MitoProteomics: New Insights in Infection Biology

Mitochondria are the organelles as central hubs of energy production, apoptosis regulation, and Ca++ signaling, therefore prime targets of pathogens to destroy the signaling network as earliest for their survivability. Recently, several proteomics approaches have been undertaken for rapid identification and analyses of disease manifestation in infection biology. Here, we have summarized the mostly popular topdown proteomics approaches including methodology to available database for mitochondrial signaling network analyses during host-pathogen interaction and a key tool for rapid biomarker identification.

Proteomic Analysis of Soybean Leaves in a Compatible and an Incompatible Interaction with Phakopsora Pachyrhizi

Asian soybean rust (ASR), which is incited by the fungus Phakopsora pachyrhizi, is considered one of the most aggressive diseases to the soybean culture. There are no commercial cultivars immune to the pathogen and the control measure currently used is the application of fungicides that harms the environment and increases production costs. For a better understanding of the host’s response to the pathogen at the molecular level, two soybean genotypes were analyzed (PI561356, resistant to ASR and Embrapa 48, susceptible) at 72 hours and 192 hours after inoculation with spores of P. pachyrhizi. Leaf protein profiles of the plants were compared by two-dimensional electrophoresis associated with mass spectrometry (MS). Twenty-two protein spots presented different levels when the two treatments were compared (inoculated vs. non-inoculated). From those, twelve proteins were identified by MS analysis. Some of them are involved in metabolic pathways related to plant defense against pathogens, as in the case of carbonic anhydrase, 1-deoxy-D-xylulose- 5-phosphate reductoisomerase, fructose-bisphosphate aldolase and glutamine synthetase. The possible biochemical-physiological meanings of our findings are discussed.

A New Method for Proteome Screening with Two-Dimensional Urea-Sds Polyacrylamide Gel Electrophoresis

Two-dimensional gel electrophoresis (2DE) separating proteins on the basis of their pI and molecular mass remain the best available technique for protein separation and characterization to date. But due to several limitations, including streak formation in IEF gels, partial solubility of proteins, expensive running conditions and relatively longer time taken, a simple urea-SDS-2D polyacrylamide gel electrophoresis (US2DE) is described here. The system is reasonably sensitive, cost effective with good reproducibility. The method described in this paper employs a chaotropic agent, urea, in the first dimension and sodium dodecyl sulphate (SDS), like conventional system, in the second dimension with an addition of polyacrylamide to screen the liver proteome of healthy and chemically induced fibrotic rats. The system separates the protein on the basis of chargeto- mass ratio and clearly demonstrates differential expression in the liver protein repertoire of healthy and fibrotic rats. Moreover, the present system, like other 2D electrophoretic procedures revealed at least 22 novel spots in the investigated tissues. The technique may be utilized for comprehensive proteome screening of any biological sample and would provide an overview to narrow down the candidate proteins or biomarkers.

Effect of Triton X-100 and Dtt Concentrations on Wide Range Two-Dimensional Gel Electrophoresis of Tissue, Cell and Fluid Proteomes

High-resolution separation by two-dimensional gel electrophoresis (2- DE) is still challenging due to the intrinsic behavior of proteins, principally throughout isoelectric focusing separation. It is often observed low resolution of proteins in the alkaline pH region when using wide range pH gradients. Herein, we show the effect of different concentrations of Triton X‑100 and DTT in the sample buffer on wide range pH (3-10) 2-DE profiles of three different biological samples as Trypanosoma cruzi cells, honey bee brain tissue and human saliva fluid. Higher resolution, number and intensity of spots were achieved when 85 mM DTT and 2.5% Triton X‑100 were employed for cell and tissue samples. No improvement was observed for fluid proteins, probably because water-soluble proteins do not require special conditions for extraction and prevention of precipitation during isoelectric focusing.

Subcellular Proteomics for Understanding Host Defense Peptides Mechanism of Action

Host defense peptides occurring in plants, invertebrates and vertebrates, represent a primary and heterogeneous group of molecules against infectious agents which may act directly on microorganisms or exert a redirectional enhancement of the immune response to them. Such molecules are an alternative if not for substituting at least complementing the classical antibiotics in anti-infective therapies. Several mechanisms of actions have been described for the majority of host defense peptides mostly acting at level of plasma membrane but some of them interacting with intracellular targets. The elucidation of their mechanisms of action besides completing their functional characterization is important for the development of more efficient subsequent molecular variants. Proteomic analyses have been applied for identifying new host defense peptides but few of them have been described for explaining their mechanism of action. In this paper we would like to update the subject remaking the importance of organelle proteomics for such purpose.