Mitochondrial proteins are targeted in FA-C and-G cells

Mitochondrial proteins are targeted in FA-C and-G cells 1

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Christoph Campregher, Katherine Fomicheva, Alex Epanchintsev, Joseph M. Metzger, Alex Lyakhovich

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Medical University of Vienna, Austria; University of Michigan Medical School, Ann Arbor, MI; Cedex, France; University of Minnesota Medical School, Minneapolis, MN; 5Duke-NUS Graduate Medical School, Singapore

Objective: Our previous approach on DIGE-proteomics revealed that several RedOx proteins were differentially expressed in FA cells undergoing oxidative stress. In parallel, we were able to establish 2D-culture model of oxidative-stress in FA and identified some altered pathways. In this study we integrated the above approaches and attempted to identify molecular targets of ROS in FA cells. Methods: We analyzed differentially expressed proteins from FA-C, FA-G or shRNA FANCC/G depleted HeLa cells either in hyperoxia (oxidative stress from activated neutrophils - PMN) or in hypoxia conditions. Transfection of cells with sensor/effector GFP-coupled DOVOS or RFP-ARE–coupled constructs allowed monitoring cell population under hypoxia or hyperoxia. DNA damage and mitochondrial injury were measured, including mitochondrial membrane potential (ΔΨm). Results: We identified induction of several mitochondria proteins, including CDK5, HSP70B and members of P450 superfamily in both FA-C and-G cells. FANCG or FANCC shRNA depleted HeLa cells showed increased level of ROS, mitochondrial 3-nitrotyrosine and 4-hydroxynonenal protein adducts. Under hyperoxia conditions, induction of those proteins was further increased and accompanied by the decreased mitochondrial membrane potential. Interestingly, we observed dissociation of mitochondria from the vimentin filaments at the nucleus periphery and formation of an interconnected cytoplasmic reticulum. These FA-C or FA-G depleted cells undergoing oxidative stress from PMN revealed an increase number of depolarized mitochondria which was proportional to the time of PMN exposure. Importantly, these defects were rescued in hypoxia conditions. Conclusions: It seems likely that two steps are necessary to damage mitochondria in FA cells. First, genetic mutations of FA genes should be acquired. This affects several mitochondrial proteins making these organelles susceptible to oxidative stress. Once sensitized, mitochondria become fragile and endogenous or exogenous oxidative stress may provoke them to the above dramatically changes. In turn, these changes can be prevented by reducing ROS either by inhibitors or by placing the FA cells in hypoxia conditions

Change of ΔΨm

Co-culture 2D Modeling of a Mild Hyperoxia Microenvironment Plating Target Cells +/-ROS inhibitor

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Co-culture PMA activated neutrophils (PMN)

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FA-C/G or FA shRNA depleted cells

Analysis of Microarray Target Cells Proteomics 2D-DIGE Cell cycle analysis Mitochondria injurie & membrane potential (ΔΨm) Flow cytometry

semipermeable membrane

ROS-Regulated Genes and Proteins

pI3K(85) ↓ p21WAF↑ pCDC25↑ pChk2↑ pCdc2↑ Mitochondrial proteins MTATP6↑ MTCO2↑ UQCRC1↓ FSHMD1A↓ CDK5 ↑HSP70B↓ Cyclin B1↓ Akt ↑

mTOR ↓ Gadd45↑ Survivin ↑ SKBM ↑

regulated genes

Low O

PMN

PMN+NAC

Fluorescence Intensity

regulated proteins and genes

regulated proteins

Deficiency or Depletion of FA-C/-G decreases mitochondrial membrane potential and increases the number of depolarized mitochondria

Hypoxia Normoxia Hyperoxia