Pathogenic mechanisms of lysosomal disease

溶酶体疾病的发病机制

• 批准号: 9184568
• 负责人: Heather R Flanagan Steet
• 金额: $ 33.6万
• 依托单位: UNIVERSITY OF GEORGIA
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-01-01 至 2018-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9184568
• 关键词: Address; Affect; BMP2 gene; Biogenesis; Bone Morphogenetic Proteins; Carbohydrates; Cartilage; Cathepsins; Cell physiology; Cells; Chondrocytes; Chondrogenesis; Collagen; Data; Defect; Deposition; Development; Digestion; Disease; Elements; Embryo; Enzymes; Equilibrium; Exocytosis; Extracellular Space; Goals; Growth Factor; Hydrolase; Impairment; In Vitro; Lead; Link; Lysosomes; Mediating; Modeling; Molecular; Molecular Probes; Mutation; N-acetylglucopyranosylamine; Pathogenicity; Pathologic; Pathology; Pathway interactions; Patients; Peptide Hydrolases; Phase; Phenotype; Phosphotransferases; Physiological; Proteins; Regulation; Severities; Signal Pathway; Signal Transduction; Signaling Protein; Sorting - Cell Movement; Specificity; System; Testing; Transforming Growth Factor beta; Transforming Growth Factors; Transgenic Organisms; Translating; Work; Zebrafish; base; cartilage development; cathepsin K; collagenase; craniofacial; experimental study; extracellular; in vivo; insight; knock-down; mannose 6 phosphate; novel; novel therapeutics; programs; public health relevance; receptor; targeted treatment; therapeutic target

DESCRIPTION (provided by applicant): Defects in the biogenesis and function of lysosomes result in lysosomal storage disorders (LSDs). The underlying mechanisms that lead to disease pathology are poorly understood, representing a barrier in identifying therapeutic targets. In the lysosomal disease mucolipidosis II (ML-II), the enzyme (GlcNAc-1- phosphotransferase) that synthesizes the carbohydrate-based tag (mannose 6-phosphate) needed for receptor-mediated lysosomal targeting is missing. This causes hypersecretion of newly made cathepsins into the extracellular space. Cathepsin mistargeting places these enzymes in contact with the matrix-deposited growth factors that control the timing and fidelity of chondrocyte maturation. The primary objective of this proposal is to address how secreted lysosomal cathepsins modulate key growth factor pathways in chondrocytes and cause ML-II cartilage pathology. Most work on ML-II (and other LSDs) focuses on the consequences of storage on cellular function. Our approach is novel in that it focuses instead on the consequences of mistargeted lysosomal hydrolases, a unique feature of ML-II. Using a morpholino- knockdown strategy, we generated the first zebrafish model for ML-II to study the impact of impaired M6P- dependent lysosomal targeting during development. We demonstrated chondrogenesis defects accompanied by sustained expression of the transforming growth factor beta (TGFss)-regulated matrix protein, type II collagen, and sustained and elevated activity of the cathepsin K. Lowering cathepsin K activity in the ML-II background reduced this sustained expression of type II collagen and alleviated the cartilage phenotypes. The finding that inhibition of cathepsin K reduces the abundance of type II collagen was unexpected since decreased activity of this potent collagenase was predicted instead to lead to collagen accumulation. This observation supports a broader function for this protease - beyond collagen turnover - during pathogenic cartilage development. Our collective data support a model whereby ML-II chondrocytes are unable to proceed beyond early phases of chondrogenesis. Since progression through the normal chondrogenic program requires a transition from TGFss to bone morphogenetic protein (BMP) signaling, we hypothesize that hypersecretion and extracellular activity of cathepsin K disrupts the balance of these growth factor pathways, in turn causing abnormal chondrocyte differentiation. ML-II is untreatable. By addressing the molecular mechanisms that underlie abnormal chondrogenesis in ML-II, we hope to identify new downstream targets for therapy and develop a powerful system to investigate the physiological relevance of secreted cathepsins during normal cartilage formation. The proposed specific aims will 1) address how cathepsin K hypersecretion alters the balance of TGFss and BMP signaling during chondrocyte differentiation, 2) define cathepsin K regulation and activity within the stages of normal cartilag development, and 3) uncover the structural elements within the GlcNAc-1-phosphotransferase enzyme that mediate specific recognition of cathepsins.

描述（由申请方提供）：溶酶体的生物发生和功能缺陷导致溶酶体贮积症（LSD）。导致疾病病理学的潜在机制知之甚少，代表了识别治疗靶点的障碍。在溶酶体疾病粘脂沉积症II（ML-II）中，合成受体介导的溶酶体靶向所需的基于碳水化合物的标签（甘露糖6-磷酸）的酶（GlcNAc-1-磷酸转移酶）缺失。这导致新产生的组织蛋白酶分泌过多进入细胞外空间。组织蛋白酶的错误定位使这些酶与基质沉积的生长因子接触，这些生长因子控制软骨细胞成熟的时间和保真度。该提案的主要目的是解决分泌的溶酶体组织蛋白酶如何调节软骨细胞中的关键生长因子途径并引起ML-II软骨病理学。大多数关于ML-II（和其他LSD）的工作都集中在储存对细胞功能的影响上。我们的方法是新颖的，因为它侧重于错误的溶酶体水解酶的后果，ML-II的一个独特的功能。使用吗啉代敲低策略，我们产生了ML-II的第一个斑马鱼模型，以研究发育期间受损的M6 P依赖性溶酶体靶向的影响。我们证明了软骨形成缺陷伴随着转化生长因子β（TGF β）调节的基质蛋白、II型胶原蛋白的持续表达以及组织蛋白酶K的持续和升高的活性。降低ML-II背景中的组织蛋白酶K活性降低了II型胶原蛋白的持续表达，并缓解了软骨表型。组织蛋白酶K的抑制降低II型胶原的丰度的发现是出乎意料的，因为预测这种有效的胶原酶的活性降低反而导致胶原积累。这一观察结果支持了这种蛋白酶在致病性软骨发育过程中的更广泛功能-超越胶原蛋白周转。我们的集体数据支持ML-II软骨细胞无法进行软骨形成的早期阶段以外的模型。由于通过正常软骨形成程序的进展需要从TGF β到骨形态发生蛋白（BMP）信号传导的转变，我们假设组织蛋白酶K的分泌过多和细胞外活性破坏了这些生长因子途径的平衡，进而导致异常的软骨细胞分化。ML-II是无法治疗的。通过解决ML-II中异常软骨形成的分子机制，我们希望确定新的下游治疗靶点，并开发一个强大的系统来研究正常软骨形成过程中分泌的组织蛋白酶的生理相关性。提出的具体目标将1）解决组织蛋白酶K分泌过多如何改变软骨细胞分化期间TGF β和BMP信号传导的平衡，2）定义正常软骨发育阶段内的组织蛋白酶K调节和活性，以及3）揭示介导组织蛋白酶特异性识别的GlcNAc-1-磷酸转移酶内的结构元件。