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dc.contributor.authorAarnio, Megan Christine
dc.description.abstractLysosomal hydrolases are targeted to lysosomes by a carbohydrate-dependent mechanism. Following protein synthesis, mannose-6-phosphate (M6P) tags are added to N-glycans on lysosomal hydrolases by GlcNAc-1-phosphotransferase, allowing hydrolases to bind M6P receptors in the Golgi for trafficking to lysosomes. Mutations in GNPTAB, the gene encoding GlcNAc-1-phosphotransferase, cause the lysosomal storage disorder, mucolipidosis II (ML-II). Patients have abnormal skeletal, cartilage, and craniofacial development. While the genetic basis of ML-II is well defined, the drivers of early pathogenesis are not. Our laboratory has developed a zebrafish model for ML-II to explore this question. ML-II zebrafish embryos have altered craniofacial development, increased col2a1 expression and elevated activity of the protease cathepsin K (CtsK). Recent work links the extracellular activity of CtsK to abnormal chondrogenesis by demonstrating that CtsK causes an imbalance in TGFβ and BMP growth factor signaling. Inhibition of CtsK activity normalizes this imbalance. Chapter two defines the mechanism whereby CtsK can cause altered growth factor signaling in ML-II cartilage. Using in vitro studies, I show that CtsK directly cleaves and activates latent TGFβ. Conversely, CtsK is capable of cleaving and degrading mature BMP ligand. This signaling imbalance can be rescued by TGFβ inhibition, indicating that elevated TGFβ is likely the main driver of pathogenesis. In parallel studies, I used a HeLa-based model for ML-II to investigate how loss of lysosomal targeting impacts cell surface glycoproteins. This revealed changes in the abundance of multiple glycoproteins including uptake receptors, tyrosine kinases, and phosphatases. Further, loss of GNPTAB results in increased c-Met phosphorylation and localization to lysosomes. Normally, dephosphorylation of Met is controlled by protein tyrosine phosphatases (PTPs), which themselves are inactivated under oxidative conditions. GNPTAB-null cells have increased oxidative stress levels as measured by reactive oxygen species (ROS), likely due to impaired clearance of damaged mitochondria. Consistent with an ROS-dependent mechanism, antioxidant treatment restores phospho-Met levels whereas hydrogen peroxide treatment of HeLa cells elevates phospho-Met levels. Collectively, these results highlight two mechanisms whereby loss of carbohydrate-dependent lysosomal targeting alters signaling – one that arises from the action of secreted cathepsin proteases and one that arises from impaired cellular clearance and lysosomal storage.
dc.subjectLysosomal storage disorder
dc.subjectgrowth factors
dc.subjectreceptor tyrosine kinase
dc.subjectreactive oxygen species
dc.titleDefects in carbohydrate-dependent lysosomal targeting lead to alterations in growth factor signaling
dc.description.departmentBiochemistry and Molecular Biology
dc.description.majorBiochemistry and Molecular Biology
dc.description.advisorRichard Steet
dc.description.committeeRichard Steet
dc.description.committeeMichael Tiemeyer
dc.description.committeeKelley Moremen
dc.description.committeeJames D. Lauderdale
dc.description.committeeHeather Flanagan-Steet

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