Elucidating pro-metastatic collagen modifying activities of lysyl hydroxylase 2

阐明赖氨酰羟化酶 2 的促转移胶原蛋白修饰活性

基本信息

批准号：
10376870
负责人：
Jonathan M Kurie
金额：
$ 52.79万
依托单位：
UNIVERSITY OF TX MD ANDERSON CAN CTR
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-04-01 至 2026-03-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10376870
关键词：
2-Oxoglutarate 5-Dioxygenase Procollagen-Lysine Acidic Amino Acids Active Sites Address Affect Affinity Aldehydes Alternative Splicing Arginine Basic Amino Acids Binding Biological Assay Cancer Model Cancer Patient Cells Coculture Techniques Collagen Collagen Fibril Collagen Receptors Crystallization Electrostatics Enzymes Exons Family member Fibroblasts Future Glucose Glucosyltransferase Hydroxylysine Immune Immunocompetent Immunosuppression KRAS2 gene Knowledge Lysine Malignant Neoplasms Malignant neoplasm of lung Mediating Modification Mus Myeloid Cells Natural Killer Cells Neoplasm Metastasis Population Production Property Protein Isoforms RNA Splicing Receptor Activation Reporting Rest Source Specific qualifier value Structure Structure-Activity Relationship T-Lymphocyte Testing The Cancer Genome Atlas Tumor Immunity antagonist base cancer cell cell behavior cohort crosslink differential expression efficacy testing glycosyltransferase high throughput screening insight lung cancer cell mechanical force migration mutant novel therapeutics tool tumor tumor progression tumor-immune system interactions

2-Oxoglutarate 5-Dioxygenase Procollagen-Lysine Acidic Amino Acids Active Sites Address Affect Affinity Aldehydes Alternative Splicing Arginine Basic Amino Acids Binding Biological Assay Cancer Model Cancer Patient Cells Coculture Techniques Collagen Collagen Fibril Collagen Receptors Crystallization Electrostatics Enzymes Exons Family member Fibroblasts Future Glucose Glucosyltransferase Hydroxylysine Immune Immunocompetent Immunosuppression KRAS2 gene Knowledge Lysine Malignant Neoplasms Malignant neoplasm of lung Mediating Modification Mus Myeloid Cells Natural Killer Cells Neoplasm Metastasis Population Production Property Protein Isoforms RNA Splicing Receptor Activation Reporting Rest Source Specific qualifier value Structure Structure-Activity Relationship T-Lymphocyte Testing The Cancer Genome Atlas Tumor Immunity antagonist base cancer cell cell behavior cohort crosslink differential expression efficacy testing glycosyltransferase high throughput screening insight lung cancer cell mechanical force migration mutant novel therapeutics tool tumor tumor progression tumor-immune system interactions

展开

项目摘要

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.

随着他们的进展，恶性肿瘤会积累交联的胶原蛋白原纤维，从而增强基质刚度，从而激活引发癌细胞侵袭和传播的胶原蛋白受体。我们以前据报道，一种称为赖氨基羟化酶2（LH2）的胶原蛋白修饰酶在转移性中高度表达肺癌细胞并通过增加特别稳定的胶原蛋白的量来促进转移交联称为羟基乙醛醛衍生的胶原蛋白交联（HLCC）。尽管LH家族成员（LH1-3）具有高度构成LH和葡萄糖基乳糖基转移酶（GGT）域，据报道缺乏LH2 GGT活性，在LHS之间是独一无二的，其在胶原蛋白N和C-上保留了羟基赖氨酸（LYS）的能力生成HLCC所需的末端（“端肽”）。 LH2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知识差距。基于我们最近解决的胶原蛋白LH和GGT结构域结构由于独特的基本居住簇，LH（T-LH）活性，该群集与酸性产生静电相互作用胶原蛋白的残留物。此外，通过使用新的胶原蛋白GGT活动测定法，我们开发了更多比以前报道的敏感的外显子13A编码的环位于GGT活动位点的入口处。我们证明LH2同工型缺乏（LH2A）或包含（LH2B）外显子13A在TCGA肺癌队列中表达不同，并且 LH2B是在原位KMLC模型中表达的主要同工型，其中LH2促进转移并引起肿瘤内免疫的宽度改变。我们开发了定义的胶原矩阵在LH2介导的HLCC（总或葡萄糖基化）中缺乏或替换，并使用这些矩阵作为显示的工具 HLCC会影响肺癌细胞行为。根据这些初步结果，我们假设 LH2通过双重（LH-和GGT介导的）胶原蛋白修饰驱动肺癌转移，并将测试通过完成3个具体目标，该假设。目标1：展示LH2之间的因果关系与胶原蛋白，HLCC形成和肺癌转移的静电相互作用。目标2：展示目标3：到展示了LH2的双重（T-LH和GGT介导的）胶原蛋白修饰与 LAIR-1介导的免疫抑制。总而言之，我们提案的新颖性在于一个假设基于对LH2的双重酶促活动的独特见解，以及我们开发的工具假设（例如，晶体结构，酶测定和固定的胶原蛋白材料）。这些发现研究将为我们已经从中确定的选择性LH2拮抗剂的未来测试提供基础高通量屏幕并已开始在此应用程序范围之外进行优化。