Hyperthermia Induces the ER Stress Pathway

The ER chaperone GRP78/BiP is a homolog of the Hsp70 family of heat shock proteins, yet GRP78/BiP is not induced by heat shock but instead by ER stress. However, previous studies had not considered more physiologically relevant temperature elevation associated with febrile hyperthermia. In this report we examine the response of GRP78/BiP and other components of the ER stress pathway in cells exposed to 40°C.AD293 cells were exposed to 43°C heat shock to confirm inhibition of the ER stress response genes. Five mammalian cell types, including AD293 cells, were then exposed to 40°C hyperthermia for various time periods and induction of the ER stress pathway was assessed.The inhibition of the ER stress pathway by heat shock (43°C) was confirmed. In contrast cells subjected to more mild temperature elevation (40°C) showed either a partial or full ER stress pathway induction as determined by downstream targets of the three arms of the ER stress pathway as well as a heat shock response. Cells deficient for Perk or Gcn2 exhibit great sensitivity to ER stress induction by hyperthermia.The ER stress pathway is induced partially or fully as a consequence of hyperthermia in parallel with induction of Hsp70. These findings suggest that the ER and cytoplasm of cells contain parallel pathways to coordinately regulate adaptation to febrile hyperthermia associated with disease or infection

TISs-ST: a web server to evaluate polymorphic translation initiation sites and their reflections on the secretory targets

<p>Abstract</p> <p>Background</p> <p>The nucleotide sequence flanking the translation initiation codon (start codon context) affects the translational efficiency of eukaryotic mRNAs, and may indicate the presence of an alternative translation initiation site (TIS) to produce proteins with different properties. Multi-targeting may reflect the translational variability of these other protein forms. In this paper we present a web server that performs computations to investigate the usage of alternative translation initiation sites for the synthesis of new protein variants that might have different functions.</p> <p>Results</p> <p>An efficient web-based tool entitled TISs-ST (Translation Initiation Sites and Secretory Targets) evaluates putative translation initiation sites and indicates the prediction of a signal peptide of the protein encoded from this site. The TISs-ST web server is freely available to both academic and commercial users and can be accessed at <url>http://ipe.cbmeg.unicamp.br/pub/TISs-ST</url>.</p> <p>Conclusion</p> <p>The program can be used to evaluate alternative translation initiation site consensus with user-specified sequences, based on their composition or on many position weight matrix models. TISs-ST provides analytical and visualization tools for evaluating the periodic frequency, the consensus pattern and the total information content of a sequence data set. A search option allows for the identification of signal peptides from predicted proteins using the PrediSi software.</p

MetWAMer: eukaryotic translation initiation site prediction

<p>Abstract</p> <p>Background</p> <p>Translation initiation site (TIS) identification is an important aspect of the gene annotation process, requisite for the accurate delineation of protein sequences from transcript data. We have developed the MetWAMer package for TIS prediction in eukaryotic open reading frames of non-viral origin. MetWAMer can be used as a stand-alone, third-party tool for post-processing gene structure annotations generated by external computational programs and/or pipelines, or directly integrated into gene structure prediction software implementations.</p> <p>Results</p> <p>MetWAMer currently implements five distinct methods for TIS prediction, the most accurate of which is a routine that combines weighted, signal-based translation initiation site scores and the contrast in coding potential of sequences flanking TISs using a perceptron. Also, our program implements clustering capabilities through use of the <it>k</it>-medoids algorithm, thereby enabling cluster-specific TIS parameter utilization. In practice, our static weight array matrix-based indexing method for parameter set lookup can be used with good results in data sets exhibiting moderate levels of 5'-complete coverage.</p> <p>Conclusion</p> <p>We demonstrate that improvements in statistically-based models for TIS prediction can be achieved by taking the class of each potential start-methionine into account pending certain testing conditions, and that our perceptron-based model is suitable for the TIS identification task. MetWAMer represents a well-documented, extensible, and freely available software system that can be readily re-trained for differing target applications and/or extended with existing and novel TIS prediction methods, to support further research efforts in this area.</p

Characterization of the Drosophila Ortholog of the Human Usher Syndrome Type 1G Protein Sans

BACKGROUND: The Usher syndrome (USH) is the most frequent deaf-blindness hereditary disease in humans. Deafness is attributed to the disorganization of stereocilia in the inner ear. USH1, the most severe subtype, is associated with mutations in genes encoding myosin VIIa, harmonin, cadherin 23, protocadherin 15, and sans. Myosin VIIa, harmonin, cadherin 23, and protocadherin 15 physically interact in vitro and localize to stereocilia tips in vivo, indicating that they form functional complexes. Sans, in contrast, localizes to vesicle-like structures beneath the apical membrane of stereocilia-displaying hair cells. How mutations in sans result in deafness and blindness is not well understood. Orthologs of myosin VIIa and protocadherin 15 have been identified in Drosophila melanogaster and their genetic analysis has identified essential roles in auditory perception and microvilli morphogenesis, respectively. PRINCIPAL FINDINGS: Here, we have identified and characterized the Drosophila ortholog of human sans. Drosophila Sans is expressed in tubular organs of the embryo, in lens-secreting cone cells of the adult eye, and in microvilli-displaying follicle cells during oogenesis. Sans mutants are viable, fertile, and mutant follicle cells appear to form microvilli, indicating that Sans is dispensable for fly development and microvilli morphogenesis in the follicle epithelium. In follicle cells, Sans protein localizes, similar to its vertebrate ortholog, to intracellular punctate structures, which we have identified as early endosomes associated with the syntaxin Avalanche. CONCLUSIONS: Our work is consistent with an evolutionary conserved function of Sans in vesicle trafficking. Furthermore it provides a significant basis for further understanding of the role of this Usher syndrome ortholog in development and disease

γCOP Is Required for Apical Protein Secretion and Epithelial Morphogenesis in Drosophila melanogaster

Background: There is increasing evidence that tissue-specific modifications of basic cellular functions play an important role in development and disease. To identify the functions of COPI coatomer-mediated membrane trafficking in Drosophila development, we were aiming to create loss-of-function mutations in the γCOP gene, which encodes a subunit of the COPI coatomer complex. Principal Findings: We found that γCOP is essential for the viability of the Drosophila embryo. In the absence of zygotic γCOP activity, embryos die late in embryogenesis and display pronounced defects in morphogenesis of the embryonic epidermis and of tracheal tubes. The coordinated cell rearrangements and cell shape changes during tracheal tube morphogenesis critically depend on apical secretion of certain proteins. Investigation of tracheal morphogenesis in γCOP loss-of-function mutants revealed that several key proteins required for tracheal morphogenesis are not properly secreted into the apical lumen. As a consequence, γCOP mutants show defects in cell rearrangements during branch elongation, in tube dilation, as well as in tube fusion. We present genetic evidence that a specific subset of the tracheal defects in γCOP mutants is due to the reduced secretion of the Zona Pellucida protein Piopio. Thus, we identified a critical target protein of COPI-dependent secretion in epithelial tube morphogenesis. Conclusions/Significance: These studies highlight the role of COPI coatomer-mediated vesicle trafficking in both general and tissue-specific secretion in a multicellular organism. Although COPI coatomer is generally required for protein secretion, we show that the phenotypic effect of γCOP mutations is surprisingly specific. Importantly, we attribute a distinct aspect of the γCOP phenotype to the effect on a specific key target protein

In Vivo Monitoring of mRNA Movement in Drosophila Body Wall Muscle Cells Reveals the Presence of Myofiber Domains

Background: In skeletal muscle each muscle cell, commonly called myofiber, is actually a large syncytium containing numerous nuclei. Experiments in fixed myofibers show that mRNAs remain localized around the nuclei in which they are produced. Methodology/Principal Findings: In this study we generated transgenic flies that allowed us to investigate the movement of mRNAs in body wall myofibers of living Drosophila embryos. We determined the dynamic properties of GFP-tagged mRNAs using in vivo confocal imaging and photobleaching techniques and found that the GFP-tagged mRNAs are not free to move throughout myofibers. The restricted movement indicated that body wall myofibers consist of three domains. The exchange of mRNAs between the domains is relatively slow, but the GFP-tagged mRNAs move rapidly within these domains. One domain is located at the centre of the cell and is surrounded by nuclei while the other two domains are located at either end of the fiber. To move between these domains mRNAs have to travel past centrally located nuclei. Conclusions/Significance: These data suggest that the domains made visible in our experiments result from prolonged interactions with as yet undefined structures close to the nuclei that prevent GFP-tagged mRNAs from rapidly moving between the domains. This could be of significant importance for the treatment of myopathies using regenerative cellbase

Plasmid pP62BP1 isolated from an Arctic Psychrobacter sp. strain carries two highly homologous type II restriction-modification systems and a putative organic sulfate metabolism operon

The complete nucleotide sequence of plasmid pP62BP1 (34,467 bp), isolated from Arctic Psychrobacter sp. DAB_AL62B, was determined and annotated. The conserved plasmid backbone is composed of several genetic modules, including a replication system (REP) with similarities to the REP region of the iteron-containing plasmid pPS10 of Pseudomonas syringae. The additional genetic load of pP62BP1 includes two highly related type II restriction-modification systems and a set of genes (slfRCHSL) encoding enzymes engaged in the metabolism of organic sulfates, plus a putative transcriptional regulator (SlfR) of the AraC family. The pP62BP1 slflocus has a compact and unique structure. It is predicted that the enzymes SlfC, SlfH, SlfS and SlfL carry out a chain of reactions leading to the transformation of alkyl sulfates into acyl-CoA, with dodecyl sulfate (SDS) as a possible starting substrate. Comparative analysis of the nucleotide sequences of pP62BP1 and other Psychrobacter spp. plasmids revealed their structural diversity. However, the presence of a few highly conserved DNA segments in pP62BP1, plasmid 1 of P. cryohalolentis K5 and pRWF-101 of Psychrobacter sp. PRwf-1 is indicative of recombinational shuffling of genetic information, and is evidence of lateral gene transfer in the Arctic environment

Interaction of SET domains with histones and nucleic acid structures in active chromatin

Changes in the normal program of gene expression are the basis for a number of human diseases. Epigenetic control of gene expression is programmed by chromatin modifications—the inheritable “histone code”—the major component of which is histone methylation. This chromatin methylation code of gene activity is created upon cell differentiation and is further controlled by the “SET” (methyltransferase) domain proteins which maintain this histone methylation pattern and preserve it through rounds of cell division. The molecular principles of epigenetic gene maintenance are essential for proper treatment and prevention of disorders and their complications. However, the principles of epigenetic gene programming are not resolved. Here we discuss some evidence of how the SET proteins determine the required states of target genes and maintain the required levels of their activity. We suggest that, along with other recognition pathways, SET domains can directly recognize the nucleosome and nucleic acids intermediates that are specific for active chromatin regions

Tempo and Mode in Evolution of Transcriptional Regulation

Perennial questions of evolutionary biology can be applied to gene regulatory systems using the abundance of experimental data addressing gene regulation in a comparative context. What is the tempo (frequency, rate) and mode (way, mechanism) of transcriptional regulatory evolution? Here we synthesize the results of 230 experiments performed on insects and nematodes in which regulatory DNA from one species was used to drive gene expression in another species. General principles of regulatory evolution emerge. Gene regulatory evolution is widespread and accumulates with genetic divergence in both insects and nematodes. Divergence in cis is more common than divergence in trans. Coevolution between cis and trans shows a particular increase over greater evolutionary timespans, especially in sex-specific gene regulation. Despite these generalities, the evolution of gene regulation is gene- and taxon-specific. The congruence of these conclusions with evidence from other types of experiments suggests that general principles are discoverable, and a unified view of the tempo and mode of regulatory evolution may be achievable