Date of Award

2006

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Molecular and Cellular Biology and Pathobiology

College

College of Graduate Studies

First Advisor

Rick Schnellmann

Second Advisor

Craig Beeson

Third Advisor

Tom Dix

Fourth Advisor

Douglas Sweet

Fifth Advisor

Lina Obeid

Abstract

The calpain family of cysteine proteases has undergone intense study since its discovery in 1976. Although numerous functions and proteolytic substrates have been linked to this 14-member family, little is known about the five atypical calpain isoforms. Over the past 25 years, investigators have identified ‘calpain-like’ activities in mitochondrial fractions isolated from various tissues. The studies undertaken in this dissertation were designed to positively identify the mitochondrial calpain present in renal cortical mitochondria (RCM) and to elucidate its function and proteolytic targets under both basal and CA2- -challenged conditions. We identified the RCM calpain as calpain 10 by both immunoblot analysis and via confocal microscopy. We also found that Ca2+-induced mitochondrial respiratory dysfunction was the result of calpain-mediated proteolysis of the Complex I electron transport chain subunits NDUFV2 and ND6. In addition, we demonstrated that calpain 10 plays a role in the formation of the membrane permeability transition (MPT) pore as evaluated by confocal analysis and mitochondrial swelling assays. The infection of primary renal proximal tubule cells (RPTC) using an adenoviral cassette expressing calpain 10-specific shRNA produced a variety of phenotypes involving both mitochondrial physiology and cell survival. Mitochondrial respiration and membrane potential were unchanged in virally-treated cells while ATP levels were increased 2-fold by day five post-infection. Increases in cellular ATP were associated with decreased ATP synthase β proteolysis and increased synthesis of the same protein. In addition, cell survival was significantly decreased by day 5 post-infection and was a result of caspase-dependent apoptosis. Calpain 10 is the only gene that has been genetically linked to Type II diabetes and its role in this disease process is believed to be associated with calpain 10's function as a mediator of glucose metabolism and insulin handling. We identified the Type II diabetes susceptibility gene ORP150 as a putative calpain 10 substrate. ORP150 was found to be dually targeted to both the ER and mitochondrial fractions of rat RPTC. Characterization of this molecule demonstrated that its induction is mediated by both global and mitochondrial-specific insults and that ORP150 is a proteolytic target for calpain 10. In summary, these results demonstrate the importance of calpain 10 under both physiological and pathological conditions. Our data also suggest that calpain 10 may serve to integrate multiple signaling events, thereby regulating cellular ATP levels and survival mechanisms. These studies provide further insight into the biology of calpain 10 and its role in Ca2+ -induced mitochondrial dysfunction, and may provide biochemical evidence sufficient to define calpain I0 as a potential target for therapeutic intervention in disease states mediated by the dysregulation of cel1ular Ca2+ homeostasis.

Rights

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