Poonam Gautam

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.

LC-MS/MS Analysis of Differentially Expressed Glioblastoma Membrane Proteome Reveals Altered Calcium Signaling and Other Protein Groups of Regulatory Functions

Membrane proteins play key roles in the development and progression of cancer. We have studied differentially expressed membrane proteins in glioblastoma multiforme (GBM), the most common and aggressive type of primary brain tumor, by high resolution LC-MS/MS mass spectrometry and quantitation by iTRAQ. A total of 1834 membrane proteins were identified with high confidence, of which 356 proteins were found to be altered by 2-fold change or more (198 up- and 158 down-regulated); 56% of them are known membrane proteins associated with major cellular processes. Mass spectrometry results were confirmed for representative proteins on individual specimens by immunohistochemistry. On mapping of the differentially expressed proteins to cellular pathways and functional networks, we notably observed many calcium-binding proteins to be altered, implicating deregulation of calcium signaling and homeostasis in GBM, a pathway also found to be enriched in the report (Dong, H., Luo, L., Hong, S., Siu, H., Xiao, Y., Jin, L., Chen, R., and Xiong, M. (2010) Integrated analysis of mutations, miRNA and mRNA expression in glioblastoma. BMC Syst. Biol. 4, 163) based on The Cancer Genome Atlas analysis of GBMs. Annotations of the 356 proteins identified by us with The Cancer Genome Atlas transcriptome data set indicated overlap with 295 corresponding transcripts, which included 49 potential miRNA targets; many transcripts correlated with proteins in their expression status. Nearly 50% of the differentially expressed proteins could be classified as transmembrane domain or signal sequence-containing proteins (159 of 356) with potential of appearance in cerebrospinal fluid or plasma. Interestingly, 75 of them have been already reported in normal cerebrospinal fluid or plasma along with other proteins. This first, in-depth analysis of the differentially expressed membrane proteome of GBM confirms genes/proteins that have been implicated in earlier studies, as well as reveals novel candidates that are being reported for the first time in GBM or any other cancer that could be investigated further for clinical applications.

Proteins with altered levels in plasma from glioblastoma patients as revealed by iTRAQ-based quantitative proteomic analysis.

Glioblastomas (GBMs) are the most common and lethal primary tumors of the central nervous system with high level of recurrence despite aggressive therapy. Tumor-associated proteins/peptides may appear in the plasma of these patients as a result of disruption of the blood-brain barrier in them, raising the scope for development of plasma-based tests for diagnosis and monitoring the disease. With this objective, we analyzed the levels of proteins present in the plasma from GBM patients using an iTRAQ based LC-MS/MS approach. Analysis with pooled plasma specimens from the patient and healthy control samples revealed high confidence identification of 296 proteins, of which 61 exhibited a fold-change ≥1.5 in the patient group. Forty-eight of them contained signal sequence. A majority have been reported in the differentially expressed transcript or protein profile of GBM tissues; 6 have been previously studied as plasma biomarkers for GBM and 16 for other types of cancers. Altered levels of three representative proteins–ferritin light chain (FTL), S100A9, and carnosinase 1 (CNDP1)–were verified by ELISA in a test set of ten individual plasma specimens. FTL is an inflammation marker also implicated in cancer, S100A9 is an important member of the Ca2+ signaling cascade reported to be altered in GBM tissue, and CNDP1 has been reported for its role in the regulation of the levels of carnosine, implicated as a potential drug for GBM. These and other proteins in the dataset may form useful starting points for further clinical investigations for the development of plasma-based biomarker panels for GBM.

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.

Surfactant protein SP-D modulates activity of immune cells: proteomic profiling of its interaction with eosinophilic cells

Surfactant protein D (SP-D), a C-type lectin, is known to protect against lung infection, allergy and inflammation. Its recombinant truncated form comprising homotrimeric neck and CRD region (rhSP-D) has been shown to bring down specific IgE levels, eosinophilia and restore Th2–Th1 homeostasis in murine models of lung hypersensitivity. SP-D knockout mice show intrinsic hypereosinophilia and airway hyper-responsiveness that can be alleviated by rhSP-D. The rhSP-D can bind activated eosinophils, inhibit chemotaxis and degranulation, and selectively induce oxidative burst and apoptosis in sensitized eosinophils. A global proteomics study of rhSP-D-treated eosinophilic cell line AML14.3D10 identified large-scale molecular changes associated with oxidative burst, cell stress and survival-related proteins potentially responsible for apoptosis induction. The data also suggested an involvement of RNA binding- and RNA splicing-related proteins. Thus, the proteomics approach yielded a catalog of differentially expressed proteins that may be protein signatures defining mechanisms of SP-D-mediated maintenance of homeostasis during allergy.

Analysis of Human Blood Plasma Proteome from Ten Healthy Volunteers from Indian Population

Analysis of any mammalian plasma proteome is a challenge, particularly by mass spectrometry, due to the presence of albumin and other abundant proteins which can mask the detection of low abundant proteins. As detection of human plasma proteins is valuable in diagnostics, exploring various workflows with minimal fractionation prior to mass spectral analysis, is required in order to study population diversity involving analysis in a large cohort of samples. Here, we used ‘reference plasma sample’, a pool of plasma from 10 healthy individuals from Indian population in the age group of 25–60 yrs including 5 males and 5 females. The 14 abundant proteins were immunodepleted from plasma and then evaluated by three different workflows for proteome analysis using a nanoflow reverse phase liquid chromatography system coupled to a LTQ Orbitrap Velos mass spectrometer. The analysis of reference plasma sample a) without prefractionation, b) after prefractionation at peptide level by strong cation exchange chromatography and c) after prefractionation at protein level by sodium dodecyl sulfate polyacrylamide gel electrophoresis, led to the identification of 194, 251 and 342 proteins respectively. Together, a comprehensive dataset of 517 unique proteins was achieved from all the three workflows, including 271 proteins with high confidence identified by≥2 unique peptides in any of the workflows or identified by single peptide in any of the two workflows. A total of 70 proteins were common in all the three workflows. Some of the proteins were unique to our study and could be specific to Indian population. The high-confidence dataset obtained from our study may be useful for studying the population diversity, in discovery and validation process for biomarker identification.

Heterogeneous nuclear ribonucleoproteins and their interactors are a major class of deregulated proteins in anaplastic astrocytoma: A grade III malignant glioma

Anaplastic astrocytoma is a high grade malignant glioma (WHO grade III) of the central nervous system which arises from a low grade II tumor and invariably progresses into lethal glioblastoma (WHO grade IV). We have studied differentially expressed proteins from the microsomal fraction of the clinical specimens of these tumors, using iTRAQ and high-resolution mass spectrometry followed by immunohistochemistry for representative proteins on tissue sections. A total of 2642 proteins were identified, 266 of them with minimum 2 peptide signatures and 2-fold change in expression. The major groups of proteins revealed to be differentially expressed were associated with key cellular processes such as post transcriptional processing, protein translation, and acute phase response signaling. A distinct inclusion among these important proteins is 10 heterogeneous nuclear ribonucleoproteins (hnRNPs) and their interacting partners which have regulatory functions in the cell. hnRNP-mediated post transcriptional events are known to play a major role in mRNA processing, stability, and distribution. Their altered levels have also been observed by us in lower (diffused astrocytoma) and higher (glioblastoma) grades of gliomas, and membrane localization of hnRNPs has also been documented in the literature. hnRNPs may thus be major factors underlying global gene expression changes observed in glial tumors while their differential presence in the microsomal fraction suggests yet additional and unknown roles in tumorigenesis.

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.

Immunoproteomic analysis of secretory proteins ofAspergillus fumigatus with specific IGE immunoreactivity.

Allergenic/antigenic proteins are known to induce Type I and Type III hypersensitivity reactions leading to allergic bronchopulmonary aspergillosis (ABPA) in immunocompetent host. The common structural features or intrinsic properties of the allergens/antigens leading to allergenicity in a host are not well understood. In the current report, comparative analysis of proteins on two dimensional gel electrophoresis (2D-3) and specific IgE immunoblots ofA. fumigatus secretory proteins (1st, 2nd and 3rd week culture filtrate proteins) was carried out. We observed a total of 159 proteins in 1st, 2nd and 3rd week culture filtrates ofA. fumigatus. Specific IgE immunoreactivity was observed in 75 proteins with different intensity. Third week culture filtrate showed maximum number of proteins, 142, and specific IgE immunoreactive proteins, 65. MALDI-TOF analysis resulted in putative identification of two allergens as hypothetical protein YBL057c fromSaccharomyces cereviseae and unnamed protein product fromDebaryomyces hansenii (similar to IPF14568 ofCandida albicans). Identification of a repertoire of specific IgE immunoreactive proteins will facilitate the studies on structure-function relationship of these proteins relevant for diagnosis and pathogenesis.