EhMAPK, the Mitogen-Activated Protein Kinase from Entamoeba histolytica Is Associated with Cell Survival

Mitogen Activated Protein Kinases (MAPKs) are a class of serine/threonine kinases that regulate a number of different cellular activities including cell proliferation, differentiation, survival and even death. The pathogen Entamoeba histolytica possess a single homologue of a typical MAPK gene (EhMAPK) whose identification was previously reported by us but its functional implications remained unexplored. EhMAPK, the only mitogen-activated protein kinase from the parasitic protist Entamoeba histolytica with Threonine-X-Tyrosine (TXY) phosphorylation motif was cloned, expressed in E. coli and functionally characterized under different stress conditions. The expression profile of EhMAPK at the protein and mRNA level remained similar among untreated, heat shocked and hydrogen peroxide-treated samples in all cases of dose and time. But a significant difference was obtained in the phosphorylation status of the protein in response to different stresses. Heat shock at 43°C or 0.5 mM H2O2 treatment enhanced the phosphorylation status of EhMAPK and augmented the kinase activity of the protein whereas 2.0 mM H2O2 treatment induced dephosphorylation of EhMAPK and loss of kinase activity. 2.0 mM H2O2 treatment reduced parasite viability significantly but heat shock and 0.5 mM H2O2 treatment failed to adversely affect E. histolytica viability. Therefore, a distinct possibility that activation of EhMAPK is associated with stress survival in E. histolytica is seen. Our study also gives a glimpse of the regulatory mechanism of the protein under in vivo conditions. Since the parasite genome lacks any typical homologue of mammalian MEK, the dual specificity kinases which are the upstream activators of MAPK, indications of the existence of some alternate regulatory mechanisms of the EhMAPK activity is perceived. These may include the autophosphorylation activity of the protein itself in combination with some upstream phosphatases which are not yet identified

Signal duration and the time scale dependence of signal integration in biochemical pathways

Signal duration (e.g. the time scales over which an active signaling intermediate persists) is a key regulator of biological decisions in myriad contexts such as cell growth, proliferation, and developmental lineage commitments. Accompanying differences in signal duration are numerous downstream biological processes that require multiple steps of biochemical regulation. Here, we present an analysis that investigates how simple biochemical motifs that involve multiple stages of regulation can be constructed to differentially process signals that persist at different time scales. We compute the dynamic gain within these networks and resulting power spectra to better understand how biochemical networks can integrate signals at different time scales. We identify topological features of these networks that allow for different frequency dependent signal processing properties. Our studies suggest design principles for why signal duration in connection with multiple steps of downstream regulation is a ubiquitous control motif in biochemical systems.Comment: 27 pages, 4 figure

Using Network Component Analysis to Dissect Regulatory Networks Mediated by Transcription Factors in Yeast

Understanding the relationship between genetic variation and gene expression is a central question in genetics. With the availability of data from high-throughput technologies such as ChIP-Chip, expression, and genotyping arrays, we can begin to not only identify associations but to understand how genetic variations perturb the underlying transcription regulatory networks to induce differential gene expression. In this study, we describe a simple model of transcription regulation where the expression of a gene is completely characterized by two properties: the concentrations and promoter affinities of active transcription factors. We devise a method that extends Network Component Analysis (NCA) to determine how genetic variations in the form of single nucleotide polymorphisms (SNPs) perturb these two properties. Applying our method to a segregating population of Saccharomyces cerevisiae, we found statistically significant examples of trans-acting SNPs located in regulatory hotspots that perturb transcription factor concentrations and affinities for target promoters to cause global differential expression and cis-acting genetic variations that perturb the promoter affinities of transcription factors on a single gene to cause local differential expression. Although many genetic variations linked to gene expressions have been identified, it is not clear how they perturb the underlying regulatory networks that govern gene expression. Our work begins to fill this void by showing that many genetic variations affect the concentrations of active transcription factors in a cell and their affinities for target promoters. Understanding the effects of these perturbations can help us to paint a more complete picture of the complex landscape of transcription regulation. The software package implementing the algorithms discussed in this work is available as a MATLAB package upon request

Downregulation of the Hsp90 System Causes Defects in Muscle Cells of Caenorhabditis Elegans

The ATP-dependent molecular chaperone Hsp90 is required for the activation of a variety of client proteins involved in various cellular processes. Despite the abundance of known client proteins, functions of Hsp90 in the organismal context are not fully explored. In Caenorhabditis elegans, Hsp90 (DAF-21) has been implicated in the regulation of the stress-resistant dauer state, in chemosensing and in gonad formation. In a C. elegans strain carrying a DAF-21 mutation with a lower ATP turnover, we observed motility defects. Similarly, a reduction of DAF-21 levels in wild type nematodes leads to reduced motility and induction of the muscular stress response. Furthermore, aggregates of the myosin MYO-3 are visible in muscle cells, if DAF-21 is depleted, implying a role of Hsp90 in the maintenance of muscle cell functionality. Similar defects can also be observed upon knockdown of the Hsp90-cochaperone UNC-45. In life nematodes YFP-DAF-21 localizes to the I-band and the M-line of the muscular ultrastructure, but the protein is not stably attached there. The Hsp90-cofactor UNC-45-CFP contrarily can be found in all bands of the nematode muscle ultrastructure and stably associates with the UNC-54 containing A-band. Thus, despite the physical interaction between DAF-21 and UNC-45, apparently the two proteins are not always localized to the same muscular structures. While UNC-45 can stably bind to myofilaments in the muscular ultrastructure, Hsp90 (DAF-21) appears to participate in the maintenance of muscle structures as a transiently associated diffusible factor

Oculofaciocardiodental and Lenz microphthalmia syndromes result from distinct classes of mutations in BCOR.

Lenz microphthalmia is inherited in an X-linked recessive pattern and comprises microphthalmia, mental retardation, and skeletal and other anomalies. Two loci associated with this syndrome, MAA (microphthalmia with associated anomalies) and MAA2, are situated respectively at Xq27-q28 (refs. 1,2) and Xp11.4-p21.2 (ref. 3). We identified a substitution, nt 254C-->T; P85L, in BCOR (encoding BCL-6-interacting corepressor, BCOR) in affected males from the family with Lenz syndrome previously used to identify the MAA2 locus. Oculofaciocardiodental syndrome (OFCD; OMIM 300166) is inherited in an X-linked dominant pattern with presumed male lethality and comprises microphthalmia, congenital cataracts, radiculomegaly, and cardiac and digital abnormalities. Given their phenotypic overlap, we proposed that OFCD and MAA2-associated Lenz microphthalmia were allelic, and we found different frameshift, deletion and nonsense mutations in BCOR in seven families affected with OFCD. Like wild-type BCOR, BCOR P85L and an OFCD-mutant form of BCOR can interact with BCL-6 and efficiently repress transcription. This indicates that these syndromes are likely to result from defects in alternative functions of BCOR, such as interactions with transcriptional partners other than BCL-6. We cloned the zebrafish (Danio rerio) ortholog of BCOR and found that knock-down of this ortholog caused developmental perturbations of the eye, skeleton and central nervous system consistent with the human syndromes, confirming that BCOR is a key transcriptional regulator during early embryogenesis