Date of Award

2014

Document Type

Thesis

Degree Name

Master of Science (MS)

Department

Biology

First Advisor

Newman, Robert H.

Abstract

Meprin metalloproteinases have been shown to play a role in the progression of diabetic nephropathy (DN) in both mice and humans. However, the mechanisms involved are not fully understood. Protein Kinase C (PKC) family members are a group of phospholipid-dependent serine/threonine kinases that regulate signaling pathways involved in cellular growth, proliferation, differentiation, and cell death. PKC signaling pathways have also been implicated in diseases such as DN. Specifically, PKCα has been shown to play a role in the development of albuminuria in mice with streptozotocin (STZ)-induced type 1 diabetes. The objective of this study was to determine whether PKCα is a meprin substrate. To this end, activated forms of purified recombinant meprin A and B were incubated for 0-4 h with cytosolic-enriched proteins from either protein lysates extracted from Mardin-Darby canine kidney (MDCK) cells (which do not express meprins) or meprin αβ double knockout mouse kidneys (which lack endogenous meprins). The levels of PKCα were determined by Western blot analysis using anti-PKCα specific antibodies. Incubation with meprin B significantly reduced the levels of PKCα present in the kidney and MDCK lysates. This decrease was not observed in proteins incubated with meprin A or control reactions lacking meprins, suggesting isoform-specific degradation of PKCα. To confirm the degradation, human PKCα was expressed in yeast, purified by GST-affinity chromatography, and incubated with the activated meprins. To determine whether meprin cleavage of PKCα occurs in models of diabetes, immunohistological analysis was conducted using mouse kidney tissues from STZ-diabetes induced mice. These data suggest that meprin B may impact kidney function via proteolysis of PKCα.

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