Collagen folding and Interactions: from basic principles to bone disorders

胶原蛋白折叠和相互作用：从基本原理到骨骼疾病

• 批准号: 7734679
• 负责人: Sergey Leikin
• 金额: $ 58.63万
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7734679
• 关键词: 2-oxoglutarate 3-dioxygenase proline; Affect; Affinity; Age-Related Bone Loss; Amino Acid Substitution; Area; Binding; Binding Sites; Biological Assay; Bone Diseases; Bone Matrix; Breast Cancer Cell; Caliber; Cartilage; Cells; Clinical; Collaborations; Collagen; Collagen Fiber; Collagen Fibril; Collagen Type I; Collagen Type III; Complex; Confocal Microscopy; Connective Tissue; Connective Tissue Diseases; Data; Defect; Degenerative polyarthritis; Dermal; Development; Diagnostic; Disease; Disruption; Ehlers-Danlos Syndrome; Endoplasmic Reticulum; Extracellular Matrix; Extracellular Matrix Proteins; Fiber; Fibroblasts; Fluorescence; Glycine; Goals; Helix (Snails); Homo; Hydrogen Bonding; Image; Individual; Interstitial Collagenase; Label; Life; Ligand Binding; Ligands; Malignant Neoplasms; Maps; Matrix Metalloproteinases; Measurement; Measures; Microfibrils; Modeling; Molecular; Mutation; National Institute of Child Health and Human Development; Osteogenesis Imperfecta; Osteoporosis; Outcome; Pathology; Patients; Peptides; Phenotype; Play; Process; Proteins; Proteoglycan; Rate; Reporting; Research Personnel; Role; Site; Skeletal system; Skin; Stress; Structure; Surface; Swelling; Symptoms; Technology; Temperature; Testing; Time; Tissues; Type I Procollagen; Variant; base; biglycan; bone; bone cell; collagenase; collagenase 3; cyclophilin B; decorin; ear helix; interest; melting; mutant; novel; null mutation; reconstitution; research study; size; spectroscopic imaging; triple helix

Type I collagen is a long, triple helical protein, which forms the matrix of bone, skin and other tissues. During the last year, we continued characterization of structural consequences of Osteogenesis Imperfecta (OI) mutations in the type I triple helix. We expanded the map of changes in the triple helix stability (melting temperature), which now includes over four dozen mutations at different sites along the helix. To relate this map to peptide-based stability predictions, we proposed a model for extracting the activation energy of local helix unfolding from the reported peptide data. We tested the model and determined its parameters by measuring the H-D exchange rate for glycine NH residues involved in inter-chain hydrogen bonds in collagen. From comparison of the peptide-based and expanded mutation-based stability maps, we refined our model of regional variations in the triple helix structure. The refined regions align with regions important for collagen fibril assembly and ligand binding, and they appear to contribute to at least some of the observed regional variations in OI phenotype. Disruption of collagen interactions with ligands, particularly other extracellular matrix proteins and proteoglycans, is one of possible mechanisms relating structural defects in collagen triple helix to functional abnormalities in OI. In the last year, we completed development of a novel confocal microscopy assay for visualizing and quantitatively measuring binding of various matrix proteins to individual collagen fibrils based on differential fluorescent labeling of collagen and the ligand. With this assay, e.g., we found that mammalian collagenases attack damaged and poorly assembled collagen fibers via preferential binding to microfibrils, which become more exposed at fiber defects. Another study revealed that decorin and biglycan bind to surfaces of collagen fibrils of different size with the same affinity, suggesting that they regulate the fibril size by favoring larger surface area rather than a specific fibril diameter. These measurements also demonstrated similar binding of decorin and biglycan to fibrils reconstituted from collagen with truncated terminal peptides (telopeptides), suggesting that the telopeptides do not contain or block important binding sites on fibril surfaces. We are now focusing on measurement of decorin and biglycan interactions with fibers containing mutant collagen molecules that cause OI and other connective tissue disorders. The overwhelming majority of severe OI cases are caused by single amino acid substitutions. However, several recessive OI and EDS cases were described, in which all type I collagen was synthesized in the form of alpha-1 homotrimer rather than the normal heterotrimer of two alpha-1 and one alpha-2 chains. Formation of type I homotrimers was also observed in cultures of breast cancer cells and in cultures of normal bone cells from individuals predisposed to common, age-related osteoporosis. Our previous studies revealed altered regional stability of the homotrimer triple helix and a small increase in the denaturation temperature of the whole molecule, but they did not offer clues to potential mechanisms of pathology. Over the last two years, we made a potential breakthrough. We discovered that while hetero- and homotrimers coassemble within the same fibrils, they segregate at a subfibrillar level, potentially forming separate microfibrils. Our measurements revealed a significant reduction in the homotrimer cleavage rate by major tissue collagenases, MMP-1 and MMP-13. More detailed examination suggested that the initial MMP binding to collagen is not affected by the lack of the alpha-2 chain, but that this chain is essential for the next step of triple helix unwinding and opening, preceding the cleavage. In tissues with mixed collagen composition, e.g., containing type I homo- and heterotrimers or type I homotrimers and type III collagen, the abnormal cleavage rate of one component will alter the normal remodeling process. In preliminary experiments, live imaging of the degradation of mixed hetero/homotrimer fibrils by confocal microscopy with differential fluorescence labeling provided first direct evidence of such abnormal remodeling. We believe that it may play an important role in various pathologies associated with homotrimer synthesis and hope that better understanding of the underlying molecular mechanism may help to develop new treatment strategies. Most but not all cases with clinical symptoms of OI are caused by mutations in type I collagen. In collaboration with clinical researchers from Bone and Extracellular Matrix Branch of NICHD, we reported that severe/lethal skeletal pathology reminiscent of OI is also caused by recessive null mutations in the cartilage associated protein (CRTAP) or prolyl-3-hydroxylase (P3H1). CRTAP and P3H1 form a tight three-protein complex with cyclophilin B in the Endoplasmic Reticulum (ER). We found that the disruption of this complex significantly delays type I procollagen folding, resulting in overhydroxylation and overglycosylation of Lys residues, which is also observed in many OI cases. Further measurements conducted during the last year revealed abnormally high rate of synthesis of the collagen chains and delayed secretion of the mature protein by dermal fibroblasts from patients lacking P3H1. The combination of faster chain synthesis, slower triple helix folding, and delayed secretion causes accumulation of up to five times the normal amount of collagen within cells. The resulting ER swelling likely leads to ER stress contributing to the severe/lethal outcome in the patients.

I型胶原蛋白是一种长的三螺旋蛋白质，形成骨、皮肤和其他组织的基质。在过去的一年中，我们继续表征I型三螺旋中成骨不全（OI）突变的结构后果。我们扩展了三螺旋稳定性（解链温度）的变化图，现在包括螺旋沿着不同位点的四十多个突变。为了将这张图与基于肽的稳定性预测联系起来，我们提出了一个模型，用于从报告的肽数据中提取局部螺旋展开的活化能。我们测试了模型，并确定其参数，通过测量的H-D交换率的甘氨酸NH残基参与链间氢键的胶原蛋白。通过比较基于肽和扩展的基于突变的稳定性图，我们改进了三螺旋结构中区域变化的模型。精细区域与胶原纤维组装和配体结合的重要区域对齐，并且它们似乎有助于至少一些观察到的OI表型的区域变化。 胶原蛋白与配体，特别是其他细胞外基质蛋白和蛋白聚糖的相互作用的破坏，是一个可能的机制，在OI的功能异常的胶原蛋白三螺旋结构缺陷。在过去的一年中，我们完成了一种新的共聚焦显微镜检测可视化和定量测量结合的各种基质蛋白质的基础上差异荧光标记的胶原蛋白和配体的个别胶原纤维的发展。通过该测定，例如，我们发现哺乳动物胶原酶通过优先结合微纤维攻击受损和组装不良的胶原纤维，微纤维在纤维缺陷处变得更加暴露。另一项研究表明，核心蛋白聚糖和双糖蛋白聚糖以相同的亲和力结合到不同大小的胶原纤维的表面，这表明它们通过有利于更大的表面积而不是特定的纤维直径来调节纤维大小。这些测量还证明了核心蛋白聚糖和双糖蛋白聚糖与从具有截短的末端肽（端肽）的胶原重构的原纤维的类似结合，表明端肽不包含或阻断原纤维表面上的重要结合位点。我们现在专注于测量核心蛋白聚糖和双糖链聚糖与含有突变胶原分子的纤维的相互作用，这些突变胶原分子会导致OI和其他结缔组织疾病。 绝大多数严重的OI病例是由单个氨基酸取代引起的。然而，几个隐性OI和EDS的情况下，其中所有的I型胶原蛋白合成的α-1同源三聚体的形式，而不是两个α-1和一个α-2链的正常异源三聚体。在乳腺癌细胞培养物和易患常见的年龄相关性骨质疏松症的个体的正常骨细胞培养物中也观察到I型同源三聚体的形成。我们先前的研究揭示了同源三聚体三螺旋的区域稳定性改变和整个分子的变性温度的小幅增加，但它们没有提供潜在病理机制的线索。在过去的两年里，我们取得了一个潜在的突破。我们发现，虽然异三聚体和同三聚体在相同的原纤维内共同组装，但它们在亚原纤维水平上分离，可能形成单独的微纤维。我们的测量结果显示，主要组织胶原酶MMP-1和MMP-13的同源三聚体裂解率显著降低。更详细的检查表明，最初的MMP与胶原蛋白的结合不受缺乏α-2链的影响，但该链对于裂解前三螺旋解旋和打开的下一步是必不可少的。在具有混合胶原成分的组织中，例如，含有I型同源三聚体和异源三聚体或I型同源三聚体和III型胶原蛋白，一种组分的异常裂解率将改变正常的重塑过程。在初步的实验中，混合异/同三聚体原纤维的降解的共聚焦显微镜与差分荧光标记的实时成像提供了这种异常重塑的第一个直接证据。我们相信它可能在与同源三聚体合成相关的各种病理中发挥重要作用，并希望更好地了解潜在的分子机制可能有助于开发新的治疗策略。 大多数但不是所有具有OI临床症状的病例是由I型胶原突变引起的。与NICHD骨和细胞外基质分支的临床研究人员合作，我们报告了软骨相关蛋白（CRTAP）或脯氨酰-3-羟化酶（P3 H1）的隐性无效突变也会引起严重/致死性骨骼病理学，使人联想到OI。CRTAP和P3 H1在内质网（ER）中与亲环素B形成紧密的三蛋白复合物。我们发现，这种复合物的破坏显着延迟I型前胶原折叠，导致过度羟基化和过度糖基化的赖氨酸残基，这也是在许多OI的情况下观察到的。在过去一年中进行的进一步测量显示，缺乏P3 H1的患者的真皮成纤维细胞合成胶原蛋白链的速率异常高，并且成熟蛋白的分泌延迟。更快的链合成、更慢的三螺旋折叠和延迟的分泌的组合导致细胞内胶原蛋白的积累高达正常量的五倍。由此产生的ER肿胀可能导致ER应激，从而导致患者的重度/致死性结局。