Conserved and essential transcription factors for cellulase gene expression in ascomycete fungi.

Rational engineering of filamentous fungi for improved cellulase production is hampered by our incomplete knowledge of transcriptional regulatory networks. We therefore used the model filamentous fungus Neurospora crassa to search for uncharacterized transcription factors associated with cellulose deconstruction. A screen of a N. crassa transcription factor deletion collection identified two uncharacterized zinc binuclear cluster transcription factors (clr-1 and clr-2) that were required for growth and enzymatic activity on cellulose, but were not required for growth or hemicellulase activity on xylan. Transcriptional profiling with next-generation sequencing methods refined our understanding of the N. crassa transcriptional response to cellulose and demonstrated that clr-1 and clr-2 were required for the bulk of that response, including induction of all major cellulase and some major hemicellulase genes. Functional CLR-1 was necessary for expression of clr-2 and efficient cellobiose utilization. Phylogenetic analyses showed that CLR-1 and CLR-2 are conserved in the genomes of most filamentous ascomycete fungi capable of degrading cellulose. In Aspergillus nidulans, a strain carrying a deletion of the clr-2 homolog (clrB) failed to induce cellulase gene expression and lacked cellulolytic activity on Avicel. Further manipulation of this control system in industrial production strains may significantly improve yields of cellulases for cellulosic biofuel production.

Multisite phosphorylation of the Saccharomyces cerevisiae filamentous growth regulator Tec1 is required for its recognition by the E3 ubiquitin ligase adaptor Cdc4 and its subsequent destruction in vivo.

In Saccharomyces cerevisiae, the pheromone-induced ubiquitylation and degradation of the filamentation pathway-specific activator, Tec1, suppresses cross talk between the mating and filamentous growth mitogen-activated protein kinase (MAPK) pathways. The mating pathway MAPK, Fus3, phosphorylates Tec1, resulting in its recognition by the SCF (for Skp1, Cullin, F-box containing) E3 ubiquitin ligase complex, leading to its proteolysis. Previously, it was found that Tec1 destruction requires phosphorylation on threonine 273 (T273). T273 is embedded in the sequence LLpTP, which is identical to the canonical binding site for Cdc4, a conserved F-box substrate adaptor for the SCF complex. However, recent work on both Cdc4 and the human Cdc4 ortholog Fbw7 has shown that a second substrate phosphorylation can be required for optimal Cdc4 binding in vitro. We report here that high-affinity binding of recombinant Cdc4 to Tec1 phosphopeptides requires phosphorylation of not only T273 but also a second site, T276. Significantly, both phospho-sites on Tec1 and a conserved basic pocket on Cdc4 are critical for Tec1 proteolysis in response to pheromone treatment of cells, establishing a role for two-phosphate recognition by yeast Cdc4 in substrate targeting in vivo.

Hog1 mitogen-activated protein kinase (MAPK) interrupts signal transduction between the Kss1 MAPK and the Tec1 transcription factor to maintain pathway specificity.

In Saccharomyces cerevisiae, the mating, filamentous growth (FG), and high-osmolarity glycerol (HOG) mitogen-activated protein kinase (MAPK) signaling pathways share components and yet mediate distinct responses to different extracellular signals. Cross talk is suppressed between the mating and FG pathways because mating signaling induces the destruction of the FG transcription factor Tec1. We show here that HOG pathway activation results in phosphorylation of the FG MAPK, Kss1, and the MAPKK, Ste7. However, FG transcription is not activated because HOG signaling prevents the activation of Tec1. In contrast to the mating pathway, we find that the mechanism involves the inhibition of DNA binding by Tec1 rather than its destruction. We also find that nuclear accumulation of Tec1 is not affected by HOG signaling. Inhibition by Hog1 is apparently indirect since it does not require any of the consensus S/TP MAPK phosphorylation sites on Tec1, its DNA-binding partner Ste12, or the associated regulators Dig1 or Dig2. It also does not require the consensus MAPK sites of the Ste11 activator Ste50, in contrast to a recent proposal for a role for negative feedback in specificity. Our results demonstrate that HOG signaling interrupts the FG pathway signal transduction between the phosphorylation of Kss1 and the activation of DNA binding by Tec1.

Optimizing TiO2-based phosphopeptide enrichment for automated multidimensional liquid chromatography coupled to tandem mass spectrometry.

An automated online multidimensional liquid chromatography system coupled to ESI-based tandem mass spectrometry was used to assess the effectiveness of TiO2 in the enrichment of phosphopeptides from tryptic digests of protein mixtures. By monitoring the enrichment of phosphopeptides, an optimized set of loading, wash, and elution conditions were realized for TiO2. A comparison of TiO2 with other resins used for phosphopeptide enrichment, Fe(III)-IMAC and ZrO2, was also carried out using tryptic digests of both simple and moderately complex protein mixtures; where TiO2 was shown to be superior in performance.

Remodeling of the extracellular matrix through overexpression of collagen VI contributes to cisplatin resistance in ovarian cancer cells.

The mechanisms of drug resistance in cancer are poorly understood. Serial analysis of gene expression (SAGE) profiling of cisplatin-resistant and sensitive cells revealed many differentially expressed genes. Remarkably, many ECM genes were elevated in cisplatin-resistant cells. COL6A3 was one of the most highly upregulated genes, and cultivation of cisplatin-sensitive cells in the presence of collagen VI protein promoted resistance in vitro. Staining of ovarian tumors with collagen VI antibodies confirmed collagen VI expression in vivo and suggested reorganization of the extracellular matrix in the vicinity of the tumor. Furthermore, the presence of collagen VI correlated with tumor grade, an ovarian cancer prognostic factor. These results suggest that tumor cells may directly remodel their microenvironment to increase their survival in the presence of chemotherapeutic drugs.