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 TGF and BMP signaling in developing cartilage. This imbalance in growth factor signaling ? caused by Ctsk-mediated increases in latent TGF 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 TGF 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 physiological relevance of secreted cathepsins during tissue development. Much of our future work will focus on exploiting our powerful experimental platform to develop mechanism-based therapies for MLII and probe cathepsin function in vivo.

Public Health Relevance

The overall goal of this proposal is to address how secreted cathepsin proteases drive disease pathogenesis outside the cell, and how interaction with glycosaminoglycans (GAGs) might mediate this process. This work will leverage multiple transgenic zebrafish lines along with novel chemical biology tools to explore the interplay between cathepsin proteases and GAGs in vivo, uncovering new mechanistic insight into lysosomal diseases that can be translated into therapies.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM086524-10A1
Application #
9590287
Study Section
Intercellular Interactions Study Section (ICI)
Program Officer
Marino, Pamela
Project Start
2009-01-01
Project End
2022-06-30
Budget Start
2018-09-15
Budget End
2019-06-30
Support Year
10
Fiscal Year
2018
Total Cost
Indirect Cost
Name
Greenwood Genetic Center
Department
Type
DUNS #
078047966
City
Greenwood
State
SC
Country
United States
Zip Code
29646
Flanagan-Steet, Heather; Christian, Courtney; Lu, Po-Nien et al. (2018) TGF-ß Regulates Cathepsin Activation during Normal and Pathogenic Development. Cell Rep 22:2964-2977
Aarnio-Peterson, Megan; Zhao, Peng; Yu, Seok-Ho et al. (2017) Altered Met receptor phosphorylation and LRP1-mediated uptake in cells lacking carbohydrate-dependent lysosomal targeting. J Biol Chem 292:15094-15104
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Flanagan-Steet, Heather; Matheny, Courtney; Petrey, Aaron et al. (2016) Enzyme-specific differences in mannose phosphorylation between GlcNAc-1-phosphotransferase ?? and ? subunit deficient zebrafish support cathepsin proteases as early mediators of mucolipidosis pathology. Biochim Biophys Acta 1860:1845-53
Han, Sang-Oh; Pope, Rand; Li, Songtao et al. (2016) A beta-blocker, propranolol, decreases the efficacy from enzyme replacement therapy in Pompe disease. Mol Genet Metab 117:114-9
Flanagan-Steet, Heather; Aarnio, Megan; Kwan, Brian et al. (2016) Cathepsin-Mediated Alterations in TGFß-Related Signaling Underlie Disrupted Cartilage and Bone Maturation Associated With Impaired Lysosomal Targeting. J Bone Miner Res 31:535-48
Qian, Yi; van Meel, Eline; Flanagan-Steet, Heather et al. (2015) Analysis of mucolipidosis II/III GNPTAB missense mutations identifies domains of UDP-GlcNAc:lysosomal enzyme GlcNAc-1-phosphotransferase involved in catalytic function and lysosomal enzyme recognition. J Biol Chem 290:3045-56
Bammens, Riet; Mehta, Nickita; Race, Valérie et al. (2015) Abnormal cartilage development and altered N-glycosylation in Tmem165-deficient zebrafish mirrors the phenotypes associated with TMEM165-CDG. Glycobiology 25:669-82
Leroy, Jules G; Sillence, David; Wood, Tim et al. (2014) A novel intermediate mucolipidosis II/III?? caused by GNPTAB mutation in the cytosolic N-terminal domain. Eur J Hum Genet 22:594-601
Boccuto, Luigi; Aoki, Kazuhiro; Flanagan-Steet, Heather et al. (2014) A mutation in a ganglioside biosynthetic enzyme, ST3GAL5, results in salt & pepper syndrome, a neurocutaneous disorder with altered glycolipid and glycoprotein glycosylation. Hum Mol Genet 23:418-33

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