BackgroundThe main technological impediment to widespread utilization of lignocellulose for the production of fuels and chemicals is the lack of low-cost technologies to overcome its recalcitrance. Organisms that hydrolyze lignocellulose and produce a valuable product such as ethanol at a high rate and titer could significantly reduce the costs of biomass conversion technologies, and will allow separate conversion steps to be combined in a consolidated bioprocess (CBP). Development of Saccharomyces cerevisiae for CBP requires the high level secretion of cellulases, particularly cellobiohydrolases.ResultsWe expressed various cellobiohydrolases to identify enzymes that were efficiently secreted by S. cerevisiae. For enhanced cellulose hydrolysis, we engineered bimodular derivatives of a well secreted enzyme that naturally lacks the carbohydrate-binding module, and constructed strains expressing combinations of cbh1 and cbh2 genes. Though there was significant variability in the enzyme levels produced, up to approximately 0.3 g/L CBH1 and approximately 1 g/L CBH2 could be produced in high cell density fermentations. Furthermore, we could show activation of the unfolded protein response as a result of cellobiohydrolase production. Finally, we report fermentation of microcrystalline cellulose (Avicel™) to ethanol by CBH-producing S. cerevisiae strains with the addition of beta-glucosidase.ConclusionsGene or protein specific features and compatibility with the host are important for efficient cellobiohydrolase secretion in yeast. The present work demonstrated that production of both CBH1 and CBH2 could be improved to levels where the barrier to CBH sufficiency in the hydrolysis of cellulose was overcome.

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Dendritic cells provide a critical link between innate and adaptive immunity and are essential to prime a naive T-cell response. The transition from immature dendritic cells to mature dendritic cells involves numerous changes in gene expression; however, the role of post-transcriptional changes in this process has been largely ignored. Tristetraprolin is an AU-rich element mRNAbinding protein that has been shown to regulate the stability of a number of cytokines and chemokines of mRNAs. Using TTP immunoprecipitations and Affymetrix GeneChips, we identified 393 messages as putative TTP mRNA targets in human dendritic cells. Gene ontology analysis revealed that ;25% of the identified mRNAs are associated with protein synthesis. We also identified six MHC Class I alleles, five MHC Class II alleles, seven chemokine and chemokine receptor genes, indoleamine 2,3 dioxygenase, and CD86 as putative TTP ligands. Real-time PCR was used to validate the GeneChip data for 15 putative target genes and functional studies performed for six target genes. These data establish that TTP regulates the expression of DUSP1, IDO, SOD2, CD86, and MHC Class I-B and F via the 39-untranslated region of each gene. A novel finding is the demonstration that TTP can interact with and regulate the expression of non-AU-rich element-containing messages. The data implicate TTP as having a broader role in regulating and limiting the immune response than previously suspected.

Tumor necrosis factor alpha (TNF-α) is a critical mediator of inflammation, and its production is tightly regulated, with control points operating at nearly every step of its biosynthesis. We sought to identify uncharacterized TNF-α 3′ untranslated region (3′UTR)-interacting proteins utilizing a novel screen, termed the RNA capture assay. We identified CARHSP1, a cold-shock domain-containing protein. Knockdown of CARHSP1 inhibits TNF-α protein production in lipopolysaccharide (LPS)-stimulated cells and reduces the level of TNF-α mRNA in both resting and LPS-stimulated cells. mRNA stability assays demonstrate that CARHSP1 knockdown decreases TNF-α mRNA stability from a half-life ( t 1/2 ) of 49 min to a t 1/2 of 22 min in LPS-stimulated cells and from a t 1/2 of 29 min to a t 1/2 of 24 min in resting cells. Transfecting CARHSP1 into RAW264.7 cells results in an increase in TNF-α 3′UTR luciferase expression in resting cells and CARHSP1 knockdown LPS-stimulated cells. We examined the functional effect of inhibiting Akt, calcineurin, and protein phosphatase 2A and established that inhibition of Akt or calcineurin but not PP2A inhibits CARHSP1 function. Subcellular analysis establishes CARHSP1 as a cytoplasmic protein localizing to processing bodies and exosomes but not on translating mRNAs. We conclude CARHSP1 is a TNF-α mRNA stability enhancer required for effective TNF-α production, demonstrating the importance of both stabilization and destabilization pathways in regulating the TNF-α mRNA half-life.

Tumor necrosis factor-␣ (TNF-␣) production is regulated by transcriptional and posttranscriptional mechanisms. Lipopolysaccharide activates the NFB pathway increasing TNF-␣ transcription. Lipopolysaccharide also activates the mitogenactivated protein kinase pathways, resulting in stabilization and enhanced translation of the TNF-␣ message. In addition, nuclear export of the TNF-␣ mRNA is a posttranscriptionally regulated process involving the Tpl2-ERK pathway and requiring the presence of the TNF-␣ AU-rich element (ARE). We demonstrate that nuclear export of the TNF-␣ message requires not only the TNF-␣ ARE but also the interaction of the proteins TAP and NxT1, both of which are involved in nucleocytoplasmic transport of mRNA. Through the use of dominant negative ERK1 and ERK2, we establish that control of TNF-␣ mRNA nuclear export operates specifically through ERK1. Finally, we examined the role of two established TNF-␣ ARE-binding proteins, HuR and tristetraprolin, that shuttle between the nucleus and cytoplasm. These data demonstrate that neither tristetraprolin nor HuR is required for TNF-␣ mRNA export. It is unclear at this time if ARE-binding protein(s) directly interact with the TAP-NxT1 complex, if each complex is independently targeted by ERK1, or if only one complex is targeted.