Proteomic Analysis of Endothelial Cold-Adaptation. [2011](IR92)

Proteomic Analysis of Endothelial Cold-Adaptation. [2011](IR92)

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(Memo Item created on June 9, 2012 01:40 PM)
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Proteomic Analysis of Endothelial Cold-Adaptation. [2011](IR92)

http://www.ncbi.nlm.nih.gov/pubmed/22192797
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BMC Genomics. 2011 Dec 22;12:630.
Proteomic Analysis of Endothelial Cold-Adaptation.
Zieger MA, Gupta MP, Wang M.

Source
Methodist Research Institute, Indiana University Health, Indianapolis, IN 46202, USA. mzieger@iuhealth.org

Abstract
BACKGROUND:
Understanding how human cells in tissue culture adapt to hypothermia may aid in developing new clinical procedures for improved ischemic and hypothermic protection. Human coronary artery endothelial cells grown to confluence at 37°C and then transferred to 25°C become resistant over time to oxidative stress and injury induced by 0°C storage and rewarming. This protection correlates with an increase in intracellular glutathione at 25°C. To help understand the molecular basis of endothelial cold-adaptation, isolated proteins from cold-adapted (25°C/72 h) and pre-adapted cells were analyzed by quantitative proteomic methods and differentially expressed proteins were categorized using the DAVID Bioinformatics Resource.

RESULTS:
Cells adapted to 25°C expressed changes in the abundance of 219 unique proteins representing a broad range of categories such as translation, glycolysis, biosynthetic (anabolic) processes, NAD, cytoskeletal organization, RNA processing, oxidoreductase activity, response-to-stress and cell redox homeostasis. The number of proteins that decreased significantly with cold-adaptation exceeded the number that increased by 2:1. Almost half of the decreases were associated with protein metabolic processes and a third were related to anabolic processes including protein, DNA and fatty acid synthesis. Changes consistent with the suppression of cytoskeletal dynamics provided further evidence that cold-adapted cells are in an energy conserving state. Among the specific changes were increases in the abundance and activity of redox proteins glutathione S-transferase, thioredoxin and thioredoxin reductase, which correlated with a decrease in oxidative stress, an increase in protein glutathionylation, and a recovery of reduced protein thiols during rewarming from 0°C. Increases in S-adenosylhomocysteine hydrolase and nicotinamide phosphoribosyltransferase implicate a central role for the methionine-cysteine transulfuration pathway in increasing glutathione levels and the NAD salvage pathway in increasing the reducing capacity of cold-adapted cells.

CONCLUSIONS:
Endothelial adaptation to mild-moderate hypothermia down-regulates anabolic processes and increases the reducing capacity of cells to enhance their resistance to oxidation and injury associated with 0°C storage and rewarming. Inducing these characteristics in a clinical setting could potentially limit the damaging effects of energy insufficiency due to ischemia and prevent the disruption of integrated metabolism at low temperatures.

PMID: 22192797 [PubMed - indexed for MEDLINE] PMCID: PMC3270058

Free PMC Article
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(Memo Item created on June 9, 2012 01:48 PM)
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NAD Metabolic Proteins
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NAD Metabolic Proteins

The increase in GSH/GST and Trx/TrxR activities requires the availability of reducing equivalents from NADPH. Cold-adapted cells had a significant increase in Nampt, the rate-limiting enzyme in NAD synthesis from nicotinamide [30]. NAD kinase, which phosphorylates NAD+(H) to NADP+(H), was not identified in our study. The enzymes of the pentose phosphate pathway (PPP) that reduce NADP+ to NADPH, glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase, were unchanged in abundance. However, the decrease in phosphofructokinase (PFK), the rate-limiting enzyme in the glycolytic pathway, potentially redirects glucose catabolism from glycolysis to the alternative PPP in a manner similarly induced by down-regulating the activity of other downstream glycolytic enzymes such as GAPDH or triosephosphate isomerase (TPI) [57]. The proteomic data indicate that there may be two additional mechanisms in cold-adapted cells to bolster NADPH availability. Aldehyde dehydrogenase (ALDH1A1- Figure Figure2),2), which oxidizes the lipid peroxidation products 4-hydroxynonenal and malondialdehyde using NADP+ as a cofactor [34], increased at 25°C. Lipid peroxidation is an important mechanism of injury during 0°C storage and rewarming [50,51]. An increase in ALDH1A1 would therefore potentially protect cell membranes from oxidative damage and simultaneously regenerate NADPH for GSSG and Trx reduction. A second approach to ensuring NADPH availability relates to the apparent decrease in NADPH-dependent biosyntheses at 25°C. Ribonucleotide reductase (RRM1), a Trx-dependent enzyme [35] that catalyzes deoxyribonucleotide formation and controls the rate of DNA synthesis at 37°C [36], decreased significantly at 25°C (Figure (Figure2).2). Inosine monophosphate dehydrogenase (IMPDH2), the rate limiting enzyme in de novo guanine nucleotide synthesis, also decreased at 25°C. Inhibition of IMPDH2 decreases guanine nucleotide pools and interrupts both DNA and RNA synthesis [58]. Down-regulating these two enzymes may therefore suppress DNA synthesis independently of the Q10 effects [59] and increase the availability of NADPH for GSSG and Trx reduction. A significant decrease in the NADPH-dependent fatty acid synthase (Figure (Figure2),2), and presumably fatty acid synthesis, at 25°C may have a similar NADPH-sparing effect and thereby contribute to the protective phenotype of cold-adapted cells.
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