Curam Sreenivasacharlu Sundaram

Proteomic and Transcriptomic Analysis of Aspergillus fumigatus on Exposure to Amphotericin B

ABSTRACT Amphotericin B (AMB) is the most widely used polyene antifungal drug for the treatment of systemic fungal infections, including invasive aspergillosis. It has been our aim to understand the molecular targets of AMB in Aspergillus fumigatus by genomic and proteomic approaches. In transcriptomic analysis, a total of 295 genes were found to be differentially expressed (165 upregulated and 130 downregulated), including many involving the ergosterol pathway, cell stress proteins, cell wall proteins, transport proteins, and hypothetical proteins. Proteomic profiles of A. fumigatus alone or A. fumigatus treated with AMB showed differential expression levels for 85 proteins (76 upregulated and 9 downregulated). Forty-eight of them were identified with high confidence and belonged to the above-mentioned categories. Differential expression levels for Rho-GDP dissociation inhibitor (Rho-GDI), secretory-pathway GDI, clathrin, Sec 31 (a subunit of the exocyst complex), and RAB GTPase Ypt51 in response to an antifungal drug are reported here for the first time and may represent a specific response of A. fumigatus to AMB. The expression of some of these genes was validated by real-time reverse transcription-PCR. The AMB responsive genes/proteins observed to be differentially expressed in A. fumigatus may be further explored for novel drug development.

Identification of novel allergens of Aspergillus fumigatus using immunoproteomics approach

Background Approximately 20% of the worlds asthmatics are suffering from Aspergillus fumigatus (Afu)‐induced allergies. The characterization of specific IgE‐inducing allergens in allergic aspergillosis patients is fundamental for clinical diagnosis and for immunotherapy.

Human surfactant protein D alters oxidative stress and HMGA1 expression to induce p53 apoptotic pathway in eosinophil leukemic cell line

Surfactant protein D (SP-D), an innate immune molecule, has an indispensable role in host defense and regulation of inflammation. Immune related functions regulated by SP-D include agglutination of pathogens, phagocytosis, oxidative burst, antigen presentation, T lymphocyte proliferation, cytokine secretion, induction of apoptosis and clearance of apoptotic cells. The present study unravels a novel ability of SP-D to reduce the viability of leukemic cells (eosinophilic leukemic cell line, AML14.3D10; acute myeloid leukemia cell line, THP-1; acute lymphoid leukemia cell lines, Jurkat, Raji; and human breast epithelial cell line, MCF-7), and explains the underlying mechanisms. SP-D and a recombinant fragment of human SP-D (rhSP-D) induced G2/M phase cell cycle arrest, and dose and time-dependent apoptosis in the AML14.3D10 eosinophilic leukemia cell line. Levels of various apoptotic markers viz. activated p53, cleaved caspase-9 and PARP, along with G2/M checkpoints (p21 and Tyr15 phosphorylation of cdc2) showed significant increase in these cells. We further attempted to elucidate the underlying mechanisms of rhSP-D induced apoptosis using proteomic analysis. This approach identified large scale molecular changes initiated by SP-D in a human cell for the first time. Among others, the proteomics analysis highlighted a decreased expression of survival related proteins such as HMGA1, overexpression of proteins to protect the cells from oxidative burst, while a drastic decrease in mitochondrial antioxidant defense system. rhSP-D mediated enhanced oxidative burst in AML14.3D10 cells was confirmed, while antioxidant, N-acetyl-L-cysteine, abrogated the rhSP-D induced apoptosis. The rhSP-D mediated reduced viability was specific to the cancer cell lines and viability of human PBMCs from healthy controls was not affected. The study suggests involvement of SP-D in host’s immunosurveillance and therapeutic potential of rhSP-D in the eosinophilic leukemia and cancers of other origins.

Comparative proteomic analysis of differentially expressed proteins in primary retinoblastoma tumors

Purpose: To understand the disease mechanism and to identify the potential tumor markers that would help in therapeutics, comparative proteomic analysis of 29 retinoblastoma (RB) tumors was performed using 14 non‐neoplastic retinas (age ranged from 45 to 89 years) as control tissues.

Reversal of Stathmin-Mediated Microtubule Destabilization Sensitizes Retinoblastoma Cells to A Low Dose of Antimicrotubule Agents: A Novel Synergistic Therapeutic Intervention

PURPOSE To explore the possibility of stathmin as an effective therapeutic target and to evaluate the synergistic combination of stathmin RNAi and the antimicrotubule agents paclitaxel and vincristine to retinoblastoma Y79 cells. METHODS RNAi-mediated specific inhibition of stathmin expression in Y79 cells was shown by real-time quantitative RT-PCR (RT-Q-PCR), its effect on cell proliferation by MTT assay, cell invasion using matrigel, microtubule polymerization by immunohistochemistry, apoptosis, cell cycle analysis by flow cytometry analysis, and the changes in FOXM1 protein expression were studied by Western blot. The effect of combination treatment of stathmin siRNA and paclitaxel/vincristine was studied by assessing cell viability and apoptosis. RESULTS Short interfering RNA-mediated transient stathmin downregulation resulted in a marked inhibition of retinoblastoma cell proliferation and cell invasion in vitro. Stathmin inhibition promoted Y79 cells to G2/M phase, and ultimately there were increased apoptotic events as evidenced by higher caspase-3 activation and cleaved poly(ADP-ribose) polymerase expression. Cells transfected with stathmin siRNA showed long and bundled microtubule polymers and sensitized the Y79 cells significantly to paclitaxel and vincristine. CONCLUSIONS Stathmin may be a pivotal determinant for retinoblastoma tumorigenesis and chemosensitivity. Strategies to inhibit stathmin will help to enhance the cytotoxic effect of paclitaxel while reducing toxicity (or side effects) to normal cells caused by high doses.

Proteome of human endometrium: Identification of differentially expressed proteins in proliferative and secretory phase endometrium

Purpose: To exploit the potential of proteomics to identify and study additional yet‐unidentified important proteins present in human endometrium.

Proteomic profiling of cancer of the gingivo-buccal complex: Identification of new differentially expressed markers

Tobacco‐related oral cancer is the most common cancer among Indian males, gingivo‐buccal complex (GBC) being the most affected subsite due to the habit of chewing tobacco. Proteins from the lysates of microdissected normal and transformed epithelium from clinically well‐characterized tissue samples of the GBC were separated by two‐dimensional gel electrophoresis to identify differentially expressed proteins. Eleven protein spots showed differential expression, which could withstand the stringency of statistical evaluation. The observations were confirmed with additional tissues. Nine of these differentiators were identified by MS as lactate dehydrogenase B, α‐enolase, prohibitin, cathepsin D, apolipoprotein A‐I, tumor protein translationally controlled‐1, an SFN family protein, 14‐3‐3σ and tropomyosin. Cluster analysis indicated that these proteins, as a coexpressed set, could distinguish normal and transformed epithelium. Functionally, these differentiator molecules are relevant to the pathways and processes that have been previously implicated in oral carcinogenesis and could therefore be investigated further as a panel of markers for management of cancer of the GBC.

Tumor antigens eliciting autoantibody response in cancer of gingivo-buccal complex.

Cancer of the gingivo‐buccal complex (GBC) is a major cancer in Indian men. This study reports the identification of tumor antigens, which elicit an antibody response in cancer of GBC using immunoproteomics. Proteins from KB cells separated by 2‐D PAGE, were immunoblotted with IgG from sera of 28 cancer patients, 12 patients with leukoplakia, and 28 healthy individuals. Antigens detected by the IgGs from the patients sera were different among different individuals with presence of any single antigen ranging from 7 to 79%. Several of these antigens have been identified by MS and confirmed by immunostaining. They are three forms of α‐enolase, peroxiredoxin‐VI, annexin‐II, HSP70, pyruvate kinase, α‐tubulin, β‐tubulin, ATP‐synthase, phosphoglycerate mutase (PGM), aldose reductase, triosephosphate isomerase, and cyclophilin‐A. Except, HSP70, these antigens are being reported in cancer of GBC for the first time. Pyruvate kinase and aldose reductase have not been reported to elicit autoantibody response in any other cancer earlier. Initial results show that autoantibody response against α‐enolase, HSP70, annexin‐II, peroxiredoxin‐VI, and aldose reductase are also seen in patients with leukoplakia of GBC, which suggest early occurrence of these autoantibodies during the process of oral carcinogenesis. These antigens can be further validated for their use in cancer management by immune intervention.

Expanded protein expression profile of human placenta using two-dimensional gel electrophoresis

Studying proteins expressed in placenta is important to understand its function in pregnancy and fetal growth. Here, we present protein expression profiling from normal human placenta by 2-D gel - MS/MS approach that resulted in identification of 117 unique proteins. Integration with earlier analyses resulted in a profile of 423 non-redundant proteins, 75 of them being new identifications unique to this study including their isoforms. We present a compilation of placental protein expressions identified by proteomic approaches, their functions and known clinical implications. We believe that our dataset would be a useful resource for studies related to placental dysfunction.

In-depth 2-DE reference map of Aspergillus fumigatus and its proteomic profiling on exposure to itraconazole

Aspergillus fumigatus (A. fumigatus) is a medically important opportunistic fungus that may lead to invasive aspergillosis in humans with weak immune system. Proteomic profiling of this fungus on exposure to itraconazole (ITC), an azole antifungal drug, may lead to identification of its molecular targets and better understanding on the development of drug resistance against ITC in A. fumigatus. Here, proteome analysis was performed using 2-DE followed by mass spectrometric analysis which resulted in identification of a total of 259 unique proteins. Further, proteome profiling of A. fumigatus was carried out on exposure to ITC, 0.154 μg/ml, the minimum inhibitory concentration (MIC50). Image analysis showed altered levels of 175 proteins (66 upregulated and 109 downregulated) of A. fumigatus treated with ITC as compared to the untreated control. Peptide mass fingerprinting led to the identification of 54 proteins (12 up-regulated and 42 down-regulated). The differentially expressed proteins include proteins related to cell stress, carbohydrate metabolism and amino acid metabolism. We also observed four proteins, including nucleotide phosphate kinase (NDK), that are reported to interact with calcineurin, a protein involved in regulation of cell morphology and fungal virulence. Comparison of differentially expressed proteins on exposure to ITC with artemisinin (ART), an antimalarial drug with antifungal activity(1), revealed a total of 26 proteins to be common among them suggesting that common proteins and pathways are targeted by these two antifungal agents. The proteins targeted by ITC may serve as important leads for development of new antifungal drugs.