Collagen Assembly in Intervertebral disc

摘要 在AR057025的这一竞争性更新中，我们扩大了范围，现在专注于细胞核中的胶原 椎间盘的髓(NP)组织。我们建议1)界定特定于NP的后语的作用-- II型胶原链中调节纤维直径的翻译修饰。2)建立 交联型胶原异质原集合体的分子指纹图谱作为天然和体外生物标志物 产生了NP组织。到了50岁，85%的美国人有证据表明 胶原网和椎间盘突出。随着人口老龄化，这种生物标志物可以用来评估 再生的NP新生组织意义重大，为椎间盘疾病的治疗提供了新的希望。 包裹果冻状髓核的纤维网络由II型、II型和XI型胶原组成， 具有透明软骨特征的相同基因产物。驱动这些分子的机制 在髓核中异聚为直径较小的纤维，而在透明软骨中为直径较大的纤维 目前仍不清楚。最近的证据令人信服地将NP中薄(20 Nm)胶原纤维的组装与 一种独特的II型胶原蛋白翻译后修饰后水平升高，即3-羟化脯氨酸 残留量944(P944)。在透明软骨中，P944的3-羟基化几乎缺失，较厚(20- 100 nm)的纤维。我们将使用RCS-LTC细胞系作为模型系统来解决这一机制。 这种细胞系最初来自脊柱肿瘤，它将II，IX，XI型胶原组装成交叉- 胶状细胞外基质中连接的细小直径的胶原纤维。3-羟基脯氨酸酶水平升高 2(P3H2)酶与II型胶原链中高度3-羟基化的P944残基相关 在矩阵中。我们将使用CRISPR/Cas9基因编辑系统联合敲除P3H2基因 用质谱仪和电子显微镜确定3-羟基脯氨酸残基在II型中的作用 胶原纤维直径调节。我们打算积极地追求这一概念，以便了解如何 在NP和其他以II型胶原为基础的组织中，细胞调节胶原纤维的厚度。这事很重要 从基础生物学和组织再生的角度来看。此外，使用生化 方法由我们最初的RO1拨款产生，我们的目标是对II型胶原间质的模式进行指纹识别- IX-XI在天然和体外培养的髓核新生组织中的交联性。这将为 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 光谱学和生物力学将被用来确定厚度和翻译后质量 纤维和功能。我们的目标是确定新组织组装组织特异性3- 羟脯氨酸将II型胶原分子修饰成典型的天然髓核网络。基本要素 将获得高胶原蛋白含量的下一代NP新生组织的信息。这一点意义重大，因为 新生的、精确交联的II-IX-XI型胶原模板对纤维的生长至关重要。

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.