Collagen Assembly in Intervertebral disc

椎间盘中的胶原蛋白组装

• 批准号: 9753934
• 负责人: RUSSELL J FERNANDES
• 金额: $ 34.21万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-04-01 至 2022-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9753934
• 关键词: 2-oxoglutarate 3-dioxygenase proline; Address; Adult; Age; Aging; Architecture; Back; Back Pain; Biochemical; Biological; Biological Markers; Biological Models; Biology; Biomechanics; CRISPR/Cas technology; Caliber; Cell Line; Cells; Collagen; Collagen Fibril; Collagen Type II; Collagen Type IX; Collagen Type XI; Complex; Deposition; Disease; Electron Microscopy; Enzymes; Extracellular Matrix; Fingerprint; Functional disorder; Future; Genes; Goals; Grant; Growth; Health Care Costs; Hyaline Cartilage; Hydroxylation; Hydroxyproline; In Vitro; Intervertebral disc structure; Investigation; Knock-out; Knowledge; Low Back Pain; Mass Spectrum Analysis; Methodology; Methods; Modification; Molecular Profiling; Natural regeneration; Pattern; Phenotype; Physiology; Polymers; Population; Post-Translational Protein Processing; Proline; Property; Rattus; Regulation; Role; Shock; Spinal; Spinal Neoplasms; Structure; System; Thick; Thinness; Time; Tissues; Trauma; Vertebral column; Width; base; covalent bond; crosslink; gene product; interest; intervertebral disk degeneration; molecular assembly/self assembly; next generation; normal aging; nucleus pulposus; pyridinoline; regenerative; repaired; symptom treatment; tissue regeneration

ABSTRACT In this competing renewal of AR057025, we expand the scope and now focus on collagen in nucleus pulposus (NP) tissue of the interertebral disc. We propose to 1) define the role of NP-specific post- translational modifications within type II collagen chains in regulating the diameter of fibrils. 2) establish a molecular fingerprint for cross-linked collagen heterofibril assembly as a biomarker for native and in vitro generated NP tissue. By the age of fifty, 85 percent of the US population shows evidence of a compromised collagen network and disc herniation. As the population ages, such biomarkers to evaluate the quality of regenerated NP neo-tissue is significant, offering new hope in the treatment of disc disease. The fibrillar network that frames the jelly-like nucleus pulposus is made up of types II, IX and XI collagen, the same gene products that characterize hyaline cartilage. The mechanism that drives these molecules to heteropolymerize as thin diameter fibrils in nucleus pulposus but as thicker diameter fibrils in hyaline cartilage is still unclear. Recent evidence convincingly correlates the assembly of thin (<20nm) collagen fibrils in NP with elevated levels of an unique type II collagen post-translational modification, the 3-hydroxylation of proline residue 944 (P944). In hyaline cartilage where the 3-hydroxylation of P944 is nearly lacking, thicker (20- 100nm) fibrils are observed. We will use the RCS-LTC cell line as a model system to address this mechanism. This cell line, originally derived from a spinal neoplasm, assembles types II, IX, XI collagens into cross- linked thin diameter collagen fibrils in a jelly-like extracellular matrix. Elevated levels of the prolyl 3-hydroxylase 2 (P3H2) enzyme correlated with the highly 3-hydroxylated P944 residues in type II collagen chains deposited in the matrix. We will use the CRISPR/Cas9 gene editing system to knock out the P3H2 gene, in combination with mass spectrometry and electron microscopy to define a role for 3-hydroxyproline residues in type II collagen fibril diameter regulation. We intend to aggressively pursue this concept in order to understand how cells modulate the thickness of collagen fibrils in NP and other type II collagen based tissues. This is important from both a basic biology and tissue regeneration perspective. Furthermore, employing biochemical methodology generated from our original RO1 grant, we aim to fingerprint the pattern of collagen inter-type II- IX-XI cross-linking in native and in vitro cultured nucleus pulposus neo-tissue. This will provide a screen for normal matrix assembly and serve as a basis for future regeneration studies. Electron microscopy, mass spectrometry and biomechanics will be used to determine the thickness and post-translational quality of the fibrils and function. The goal is to ascertain the ability of the neo-tissues to assemble tissue-specific 3- hydroxyproline modified type II collagen molecules into a network typical of native nucleus pulposus. Essential information for next-generation NP neo-tissue with high collagen content will be gained. This is significant as a nascent, accurately cross-linked, type II-IX-XI collagen template is crucial to the growth of the fibril.

抽象的 在 AR057025 的竞争性更新中，我们扩大了范围，现在重点关注细胞核中的胶原蛋白 椎间盘的髓（NP）组织。我们建议 1）定义 NP 特定后的作用 II 型胶原链内的翻译修饰可调节原纤维的直径。 2）建立一个 交联胶原异原纤维组装的分子指纹作为天然和体外生物标志物 生成NP组织。到了 50 岁，85% 的美国人都表现出了受到损害的迹象 胶原网络和椎间盘突出。随着人口老龄化，此类生物标志物来评估人口的质量 再生的椎间盘新组织意义重大，为椎间盘疾病的治疗带来了新的希望。 构成果冻样髓核的纤维网络由 II、IX 和 XI 型胶原蛋白组成， 具有透明软骨特征的相同基因产物。驱动这些分子的机制 在髓核中异聚为细直径原纤维，但在透明软骨中异聚为粗直径原纤维 目前还不清楚。最近的证据令人信服地将 NP 中薄（<20nm）胶原纤维的组装与 独特的 II 型胶原蛋白翻译后修饰（脯氨酸的 3-羟基化）水平升高 残基944(P944)。在透明软骨中，P944 的 3-羟基化几乎缺失，较厚（20- 100nm) 观察到原纤维。我们将使用 RCS-LTC 细胞系作为模型系统来解决这一机制。 该细胞系最初源自脊柱肿瘤，可将 II、IX、XI 型胶原组装成交叉 在果冻状的细胞外基质中连接细直径的胶原纤维。脯氨酰 3-羟化酶水平升高 2 (P3H2) 酶与沉积的 II 型胶原链中高度 3-羟基化的 P944 残基相关 在矩阵中。我们将利用CRISPR/Cas9基因编辑系统敲除P3H2基因，结合 使用质谱和电子显微镜确定 3-羟脯氨酸残基在 II 型中的作用 胶原纤维直径调节。我们打算积极追求这个概念，以了解如何 细胞调节 NP 和其他基于 II 型胶原蛋白的组织中胶原蛋白原纤维的厚度。这很重要 从基础生物学和组织再生的角度来看。此外，采用生化 我们最初的 RO1 资助产生的方法，我们的目标是对 II 型胶原蛋白的模式进行指纹识别 天然和体外培养的髓核新组织中的 IX-XI 交联。这将提供一个屏幕 正常的基质组装并作为未来再生研究的基础。电子显微镜，质量 光谱测定法和生物力学将用于确定厚度和翻译后质量 原纤维和功能。目标是确定新组织组装组织特异性 3- 羟脯氨酸将 II 型胶原蛋白分子修饰成天然髓核的典型网络。基本的 将获得具有高胶原蛋白含量的下一代 NP 新生组织的信息。这具有重要意义 新生的、精确交联的 II-IX-XI 型胶原蛋白模板对于原纤维的生长至关重要。