Pathogenic mechanisms of lysosomal disease

描述（由申请人提供）：溶酶体的生物发生和功能缺陷导致溶酶体储存障碍（lsd）。导致疾病病理的潜在机制尚不清楚，这是确定治疗靶点的障碍。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糖基标签（甘露糖6-磷酸）的酶（GlcNAc-1-磷酸转移酶）缺失。这导致新生成的组织蛋白酶大量分泌到细胞外空间。组织蛋白酶错标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。本提案的主要目的是解决分泌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导致ML-II软骨病理。大多数关于ML-II（和其他lsd）的研究都集中在储存对细胞功能的影响上。我们的方法是新颖的，因为它侧重于错误靶向溶酶体水解酶的后果，这是ML-II的独特特征。使用morpholino- knockdown策略，我们创建了ML-II的第一个斑马鱼模型，以研究发育过程中M6P依赖性溶酶体靶向受损的影响。我们证明，软骨形成缺陷伴随着转化生长因子β (TGFss)调控的基质蛋白、II型胶原的持续表达，以及组织蛋白酶K活性的持续和升高。在ML-II背景下，组织蛋白酶K活性的降低减少了II型胶原的持续表达，并减轻了软骨表型。抑制组织蛋白酶K降低II型胶原丰度的发现是出乎意料的，因为预测这种强效胶原酶活性的降低反而会导致胶原积累。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的功能——超越胶原蛋白的转换。我们的集体数据支持一种模型，即ML-II软骨细胞不能超过软骨形成的早期阶段。由于正常的软骨形成过程需要从TGFss到骨形态发生蛋白（BMP）信号的转变，我们假设组织蛋白酶K的高分泌和细胞外活性破坏了这些生长因子通路的平衡，进而导致异常的软骨细胞分化。ML-II是无法治疗的。通过研究ML-II异常软骨形成的分子机制，我们希望找到新的下游治疗靶点，并开发一个强大的系统来研究正常软骨形成过程中分泌组织蛋白酶的生理相关性。提出的具体目标将包括：1)研究组织蛋白酶K高分泌如何改变软骨细胞分化过程中tgf - ss和BMP信号的平衡；2)确定组织蛋白酶K在正常软骨发育阶段的调节和活性；3)揭示介导组织蛋白酶特异性识别的glcnac -1磷酸转移酶的结构元件。

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.