Multi-site phosphorylation regulates NeuroD4 activity during primary neurogenesis: a conserved mechanism amongst proneural proteins.

BACKGROUND: Basic Helix Loop Helix (bHLH) proneural transcription factors are master regulators of neurogenesis that act at multiple stages in this process. We have previously demonstrated that multi-site phosphorylation of two members of the proneural protein family, Ngn2 and Ascl1, limits their ability to drive neuronal differentiation when cyclin-dependent kinase levels are high, as would be found in rapidly cycling cells. Here we investigate potential phospho-regulation of proneural protein NeuroD4 (also known as Xath3), the Xenopus homologue of Math3/NeuroM, that functions downstream of Ngn2 in the neurogenic cascade. RESULTS: Using the developing Xenopus embryo system, we show that NeuroD4 is expressed and phosphorylated during primary neurogenesis, and this phosphorylation limits its ability to drive neuronal differentiation. Phosphorylation of up to six serine/threonine-proline sites contributes additively to regulation of NeuroD4 proneural activity without altering neuronal subtype specification, and number rather than location of available phospho-sites is the key for limiting NeuroD4 activity. Mechanistically, a phospho-mutant NeuroD4 displays increased protein stability and enhanced chromatin binding relative to wild-type NeuroD4, resulting in transcriptional up-regulation of a range of target genes that further promote neuronal differentiation. CONCLUSIONS: Multi-site phosphorylation on serine/threonine-proline pairs is a widely conserved mechanism of limiting proneural protein activity, where it is the number of phosphorylated sites, rather than their location that determines protein activity. Hence, multi-site phosphorylation is very well suited to allow co-ordination of proneural protein activity with the cellular proline-directed kinase environment.This work was supported by UK Medical Research Council (MRC) Research Grant MR/L021129/1. LH is supported by an MRC Doctoral Training Award.This is the final version of the article. It first appeared from BioMed Central via http://dx.doi.org/10.1186/s13064-015-0044-

Dynamic Modeling of Apoptosis and its Interaction with Cell Growth in Mammalian Cell Culture

In order to optimize productivity of a cell culture it is necessary to understand growth and productivity and couple these features of the culture to extracellular nutrients whose profiles can be manipulated. Also, since growth and productivity are directly affected by cell death mechanisms such as apoptosis, it is imperative to understand these mechanisms. This work describes the development of a differential equation based population balance model of apoptosis in a Chinese Hamster Ovary cell culture producing Anti-RhD monoclonal antibody (mAb). The model was verified in isolation and was then coupled to a metabolic flux model. The model distinguishes between various subpopulations at normal healthy states and at various stages of apoptosis. After finding that glucose and glutamine are not limiting nutrients for this culture, different hypotheses were explored to explain growth arrest. Initially, it was hypothesized that there is some unknown nutrient in either media or serum which is depleted, thus causing growth arrest. Accordingly a first model was developed assuming depletion of this nutrient. Subsequent experiments with different additions of media and serum showed that there is no such nutrient limitation for the media and serum conditions used in most of the experiments. Additional experiments with different culture volumes showed that cell growth was actually controlled by a compound that accumulates and causes pH deviation from its optimal range of operation. Since strong correlations were found between culture volume and growth, it was hypothesized that the compound may be carbon dioxide (CO2), which is inhibitory for growth and may accumulate due to mass transfer limitations. Following this finding, a second model was proposed to take into account the accumulation of this inhibitor, although the specific inhibiting compound could not be exactly identified. This second mathematical model of cell growth was then integrated with a metabolic flux model to provide for a link between intracellular and extracellular species balances, since the latter are the ones to be manipulated for increasing productivity. This final model formulation was then used to describe mAb productivity. The model was also able to reasonably predict all cell subpopulations, nutrients, metabolites and mAb. In an attempt to mitigate the effect of CO2 accumulation and renew the cell growth, culture perfusions were performed. Although this approach resulted in some renewal of growth, the cell concentration progressively decreased after each successive perfusion event. This suggests that irreversible cell damage occurs because of CO2 accumulation. The model was used to describe the perfusion experiments. Agreement between data and model predictions were reasonable. In addition, it was shown that operation with successive perfusions results in a significant increase in productivity and therefore it can be used for further process optimization.1 yea

Cell-Cycle Inhibition by Helicobacter pylori L-Asparaginase

Helicobacter pylori (H. pylori) is a major human pathogen causing chronic gastritis, peptic ulcer, gastric cancer, and mucosa-associated lymphoid tissue lymphoma. One of the mechanisms whereby it induces damage depends on its interference with proliferation of host tissues. We here describe the discovery of a novel bacterial factor able to inhibit the cell-cycle of exposed cells, both of gastric and non-gastric origin. An integrated approach was adopted to isolate and characterise the molecule from the bacterial culture filtrate produced in a protein-free medium: size-exclusion chromatography, non-reducing gel electrophoresis, mass spectrometry, mutant analysis, recombinant protein expression and enzymatic assays. L-asparaginase was identified as the factor responsible for cell-cycle inhibition of fibroblasts and gastric cell lines. Its effect on cell-cycle was confirmed by inhibitors, a knockout strain and the action of recombinant L-asparaginase on cell lines. Interference with cell-cycle in vitro depended on cell genotype and was related to the expression levels of the concurrent enzyme asparagine synthetase. Bacterial subcellular distribution of L-asparaginase was also analysed along with its immunogenicity. H. pylori L-asparaginase is a novel antigen that functions as a cell-cycle inhibitor of fibroblasts and gastric cell lines. We give evidence supporting a role in the pathogenesis of H. pylori-related diseases and discuss its potential diagnostic application

Investigation of the intrinsic mechanism of drug resistance in multiple myeloma

The focus of this thesis was to evaluate the mechanisms whereby myeloma cells develop intrinsic resistance with a focus on resistance in the context of bortezomib treatment. The aims of this thesis were to examine multidrug resistance pumps as a mechanism of resistance in MM, to investigate the contribution of p53 signalling perturbations in resistance mechanism in MM, to study the AMPK pathway as an alternative target to overcome MM resistance and finally to characterise myeloma resistance to bortezomib treatment using 2D-DIGE analysis. Focussing on bortezomib resistance models, we found that that overexpression of P-gp attenuates bortezomib activity. Bortezomib is a P-gp substrate and a combination of P-gp inhibitor and bortezomib is able to overcome resistance. Bortezomib is also able to downregulate the expression and function of P-gp. Our findings therefore suggest that combination of a P-gp inhibitor and bortezomib in P-gp positive myeloma would be a reasonable treatment combination to extend use of the drug. We have shown that p53 apoptotic signalling pathways can be accentuated when bortezomib is combined with a Mdm2 inhibitor. In p53 WT cells, nutlin-3 in combination with bortezomib generates additive toxicity in MM cells but is highly synergistic in epithelial models and p53-mutated cell lines. This synergy persists in the presence of BMSCs. This observation has implications more so in epithelial cancers and p53 mutated cancers where single agent bortezomib activity is mild. We have also shown that bortezomib-treated patients who had high expression of nutlin-3-suppressed genes had significantly shorter progression-free (p=0.001, log-rank test) and overall survival (p=0.002, log-rank test) compared to those with low expression levels. AMPK activation is promising as an anticancer pathway and may also be a chemoprevention target. Metformin and AICAR, which activate this pathway, both have demonstrated useful preclinical anticancer properties and have a good therapeutic index in patients. We explored mechanism of cell death and showed that AICAR was able to activate the apoptotic pathway. These agents also synergise with glycolysis inhibitors to further increase cytotoxicity in cancer cells. Identification of proteins whose expression is altered in differing states of sensitivity and resistance provides candidates for better understanding of resistance mechanisms so we also investigated bortezomib resistance in cellular models using proteomic techniques and isolated and identified several novel proteins which may play a role in this phenomenon. Our findings are mechanistically consistent since two of the identified proteins Hsp70 and caspase-3 are known in the literature to be affected by bortezomib treatment

Doctor of Philosophy

dissertationMicroRNAs (miRNAs) are small, noncoding RNA regulators of gene expression that have many important functions within the immune system. While various critical immunologic functions for specific miRNAs have been uncovered, less is known about the roles of these molecules within the intestinal and adipose microenvironments. Recently, many studies have described the complex intestinal interface, which contains host immune cells and epithelial cells interacting with the microbiota in a manner that promotes symbiosis. Further, there is emerging evidence that miRNAs have evolved to fine tune host gene expression networks and signaling pathways that modulate cellular physiology in the intestinal tract. Here, I first review the present knowledge of the influence miRNAs have on both immune and epithelial cell biology in the mammalian intestines and the impact this has on the microbiota. Next, my work demonstrates the role of one specific miRNA, microRNA-146a (miR-146a), in intestinal homeostasis and disease. miR-146a has previously been shown to have anti-inflammatory function within the immune system and is required to downregulate inflammation in mammals. I find that this miRNA constrains multiple parameters of intestinal immunity and increases murine colitis severity. Further, because miR-146a regulates intestinal homeostasis and populations of the gut microbiota, I hypothesized that this molecule may also be important in regulating immunometabolism in a model of diet-induced obesity. I demonstrate that miR-146a is required to prevent obesity, diabetes, and metabolic disease during high-fat diet. miR-146a was found to regulate multiple networks of gene expression in adipose tissue macrophages both during dietary homeostasis and metabolic disease, and these miR-146a-dependent pathways converge upon inflammation and cell metabolism. Altogether, miR-146a constrains immune responses both within the intestine and adipose tissue, and can both prevent or promote disease, depending on disease and context. This institutes the importance of studying miRNA functions within multiple tissues types and disease contexts, as novel roles for these molecules may be established in various situations