PTM-Switchboard—a database of posttranslational modifications of transcription factors, the mediating enzymes and target genes

Gene transcription is largely regulated by sequence-specific transcription factors (TFs). The TF activity is significantly regulated by its posttranslational modifications (PTMs). TF-PTMs serve as ‘molecular switchboards’ that map multiple upstream signaling events, in response to various environmental perturbations, to the downstream transcriptional events. While many instances of TF-PTMs and their effect on gene regulation have been experimentally determined, a systematic meta-analysis or a quantitative model-based investigation of this process has not been undertaken. A prerequisite to such analyses is a database of known instances of TF-PTMs affecting transcriptional regulation. The PTM-Switchboard database meets this need by cataloging such instances in the model system Saccharomyces cerevisiae. The database stores triplets of genes such that the ability of one gene (TF) to regulate a target gene is dependent on one or more PTMs catalyzed by a third gene (modifying enzyme). The database currently includes a large sample of experimentally characterized instances curated from the literature. In addition to providing a framework for searching and analyzing the data, the database will serve to benchmark computational methods. In the future, the database will be expanded to mammalian organisms, and will also include triplets predicted from computational approaches. The database can be accessed at http://cagr.pcbi.upenn.edu/PTMswitchboard

Genomic Methods for Studying the Post-Translational Regulation of Transcription Factors

The spatiotemporal coordination of gene expression is a fundamental process in cellular biology. Gene expression is regulated, in large part, by sequence-specific transcription factors that bind to DNA regions in the proximity of each target gene. Transcription factor activity and specificity are, in turn, regulated post-translationally by protein-modifying enzymes. High-throughput methods exist to probe specific steps of this process, such as protein-protein and protein-DNA interactions, but few computational tools exist to integrate this information in a principled, model-oriented manner. In this work, I develop several computational tools for studying the functional implications of transcription factor modification. I establish the first publicly accessible database for known and predicted regulatory circuits that encompass modifying enzymes, transcription factors, and transcriptional targets. I also develop a model-based method for integrating heterogeneous genomic and proteomic data for the inference of modification-dependent transcriptional regulatory networks. The model-based method is thoroughly validated as a reliable and accurate computational genomic tool. Additionally, I propose and demonstrate fundamental improvements to computational proteomic methods for identifying modified protein forms. In summary, this work contributes critical methodological advances to the field of regulatory network inference

Coordination of Plastid and Light Signaling Pathways upon Development of Arabidopsis Leaves under Various Photoperiods

Plants synchronize their cellular and physiological functions according to the photoperiod ( the length of the light period) in the cycle of 24 h. Photoperiod adjusts several traits in the plant life cycle, including flowering and senescence in annuals and seasonal growth cessation in perennials. Photoperiodic development is controlled by the coordinated action of photoreceptors and the circadian clock. During the past 10 years, remarkable progress has been made in understanding the molecular mechanism of the circadian clock, especially with regard to the transition of Arabidopsis from the vegetative growth to the reproductive phase. Besides flowering photoperiod also modifies plant photosynthetic structures and traits. Light signals controlling biogenesis of chloroplasts and development of leaf photosynthetic structures are perceived both by photoreceptors and in chloroplasts. In this review, we provide evidence suggesting that the photoperiodic development of Arabidopsis leaves mimics the acclimation of plant to various light intensities. Furthermore, the chloroplast-to-nucleus retrograde signals that adjust acclimation to light intensity are proposed to contribute also to the signaling pathways that control photoperiodic acclimation of leaves.</p

POST-TRANSCRIPTIONAL AND POST-TRANSLATIONAL REGULATION OF LSD1 IN MAMMALIAN BRAIN

Epigenetic mechanisms play important roles in brain development, orchestrating proliferation, differentiation, and morphogenesis. Lysine-Specific Demethylase 1 (LSD1 also known as KDM1A and AOF2) is a histone modifier involved in transcriptional repression, forming a stable core complex with the corepressors corepressor of REST (CoREST) and histone deacetylases (HDAC1/2). Importantly, in the mammalian CNS, neuronal neuroLSD1, an alternative splicing isoform of LSD1 including the microexon E8a, sets alongside LSD1 and is capable of enhancing neurite growth and morphogenesis. Here, we describe that the morphogenic properties of neuronal neuroLSD1 require switching off repressive activity and this negative modulation is mediated in vivo by phosphorylation of the Thr369b residue coded by exon E8a. Three-dimensional crystal structure analysis using a phospho-mimetic mutant (Thr369bAsp), indicate that phosphorylation affects the residues surrounding the exon E8a-coded amino acids, causing a local conformational change. We suggest that phosphorylation, without affecting demethylase activity, causes in neurons CoREST and HDAC1/2 corepressors detachment from LSD1-8a and impairs neuroLSD1 repressive activity. In neurons, Thr369b phosphorylation is required for morphogenic activity, converting neuronal LSD1-8a in a dominant-negative isoform, challenging LSD1-mediated transcriptional repression on target genes. We show that in the hippocampus LSD1 together with HDAC2 are co-repressors of SRF and involved in the transcriptional regulation of egr1 and c-fos. Consistent with neuroLSD1 dominant negative function, neuroLSD1KO mice display a more repressed epigenetic landscape in terms of reduced histone H3K4 methylation and H3 acetylation levels at egr1 and c-fos promoters

Redox Regulation of Protein Kinase B/Akt Function by an Allosteric Disulphide Bond

Most proteins in nature are chemically modified after they are made to control how, when and where they function. One type of chemical modification is the cleavage of disulphide bonds that link pairs of cysteine residues in the polypeptide chain. These cleavable bonds are known as allosteric disulphides. From an analysis of labile disulphide bonds in all protein structures from the Protein Data Bank (PDB), my colleagues and I identified a potential allosteric disulphide in the serine/threonine protein kinase B/Akt; linking cysteine residues 60 and 77 in the N-terminus pleckstrin homology (PH) domain. Akt plays a central role in glucose metabolism, cell survival and angiogenesis and is often hyper-activated in cancer cells. Akt is activated at the plasma membrane via binding to phosphatidylinositol-3,4,5-trisphosphate (PIP3) through its PH domain. Dissociation of Akt from the plasma membrane leads to PH domain-mediated autoinhibition of the kinase by a mechanism that is currently unknown. I hypothesised that the PH domain Cys60–Cys77 disulphide is an allosteric bond that regulates autoinhibition and inactivation of the kinase. To elucidate the role of the Cys60–Cys77 disulphide bond in Akt function, wild-type and reduced (Cys60 and/or Cys77 substituted with Ser) PH domain or full-length Akt mutants were analysed for PIP3 plasma membrane binding, Akt phosphorylation and Akt downstream substrate activation, transformation of fibroblasts, and angiogenesis, survival and development of zebrafish. Ablation of the Cys60–Cys77 disulphide bond did not appreciably affect binding of recombinant PH domain to PIP3, but markedly impaired insulin-stimulated binding of full-length Akt to the plasma membrane of adipocytes. Ablation of the Cys60–Cys77 disulphide bond had mixed effects on insulin-stimulated phosphorylation of Akt in fibroblasts. The Cys60Ser mutant was phosphorylated to the same extent as the wild-type, while the Cys77Ser mutant was poorly phosphorylated. Wild-type but not disulphide mutant Akt induced transformation of fibroblasts, indicating an oncogenic role for oxidised but not reduced Akt. Expression of disulphide mutant Akt in zebrafish increased the induction of angiogenesis and development of embryos but did not affect zebrafish survival. My findings imply that the Cys60–Cys77 disulphide bond in the PH domain of Akt is an allosteric disulphide involved in autoinhibition and functioning of the kinase

Oxidative, inflammatory and vascular factors in Alzheimer's disease

In spite of impressive recent progress, the aetiopathogenesis of Alzheimer’s disease (AD) remains incompletely understood. The distinctive neuropathological features of AD, in particular the plaques and tangles, have been the particular focus of most aetiological theories. It is well accepted that AD is a multifactorial disease, with alterations to a variety of brain structures and cell types, including neurons, glia and the brain vasculature. Studies of risk factors have revealed a diversity of genetic variables that interact with health, diet and lifestyle-related factors in the causation of AD. These factors influence the structure, aggregation and function of a set of proteins that are increasingly the focus of research. The work in this thesis has focused on the pathophysiological aspects of some of these proteins in a number of cellular compartments and brain. Several assays have been established and techniques utilized in the completion of this work, including; differential detergent fractionation of brain tissue, 1D and 2D PAGE, western blotting with chemiluminescence detection, ELISA assays of Abeta 1-40 and 1-42, quantitative ECNI GCMS of o- and m-tyrosine as well as metabolites of the kynurenine pathway, quantitative MALDI-TOF assay of hemorphins and LCMSMS based proteomics, to identify proteins with altered expression levels in AD relative to control brain tissue. A variety of regional differences have been observed in the biochemistry of the AD cortex which are probably the outcome of local response variations to AD pathology. One of the most consistent threads throughout this work has been an apparent resilience of the occipital lobe relative to the other brain regions, as reflected in lower overall levels of oxidative stress and increased levels of proteins associated with metabolic processes, neuronal remodeling and stress reduction