Extracting proteins involved in disease progression using temporally connected networks

Background

Metabolic disorders such as obesity and diabetes are diseases which develop gradually over time in an individual and through the perturbations of genes. Systematic experiments tracking disease progression at gene level are usually conducted giving a temporal microarray data. There is a need for developing methods to analyze such complex data and extract important proteins which could be involved in temporal progression of the data and hence progression of the disease.

Results

In the present study, we have considered a temporal microarray data from an experiment conducted to study development of obesity and diabetes in mice. We have used this data along with an available Protein-Protein Interaction network to find a network of interactions between proteins which reproduces the next time point data from previous time point data. We show that the resulting network can be mined to identify critical nodes involved in the temporal progression of perturbations. We further show that published algorithms can be applied on such connected network to mine important proteins and show an overlap between outputs from published and our algorithms. The importance of set of proteins identified was supported by literature as well as was further validated by comparing them with the positive genes dataset from OMIM database which shows significant overlap.

Conclusions

The critical proteins identified from algorithms can be hypothesized to play important role in temporal progression of the data.

     

Phenylalanine-Rich Peptides Potently Bind ESAT6, a Virulence Determinant of Mycobacterium tuberculosis,and Concurrently Affect the Pathogen's Growth

Background

The secretory proteins of Mycobacterium tuberculosis (M. tuberculosis) have been known to be involved in the virulence, pathogenesis as well as proliferation of the pathogen. Among this set, many proteins have been hypothesized to play a critical role at the genesis of the onset of infection, the primary site of which is invariably the human lung.

Methodology/Principal Findings

During our efforts to isolate potential binding partners of key secretory proteins of M. tuberculosis from a human lung protein library, we isolated peptides that strongly bound the virulence determinant protein Esat6. All peptides were less than fifty amino acids in length and the binding was confirmed by in vivo as well as in vitro studies. Curiously, we found all three binders to be unusually rich in phenylalanine, with one of the three peptides a short fragment of the human cytochrome c oxidase-3 (Cox-3). The most accessible of the three binders, named Hcl1, was shown also to bind to the Mycobacterium smegmatis (M. smegmatis) Esat6 homologue. Expression of hcl1 in M. tuberculosis H37Rv led to considerable reduction in growth. Microarray analysis showed that Hcl1 affects a host of key cellular pathways in M. tuberculosis. In a macrophage infection model, the sets expressing hcl1 were shown to clear off M. tuberculosis in much greater numbers than those infected macrophages wherein the M. tuberculosis was not expressing the peptide. Transmission electron microscopy studies of hcl1 expressing M. tuberculosis showed prominent expulsion of cellular material into the matrix, hinting at cell wall damage.

Conclusions/Significance

While the debilitating effects of Hcl1 on M. tuberculosis are unrelated and not because of the peptide''s binding to Esat6–as the latter is not an essential protein of M. tuberculosis–nonetheless, further studies with this peptide, as well as a closer inspection of the microarray data may shed important light on the suitability of such small phenylalanine-rich peptides as potential drug-like molecules against this pathogen.     

Inhibition of proteasomal function by curcumin induces apoptosis through mitochondrial pathway

Curcumin is a natural polyphenolic compound having an antiproliferative property, which recent evidence suggests is due to its ability to induce apoptosis. However, the molecular mechanisms through which curcumin induces apoptosis are not fully understood. Here, we report that the curcumin-induced apoptosis is mediated through the impairment of the ubiquitin-proteasome system. Exposure of curcumin to the mouse neuro 2a cells causes a dose-dependent decrease in proteasome activity and an increase in ubiquitinated proteins. Curcumin exposure also decreases the turnover of the destabilized enhanced green fluorescence protein, a model substrate for proteasome and cellular p53 protein. Like other proteasome inhibitors, curcumin targets proliferative cells more efficiently than differentiated cells and induces apoptosis via mitochondrial pathways. Addition of curcumin to neuro 2a cells induces a rapid decrease in mitochondrial membrane potential and the release of cytochrome c into cytosol, followed by activation of caspase-9 and caspase-3.     

Mn‐catalase (Alr0998) protects the photosynthetic,nitrogen‐fixing cyanobacterium Anabaena PCC7120 from oxidative stress

Role of the non‐haem, manganese catalase (Mn‐catalase) in oxidative stress tolerance is unknown in cyanobacteria. The ORF alr0998 from the Anabaena PCC7120, which encodes a putative Mn‐catalase, was constitutively overexpressed in Anabaena PCC7120 to generate a recombinant strain, AnKat⁺. The Alr0998 protein could be immunodetected in AnKat⁺ cells and zymographic analysis showed a distinct thermostable catalase activity in the cytosol of AnKat⁺ cells but not in the wild‐type Anabaena PCC7120. The observed catalase activity was insensitive to inhibition by azide indicating that Alr0998 protein is indeed a Mn‐catalase. In response to oxidative stress, the AnKat⁺ showed reduced levels of intracellular ROS which was also corroborated by decreased production of an oxidative stress‐inducible 2‐Cys‐Prx protein. Treatment of wild‐type Anabaena PCC7120 with H₂O₂ caused (i) RNA degradation in vivo, (ii) severe reduction of photosynthetic pigments and CO₂ fixation, (iii) fragmentation and lysis of filaments and (iv) loss of viability. In contrast, the AnKat⁺ strain was protected from all the aforesaid deleterious effect under oxidative stress. This is the first report on protection of an organism from oxidative stress by overexpression of a Mn‐catalase.     

Crystal structure of uncleaved L-aspartate-alpha-decarboxylase from Mycobacterium tuberculosis

L-aspartate-alpha-decarboxylase (ADC) is a critical regulatory enzyme in the pantothenate biosynthetic pathway and belongs to a small class of self-cleaving and pyruvoyl-dependent amino acid decarboxylases. The expression level of ADC in Mycobacterium tuberculosis (Mtb) was confirmed by cDNA analysis, immunoblotting with an anti-ADC polyclonal antibody using whole cell lysate and immunoelectron microscopy. The recombinant ADC proenzyme from Mycobacterium tuberculosis (MtbADC) was overexpressed in E. coli and the protein structure was determined at 2.99 A resolution. The proteins fold into the double-psi beta-barrel structure. The subunits of the two tetramers (there are eight ADC molecules in the asymmetric unit) form pseudo fourfold rotational symmetry, similar to the E. coli ADC proenzyme structure. As pantothenate is synthesized in microorganisms, plants, and fungi but not in animals, structure elucidation of Mtb ADC is of substantial interest for structure-based drug development.     

Heterotopic Renal Autotransplantation in a Porcine Model: A Step-by-Step Protocol

Kidney transplantation is the treatment of choice for patients suffering from end-stage renal disease. It offers better life expectancy and higher quality of life when compared to dialysis. Although the last few decades have seen major improvements in patient outcomes following kidney transplantation, the increasing shortage of available organs represents a severe problem worldwide. To expand the donor pool, marginal kidney grafts recovered from extended criteria donors (ECD) or donated after circulatory death (DCD) are now accepted for transplantation. To further improve the postoperative outcome of these marginal grafts, research must focus on new therapeutic approaches such as alternative preservation techniques, immunomodulation, gene transfer, and stem cell administration.Experimental studies in animal models are the final step before newly developed techniques can be translated into clinical practice. Porcine kidney transplantation is an excellent model of human transplantation and allows investigation of novel approaches. The major advantage of the porcine model is its anatomical and physiological similarity to the human body, which facilitates the rapid translation of new findings to clinical trials. This article offers a surgical step-by-step protocol for an autotransplantation model and highlights key factors to ensure experimental success. Adequate pre- and postoperative housing, attentive anesthesia, and consistent surgical techniques result in favorable postoperative outcomes. Resection of the contralateral native kidney provides the opportunity to assess post-transplant graft function. The placement of venous and urinary catheters and the use of metabolic cages allow further detailed evaluation. For long-term follow-up studies and investigation of alternative graft preservation techniques, autotransplantation models are superior to allotransplantation models, as they avoid the confounding bias posed by rejection and immunosuppressive medication.     

Molecular Changes in Entamoeba histolytica in Response to Bacteria1

Entamoeba histolytica, the protozoan parasite, is the causative agent of amoebiasis. The degree of virulence, as inferred from invasiveness, of potentially pathogenic strains may be regulated by both host and parasite factors that determine the gut environment. One such factor that plays an important role is the bacterial flora in the gut. Previous studies have clearly shown that bacterial flora is an important determinant of virulence in E. histolytica. However, the exact nature of changes induced in E. histolytica in response to bacteria and their role in virulence is not clear. In this study the levels of a number of molecules potentially important in virulence mechanisms were determined in E. histolytica cells grown with and without normal human bacterial flora, using enzyme-linked immunosorbent assay. Significant changes were observed only after the E. histolytica cells had been adapted to grow with bacterial flora for a number of generations, and not in short term culture.