Involvement of yeast HSP90 isoforms in response to stress and cell death induced by acetic acid

Acetic acid-induced apoptosis in yeast is accompanied by an impairment of the general protein synthesis machinery, yet paradoxically also by the up-regulation of the two isoforms of the heat shock protein 90 (HSP90) chaperone family, Hsc82p and Hsp82p. Herein, we show that impairment of cap-dependent translation initiation induced by acetic acid is caused by the phosphorylation and inactivation of eIF2 alpha by Gcn2p kinase. A microarray analysis of polysome-associated mRNAs engaged in translation in acetic acid challenged cells further revealed that HSP90 mRNAs are over-represented in this polysome fraction suggesting preferential translation of HSP90 upon acetic acid treatment. The relevance of HSP90 isoform translation during programmed cell death (PCD) was unveiled using genetic and pharmacological abrogation of HSP90, which suggests opposing roles for HSP90 isoforms in cell survival and death. Hsc82p appears to promote survival and its deletion leads to necrotic cell death, while Hsp82p is a pro-death molecule involved in acetic acid-induced apoptosis. Therefore, HSP90 isoforms have distinct roles in the control of cell fate during PCD and their selective translation regulates cellular response to acetic acid stress.This work was supported by Fundacao para a Ciencia e Tecnologia and COMPETE/QREN/EU (PTDC/BIA-MIC/114116/2009), and by the Canadian Institute for Health Research (MOP 89737 to MH). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Different Effect of Proteasome Inhibition on Vesicular Stomatitis Virus and Poliovirus Replication

Proteasome activity is an important part of viral replication. In this study, we examined the effect of proteasome inhibitors on the replication of vesicular stomatitis virus (VSV) and poliovirus. We found that the proteasome inhibitors significantly suppressed VSV protein synthesis, virus accumulation, and protected infected cells from toxic effect of VSV replication. In contrast, poliovirus replication was delayed, but not diminished in the presence of the proteasome inhibitors MG132 and Bortezomib. We also found that inhibition of proteasomes stimulated stress-related processes, such as accumulation of chaperone hsp70, phosphorylation of eIF2α, and overall inhibition of translation. VSV replication was sensitive to this stress with significant decline in replication process. Poliovirus growth was less sensitive with only delay in replication. Inhibition of proteasome activity suppressed cellular and VSV protein synthesis, but did not reduce poliovirus protein synthesis. Protein kinase GCN2 supported the ability of proteasome inhibitors to attenuate general translation and to suppress VSV replication. We propose that different mechanisms of translational initiation by VSV and poliovirus determine their sensitivity to stress induced by the inhibition of proteasomes. To our knowledge, this is the first study that connects the effect of stress induced by proteasome inhibition with the efficiency of viral infection

Proteomic Analysis of Chikungunya Virus Infected Microgial Cells

Chikungunya virus (CHIKV) is a recently re-emerged public health problem in many countries bordering the Indian Ocean and elsewhere. Chikungunya fever is a relatively self limiting febrile disease, but the consequences of chikungunya fever can include a long lasting, debilitating arthralgia, and occasional neurological involvement has been reported. Macrophages have been implicated as an important cell target of CHIKV with regards to both their role as an immune mediator, as well evidence pointing to long term viral persistence in these cells. Microglial cells are the resident brain macrophages, and so this study sought to define the proteomic changes in a human microglial cell line (CHME-5) in response to CHIKV infection. GeLC-MS/MS analysis of CHIKV infected and mock infected cells identified some 1455 individual proteins, of which 90 proteins, belonging to diverse cellular pathways, were significantly down regulated at a significance level of p<0.01. Analysis of the protein profile in response to infection did not support a global inhibition of either normal or IRES-mediated translation, but was consistent with the targeting of specific cellular pathways including those regulating innate antiviral mechanisms

Extensive Translatome Remodeling during ER Stress Response in Mammalian Cells

In this work we have described the translatome of two mammalian cell lines, NIH3T3 and Jurkat, by scoring the relative polysome association of ∼10,000 mRNA under normal and ER stress conditions. We have found that translation efficiencies of mRNA correlated poorly with transcript abundance, although a general tendency was observed so that the highest translation efficiencies were found in abundant mRNA. Despite the differences found between mouse (NIH3T3) and human (Jurkat) cells, both cell types share a common translatome composed by ∼800–900 mRNA that encode proteins involved in basic cellular functions. Upon stress, an extensive remodeling in translatomes was observed so that translation of ∼50% of mRNA was inhibited in both cell types, this effect being more dramatic for those mRNA that accounted for most of the cell translation. Interestingly, we found two subsets comprising 1000–1500 mRNA whose translation resisted or was induced by stress. Translation arrest resistant class includes many mRNA encoding aminoacyl tRNA synthetases, ATPases and enzymes involved in DNA replication and stress response such as BiP. This class of mRNA is characterized by high translation rates in both control and stress conditions. Translation inducible class includes mRNA whose translation was relieved after stress, showing a high enrichment in early response transcription factors of bZIP and zinc finger C2H2 classes. Unlike yeast, a general coordination between changes in translation and transcription upon stress (potentiation) was not observed in mammalian cells. Among the different features of mRNA analyzed, we found a relevant association of translation efficiency with the presence of upstream ATG in the 5′UTR and with the length of coding sequence of mRNA, and a looser association with other parameters such as the length and the G+C content of 5′UTR. A model for translatome remodeling during the acute phase of stress response in mammalian cells is proposed

Recombinase technology: applications and possibilities

The use of recombinases for genomic engineering is no longer a new technology. In fact, this technology has entered its third decade since the initial discovery that recombinases function in heterologous systems (Sauer in Mol Cell Biol 7(6):2087–2096, 1987). The random insertion of a transgene into a plant genome by traditional methods generates unpredictable expression patterns. This feature of transgenesis makes screening for functional lines with predictable expression labor intensive and time consuming. Furthermore, an antibiotic resistance gene is often left in the final product and the potential escape of such resistance markers into the environment and their potential consumption raises consumer concern. The use of site-specific recombination technology in plant genome manipulation has been demonstrated to effectively resolve complex transgene insertions to single copy, remove unwanted DNA, and precisely insert DNA into known genomic target sites. Recombinases have also been demonstrated capable of site-specific recombination within non-nuclear targets, such as the plastid genome of tobacco. Here, we review multiple uses of site-specific recombination and their application toward plant genomic engineering. We also provide alternative strategies for the combined use of multiple site-specific recombinase systems for genome engineering to precisely insert transgenes into a pre-determined locus, and removal of unwanted selectable marker genes