Bulbul Chakravarti

Oxidative Modification of Proteins: Age-Related Changes

Aging is a complex biological phenomenon which involves progressive loss of different physiological functions of various tissues of living organisms. It is the inevitable fate of life and is a major risk factor for death and different pathological disorders. Based on a wide variety of studies performed in humans as well as in various animal models and microbial systems, reactive oxygen species (ROS) are believed to play a key role in the aging process. The production of ROS is influenced by cellular metabolic activities as well as environmental factors. ROS can react with all major biological macromolecules such as carbohydrates, nucleic acids, lipids, and proteins. Since, in general, proteins are the key molecules that play the ultimate role in various structural and functional aspects of living organisms, this review will focus on the age-related oxidative modifications of proteins as well as on mechanism for removal or repair of the oxidized proteins. The topics covered include protein oxidation as a marker of oxidative stress, experimental evidence indicating the role of ROS in protein oxidation, protein carbonyl content, enzymatic degradation of oxidized proteins, and effects of caloric restriction on protein oxidation in the context of aging. Finally, we will discuss different strategies which have been or can be undertaken to slow down the oxidative damage of proteins and the aging process.

Proteomic profiling of aging in the mouse heart: Altered expression of mitochondrial proteins.

Using two-dimensional gel electrophoresis and liquid chromatography-tandem mass spectrometry, we have used a systems biology approach to study the molecular basis of aging of the mouse heart. We have identified 8 protein spots whose expression is up-regulated due to aging and 36 protein spots whose expression is down-regulated due to aging (p0.05 as judged by Wilcoxon Rank Sum test). Among the up-regulated proteins, we have characterized 5 protein spots and 2 of them, containing 3 different enzymes, are mitochondrial proteins. Among the down-regulated proteins, we have characterized 27 protein spots and 16 of them are mitochondrial proteins. Mitochondrial damage is believed to be a key factor in the aging process. Our current study provides molecular evidence at the level of the proteome for the alteration of structural and functional parameters of the mitochondria that contribute to impaired activity of the mouse heart due to aging.

Proteome profiling of aging in mouse models : Differential expression of proteins involved in metabolism, transport, and stress response in kidney

Aging is a time‐dependent complex biological phenomenon observed in various organs and organelles of all living organisms. To understand the molecular mechanism of age‐associated functional loss in aging kidneys, we have analyzed the expression of proteins in the kidneys of young (19–22 wk) and old (24 months) C57/BL6 male mice using 2‐DE followed by LC‐MS/MS. We found that expression levels of 49 proteins were upregulated (p ≤ 0.05), while that of only ten proteins were downregulated (p ≤ 0.05) due to aging. The proteins identified belong to three broad functional categories: (i) metabolism (e.g., aldehyde dehydrogenase family, ATP synthase β‐subunit, malate dehydrogenase, NADH dehydrogenase (ubiquinone), hydroxy acid oxidase 2), (ii) transport (e.g., transferrin), and (iii) chaperone/stress response (e.g., Ig‐binding protein, low density lipoprotein receptor‐related protein associated protein 1, selenium‐binding proteins (SBPs)). Some proteins with unknown functions were also identified as being differentially expressed. ATP synthase β subunit, transferrin, fumarate hydratase, SBPs, and albumin are present in multiple forms, possibly arising due to proteolysis or PTMs. The above functional categories suggest specific mechanisms and pathways for age‐related kidney degeneration.

Glucocorticoid-stimulated, transcription-independent release of annexin A1 by cochlear Hensen cells

Background and purpose:  The current clinical strategy to protect the auditory organ against inflammatory damage by migrating leukocytes is the local delivery of glucocorticoids. However, the mechanism by which glucocorticoids confer this protection remains unknown. Therefore, we investigated the cellular and molecular targets of glucocorticoids in the cochlea that could be involved in preventing leukocyte migration.

Differential hepatic protein tyrosine nitration of mouse due to aging - effect on mitochondrial energy metabolism, quality control machinery of the endoplasmic reticulum and metabolism of drugs.

Aging is the inevitable fate of life which leads to the gradual loss of functions of different organs and organelles of all living organisms. The liver is no exception. Oxidative damage to proteins and other macromolecules is widely believed to be the primary cause of aging. One form of oxidative damage is tyrosine nitration of proteins, resulting in the potential loss of their functions. In this study, the effect of age on the nitration of tyrosine in mouse liver proteins was examined. Liver proteins from young (19-22 weeks) and old (24 months) C57/BL6 male mice were separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and electroblotted onto nitrocellulose membranes. Proteins undergoing tyrosine nitration were identified using anti-nitrotyrosine antibody. Three different protein bands were found to contain significantly increased levels of nitrotyrosine in old mice (Wilconxon rank-sum test, p<0.05). Electrospray ionization liquid chromatography tandem mass spectrometry (ESI-LC-MS/MS) was used to identify the proteins in these bands, which included aldehyde dehydrogenase 2, Aldehyde dehydrogenase family 1, subfamily A1, ATP synthase, H(+) transporting, mitochondrial F1 complex, β subunit, selenium-binding protein 2, and protein disulfide-isomerase precursor. The possible impairment of their functions can lead to altered hepatic activity and have been discussed.

Proteomics and systems biology: application in drug discovery and development.

Studies of complex biological systems aimed at understanding their functions at a global level are the goals of systems biology. Proteomics, generally regarded as the comprehensive study of the expression of all the proteins at a particular time in different organs, tissues, and cell types is a key enabling technology for the systems biology approach. Rapid advances in this regard have been made following the success of the human genome project as well as those of various animals and microorganisms. Possibly, one of the most promising outcomes from studies on the human genome and proteome is the identification of potential new drugs for the treatment of different diseases and tailoring the drugs for individualized patient therapy. Following the identification of a new drug candidate, knowledge on organ and system-level responses helps prioritize the drug targets and design clinical trials based on their efficacy and safety. Toxicoproteomics is playing an important role in that respect. In essence, over the past decade, proteomics has played a major role in drug discovery and development. In this review article, we explain systems biology, discuss the current proteomic technologies, and highlight some important applications of proteomics and systems biology approaches in drug discovery and development.

In vivo molecular analysis of cytokines in a murine model of ocular onchocerciasis I. Up-regulation of IL-4 and IL-5 mRNAs and not IL-2 and IFNγ mRNAs in the cornea due to experimental interstitial keratitis

Sclerosing keratitis is the major cause of blindness due to onchocerciasis which results from chronic infection with the filarial parasite Onchocerca volvulus. Using a murine model of onchocercal sclerosing keratitis, we have demonstrated previously that predominantly (> 85%) CD3 + /CD4+ T-cells as well as the IL-2 receptor bearing cells infiltrate into the cornea in vivo during development and progress of the disease. The identification of CD4+ subsets TH1 and TH2 based on the cytokine secretion patterns of murine T-lymphocytes has been useful for understanding the immune basis of resistance and pathogenesis in murine models of several parasitic diseases. The present investigation was carried out to demonstrate whether the local immune response at the corneal lesion due to onchocercal interstitial keratitis correlated with such distinct patterns of cytokine production. For that purpose, mRNA was extracted separately from corneas obtained from the diseased eyes and the normal eyes of A/J mice with onchocercal interstitial keratitis, reverse transcribed and amplified by the polymerase chain reaction with four different cytokine specific primers. In corneas obtained from the eyes affected with onchocercal interstitial keratitis, mRNAs coding for IL-4 and IL-5 were up-regulated compared to the normal eyes having no lesions from the same animals. However, the levels of mRNAs for IL-2 and IFN gamma were found to be the same in the diseased and normal eyes. Taken together, these data suggest that IL-4 and IL-5 producing TH2-lymphocytes are active at the corneal lesion due to onchocercal interstitial keratitis.

A highly uniform UV transillumination imaging system for quantitative analysis of nucleic acids and proteins

The digital fluorescent imaging for documentation and analysis of gel electrophoretic separations of nucleic acids and proteins is widely used in quantitative biology. Most fluorescent stains used in postelectrophoretic analysis of proteins and nucleic acids have significant excitation peaks with UV light (300–365 nm), making midrange UV (UV‐B) as the excitation source of choice. However, coupling quantitative CCD imaging with UV is difficult due to lack of uniformity found in typical UV transilluminators. The apparent amount of those macromolecules depends on the position of the gel band on the imaging surface of the transilluminator. Here, we report the development and validation of a highly uniform UV transillumination system. Using a novel high density lighting system containing a single lamp formed into a high density grid, an electronic ballast, a phosphor coating, and a bandpass filter to convert 254 nm light produced to 300–340 nm, uniformity of ≤5% CV has been achieved, compared to >80% CV observed in typical UV transilluminators. This system has been used for the quantitative analysis of electrophoretically separated nucleic acids and proteins (CV≤8%) without uniformity correction by software, and continues to outperform typical transilluminators when a uniformity correction is applied.

Comparison of SYPRO Ruby and Flamingo fluorescent stains for application in proteomic research.

Fluorescent dyes are widely used for the detection and quantitation of proteins separated by polyacrylamide gel electrophoresis. SYPRO Ruby is one such fluorescent dye widely used for this purpose. More recently, another fluorescent dye, Flamingo, is available for expression proteomic research. Using a standard ultraviolet (UV) transilluminator and a charge-coupled device (CCD)-based imaging system, the relative sensitivity of these two different fluorescent stains with regard to detection of protein spots separated by two-dimensional gel electrophoresis (2D-GE) and identification by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) were compared. Using mouse kidney and liver homogenates as well as Escherichia coli extract, we detected a greater number of protein spots using Flamingo compared with SYPRO Ruby. In addition, when we compared the number of matched peptides and the percentage of amino acid residues identified for 22 different protein spots of mouse kidney proteome, we observed a higher number of matched peptides and a higher percentage of amino acid residues for the majority of the proteins using Flamingo compared with SYPRO Ruby. Also, we were able to characterize a protein spot that can be detected by Flamingo only. Therefore, we recommend Flamingo over SYPRO Ruby to be used for studies on expression proteomics.

Studies on phospholipase activities in Neurospora crassa conidia

Abstract Phospholipase A activity has been characterized in the conidia of Neurospora crassa . It has been observed that almost the entire phospholipase A activity in the conidia could be leached out by washing these with aqueous solution. The properties of this exocellular phospholipase A have been determined. Two distinct phospholipases have been fractionated from the “conidial water washing” by gel chromatography using Sephadex G-100. Using specific substrate, 1-stearyl-2-oleoyl lecithin, the predominant phospholipase activity in the fraction with higher molecular weight has been found to be due to phospholipase A 1 and that in fraction with lower molecular weight has been found to be due to phospholipase A 2 . Properties of these two different enzyme fractions have been studied. The most active fraction in the latter gives a single band in 7.5% polyacrylamide gel. Lysophospholipase activity and detergent-stimulated phospholipase D activity have been detected also in the conidia. The effects of different physical and chemical agents on phospholipase A activity in the “conidial water washing” as well as in the purified fractions have been studied.