Nikhat Ahmed

Differential S-nitrosylation of proteins in Alzheimer’s disease

Numerous studies have provided evidence regarding the involvement of protein S-nitrosylation in the progression of Alzheimers disease (AD) pathology and its implication in the formation and accumulation of misfolded protein aggregates. The identification of S-nitrosylated proteins can be a major step toward the understanding of mechanisms leading to neuronal degeneration. The present study targeted S-nitrosylated proteins in AD hippocampus, substantia nigra and cortex using the following work-flow that combines S-nitrosothiol-specific antibody detection, classical biotin switch method labeled with fluorescence dye followed by electrospray ionization quadrupole time of flight tandem MS (ESI-QTOF MS/MS) identification. Endogenous nitrosocysteines were identified in 45 proteins, mainly involved in metabolism, signaling pathways, apoptosis and redox regulation as assigned by REACTOME and KEGG pathway database analysis. Superoxide dismutase (SOD2) [Mn], fructose-bisphosphate aldolase C (ALDOC) and voltage-dependent anion-selective channel protein 2 (VDAC2) showed differential S-nitrosylation signal, not previously reported in AD regions. Extensive neuronal atrophy with increased protein S-nitrosylation in AD regions is also evident from immunofluorescence studies using S-nitrosocysteine antibody. A number of plausible cysteine modification sites were predicted via Group-based Prediction System-S-nitrosothiols (GPS-SNO) 1.0 while STRING 8.3 analysis revealed functional annotations in the modified proteins. The findings are helpful in characterization of functional abnormalities and may facilitate the understanding of molecular mechanisms and biological function of S-nitrosylation in AD pathology.

Phosphoproteome profiling of substantia nigra and cortex regions of Alzheimer’s disease patients

J. Neurochem. (2012) 121, 954–963.

The Asia Oceania Human Proteome Organisation Membrane Proteomics Initiative. Preparation and characterisation of the carbonate‐washed membrane standard

The Asia Oceania Human Proteome Organisation (AOHUPO) has embarked on a Membrane Proteomics Initiative with goals of systematic comparison of strategies for analysis of membrane proteomes and discovery of membrane proteins. This multilaboratory project is based on the analysis of a subcellular fraction from mouse liver that contains endoplasmic reticulum and other organelles. In this study, we present the strategy used for the preparation and initial characterization of the membrane sample, including validation that the carbonate‐washing step enriches for integral and lipid‐anchored membrane proteins. Analysis of 17 independent data sets from five types of proteomic workflows is in progress.

Differential Expression of Proteins in Brain Regions of Alzheimer’s Disease Patients

Alzheimer’s disease (AD), a progressive neurodegenerative disorder and the most common form of dementia and cognitive impairment is usually characterized by neuritic amyloid plaques, cerebrovascular amyloidosis and neurofibrillary tangles. In order to find out the pathological protein expression, a quantitative proteome analysis of AD hippocampus, substantia nigra and cortex was performed and the extent of protein expression variation not only in contrast to age-matched controls but also among the understudied regions was analyzed. Expression alterations of 48 proteins were observed in each region along with significant co/contra regulation of malate dehydrogenase, lactate dehydrogenase B chain, aconitate hydratase, protein NipSnap homolog 2, actin cytoplasmic 1, creatine kinase U-type and glyceraldehyde-3-phosphate dehydrogenase. These differentially expressed proteins are mainly involved in energy metabolism, cytoskeleton integration, apoptosis and several other potent cellular/molecular processes. Interaction association network analysis further confirms the close interacting relationship between the co/contra regulated differentially expressed proteins among all the three regions. Elucidation of co/contra regulation of differentially expressed proteins will be helpful to understand disease progression and functional alterations associated with AD.

Albumin precursor and Hsp70 modulate corneal wound healing in an organ culture model

In order to investigate the role of albumin precursor and Hsp70 in corneal wound healing, we have analyzed the distribution of these proteins in wounded and non-wounded corneas of rabbits and the effects of topical applications of anti-albumin precursor and anti-Hsp70 antibodies on wound healing. Anti-albumin precursor and anti-Hsp70 antibodies were topically applied in healing corneal epithelium of rabbit eyes in organ culture. Corneas were allowed to heal in vitro for up to 120 h in serum-free medium with 5 and 10 μg/ml or without (migrating control) anti-albumin precursor/ or anti-Hsp70 antibodies. Fibronectin (Fb) (5 μg/ml) was used as a positive control. Immunofluorescence labelling was used to detect proteins in corneal epithelium at various time intervals following an epithelial defect. Delay in wound healing (p<0.005) was observed with 10 μg/ml anti-albumin antibody labelling. A similar pattern was observed when anti-fibronectin antibody (5 μg/ml) alone and in combination with anti-albumin (10 μg/ml) was ectopically added with wound closure occurring at 120 h. However with anti-Hsp70 antibody (5 μg/ml) slightly delayed (p<0.005) wound closure was observed at 96 h and considerable retardation >120 h with 10 μg/ml. Additionally, immunofluoresence showed a strong co-localization of Hsp70 and albumin precursor during the active phase of wound healing. The presence of albumin precursor and Hsp70 in the epithelial compartment of the cornea indicates a role for these proteins in modulating cell behavior such as epithelial growth, adhesion or regeneration, thus contributing to corneal epithelial wound healing.

Protein expression profiling of nuclear membrane protein reveals potential biomarker of human hepatocellular carcinoma

BackgroundComplex molecular events lead to development and progression of liver cirrhosis to HCC. Differentially expressed nuclear membrane associated proteins are responsible for the functional and structural alteration during the progression from cirrhosis to carcinoma. Although alterations/ post translational modifications in protein expression have been extensively quantified, complementary analysis of nuclear membrane proteome changes have been limited. Deciphering the molecular mechanism that differentiate between normal and disease state may lead to identification of biomarkers for carcinoma.ResultsMany proteins displayed differential expression when nuclear membrane proteome of hepatocellular carcinoma (HCC), fibrotic liver, and HepG2 cell line were assessed using 2-DE and ESI-Q-TOF MS/MS. From the down regulated set in HCC, we have identified for the first time a 15 KDa cytochrome b5A (CYB5A), ATP synthase subunit delta (ATPD) and Hemoglobin subunit beta (HBB) with 11, 5 and 22 peptide matches respectively. Furthermore, nitrosylation studies with S-nitrosocysteine followed by immunoblotting with anti SNO-cysteine demonstrated a novel and biologically relevant post translational modification of thiols of CYB5A in HCC specimens only. Immunofluorescence images demonstrated increased protein S-nitrosylation signals in the tumor cells and fibrotic region of HCC tissues. The two other nuclear membrane proteins which were only found to be nitrosylated in case of HCC were up regulated ATP synthase subunit beta (ATPB) and down regulated HBB. The decrease in expression of CYB5A in HCC suggests their possible role in disease progression. Further insight of the functional association of the identified proteins was obtained through KEGG/ REACTOME pathway analysis databases. String 8.3 interaction network shows strong interactions with proteins at high confidence score, which is helpful in characterization of functional abnormalities that may be a causative factor of liver pathology.ConclusionThese findings may have broader implications for understanding the mechanism of development of carcinoma. However, large scale studies will be required for further verification of their critical role in development and progression of HCC.