Sulphur metabolism and cellulase gene expression are connected processes in the filamentous fungus Hypocrea jecorina (anamorph Trichoderma reesei)

<p>Abstract</p> <p>Background</p> <p>Sulphur compounds like cysteine, methionine and S-adenosylmethionine are essential for the viability of most cells. Thus many organisms have developed a complex regulatory circuit that governs the expression of enzymes involved in sulphur assimilation and metabolism. In the filamentous fungus <it>Hypocrea jecorina </it>(anamorph <it>Trichoderma reesei</it>) little is known about the participants in this circuit.</p> <p>Results</p> <p>Analyses of proteins binding to the cellulase activating element (CAE) within the promotor of the cellobiohydrolase <it>cbh2 </it>gene led to the identification of a putative E3 ubiquitin ligase protein named LIMPET (LIM1), which is an orthologue of the sulphur regulators SCON-2 of <it>Neurospora crassa </it>and Met30p of <it>Saccharomyces cerevisiae</it>. Transcription of <it>lim1 </it>is specifically up-regulated upon sulphur limitation and responds to cellulase inducing conditions. In addition, light dependent stimulation/shut down of cellulase gene transcription by methionine in the presence of sulphate was observed. Further, <it>lim1 </it>transcriptionally reacts to a switch from constant darkness to constant light and is subject to regulation by the light regulatory protein ENVOY. Thus <it>lim1</it>, despite its function in sulphur metabolite repression, responds both to light as well as sulphur- and carbon source. Upon growth on cellulose, the uptake of sulphate is dependent on the light status and essential for growth in light. Unlike other fungi, growth of <it>H. jecorina </it>is not inhibited by selenate under low sulphur conditions, suggesting altered regulation of sulphur metabolism. Phylogenetic analysis of the five sulphate permeases found in the genome of <it>H. jecorina </it>revealed that the predominantly mycelial sulphate permease is lacking, thus supporting this hypothesis.</p> <p>Conclusion</p> <p>Our data indicate that the significance of the sulphate/methionine-related signal with respect to cellulase gene expression is dependent on the light status and reaches beyond detection of sulphur availability.</p

Light-dependent roles of the G-protein α subunit GNA1 of Hypocrea jecorina (anamorph Trichoderma reesei)

<p>Abstract</p> <p>Background</p> <p>The filamentous ascomycete <it>Hypocrea jecorina </it>(anamorph <it>Trichoderma reesei</it>) is primarily known for its efficient enzymatic machinery that it utilizes to decompose cellulosic substrates. Nevertheless, the nature and transmission of the signals initiating and modulating this machinery are largely unknown. Heterotrimeric G-protein signaling represents one of the best studied signal transduction pathways in fungi.</p> <p>Results</p> <p>Analysis of the regulatory targets of the G-protein α subunit GNA1 in <it>H. jecorina </it>revealed a carbon source and light-dependent role in signal transduction. Deletion of <it>gna1 </it>led to significantly decreased biomass formation in darkness in submersed culture but had only minor effects on morphology and hyphal apical extension rates on solid medium. Cellulase gene transcription was abolished in Δ<it>gna1 </it>on cellulose in light and enhanced in darkness. However, analysis of strains expressing a constitutively activated GNA1 revealed that GNA1 does not transmit the essential inducing signal. Instead, it relates a modulating signal with light-dependent significance, since induction still required the presence of an inducer. We show that regulation of transcription and activity of GNA1 involves a carbon source-dependent feedback cycle. Additionally we found a function of GNA1 in hydrophobin regulation as well as effects on conidiation and tolerance of osmotic and oxidative stress.</p> <p>Conclusion</p> <p>We conclude that GNA1 transmits a signal the physiological relevance of which is dependent on both the carbon source as well as the light status. The widespread consequences of mutations in GNA1 indicate a broad function of this Gα subunit in appropriation of intracellular resources to environmental (especially nutritional) conditions.</p

A selective and orally bioavailable VHL-recruiting PROTAC achieves SMARCA2 degradation in vivo

Targeted protein degradation offers an alternative modality to classical inhibition and holds the promise of addressing previously undruggable targets to provide novel therapeutic options for patients. Heterobifunctional molecules co-recruit a target protein and an E3 ligase, resulting in ubiquitylation and proteosome-dependent degradation of the target. In the clinic, the oral route of administration is the option of choice but has only been achieved so far by CRBN- recruiting bifunctional degrader molecules. We aimed to achieve orally bioavailable molecules that selectively degrade the BAF Chromatin Remodelling complex ATPase SMARCA2 over its closely related paralogue SMARCA4, to allow in vivo evaluation of the synthetic lethality concept of SMARCA2 dependency in SMARCA4-deficient cancers. Here we outline structure- and property-guided approaches that led to orally bioavailable VHL-recruiting degraders. Our tool compound, ACBI2, shows selective degradation of SMARCA2 over SMARCA4 in ex vivo human whole blood assays and in vivo efficacy in SMARCA4-deficient cancer models. This study demonstrates the feasibility for broadening the E3 ligase and physicochemical space that can be utilised for achieving oral efficacy with bifunctional molecules

Homeobox transcription factor muscle segment homeobox 2 (Msx2) correlates with good prognosis in breast cancer patients and induces apoptosis in vitro

Introduction: The homeobox-containing transcription factor muscle segment homeobox 2 (Msx2) plays an important role in mammary gland development. However, the clinical implications of Msx2 expression in breast cancer are unclear. The aims of this study were to investigate the potential clinical value of Msx2 as a breast cancer biomarker and to clarify its functional role in vitro. Methods: Msx2 gene expression was first examined in a well-validated breast cancer transcriptomic dataset of 295 patients. Msx2 protein expression was then evaluated by immunohistochemistry in a tissue microarray (TMA) containing 281 invasive breast tumours. Finally, to assess the functional role of Msx2 in vitro, Msx2 was ectopically expressed in a highly invasive breast tumour cell line (MDA-MB-231) and an immortalised breast cell line (MCF10a), and these cell lines were examined for changes in growth rate, cell death and cell signalling. Results: Examination of Msx2 mRNA expression in a breast cancer transcriptomic dataset demonstrated that increased levels of Msx2 were associated with good prognosis (P = 0.011). Evaluation of Msx2 protein expression on a TMA revealed that Msx2 was detectable in both tumour cell nuclei and cytoplasm. Cytoplasmic Msx2 expression was associated with low grade tumours (P = 0.012) and Ki67 negativity (P = 0.018). Nuclear Msx2 correlated with low-grade tumours (P = 0.015), estrogen receptor positivity (P = 0.038), low Ki67 (P = 0.005) and high cyclin D1 expression (P = 0.037). Increased cytoplasmic Msx2 expression was associated with a prolonged breast cancer-specific survival (P = 0.049), recurrence-free survival (P = 0.029) and overall survival (P = 0.019). Ectopic expression of Msx2 in breast cell lines resulted in radically decreased cell viability mediated by induction of cell death via apoptosis. Further analysis of Msx2-expressing cells revealed increased levels of p21 and phosphorylated extracellular signal-regulated kinase (ERK) and decreased levels of Survivin and the 'split ends' (SPEN) protein family member RBM15. Conclusions: We conclude that increased Msx2 expression results in improved outcome for breast cancer patients, possibly by increasing the likelihood of tumour cell death by apoptosis

ANLN is a prognostic biomarker independent of Ki-67 and essential for cell cycle progression in primary breast cancer

Background: Anillin (ANLN), an actin-binding protein required for cytokinesis, has recently been presented as part of a prognostic marker panel in breast cancer. The objective of the current study was to further explore the prognostic and functional value of ANLN as a single biomarker in breast cancer. Methods: Immunohistochemical assessment of ANLN protein expression was performed in two well characterized breast cancer cohorts (n = 484) with long-term clinical follow-up data and the results were further validated at the mRNA level in a publicly available transcriptomics dataset. The functional relevance of ANLN was investigated in two breast cancer cell lines using RNA interference. Results: High nuclear fraction of ANLN in breast tumor cells was significantly associated with large tumor size, high histological grade, high proliferation rate, hormone receptor negative tumors and poor prognosis in both examined cohorts. Multivariable analysis showed that the association between ANLN and survival was significantly independent of age in cohort I and significantly independent of proliferation, as assessed by Ki-67 expression in tumor cells, age, tumor size, ER and PR status, HER2 status and nodal status in cohort II. Analysis of ANLN mRNA expression confirmed that high expression of ANLN was significantly correlated to poor overall survival in breast cancer patients. Consistent with the role of ANLN during cytokinesis, transient knock-down of ANLN protein expression in breast cancer cell lines resulted in an increase of senescent cells and an accumulation of cells in the G2/M phase of the cell cycle with altered cell morphology including large, poly-nucleated cells. Moreover, ANLN siRNA knockdown also resulted in decreased expression of cyclins D1, A2 and B1. Conclusions: ANLN expression in breast cancer cells plays an important role during cell division and a high fraction of nuclear ANLN expression in tumor cells is correlated to poor prognosis in breast cancer patients, independent of Ki-67, tumor size, hormone receptor status, HER2 status, nodal status and age

Additional file 1: Figure S1. of ANLN is a prognostic biomarker independent of Ki-67 and essential for cell cycle progression in primary breast cancer

Distribution of ANLN nuclear fraction with regard to tamoxifen treatment. Distribution of ANLN nuclear fraction was analyzed with regard to tamoxifen treatment in cohort II, a randomized prospective tamoxifen trial. The distribution of ANLN nuclear fraction was similar in the treatment and control arms with the majority of tumors expressing less than 25% of ANLN nuclear staining. (PDF 25 kb