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PubMed2025

Balancing nutrient remobilization and photosynthesis: proteomic insights into the dual role of lupin cotyledons after germination.

Efficient nutrient mobilization from seed storage tissues is essential for seedling establishment, particularly in legumes such as Lupinus albus (white lupin), which thrive in nutrient-poor soils. This study investigates the role of cotyledons in nitrogen (N) and mineral remobilization after germination during their transition from storage organs to photosynthetically active tissues, including the metabolic challenges posed by the coexistence of these two functions in epigeal germination. We cultivated white lupin seedlings under nitrogen-deficient conditions, analyzing cotyledon composition and function over 28 days. Our results indicate that 60% of cotyledon-stored proteins are degraded within the first 8 days, with free amino acids transiently accumulating before being redistributed to support growth. The progressive depletion of cotyledon reserves was accompanied by structural and metabolic changes, including an increase in photosynthetic proteins. However, cotyledon photosynthetic capacity remained lower than that of true leaves, suggesting a transient role in energy metabolism. The loss of cotyledons before day 12 significantly impaired seedling development, emphasizing their critical contribution to nitrogen, phosphate, and micronutrient supply during early growth. Comparative proteomic analysis revealed dynamic shifts in nutrient transport, amino acid metabolism, and stress response pathways following cotyledon removal. These findings underscore the significance of cotyledon nutrient remobilization in legume adaptation to low-fertility soils and highlight potential targets for breeding strategies aimed at improving nutrient use efficiency. By optimizing cotyledon nutrient composition and function, future breeding efforts could enhance seedling vigor, reduce fertilizer dependency, and improve the nutritional value of lupin-based foods.

Lupinus↗
PubMed2006

Hypusination of eukaryotic initiation factor 5A (eIF5A): a novel therapeutic target in BCR-ABL-positive leukemias identified by a proteomics approach.

Inhibition of BCR-ABL tyrosine kinase with imatinib represents a major breakthrough in the treatment of patients with chronic myeloid leukemia (CML). However, resistance to imatinib develops frequently, particularly in late-stage disease. To identify new cellular BCR-ABL downstream targets, we analyzed differences in global protein expression in BCR-ABL-positive K562 cells treated with or without imatinib in vitro. Among the 19 proteins found to be differentially expressed, we detected the down-regulation of eukaryotic initiation factor 5A (eIF5A), a protein essential for cell proliferation. eIF5A represents the only known eukaryotic protein activated by posttranslational hypusination. Hypusination inhibitors (HIs) alone exerted an antiproliferative effect on BCR-ABL-positive and -negative leukemia cell lines in vitro. However, the synergistic dose-response relationship found for the combination of imatinib and HI was restricted to Bcr-Abl-positive cells. Furthermore, this synergistic effect was confirmed by cytotoxicity assays, cell-cycle analysis, and CFSE labeling of primary CD34+ CML cells. Specificity of this effect could be demonstrated by cotreatment of K562 cells with imatinib and siRNA against eIF5. In conclusion, through a comparative proteomics approach and further functional analysis, we identified the inhibition of eIF5A hypusination as a promising new approach for combination therapy in BCR-ABL-positive leukemias.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed2002

Proteome study of Francisella tularensis live vaccine strain-containing phagosome in Bcg/Nramp1 congenic macrophages: resistant allele contributes to permissive environment and susceptibility to infection.

The phagocytosis of pathogens by macrophages classically initiates maturation of the phagosome that involves a dynamic exchange of phagosomal components with intracellular compartments of the endocytic pathway. The intracellular microorganisms have developed sophisticated mechanisms to sense environmental conditions and respond to them by phenotypic alterations that ensure their adaptation, survival and proliferation inside the cell. They have learned also to utilise host cellular components to ensure own survival. Recent results suggest that the Bcg locus/Nramp1 gene (natural resistance-associated macrophage protein 1) controls natural resistance to infection by Francisella tularensis LVS (live vaccine strain) and its effect is opposite to that observed for other Bcg/Nramp1-controlled pathogens such as several mycobacterial species, Leischmania donovani, and Salmonella typhimurium. In the case of F. tularensis LVS infection, the mutant allele of the Bcg locus (Bcg(s)/Nramp1(s)) is associated with natural resistance and, inversely, the wild type allele (Bcg(r)/Nramp1(r)) confers susceptibility. To determine whether differential allelic expression of the Bcg locus/Nramp1 gene modifies the composition of F. tularensis LVS-containing phagosomes (FCP), we have utilised an approach where we isolated FCP from infected Bcg congenic B10R (Bcg(r)/Nramp1(r)) and B10S (Bcg(s)/Nramp1(s)) macrophages of susceptible and resistant phenotype, respectively. Comparative proteomic analysis of the two phagosomal compartments with subsequent mass spectrometric analysis allowed identification of several proteins typical for FCP from B10R macrophages. They include a bacterial hypothetical 23 kDa protein, 60 kDa chaperonin GroEL, and host putative proteins that appeared to be mitochondrial ATP synthase beta-chain and NADH-ubiquinone oxidoreductase based on high cross-species homology. High abundance of the hypothetical 23 kDa protein correlates with the susceptible phenotype and, possibly, pathogenicity of F. tularensis LVS. The results demonstrate that F. tularensis LVS can exploit ion transport function of Bcg/Nramp1 to its own advantage.

Alleles↗
PubMed2002

Analytical and biological variances associated with proteomic studies of Medicago truncatula by two-dimensional polyacrylamide gel electrophoresis.

Two-dimensional polyacrylamide gel electrophoresis (2-DE) and mass spectrometry are being used as proteomic tools in an integrated functional genomics program focused on the model legume Medicago truncatula. Due to the perceived high levels of indeterminate error associated with 2-DE we deemed it necessary to quantify the coefficient of variance (or relative standard deviation) for both analytical and biological sources associated with 2-DE of Medicago truncatula leaf protein extracts. Leaf protein extracts were chosen because of their biological significance and due to the more challenging nature of green tissues. Analytical variance was calculated for fifty proteins from ten replicate 2-DE gels of the same protein extract. Biological variance was calculated for the same fifty proteins from ten independent 2-DE gel analyses of ten independent but similar plants grown under identical conditions. Average analytical and biological variances were calculated for both data sets and represent the average variance of approximately 500 independent measurements of protein concentration. Analytical variance was determined to be 16.2% and biological variance was determined to be 24.2%. These average variances provide a quantified and statistical basis for evaluation of protein expression changes in future comparative proteomic investigations. It is proposed that 2-DE measured protein expression levels should differ by a minimum of 3.92sigma (i.e. /+/-2sigma/ and sigma = standard deviation), or 94.7% based on our measured variances, for the difference to be significant at the 95% confidence level.

Data Interpretation, Statistical↗
PubMed2005

The holm oak leaf proteome: analytical and biological variability in the protein expression level assessed by 2-DE and protein identification tandem mass spectrometry de novo sequencing and sequence similarity searching.

As a first approach in establishing the holm oak leaf proteome, we have optimised a protocol for this plant and tissue which includes the following steps: trichloroacetic acid-acetone extraction, two-dimensional gel electrophoresis (2-DE) on pH 5 to 8 linear gradient immobilised pH gradient strips as the first dimension, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis on 13% polyacrylamide gels as the second one. Proteins were detected by Coomassie staining. Gel images were recorded and digitalized, and the protein spots quantified by using a linear regression equation of protein quantity on spot volume obtained against standard proteins. Analytical variance was calculated for one-hundred protein spots from three replicate 2-DE gels of the same protein extract. Biological variance was determined for the same protein spots from independent tissue extracts corresponding to leaves from different trees, or the same tree at different orientations or sampling times during a day. Values of 26% for the analytical variance and 58.6% for the biological variance among independent trees were obtained. These values provide a quantified and statistical basis for the evaluation of protein expression changes in comparative proteomic investigations with this species. A representative set of the major proteins, covering the isoelectric point range of 5 to 8 and the relative molecular mass(r) range of 14 to 78 kDa, were subjected to liquid chromatography-tandem mass spectrometry analysis. Due to the absence of Quercus DNA or protein sequence databases, a method based on the procedure reported by Liska and Shevchenko including de novo sequencing and BLAST similarity searching against other plant species databases was used for protein identification. Out of 43 analysed spots, 35 were positively identified. The identified proteins mainly corresponded to enzymes involved in photosynthesis and energetic metabolism, with a significant number corresponding to RubisCO.

Amino Acid Sequence↗
PubMed2003

Proteome analysis on an early transformed human bronchial epithelial cell line, BEP2D, after alpha-particle irradiation.

To probe the mechanism of carcinogenesis of lung cancer at the molecular level and to find potential protein markers involved in the early phase of tumorgenesis, differential proteome analysis on primary passage cell line R15H, and early transformed cell line R15H20 derived from (238)Pu alpha-particle irradiation of human papillomavirus (HPV) 18-immortalized human bronchial epithelial cell line (BEP2D), was carried out using two-dimensional electrophoresis (2-DE) and peptide mass fingerprinting (PMF) with matrix-assisted laser desorption/ionisation-time of flight mass spectrometry. Image analysis and Student's t-test (p < 0.05) showed that three protein spots were only expressed in R15H, intensities of 43 protein spots on the gels were altered between R15H and R15H20. Two of the three spots that were only expressed in R15H were identified as high mobility group protein 1. Two proteins decreased in abundance in R15H20 were identified as maspin precursor, a tumor suppressor and aminoacylase-1. Ornithine aminotransferase and peptidyl-prolyl cis-trans isomerase A that were increased in R15H20, were also identified. Relationships between these differentially expressed proteins and the carcinogenesis mechanism of lung cancer are discussed. The protein expression profile of the R15H cell line was also constructed during the study as a reference map for further comparative proteome analysis of the irradiation induced BEP2D cell line. Of the 90 spots analyzed with PMF in the 2-DE gel of R15H cell line, 50 proteins were identified by searching the nonredundant protein database SWISS-PROT/TrEMBL.

Alpha Particles↗
PubMed2004

[Identification of colorectal cancer using proteomic patterns in serum].

BACKGROUND & OBJECTIVE: At present there is no serological parameter with high sensitivity and specificity to diagnose colorectal cancer. This study aimed at establishing a serum protein fingerprinting technique coupled with a pattern-matching algorithm to distinguish colorectal cancer from benign colorectal diseases and healthy people. METHODS: Preliminary group and test group were both random selected serum samples, which each comprises 73 cases of colorectal cancer, 31 healthy people, and 16 cases of benign colorectal diseases. The sera of the test group was combined with the surface of the IMAC3 proteinchip. Then reading data of surface enhanced laser desorption/ionization- time of flight-mass spectrometry (SELDI-TOF-MS) was analyzed by Biomarker Wizard software and Biomarker pattern software to get a classification rule of tree, which is standard configuration that can distinguish the sera of colorectal cancer patients from the sera of benign colorectal disease patients and healthy people, and the standard was approved test validity by double-blindly in the test group. RESULTS: At the key M/Z values of 4 467 Da, 8 131 Da, 8 939 Da, 9 192 Da, 9 134 Da, 8 221 Da, 5 928 Da, 8 324 Da, 11 732 Da, protein contents of the three classes in preliminary group are obviously different by the software analysis. And the classification rule of tree was discovered, and corresponding correct ratio was 98.33% (118/120), corresponding sensitivity was 97.26% (71/73) and corresponding specificity was 100% (47/47); after double-blind examining the test group with that rule, the corresponding correct ratio was 96.77% (116/120),the corresponding sensitivity was 95.89% (70/73) and the corresponding specificity was 97.87%(46/47). CONCLUSION: Via comparative proteomics research in the sera of colorectal cancer patients and benign colorectal disease patients and healthy people, the group of protein was obviously different in pathological process in discovering colorectal cancer.

Algorithms↗
PubMed2005

Two-dimensional gel electrophoresis maps of the proteome and phosphoproteome of primitively cultured rat mesangial cells.

Mesangial cells (MC) play an important role in maintaining the structure and function of the glomerulus. The proliferation of MC is a prominent feature of many kinds of glomerular disease. The first reference 2-DE maps of rat mesangial cells (RMC), stained with silver staining or Pro-Q Diamond dye, have been established here to describe the proteome and phosphoproteome of RMC, respectively. A total of 157 selected protein spots, corresponding to 118 unique proteins, have been identified by MALDI-TOF-MS or LC-ESI-IT-MS/MS, in which 37 protein spots representing 28 unique proteins have also been stained with Pro-Q Diamond, indicating that they are in phosphorylated forms. All the identified proteins were bioinformatically annotated in detail according to their physiochemical characteristics, subcellular location, and function. Most of the separated or identified protein spots are distributed in the area of mass 10-70 kDa and pI 5.0-8.0. The identified proteins include mainly cytoplasmic and nuclear proteins and some mitochondrial, endoplasmic reticulum, and membrane proteins. These proteins are classified into different functional groups such as structure and mobility proteins (21.2%), metabolic enzymes (16.9%), protein folding and metabolism proteins (13.6%), signaling proteins (14.4%), heat-shock proteins (7.6%), and other functional proteins (12.7%). While structure and mobility proteins are mostly represented by protein spots with high abundance, signaling proteins are mostly represented by protein spots with relatively low abundance. Such a 2-DE database for RMC, especially with many signaling proteins and phosphoproteins characterized, will provide a valuable resource for comparative proteomics analysis of normal and pathologic conditions affecting MC function or pathologic progress.

Animals↗
PubMed2005

Comparative genomics on SNAI1, SNAI2, and SNAI3 orthologs.

SNAI1, SNAI2, and SNAI3 genes, encoding transcriptional repressors implicated in epithelial mesenchymal transition (EMT), are human homologs of Drosophila snail (sna) and slug genes. SNAI1 represses transcription of CDH1 (E-cadherin) gene. SNAI2 induces the first phase of EMT, including desmosome dissociation, cell spreading, and initiation of cell separation. Because SNAI family proteins are implicated in EMT during embryogenesis and carcinogenesis, SNAI family genes are potent targets of pharmacogenomics. Here, comparative genomics analyses and comparative proteomics analyses on SNAI family orthologs were performed. Rat Snai3 gene, consisting of three exons, was identified within rat genome sequence AC111791.4. Zebrafish snai1a (NM_131066.1) was identified as SNAI1 ortholog. Chicken ChEST362l17 (CR407272.1), Xenopus slug (AF368041.1), and zebrafish zgc92564 (NM_001008581.1) were identified as SNAI2 orthologs. Chicken snail (NM_ 205142.1), Xenopus snail (BC056857.1), and zebrafish snai1b (NM_130989.1) were identified as SNAI3 orthologs. SNAI1 orthologs consisted of SNAG domain and four zinc finger (ZNF) domains, while SNAI2 and SNAI3 orthologs consisted of SNAG domain and five ZNF domains. Based on the integromics analyses, SNAI2 orthologs were found to be more conserved than SNAI1 and SNAI3 orthologs. SNAI1 mRNA was expressed in placenta, neuroblastoma, and diffuse type gastric cancer. SNAI2 mRNA was expressed in placenta, melanocyte, embryonic stem (ES) cells, leiomyosarcoma, neuroblastoma, and glioblastoma. SNAI3 mRNA was expressed in B cells. Expression of SNAI3 mRNA was repressed due to the existence of anti-sense single-exon transcript. SNAI1, functioning as E-cadherin repressor, is implicated in the malignant infiltrating phenotype of diffuse type gastric cancer through the induction of EMT or fibroblastoid transformation.

Amino Acid Sequence↗
PubMed2006

Genomic and proteomic approaches in comparative biochemistry and physiology.

Emerging technologies in genomics (e.g., cDNA library screening, DNA arrays), proteomics (e.g., two-dimensional gel electrophoresis and mass-spectroscopy fingerprinting), and metabolic regulation (e.g., elucidating protein-protein binding associations or signal transduction pathways) offer powerful tools for comparative biochemistry that will greatly increase our understanding of how animals adapt to environmental stress. The power of these new technologies and their potential application for physiological and ecophysiological research are illustrated with examples of recent advances in key fields. These include hypoxia/anoxia-responsive gene expression, the actions and regulation of the hypoxia-inducible transcription factor, the central role of the AMP-dependent kinase in mediating cellular responses to changing energy status, mammalian target of rapamycin mediation of nutrient signals in the control of protein synthesis and growth, proteomics approaches to identifying the physiological substrates of protein kinases, and the interactions of biochemistry and physiology in determining a net organismal response to high hematocrit values stimulated by erythropoietin. In particular, gene- and protein-screening technologies will drive a fundamental shift in the way that environmental stress effects on metabolism are evaluated by providing a holistic overview of the responses of a huge range of cell functions to stress and allowing researchers to identify multiple new areas of metabolic response that contribute to biochemical and physiological adaptation.

Adaptation, Physiological↗
PubMed2005

Edema is a precursor to central nervous system peritumoral cyst formation.

Despite the common occurrence and frequent clinical effects of peritumoral cysts in the central nervous system (CNS), the mechanism underlying their development and evolution is not understood. Because they commonly produce peritumoral cysts and because serial magnetic resonance imaging (MRI) is obtained in von Hippel-Lindau disease patients, hemangioblastomas provide an opportunity to examine the pathophysiology of CNS peritumoral cyst formation. Serial MRI was correlated with the clinical findings in 16 von Hippel-Lindau disease patients with 22 CNS hemangioblastomas (11 spinal cord; 11 cerebellar) that were associated with the appearance and evolution of peritumoral cysts. Hemangioblastoma-associated cyst wall histomorphological analysis was performed on postmortem tissues from three von Hippel-Lindau disease patients (not in the clinical series). Comparative proteomic profiling was performed on peritumoral cyst fluid and serum. Vascular endothelial growth factor levels were determined in peritumoral cysts. MRI clearly showed peritumoral edema that developed and slowly and progressively evolved into enlarging hemangioblastoma-associated cysts in all tumors (mean follow-up, 130 +/- 38 months; mean +/- standard deviation). Postcontrast MRI demonstrated convective leakage of gadolinium into cysts. Mean time required for edema to evolve into a cyst was 36 +/- 23 months (range, 8-72 months). Thirteen (59%) hemangioblastoma-cysts became symptomatic (mean time to symptom formation after cyst development, 35 +/- 32 months; range, 3-102 months) and required resection. Protein profiles of cyst fluid and serum were similar. Mean cyst fluid vascular endothelial growth factor concentration was 1.5 ng/ml (range, 0-5.4 ng/ml). Histology of the cyst walls was consistent with reactive gliosis. CNS peritumoral cyst formation is initiated by increased tumor vascular permeability, increased interstitial pressure in the tumor, and plasma extravasation with convective distribution into the surrounding tissue. When the delivery of plasma from the tumor exceeds the capacity of the surrounding tissue to absorb the extravasated fluid, edema (with its associated increased interstitial pressure) and subsequent cyst formation occur.

Adult↗
PubMed2005

Mortalin is over-expressed by colorectal adenocarcinomas and correlates with poor survival.

Using comparative proteomic analysis we have identified over-expression of mortalin in colorectal adenocarcinomas. Mortalin, also known as mitochondrial heat-shock protein 70 (mhsp 70), is involved in cell cycle regulation with important roles in cellular senescence and immortalization pathways. It is known to bind to and inactivate wild-type tumour suppressor protein p53 and influences the Ras-Raf-MAPK pathway. By immunostaining a colorectal cancer tissue microarray linked to a patient database, we further found that mortalin over-expression correlates with poor patient survival and, in multivariate analysis, is independent of standard prognostic variables (p = 0.04). Our findings demonstrate that mortalin over-expression may predict outcome in colorectal cancer and suggest that this protein is involved in colorectal neoplasia. Our experimental approach emphasises the analytical power of combining proteomics with tissue microarray analysis in the context of a well-defined tumour database.

Adenocarcinoma↗
PubMed2006

Characterization and over-expression of chaperonin t-complex proteins in colorectal cancer.

The chaperonins are key molecular complexes, which are essential in the folding of proteins to produce stable and functionally competent protein conformations. One member of the chaperonin group of proteins is TCP1 (chaperonin containing t-complex polypeptide 1, or CCT), but little is known about this protein in tumours. In this study, we used comparative proteomic analysis to show that t-complex protein subunits TCP1 beta and TCP1 epsilon are over-expressed in colorectal adenocarcinomas. Monoclonal antibodies to these proteins were developed and the expression and cellular localization of these two proteins in colorectal cancer were analysed by immunohistochemistry on a colorectal cancer tissue microarray. In colorectal cancer, TCP1 beta cellular localization was exclusively cytoplasmic, whereas TCP1 epsilon staining was seen in both the nucleus and the cytoplasm. Both cytoplasmic TCP1 beta and cytoplasmic TCP1 epsilon were significantly over-expressed (p < 0.001 for each protein) in primary colorectal cancer and also showed increased expression with advancing Dukes' stage (p = 0.018 for TCP1 beta and p = 0.045 for TCP1 epsilon). A trend was also identified between over-expression of cytoplasmic TCP1 beta and reduced patient survival (p = 0.05). These results show that both TCP1 beta and TCP1 epsilon are over-expressed in colorectal cancer and indicate a role for TCP1 beta and TCP1 epsilon in colorectal cancer progression.

Adenocarcinoma↗
PubMed2004

UDP-glucose pyrophosphorylase is upregulated in carriers of the porcine RN- mutation in the AMP-activated protein kinase.

The AMP-activated protein kinase (AMPK) plays a key role in the regulation of energy metabolism in eukaryotic cells acting as a metabolic sensor. In its activated form AMPK inhibits ATP consuming pathways and stimulates ATP generating pathways. A dominant mutation, denoted RN(-), in the porcine PRKAG3 gene, encoding the regulatory gamma3 subunit of AMPK, results in hyperaccumulation of glycogen in glycolytic skeletal muscle cells. To study the effects of this mutation on protein expression patterns in skeletal muscle, comparative proteome analysis of muscle samples from 12 animals (6 rn (+)/rn (+) and 6 RN(-)/rn (+)) was performed. The major finding of the proteome analysis was that the key enzyme in the synthesis of glycogen, UDP-glucose pyrophosphorylase, was significantly up-regulated in RN(-) carriers. This observation was subsequently supported by studies of enzyme activity and Northern blot analysis. Furthermore, the expression patterns of enzymes related to glycolysis and the citric acid cycle were also affected. Our data suggests that hyperaccumulation of glycogen mediated by the RN(-) mutation is due to an increased synthesis of glycogen.

Animals↗
PubMed2004

Differential expression profiling of human pancreatic adenocarcinoma and healthy pancreatic tissue.

Due to poor prognosis and lack of effective treatment, pancreatic carcinoma (PC) is a devastating disease. With the goal of contributing to an improved detection, prevention and treatment of the disease, a comparative proteome analysis of PC and normal tissue was carried out. Paired tissue extracts from 12 patients (pancreatic adenocarcinoma and adjacent healthy tissue) were separated by two-dimensional electrophoresis. Differential protein expression was analyzed by gel comparison with the help of image analysis software. The differentially expressed spots were identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry. Seventy proteins were more strongly expressed (mostly two-fold or more) in cancerous tissue, while 41 were stronger in normal pancreas respectively. Those spots highly expressed in PC were confirmed in gels from independent individual samples. Among them were several cytoskeletal proteins, small GTP-binding proteins, and members of the S100 protein family etc. Nine proteins had been reported in previous nuclear acid-based studies. The levels of two proteins were confirmed by immunohistochemistry. One of them, fascin, was detected in 13 out of 21 carcinoma and negative in all normal pancreas samples. Moreover, fascin expression was related to the differentiation of pancreatic carcinoma.

Adenocarcinoma↗
PubMed2006

Identification of target proteins of N-acetylglucosaminyl transferase V in human colon cancer and implications of protein tyrosine phosphatase kappa in enhanced cancer cell migration.

To gain a better understanding of the mechanism underlying colon cancer and to search for potential markers of colon cancer prognosis, a comparative proteomic analysis of colon cancer WiDr cells was conducted using 2-DE and lectin blot, followed by identification based on ESI-MS. Through these approaches 14 proteins were identified as candidate target proteins for N-acetylglucosaminyl transferase V (GnT-V) that would be expected to be implicated in the progression of colon cancer. We selected protein tyrosine phosphatase kappa (PTPkappa) as a model protein to validate this approach to the discovery of novel biomarkers in colon cancer. PTPkappa underwent an aberrant glycosylation in GnT-V-overexpressing WiDr cells, and the aberrantly glycosylated PTPkappa was vulnerable to proteolytic cleavage. The enhanced cleavage of PTPkappa in GnT-V-overexpressing cells was responsible for the mitigation of the homophilic binding capacity, resulting in an increase in cancer cell migration.

Cell Movement↗
PubMed2003

Identification of proteins highly expressed in the hyphae of Candida albicans by two-dimensional electrophoresis.

The increase in Candida albicans infections is caused by the increase in therapies resulting in immunocompromised patients. One factor required for C. albicans pathogenicity is the morphological transition from yeast to hypha. The protein profiles of whole extracts from yeasts and hyphae were examined using two-dimensional electrophoresis to identify the proteins related to the morphological transition. Over 900 protein spots were visualized by silver staining and 11 spots were increased more than three-fold reproducibly during hyphal differentiation. Six of the 11 spots were identified by peptide mass fingerprints, of which three represented PRA1, two PHR1 and the last TSA1. Vertical streak patterns of Pra1p and Phr1p indicated that post-translational modifications seem to be caused by variable glycosylation. Comparative proteome analysis between the wild-type and the deletion mutants, CAMB43 (deltapra1) and CAS10 (deltaphr1), further confirmed the identity of PRA1 and PHR1. Interestingly, Pra1p was downregulated in phr1-deleted mutants. Only PHR1 transcription was increased, indicating that PRA1 and TSA1 are controlled at the post-translational level.

Apoenzymes↗
PubMed2003

Protein microarrays: new tools for pharmaceutical development.

Protein microarrays are a relatively new technology, which will dramatically impact the pharmaceutical industry. The critical need for more rapid identification of novel drug targets, and for obtaining high-quality information early in the target validation process is a major driver for the industry. High-throughput protein analytical techniques are critical for obtaining biological information beyond that which transcript analysis can provide, given that proteins are the "worker bees" in cells. The vast complexity of proteins when compared to DNA and RNA in terms of sheer number, and structural and biochemical diversity requires a higher degree of sophistication in both assay design and data analysis. High-throughput microarray technology platforms allow for simultaneous, multi-parametric analysis of complex protein mixtures. Protein microarrays have tremendous potential as a tool for the study of protein-protein, enzyme-substrate, and antibody-antigen interactions among others. They can also be used for biomarkers and drug target identification via comparative proteomic analysis of healthy and disease tissues. More recently, cellular microarrays that enable identification of cell-surface receptors and other cell-surface proteins allowing rapid screening of cell-specific, novel drug targets, are being developed. This review will focus on the technical issues and potential applications of protein microarrays in pharmaceutical discovery.

Drugs, Investigational↗