Access Type

Open Access Dissertation

Date of Award

January 2016

Degree Type

Dissertation

Degree Name

Ph.D.

Department

Biochemistry and Molecular Biology

First Advisor

Maik Huttemann

Abstract

The long term goal of my thesis research is to understand how tissue-specific

phosphorylations on the small mitochondrial protein, cytochrome c (Cytc), regulate its

functions, under both physiologically healthy and stressed conditions, and to identify the

cell signaling pathways targeting Cytc. Cytc is a functionally diverse protein that carries

electrons in the electron transport chain and plays a critical role in cellular apoptosis, two

diverse pathways that maintain cellular health that are active under diverse conditions.

Since Cytc plays a pivotal role in both these highly divergent pathways, regulation of the

protein is very important—phosphorylation of the protein under physiological conditions

hence implies a regulation by cell signaling pathways that have yet to be identified and

studied. Previous work by our lab suggests the importance of reversible phosphorylation

of Cytc in regulating its functions

1-4

. We hypothesize that under healthy conditions,

phosphorylated Cytc partially inhibits mitochondrial respiration and maintains healthy

mitochondrial membrane potential, preventing ROS generation, while cellular stress-

mediated dephosphorylation leads to increased respiration and ROS generation, initiating

apoptosis. To further test this hypothesis and to extend our understanding of Cytc

phosphorylation on its functions, I conducted two studies. In the first study, I investigated

the physiological phosphorylation status of Cytc in mammalian kidney tissues. To begin

with, I purified bovine kidney Cytc in the presence of phosphatase inhibitors, identified

threonine phosphorylation by immunoblot analysis, and determined threonine 28

1

phosphorylation by immobilized metal affinity chromatography/nano-liquid

chromatography/electrospray ionization mass spectrometry (Nano/LC/ESI/MS/MS). To

characterize the effect of Thr28 phosphorylation on Cytc functions, I mutated Thr28 to

glutamate, a phosphomimetic mutation, and alanine, a nonphosphorylatable control. I

went on to express and purify wild-type, the phosphomimetic mutant and the non-

phosphorylatable mutant Cytc in bacterial cells. I also expressed and analyzed wild-type,

the phosphomimetic mutant and the nonphosphorylatable mutant Cytc in mammalian

cells to determine the effects of the Cytc mutations on the functions of the protein in vitro

and on overall cellular metabolism and physiology, under healthy and stressed conditions.

I also found that Thr28 phosphorylation is AMP kinase-mediated, and AMP kinase

colocalizes with Cytc to the mitochondrial intermembrane space. Our data suggest that

Thr28, conserved in mammalian Cytc, is an important regulatory site that leads to

regulation of ETC flux via ‘controlled respiration,’ preventing 

m

hyperpolarization, a

known cause of ROS and trigger of apoptosis (discussed in Chapter 2, manuscript under

preparation). In the second study, the phosphorylation status of Cytc in ischemic brain

was investigated to determine if insulin-induced neuroprotection and inhibition of Cytc

release in ischemic brain was associated with Cytc phosphorylation. We used an animal

model of global brain ischemia, and found a 50% decreased death rate of CA1

hippocampal neurons after neuroprotective post-ischemic insulin administration as

compared to untreated controls. The increased survival of CA1 neurons was correlated

to inhibition of Cytc release from mitochondria into cytosol 24 hours post reperfusion,

which in turn was mediated by Cytc phosphorylation on Tyr97. We thus propose that Cytc

is phosphorylated by an insulin-dependent signaling pathway, and this may impede with

2

its release from mitochondria and its ability to induce apoptosis (discussed in Chapter 3,

manuscript published in PLoS One, 2013 8(11):e78627).

Download

Included in

Biochemistry Commons

Share

COinS