Thirty years of molecular dynamics simulations on posttranslational modifications of proteins

Posttranslational modifications (PTMs) are an integral component to how cells respond to perturbation. While experimental advances have enabled improved PTM identification capabilities, the same throughput for characterizing how structural changes caused by PTMs equate to altered physiological function has not been maintained. In this Perspective, we cover the history of computational modeling and molecular dynamics simulations which have characterized the structural implications of PTMs. We distinguish results from different molecular dynamics studies based upon the timescales simulated and analysis approaches used for PTM characterization. Lastly, we offer insights into how opportunities for modern research efforts on in silico PTM characterization may proceed given current state-of-the-art computing capabilities and methodological advancements.Comment: 64 pages, 11 figure

Mining the TRAF6/p62 interactome for a selective ubiquitination motif

A new approach is described here to predict ubiquitinated substrates of the E3 ubiquitin ligase, TRAF6, which takes into account its interaction with the scaffold protein SQSTM1/p62. A novel TRAF6 ubiquitination motif defined as [–(hydrophobic)–k–(hydrophobic)–x–x–(hydrophobic)– (polar)–(hydrophobic)–(polar)–(hydrophobic)] was identified and used to screen the TRAF6/p62 interactome composed of 155 proteins, that were either TRAF6 or p62 interactors, or a negative dataset, composed of 54 proteins with no known association to either TRAF6 or p62. NRIF (K19), TrkA (K485), TrkB (K811), TrkC (K602 and K815), NTRK2 (K828), NTRK3 (K829) and MBP (K169) were found to possess a perfect match for the amino acid consensus motif for TRAF6/p62 ubiquitination. Subsequent analyses revealed that this motif was biased to the C-terminal regions of the protein (nearly 50% the sites), and had preference for loops (~50%) and helices (~37%) over beta-strands (15% or less). In addition, the motif was observed to be in regions that were highly solvent accessible (nearly 90%). Our findings suggest that specific Lysines may be selected for ubiquitination based upon an embedded code defined by a specific amino acid motif with structural determinants. Collectively, our results reveal an unappreciated role for the scaffold protein in targeting ubiquitination. The findings described herein could be used to aid in identification of other E3/scaffold ubiquitination sites

Analysis of the understudied parts of the phospho-signalome using machine learning methods

Abstract Analysis of the understudied parts of the phospho-signalome using machine learning methods Borgthor Petursson In order to make decisions and respond appropriately to external stimuli, cells rely on an intricate signalling system. One of the most important and best studied components of this signalling system is the phospho-signalling network. Phosphorylation relays information through adding phosphoryl groups onto substrates such as lipids or proteins, which in turn leads to changes in substrate function. Crucial components of this system include kinases, which phosphorylate on the substrate molecule and phosphatases that remove the phosphoryl group from the substrate. To date, even though >100K phosphoproteins have been identified through high throughput experiments, the vast majority of phosphosites are of unknown function, while over a third of kinases have no known substrate (Needham et al., 2019). Furthermore, there is a large study bias in our current knowledge, demonstrated by a disproportionate number of interactions between highly cited kinases and substrates Invergo and Beltrao, 2018. The vast understudied signalling space combined with this study bias make it difficult to understand the general principles underpinning cell signalling regulation and stresses the need to research the phosphoproteomic signalling system in an unbiased manner. In this thesis the central aim is to use data-driven and unbiased approaches to study the human phosphoproteomic signalling network. The first chapter describes a project where I co-developed a machine learning model to predict signed kinase-kinase regulatory circuits based on kinase specificities and high throughput phosphoproteomics and transcriptomic data. The network was validated using independent high throughput data and used to identify novel kinase-kinase regulatory interactions. This project was done in collaboration with Brandon Invergo, a postdoc in Pedro Beltrao’s research group. In the second chapter I expand upon work done in the first chapter. I used various predictors such as: Co-expression, kinase specificities and different variables characterising kinase-substrate potential target phosphosites to predict kinase-substrate relationships and their signs. I then used independent experimental kinase-substrate predictions to validate the predictions and identify high confidence kinase-substrate relationships. I then combined the kinase-substrate predictions with the kinase-kinase regulatory circuits to identify condition-specific signalling networks. To enable easy use of my method and networks and analyses of phosphoproteomics data by non-expert users I also developed the SELPHI2 server, where the user can extract biological insight from their datasets. SELPHI2 presents a substantial improvement upon the SELPHI server, which was developed in 2015 by my supervisor, Evangelia Petsalaki. Thirdly, to study the architecture of human cell signalling networks at a whole-cell level and address the limited predictive power of the current models of cell signalling such as pathways found in KEGG (Kanehisa, 2019), Reactome (Jassal et al., 2020) and WikiPathways (Slenter et al., 2018), the third chapter aims to identify signalling modules from phosphoproteomic data. These data-extracted modules were found to have a greater predictive power for independent data sets in terms of number of significant enrichments. Furthermore, we sought to predict the probability of module co-membership from predictors such as membership within data-driven modules, co-phosphorylation and co-expression. In summary, the work presented here seeks to explore the understudied phospho-signalling systems through system-wide prediction of kinase-substrate regulation and the identification of phospho-signalling modules through data-driven means

Genotype‐phenotype analysis of LMNA‐related diseases predicts phenotype‐selective alterations in lamin phosphorylation

Laminopathies are rare diseases associated with mutations in LMNA, which encodes nuclear lamin A/C. LMNA variants lead to diverse tissue‐specific phenotypes including cardiomyopathy, lipodystrophy, myopathy, neuropathy, progeria, bone/skin disorders, and overlap syndromes. The mechanisms underlying these heterogeneous phenotypes remain poorly understood, although post‐translational modifications, including phosphorylation, are postulated as regulators of lamin function. We catalogued all known lamin A/C human mutations and their associated phenotypes, and systematically examined the putative role of phosphorylation in laminopathies. In silico prediction of specific LMNA mutant‐driven changes to lamin A phosphorylation and protein structure was performed using machine learning methods. Some of the predictions we generated were validated via assessment of ectopically expressed wild‐type and mutant LMNA. Our findings indicate phenotype‐ and mutant‐specific alterations in lamin phosphorylation, and that some changes in phosphorylation may occur independently of predicted changes in lamin protein structure. Therefore, therapeutic targeting of phosphorylation in the context of laminopathies will likely require mutant‐ and kinase‐specific approaches.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/155891/1/fsb220571.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/155891/2/fsb220571_am.pd

Semaphorin Receptor Function in Hedgehog Signal Transduction

Hedgehog signaling is a conserved cell-cell communication pathway that plays essential and diverse roles during embryonic development and adult tissue homeostasis. Many cell surface-associated molecules critically regulate the Hedgehog signaling pathway, coordinating the secretion and distribution of Hedgehog ligands as well as signal reception and downstream signal transduction. Recent evidence suggests that Neuropilins, single-pass transmembrane receptors for Semaphorin and vascular endothelial growth factor ligands, positively regulate Hedgehog signaling. However, the mechanism of Neuropilin action in Hedgehog signal transduction remains unclear. Neuropilins require Plexin co-receptors to transduce Semaphorin signals, although a role for Plexins in HH signaling has not been explored. These questions are particularly interesting given overlapping expression of HH and Semaphorin components in both development and disease. Investigating how Semaphorin receptors function within the Hedgehog signaling cascade will provide important insight into the regulation of this key developmental pathway. Furthermore, therapeutic approaches targeting Semaphorin receptors may be useful to regulate deregulated Hedgehog signaling in cancer and other diseases. In this chapter, I review both the Hedgehog and Semaphorin signal transduction pathways, emphasizing areas of overlap between these two pathways which could be mediated by Neuropilin and Plexin receptors.PHDCell and Developmental BiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttps://deepblue.lib.umich.edu/bitstream/2027.42/147599/1/jpinskey_1.pd

Differences in transcription between free-living and CO_2-activated third-stage larvae of Haemonchus contortus

Background: The disease caused by Haemonchus contortus, a blood-feeding nematode of small ruminants, is of major economic importance worldwide. The infective third-stage larva (L3) of this gastric nematode is enclosed in a cuticle (sheath) and, once ingested with herbage by the host, undergoes an exsheathment process that marks the transition from the free-living (L3) to the parasitic (xL3) stage. This study explored changes in gene transcription associated with this transition and predicted, based on comparative analysis, functional roles for key transcripts in the metabolic pathways linked to larval development. Results: Totals of 101,305 (L3) and 105,553 (xL3) expressed sequence tags (ESTs) were determined using 454 sequencing technology, and then assembled and annotated; the most abundant transcripts encoded transthyretin-like, calcium-binding EF-hand, NAD(P)-binding and nucleotide-binding proteins as well as homologues of Ancylostoma-secreted proteins (ASPs). Using an in silico-subtractive analysis, 560 and 685 sequences were shown to be uniquely represented in the L3 and xL3 stages, respectively; the transcripts encoded ribosomal proteins, collagens and elongation factors (in L3), and mainly peptidases and other enzymes of amino acid catabolism (in xL3). Caenorhabditis elegans orthologues of transcripts that were uniquely transcribed in each L3 and xL3 were predicted to interact with a total of 535 other genes, all of which were involved in embryonic development. Conclusion: The present study indicated that some key transcriptional alterations taking place during the transition from the L3 to the xL3 stage of H. contortus involve genes predicted to be linked to the development of neuronal tissue (L3 and xL3), formation of the cuticle (L3) and digestion of host haemoglobin (xL3). Future efforts using next-generation sequencing and bioinformatic technologies should provide the efficiency and depth of coverage required for the determination of the complete transcriptomes of different developmental stages and/or tissues of H. contortus as well as the genome of this important parasitic nematode. Such advances should lead to a significantly improved understanding of the molecular biology of H. contortus and, from an applied perspective, to novel methods of intervention

AdaSampling for positive-unlabeled and label noise learning with bioinformatics applications

© 2018 IEEE. Class labels are required for supervised learning but may be corrupted or missing in various applications. In binary classification, for example, when only a subset of positive instances is labeled whereas the remaining are unlabeled, positive-unlabeled (PU) learning is required to model from both positive and unlabeled data. Similarly, when class labels are corrupted by mislabeled instances, methods are needed for learning in the presence of class label noise (LN). Here we propose adaptive sampling (AdaSampling), a framework for both PU learning and learning with class LN. By iteratively estimating the class mislabeling probability with an adaptive sampling procedure, the proposed method progressively reduces the risk of selecting mislabeled instances for model training and subsequently constructs highly generalizable models even when a large proportion of mislabeled instances is present in the data. We demonstrate the utilities of proposed methods using simulation and benchmark data, and compare them to alternative approaches that are commonly used for PU learning and/or learning with LN. We then introduce two novel bioinformatics applications where AdaSampling is used to: 1) identify kinase-substrates from mass spectrometry-based phosphoproteomics data and 2) predict transcription factor target genes by integrating various next-generation sequencing data

Comprehending meningioma signaling cascades using multipronged proteomics approaches & targeted validation of potential markers

Meningiomas are one of the most prevalent primary brain tumors. Our study aims to obtain mechanistic insights of meningioma pathobiology using mass spectrometry-based label-free quantitative proteome analysis to identifying druggable targets and perturbed pathways for therapeutic intervention. Label-free based proteomics study was done from peptide samples of 21 patients and 8 non-tumor controls which were followed up with Phosphoproteomics to identify the kinases and phosphorylated components of the perturbed pathways. In silico approaches revealed perturbations in extracellular matrix remodeling and associated cascades. To assess the extent of influence of Integrin and PI3K-Akt pathways, we used an Integrin Linked Kinase inhibitor on patient-derived meningioma cell line and performed a transcriptomic analysis of the components. Furthermore, we designed a Targeted proteomics assay which to the best of our knowledge for very first-time enables identification of peptides from 54 meningioma patients via SRM assay to validate the key proteins emerging from our study. This resulted in the identification of peptides from CLIC1, ES8L2, and AHNK many of which are receptors and kinases and are difficult to be characterized using conventional approaches. Furthermore, we were also able to monitor transitions for proteins like NEK9 and CKAP4 which have been reported to be associated with meningioma pathobiology. We believe, this study can aid in designing peptide-based validation assays for meningioma patients as well as IHC studies for clinical applications

Investigating the effects of Wnt/β-catenin Signalling on Melanoma Cell Metabolism and Mitochondrial Dynamics

Wnts are secreted morphogens that play pivotal roles in embryonic development, stem cell biology and a number of disease states including cancer. Most Wnts signal through a pathway that results in the stabilisation of an intracellular signalling molecule called β-catenin. In melanoma cells, Wnt/β-catenin signalling has been implicated as a key regulator of cellular invasion and metastasis. Using both transient and stable enhancement of Wnt/β-catenin signalling, I have found that mutation–based dysregulation of PI3K signalling dictates the invasive capacity of melanoma cell lines in response to Wnt3a stimulation. I demonstrate by confocal imaging that WNT3A facilitates perinuclear localisation of mitochondria with higher levels of mitochondrial networking and they show significant changes in the proteins of mitochondrial dynamics. Observed changes in mitochondrial fusion and fission proteins including MFN1, MFN2, OPA1 and DNM1L suggest that activation of Wnt/β-catenin signalling can increase mitochondrial fusion and decrease mitochondrial fission in melanoma cells. Cellular metabolic analysis using the Seahorse Bioscience XFe96 Analyzer suggests that Wnt/β-catenin mediated mitochondrial fusion may cause a global down-regulation of cellular energy metabolism in melanoma cells. This is supported by biochemical analysis of citrate synthase and lactate dehydrogenase activity. Knockout of -catenin removes the mitochondrial fusion effect in these cells and reverses any Wnt driven reduction in migration and metabolism suggesting that -catenin is able to control mitochondrial function and dynamics. We show that -catenin binds to the mitochondrial regulatory protein PARK2 in melanoma cells and subsequently blocks the autophagy dependency of melanoma cells. In summary, we demonstrate that activation of Wnt/β-catenin signalling in melanoma cells can lead to reduced cellular metabolism coupled with highly altered mitochondrial dynamics. This novel finding, controlled by -catenin, has potentially wide implications for understanding how certain context-dependent effects of Wnt/β-catenin signalling may be secondary to the regulation of mitochondrial dynamics and global cellular metabolism

T-cadherin signaling in endothelial cells

The cadherin superfamily comprises transmembrane glycoproteins that mediate calciumdependent homophilic cell-cell adhesion. In addition to their pivotal role in mechanical adhesion between cells, cadherins have multiple functions in tissue morphogenesis, cell recognition and sorting, regulated cell motility and the induction and maintenance of tissue/cell polarity. T-cadherin (T-cad) is unusual member of cadherin superfamily; while possessing the Nterminal tandem cadherin repeat structure (EC domain), it lacks both transmembrane and cytoplasmic domains, and is bound to the plasma membrane via a glycosylphosphatidylinositol (GPI) anchor. T-cad has five EC domains and a propeptide in its precursor form (130kDa), which upon cleavage gets converted to mature form (105kDa). A role for T-cad in tissue organization was first demonstrated in the avian embryonic nervous system where the protein influenced the pattern of neural crest cell migration and maintained somite polarity. Many cancer cell lines (e.g. breast, colon, lung, inter alia) display allelic loss of T-cad which is correlated with tumor progression, and hence T-cad has been described as tumor suppressor gene. T-cad is widely expressed in the vasculature and is upregulated in proliferative vascular disorders such as atherosclerosis and restenosis. GPI-anchored T-cad is not localized at adherent junctions but rather distributed globally over the cell surface. T-cad is localized within lipid rafts. In vitro data supports participation of T-cad in many cellular processes such as vascular differentiation, migration and proliferation of smooth muscle cells (SMC) and endothelial cells (EC) and angiogenesis. Adenoviral mediated overexpression of T-cad in EC and SMC results in cell cycle progression and a concomitant promotion of proliferation. T-cad exhibits deadhesive functions upon homophilic ligation with antibody against T-cad or with immobilized recombinant protein. Homophilic ligation of T-cad induces polarization and migration of endothelial cells in a RhoA/ROCK and Rac dependent fashion. T-cad stimulates in-gel outgrowth of endothelial sprouts in 3-dimensional EC-spheroid and heart tissue models of angiogenesis. In vivo, myoblast-mediated delivery of recombinant soluble T-cad to mouse skeletal muscle facilitates VEGF-induced angiogenesis, supporting a physiological role for Tcad as a proangiogenic protein. The present thesis is focused on the regulation of T-cad expression and the signaling mechanisms whereby T-cad affects vascular cell behavior. The conditions of proliferative vascular cell disorders in which T-cad is upregulated are associated with oxidative stress and cell survival/cell death. We observed an elevation in T-cad levels under condition of oxidative stress induced by serum-deprivation and H2O2; this response was normalized upon inclusion of an antioxidant, N-acetyl cysteine or NADPH oxidase inhibitor diphenyleneiodonium, suggesting T- cad induction by reactive oxygen species is NADPH oxidase dependent. Adenoviral mediated overexpression of T-cad in EC facilitated EC survival upon induction of apoptosis by serumdeprivation and various apoptosis-inducing pharmacologicals. Western blot analysis of lysates infected with Empty –adenovirus (E-EC) and T-cad adenovirus (T-cad+-EC) resulted in hyperactivity of anti-apoptotic proteins (Akt and mTOR target p70S6 kinase) and diminished activity of pro-apoptotic proteins (p38MAPK and active caspase3). PI3 kinase inhibitor, wortmannin, and mTOR inhibitor, rapamycin, normalized anti-apoptotic effects of T-cad; these data suggest that upregulation of T-cad in response to oxidative stress functions to protect EC by concomitant induction of PI3K/Akt/mTOR pathway and suppression of p38/caspase3 pathways. Subsequently we focused on identifying downstream targets of Akt and candidate proximal molecular mediators for T-cad. T-cad+-EC exhibited hyperphosphorylation of glycogen synthase kinase β (GSK3β) and concomitant nuclear accumulation of active β-catenin, a transcription factor regulating cell cycle proteins. Using various GSK3β-carrying adenovectors (kinase mutant, dominant negative or wild type) we demonstrated that T-cad induced nuclear accumulation of β-catenin is GSK3β-dependent. siRNA mediated knockdown of T-cad resulted in decreased phosphorylation of Akt and GSK3β and also in reduced nuclear accumulation of β- catenin. T cell factor (TCF) and Leukocyte enhancer factor (LEF) are co-factors for β-catenin; we found that luciferase (reporter) activity of TCF/LEF elements in T-cad+-EC was markedly increased as compared to E-EC. Cyclin D1, one of the important regulators of the cell cycle is a target of β-catenin/TCF/LEF transcription machinery; T-cad+-EC showed increased mRNA and protein levels of cyclin D1 and increased cell proliferation. In searching for molecular mediators of T-cad we considered Integrin linked kinase (ILK) as a putative candidate because both the proteins are located in rafts and ILK acts upstream of Akt and GSK3β in a PI3K-dependent fashion. T-cad+-EC exhibited increased ILK “kinase” activity in a pull-down assay. RNAimediated knockdown of ILK abrogated effects of T-cad on both phosphorylation of Akt and GSK3β and the nuclear accumulation β-catenin, suggesting involvement of ILK in T-cad signaling. Confocal microscopy studies revealed colocalisation of T-cad and ILK in EC which was most prominent within leading edges of migratory cells and at focal adhesions. Anti-ILK immunoprecipitates contained T-cad indicating the existence of T-cad/ILK complexes, and supporting our hypothesis that ILK can function as a proximal molecular mediator for T-cadelicited PI3K/Akt/GSK3β signaling. Transcriptional regulation of T-cad in endothelial cells is poorly understood. To characterize the minimal promoter region of T-cad, we cloned serially deleted fragments of Tcad promoter stretches into luciferase reporter vector (pGL3). Reporter gene analysis exhibited basal levels of luciferase activity within -285bps suggesting existence of minimal promoter region within -285bps from translational start site. Oxidative stress elevated reporter activity of -285 bps construct, suggesting the minimal promoter region might be responsible for the redox sensitivity of T-cad expression. To identify regulatory elements (transcription factors) responsible for T-cad regulation gel shift assays were performed using nuclear extracts of EC and various oligos designed from T-cad promoter region from -1 to -284 bps We identified specific binding of regulatory protein(s) between -156 to -203 bps. Nuclear extracts from serumdeprived EC exhibited increased binding to -156 to -203 bps oligo, suggesting that the identified nucleoprotein complex could function to induce T-cad expression under conditions of oxidative stress. To identify transcription factor(s) within the identified nucleoprotein complex we performed pull-down assay using nuclear extracts of EC, biotinylated -156 to -203 bps and streptavidin agarose beads. Proteins pulled down were subjected to microsequencing by mass spectrometry. Interestingly thioredoxin (TRX1) was found to be present. TRX1 is a 12kDa protein induced by NADPH oxidase under stress and it acts as an antioxidant by facilitating the reduction of other proteins by cysteine thiol-disulfide exchange. Following its translocation to the nucleus TRX1 reduces transcription factors, enabling their binding to regulatory elements. Preliminary data using RNAi-mediated knockdown of TRX1 abrogates oxidative stress-induced upregulation of T-cad in EC, suggesting that NADPH dependent-induction of T-cad involves nuclear translocation of TRX1. These data may explain the observations of upregulation of Tcad on vascular cells in atherosclerotic lesions where oxidative stress plays a key pathogenic role