗剂提供基础。 高通量筛选，并已开始在本申请范围外进行优化。

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.