Helen Kim

Modification of the Mitochondrial Proteome in Response to the Stress of Ethanol-dependent Hepatotoxicity

Mitochondria are particularly susceptible to increased formation of reactive oxygen and nitrogen species in the cell that can occur in response to pathological and xenobiotic stimuli. Proteomics can give insights into both mechanism of pathology and adaptation to stress. Herein we report the use of proteomics to evaluate alterations in the levels of mitochondrial proteins following chronic ethanol exposure in an animal model. Forty-three proteins showed differential expression, 13 increased and 30 decreased, as a consequence of chronic ethanol. Of these proteins, 25 were not previously known to be affected by chronic ethanol emphasizing the power of proteomic approaches in revealing global responses to stress. Both nuclear and mitochondrially encoded gene products of the oxidative phosphorylation complexes in mitochondria from ethanol-fed rats were decreased suggesting an assembly defect in this integrated metabolic pathway. Moreover mtDNA damage was increased by ethanol demonstrating that the effects of ethanol consumption extend beyond the proteome to encompass mtDNA. Taken together, we have demonstrated that chronic ethanol consumption extends to a modification of the mitochondrial proteome far broader than realized previously. These data also suggest that the response of mitochondria to stress may not involve non-discriminate changes in the proteome but is restricted to those metabolic pathways that have a direct role in a specific pathology.

Hepcidin revisited, disulfide connectivity, dynamics, and structure.

Hepcidin is a tightly folded 25-residue peptide hormone containing four disulfide bonds, which has been shown to act as the principal regulator of iron homeostasis in vertebrates. We used multiple techniques to demonstrate a disulfide bonding pattern for hepcidin different from that previously published. All techniques confirmed the following disulfide bond connectivity: Cys1–Cys8, Cys3–Cys6, Cys2–Cys4, and Cys5–Cys7. NMR studies reveal a new model for hepcidin that, at ambient temperatures, interconverts between two different conformations, which could be individually resolved by temperature variation. Using these methods, the solution structure of hepcidin was determined at 325 and 253 K in supercooled water. X-ray analysis of a co-crystal with Fab appeared to stabilize a hepcidin conformation similar to the high temperature NMR structure.

Chemoprevention by Grape Seed Extract and Genistein in Carcinogen-induced Mammary Cancer in Rats Is Diet Dependent

Many popular dietary supplements are enriched in polyphenols such as the soy isoflavones, tea catechins, and resveratrol (from grape skins), each of which has been shown to have chemopreventive activity in cellular models of cancer. The proanthocyanidins, which are oligomers of the catechins, are enriched in grape seeds and form the basis of the dietary supplement grape seed extract (GSE). Evidence suggests that the proanthocyanidins may be metabolized to the monomeric catechins. This study was carried out to determine whether GSE added to rodent diets protected against carcinogen-induced mammary tumorigenesis in rats and whether this was affected by the composition of the whole diet. Female rats were begun on 5%, 1.25%, or 0% (control) GSE-supplemented diets at age 35 d. At age 50 d they were administered 7,12-dimethylbenz[a]anthracene (DMBA) in sesame oil at 80 mg/kg body weight. They were weighed and monitored weekly for tumor development until 120 d after DMBA administration. Administration of GSE in AIN-76A diet did not show any protective activity of GSE against DMBA-induced breast cancer. However, administration of GSE in a laboratory dry food diet (Teklad 4% rodent diet) resulted in a 50% reduction in tumor multiplicity. In similar experiments, genistein administered in AIN-76A diet also failed to show chemopreventive activity against the carcinogen N-methyl-N-nitrosourea; however, when administered at the same dose in the Teklad 4% rodent diet, genistein exhibited significant chemopreventive activity (44-61%). These results demonstrate that GSE is chemopreventive in an animal model of breast cancer; moreover, the diet dependency of the chemopreventive activity for both GSE and genistein suggests that whether or not a compound is chemopreventive may depend on the diet in which the agent is administered.

Lack of LDL receptor aggravates learning deficits and amyloid deposits in Alzheimer transgenic mice

Emerging evidence indicates that cholesterol metabolism affects the pathogenesis of Alzheimers disease (AD). The LDL receptor (LDLR) is obligatory in maintaining cholesterol homeostasis in the periphery. To investigate the role of LDLR in the development of AD-like behavior and pathology, Tg2576 mice, a well-characterized transgenic mouse model of AD, with different genotypes of LDLR were generated. Here we show that LDLR-deficient Tg2576 mice developed hypercholesterolemia and age-dependent cerebral beta-amyloidosis. Before the manifestation of amyloid-beta (Abeta) deposition, these mice displayed hyperactivity, reduced anxiety, and impaired spatial learning regardless of LDLR genotypes. After the manifestation of Abeta deposition, LDLR-deficient Tg2576 mice showed more spatial learning deficits than LDLR-intact Tg2576 mice. Although LDLR genotypes did not affect the expression level of the amyloid-beta precursor protein transgene, there was a significant increase in Abeta deposition accompanied with an increase of apoE expression in LDLR-deficient Tg2576 mice. Our results suggest that the LDLR plays a role in the development of Alzheimer-type learning impairment and amyloidosis and can be a novel therapeutic target for AD.

The case for well-conducted experiments to validate statistical protocols for 2D gels: different pre-processing = different lists of significant proteins

BackgroundThe proteomics literature has seen a proliferation of publications that seek to apply the rapidly improving technology of 2D gels to study various biological systems. However, there is a dearth of systematic studies that have investigated appropriate statistical approaches to analyse the data from these experiments.ResultsComparison of the effects of statistical pre-processing on the results of two sample t-tests suggests that the results of 2D gel experiments and by extension the conclusions derived from these experiments are not independent of the statistical protocol used.ConclusionsThis study suggests that there is a need for well-conducted validation studies to establish optimal statistical techniques to be used on such data sets.