# PATHOGENIC MECHANISMS OF LYSOSOMAL DISEASE > **NIH NIH R01** · GREENWOOD GENETIC CENTER · 2021 · $270,408 ## Abstract PROJECT SUMMARY Defects in the biogenesis or function of lysosomes result in lysosomal storage disorders. For most of these disorders the underlying pathogenic mechanisms are poorly understood, representing a barrier in identifying therapeutic targets. In the lysosomal disease mucolipidosis II (MLII), the enzyme (GlcNAc-1- phosphotransferase) that synthesizes the carbohydrate-based tag needed for receptor-mediated lysosomal targeting is missing. This causes cathepsin proteases to be secreted outside the cell where they can become activated by an unknown mechanism. Using powerful tools in the zebrafish system, our prior studies showed that secreted cathepsin K (Ctsk) alters the balance of TGFß and BMP signaling in developing cartilage. This imbalance in growth factor signaling – caused by Ctsk-mediated increases in latent TGFß activation - disrupts the timing and fidelity of chondrocyte maturation. These findings highlight a central role for mislocalized Ctsk in the cartilage defects associated with impaired lysosomal targeting. The primary objective of the current proposal is to address how the secretion of Ctsk is linked to its promiscuous activation in vivo, and how interactions with glycosaminoglycans (GAGs) mediate this process. This new direction is premised by the in vitro studies of others that show GAGs bind to Ctsk modulating its enzymatic properties as well as our own observations that 1) secreted Ctsk is susceptible to proteolytic activation, 2) C4-S GAGs are increased in MLII cartilage, and 3) Ctsk activity is reduced when C4-S GAG formation is inhibited. Furthermore, we believe that increased TGFß signaling reciprocally promotes the formation of GAG structures that participate in Ctsk activation and stabilize its extracellular activity, creating a pathogenic feedback loop that drives abnormal chondrogenesis. The specific aims leverage an innovative chemical toolkit including activity-based probes and nanoparticles within the zebrafish system to address the relationship between cathepsin activation, GAG sulfation and growth factor signaling. AIM 1 uses multiple approaches to drive Ctsk secretion and address how its increased extracellular localization influences its activity and function. AIM 2 will utilize zebrafish lines with mutations in GAG biosynthetic enzymes to ask how different GAG compositions impact Ctsk's properties. In a more exploratory part of this second aim, we will leverage our system to test nanoparticle-mediated delivery of Ctsk inhibitors or activators as a way to manipulate Ctsk activity in vivo. Most work on lysosomal disease pathogenesis focuses on the consequences of storage in tissue homeostasis. Our conceptual framework is novel in that it focuses instead on the consequences of mistargeted lysosomal hydrolases, such as the cathepsin proteases. By addressing the molecular mechanisms that underlie abnormal chondrogenesis in MLII, we will continue to identify downstream targets for therapy and investigate the ph... ## Key facts - **NIH application ID:** 10201613 - **Project number:** 5R01GM086524-13 - **Recipient organization:** GREENWOOD GENETIC CENTER - **Principal Investigator:** Richard Steet - **Activity code:** R01 (R01, R21, SBIR, etc.) - **Funding institute:** NIH - **Fiscal year:** 2021 - **Award amount:** $270,408 - **Award type:** 5 - **Project period:** 2009-01-01 → 2022-06-30 ## Primary source NIH RePORTER: https://reporter.nih.gov/project-details/10201613 ## Citation > US National Institutes of Health, RePORTER application 10201613, PATHOGENIC MECHANISMS OF LYSOSOMAL DISEASE (5R01GM086524-13). Retrieved via AI Analytics 2026-05-21 from https://api.ai-analytics.org/grant/nih/10201613. Licensed CC0. --- *[NIH grants dataset](/datasets/nih-grants) · CC0 1.0*