Cellular phosphatases facilitate combinatorial processing of receptor-activated signals

<p>Abstract</p> <p>Background</p> <p>Although reciprocal regulation of protein phosphorylation represents a key aspect of signal transduction, a larger perspective on how these various interactions integrate to contribute towards signal processing is presently unclear. For example, a key unanswered question is that of how phosphatase-mediated regulation of phosphorylation at the individual nodes of the signaling network translates into modulation of the net signal output and, thereby, the cellular phenotypic response.</p> <p>Results</p> <p>To address the above question we, in the present study, examined the dynamics of signaling from the B cell antigen receptor (BCR) under conditions where individual cellular phosphatases were selectively depleted by siRNA. Results from such experiments revealed a highly enmeshed structure for the signaling network where each signaling node was linked to multiple phosphatases on the one hand, and each phosphatase to several nodes on the other. This resulted in a configuration where individual signaling intermediates could be influenced by a spectrum of regulatory phosphatases, but with the composition of the spectrum differing from one intermediate to another. Consequently, each node differentially experienced perturbations in phosphatase activity, yielding a unique fingerprint of nodal signals characteristic to that perturbation. This heterogeneity in nodal experiences, to a given perturbation, led to combinatorial manipulation of the corresponding signaling axes for the downstream transcription factors.</p> <p>Conclusion</p> <p>Our cumulative results reveal that it is the tight integration of phosphatases into the signaling network that provides the plasticity by which perturbation-specific information can be transmitted in the form of a multivariate output to the downstream transcription factor network. This output in turn specifies a context-defined response, when translated into the resulting gene expression profile.</p

THP1 proteomics in response to mycobacterium tuberculosis infection

Temporal data on how the mycobacterium infection establishes itself inside the host cell is not available. We differentiated human THP1 cell line with PMA and infected them with different laboratory (H37Ra and H37Rv) and clinical strains (BND433 and JAL2287) of mycobacterium tuberculosis (Mtb). Uninfected differentiated THP1 cells were used as infection control. Host proteome was investigated at four different time points to understand the dynamics of host response to mycobacterial infection with time. The investigated time points included 6 hrs, 18 hrs, 30 hrs and 42 hrs of infection with all the Mtb strains. SWATH-MS method was used to quantitate the host proteome in response to Mtb infection and the data thus obtained are available via PRIDE repository with the dataset identifier PXD022352 (https://www.ebi.ac.uk/pride/archive/projects/PXD022352)

Integration of signals from the B-cell antigen receptor and the IL-4 receptor leads to a cooperative shift in the cellular response axis.

Although intracellular signaling events activated through individual cell surface receptors have been characterized in detail, cells are often exposed to multiple stimuli simultaneously in physiological situations. The response elicited then is defined through the cooperative interactions between signals activated by these multiple stimuli. Examples of such instances include cooperativity between individual isoforms of G-protein-coupled receptors, between different growth factor receptors, or between growth factor and integrin receptors. Mechanisms by which the integration of signals emanating from independent receptors influences cellular responses, however, are unknown. In this report, we studied interactions between the antigen and the IL-4 receptors in immature B cells. While stimulation through the B-cell antigen receptor alone causes cell cycle arrest and subsequent apoptosis, the inclusion of IL-4 during stimulation provides a protective effect. We therefore sought to obtain a systems view on how crosstalk between the two respective cell surface receptors modulates the cellular response. We found that, in comparison to the effects of B-cell receptor activation alone, combined stimulation through both receptors enforced a marked reorientation in the 'survival vs. apoptosis' axis of the signaling machinery. The consequent modulation of transcription factor activities yielded an integrated network, spanning the signaling and the transcriptional regulatory components, that was now biased towards the recruitment of molecules with a pro-survival function. This alteration in network properties influenced early-induced gene expression, in a manner that could rationalize the antagonistic effect of the IL-4 receptor on B-cell receptor signaling. Importantly, this antagonism was achieved through an expansion in the repertoire of the genes expressed, wherein the newly generated products counteracted the effects of the B-cell receptor-specific subset. Thus the plasticity of the regulatory networks is also experienced at the level of gene expression, and is the resultant pattern obtained that then modulates cell-fate decisions.</p

Differential proteomics approach to identify putative protective antigens of <i>Mycobacterium tuberculosis </i> presented during early stages of macrophage infection and their evaluation as DNA vaccines

429-439<span style="font-size:11.0pt;font-family: " times="" new="" roman","serif";mso-fareast-font-family:"times="" roman";mso-bidi-font-family:="" mangal;mso-ansi-language:en-gb;mso-fareast-language:en-us;mso-bidi-language:="" hi"="" lang="EN-GB">Unsatisfactory performance of the existing BCG vaccines, especially against the adult pulmonary disease, has urged the need for an effective vaccine against tuberculosis (TB). In this study, we employed differential proteomics to obtain a list of antigens as potential vaccine candidates. Bacterial epitopes being presented at early stages on MHC class I and class II molecules of macrophages infected with <i style="mso-bidi-font-style: normal">Mycobacterium tuberculosis (M. tb) were identified using iTRAQ labelling and reverse phase LC-MS/MS. The putative vaccine candidates, thus identified, were tested as plasmid DNA vaccines in mice to ascertain their protective efficacy against the aerosolized M. tb challenge, based on their ability to reduce the bacterial load in the lungs of infected mice. Here, we observed that 4 out of the 17 selected antigens imparted significant protection against the challenge of M. tb. The four shortlisted antigens were further assessed in a more stringent guinea pig model, where too, they demonstrated significant protection. It concludes that combining a proteomics approach with the in vivo assessment of vaccine candidates in animal models can be valuable in identifying new potential candidates to expand the antigenic repertoire for novel vaccines against TB.</span