Selected Works in Bioinformatics

This book consists of nine chapters covering a variety of bioinformatics subjects, ranging from database resources for protein allergens, unravelling genetic determinants of complex disorders, characterization and prediction of regulatory motifs, computational methods for identifying the best classifiers and key disease genes in large-scale transcriptomic and proteomic experiments, functional characterization of inherently unfolded proteins/regions, protein interaction networks and flexible protein-protein docking. The computational algorithms are in general presented in a way that is accessible to advanced undergraduate students, graduate students and researchers in molecular biology and genetics. The book should also serve as stepping stones for mathematicians, biostatisticians, and computational scientists to cross their academic boundaries into the dynamic and ever-expanding field of bioinformatics

Neue aktivitäts-basierte Sonden zur funktionalen Analyse von Deubiquitinierungsenzymen

Ubiquitin ist ein kleines aus 76 Aminosäuren bestehendes Protein. Die Ubiquitinierung ist ein reversibler Prozess, wobei das Ubiquitin durch Deubiquitinierungs Enzyme entfernt wird. Demnach, sind DUBs ein Analogon zu Phosphotasen und funktionell ebenso wichtig wie Ligasen oder Kinasen und repräsentieren somit ein attraktives therapeutisches Ziel. Bedauerlicherweise ist der Mechanismus und die Funktion von DUBs nur wenig verstanden. Vielversprechend sind die Aktivität basierten Sonden (activity based probes -ABPs) dessen Aufbau ein modifiziertes Ubiquitin beinhaltet, welches am C-terminus eine reaktive Gruppe trägt. Bei der Anwendung dieser ABPs im funktionsbasierten Proteomiks Ansatz interagieren diese mit aktiven DUBs und binden kovalent an dessen katalytischen Aminosäurenrest. Ziel dieser Arbeit war es die Reaktivität der Sonden weiterhin zu verbessern und die fundamentale Frage der DUB Spezifität anhand neue entwickelter, spezifischer Sonden zu klären. Zusammengefasst, unter Verwendung des verbesserten Syntheseprotokolls ist es gelungen fünf neuartige ABPs (HAUb-VEE, HAUb-MVEE, HAUb-VA, HAUb-VFEA und HAUb-VF3EA) mit unterschiedlichen aktivitätsabhängigen DUB Bindungsprofilen zu synthetisieren. Das aktivitätsbasierte Profiling unter Verwendung des neuen hoch reaktiven HAUb-VFEA ermöglichte das detektieren von 63 aktiven DUBs in fünf verschiedenen Zelllinien (A549, EL-4, MCF-7, Jurkat E6-1 and HeLa S3). Eine weitere Intention war es eine neue Klasse von ABPs zu implementieren die es ermöglicht die DUBs Präferenz hinsichtilich der Ubiquitin- Verknüpfung in einem aktivitäts-basierten Assay zu profilen. Das Resultat war eine neue Art von reaktiven Ubiquitin-Peptid basierten Sonden (Ubiquitin Isopeptide Activity Based Probe (UIPP)). Zusammengefasst, die in dieser Arbeit neu entwickelten ABPs und UIPPs repräsentieren ein ideales Tool zur Anreicherung von aktiven DUBs und für die Charaktereisierung dessen Spezifität bezüglich Ubiquitinverknüpfungen.Ubiquitin is a 76-amino-acid protein. The Ubiquitination is a reversible process, which is enabled by deubiquitinating enzymes (DUBs) that remove the ubiquitin. Therefore, DUBs have an analogous role to phosphatases and are functionally as important as ligases or kinases, thus representing an attractive therapeutic target. Unfortunately, the mechanism and function of DUBs are poorly understood. Useful tools termed activity-based probes (ABPs) are implemented by conversion of ubiquitin into a suicide substrate, which possesses a C-terminal reactive group. By utilizing the ABPs in a functional proteomics approach, these probes interact and bind covalently to the catalytic residue of active DUBs. The aim of this study was the improvement of the ubiquitin ABP by generating specific probes to clarify DUB linkage and substrate specificity issues. To conclude, using the improved synthesis protocol, it was possible to design and synthesize a set of five new ABPs (HAUb-VEE, HAUb-MVEE, HAUb-VA, HAUb-VFEA and HAUb-VF3EA), showing distinct activity-based DUB labeling profiles. The activity-based protein profiling using the most reactive probe (HAUb-VFEA) designed in this study revealed an overall of 67 active DUBs in five different cell lines (A549, EL-4, MCF-7, Jurkat E6-1 and HeLa S3). The next intention was to develop a new class of ABPs for characterization of the Ub-linkage preference of DUBs within the activity profiling assay. The result was novel peptide branched Ubiquitin Isopeptide Activity Based Probes (UIPPs). In conclusion, the ABPs and UIPPs created in this study were found to be enabling tools to capture active DUBs and study their selectivity towards ubiquitin-linkage specificity

Discovery and Development of Small Molecule Probes for the SUMO protease SENP1, a Novel Target for Advanced Prostate Cancer Therapy

Androgen signaling through the androgen receptor (AR) is essential for normal growth and function of the prostate gland. In prostate cancer (PC), androgens provide the main proliferative drive for the disease, making androgen-deprivation one of the primary therapeutic strategies. Although initially effective, such treatments select for tumor cells that are able to sustain proliferation in a reduced androgen environment.  This allows for the emergence of castration resistant PC, an incurable disease where both the AR transcriptional program is subverted and cellular senescence is evaded. SUMOylation is a post-translational modification that regulates both of these processes. SUMOylation of AR inhibits both basal and androgen-stimulated transcription in a promoter context manner and enhanced global SUMO modification induces prostate cell senescence. Advanced PC cells evade these mechanisms at least in part through the upregulation of SENP1, a SUMO-specific cysteine protease that reverses SUMOylation. In addition, AR is a direct activator of the SENP1 gene, creating a self-reinforcing loop that promotes and sustains its own activity and PC progression. This dissertation is aimed at the discovery and development of small molecule inhibitors of SENP1 as the basis for novel prostate cancer therapeutics. Using a robust FRET-based assay, we defined the kinetic properties of SENP1 and its closest paralog SENP2. This analysis revealed significant product inhibition and a differential sensitivity to ionic strength. Using this assay, an extensive high-throughput screening campaign led to the identification of two structurally distinct inhibitor classes. Characterization of these compounds indicates that they display significant selectivity towards SENP1 relative to SENP2 and that they act in both a reversible and competitive manner. Furthermore, these compounds inhibit native full-length SENP1 acting on endogenous SUMOylated substrates. Notably, both groups of compounds are known to display activity as purinergic receptor antagonists. The remarkable parallel pharmacology to P2X1 receptors led to our discovery that ATP, the endogenous P2X ligand, is a SENP1 selective inhibitor. We have thus revealed a novel nucleotide mediated regulation of SENP1. Using a combination of mutagenesis, biochemical assays, and fluorescence and NMR spectroscopy, we have characterized the binding of inhibitors and identified key enzyme residues involved in the interactions as well as residues responsible for SENP isoform selectivity. These findings reveal that SENP1 harbors a unique binding site for nucleotides that can be targeted by small molecules. This knowledge can guide novel strategies for further inhibitor development for evaluation of the therapeutic efficacy of SENP1 inhibitors in advanced prostate cancer models.PHDChemical BiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttps://deepblue.lib.umich.edu/bitstream/2027.42/140973/1/carriemn_1.pd

Combining experiments and simulation to characterise structural and dynamical properties of intrinsically disordered peptides and regions

Intrinsically disordered proteins and regions play important roles in the regulation of protein dynamics and protein-protein interactions. In this thesis two IDPs, both of which have been implicated in neurodegenerative diseases, are explored using fully atomistic molecular dynamics simulations. The first is the N-terminal fragment of the huntingtin protein, which controls the protein’s localisation and function in vivo. The second is the disordered pro domain of the proNGF dimer, which antagonises NGF in the brain. Huntingtin is the causative agent of Huntington’s disease, which is a progres- sive neurodegenerative disease, characterised by CAG repeats in the first exon of Huntingtin, which are translated into a polyglutamine (polyQ) tract, responsible for protein aggregation and subsequent neuron death. Huntingtins poly-Q tract is preceded by a 17-residue regulatory fragment (Htt1-17), which is intrinsically dis- ordered in aqueous environments but forms an amphipathic helix in the presence of TFE or DPC micelles. Htt1-17 regulates localisation and function of the full-length protein and is subject to multiple post-translational modifications in the cell. I used molecular dynamics simulations with a novel enhanced sampling method, to study the effect of phosphorylation, phosphomimetic substitutions and acetylation on the secondary structure of Htt1-19. ProNGF is the precursor to the neurotrophin NGF, and is involved in apoptotic signalling in the brain. A disturbed proNGF:NGF was shown to lead to Alzheimer’s disease. A high-resolution structure of the pro domain has been missing so far. I modelled the proNGF dimer by combining experimental data with long MD simu- lations

Progress in Analytical Methods for the Characterization, Quality and Safety of the Beehive Products

This reprint presents some recent results from applying original analytical methods to the most renowned hive matrices. Particular consideration was given to methods devoted to the attribution of the origin of honey and propolis, but also studies dealing with the chemical characterization of honey and other hive matrices are here reported. Attention has also been paid to the use of optimized methods of elemental analysis in several hive products for quality and safety purposes, but also for environmental biomonitoring. The treatment of the data was often achieved through multivariate analysis methods, which made it possible to obtain reliable classifications of honeys and propolis according to their botanic or geographical origin

Spectroscopic Probes of Protein Structure, Dynamics, Hydration and Electrostatics

The structure, dynamics and function of a protein are intimately controlled by a large number of intermolecular and intramolecular interactions. Thus, achieving a quantitative and molecular-level understanding of how proteins fold and function requires experimental techniques that can â??senseâ?? and differentiate various molecular forces and, in many cases, in a site-specific manner. To that end, the focus of this thesis work is to develop non-natural amino acid-based infrared and fluorescence probes that can be used to assess the local hydration status and electrostatic environment of proteins. First, we expand the utility of a well-known site-specific spectroscopic probe, p-cyano-phenylalanine (PheCN), by showing that (1) its fluorescence is sensitive to the presence of various anions and can thus be used to measure the heterogeneity of the protein conformation, (2) when placed at the N-terminal end of a peptide this non-natural amino acid can be used as a pH sensor for a wide variety of applications, and (3) its nitrile stretching vibration is a microscopic reporter of how a co-solvent, such as urea and trimethylamine N-oxide, modulates the protein-water interactions. Secondly, we demonstrate that the ester carbonyl stretching vibration of the non-natural amino acids, L-aspartic acid 4-methyl ester and L-glutamic acid 5-methyl ester, can be used to site-specifically quantify the electrostatic environment of proteins as its vibrational frequency correlates linearly with the local electrostatic field. Application of this infrared probe to amyloids allows us to gain new insight into their structure and dynamics

Biophysical study of the aggregation of the androgen receptor protein in spinal bulbar muscular atrophy

[eng] Spinal bulbar muscular atrophy (SBMA) is a member of the polyglutamine (polyQ) expansion diseases family; the most famous of which is Huntington disease (HD). SBMA is caused by the expansion of the coding region for the polyQ tract in the exon 1 of androgen receptor (AR), which represents the N-terminal intrinsically disordered transactivation domain (NTD). AR is the nuclear receptor sensible to testosterone and aggregates of this polyQ-expanded protein are observed in the motor neurons of SBMA patients. The aggregation mechanism of polyQ proteins depends both on the length of the tract and on the chemical properties of the regions flanking it, that can increase or decrease the rate of aggregation depending on their secondary structure. In order to study the structure of the polyQ tract in AR and the mechanism by which this protein forms aggregates, we developed recombinant proteins designed over the N-terminal fragment of cleavage of a caspase (Caspase 3) associated to the onset of the toxicity in SBMA. We also developed a set of biophysical tools for rendering these aggregation-prone proteins monomeric and to monitor their evolution from the monomer level to the fibril. These methodologically challenging endeavor allowed us to study the secondary structure of this intrinsically disordered protein as a monomer and then to monitor what regions are important in its oligomerization and aggregation. Bulk biophysical experiments and NMR indicated that the polyQ tract of AR is in α-helical conformation, unlike other polyQ tracts described in literature, and we demonstrated that this conformation is caused by the nucleating effect of an N-terminal flanking sequence of four Leu residues (54LLLL58). We also showed that the helical conformation of this tract prevents the polyQ to acquire the ß-sheet conformation and to progress as a fibril, as a deletion mutant of the 54LLLL58 motif aggregates and forms fibrils faster than the wild type. By measuring the aggregation rates of three different AR recombinant proteins with progressively higher polyQ length (4Q, 25Q and 51Q) emerged that the polyQ is not the only region responsible for oligomerization and we identified by NMR that a second region, N- terminal and far apart from the polyQ is responsible of the early oligomerization. By analysis of the chemical shifts in different NMR experiments we obtained that this region (23FQNLF27) however not entirely helical, is prone to interact and acquire secondary structure. Furthermore, this sequence is known to bind to the ligand binding domain (LBD) of AR in an interaction critical for its dimerization and subsequent translocation into the nucleus, which is called N/C interaction. The crystal structure of this complex shows 23FQNLF27 in α-helical conformation when bound to LBD. We then investigated what amino-acids were important in the interaction stabilizing the intereaction of 23FQNLF27. By mutational analysis and measurements of aggregation rates we demonstrated that the helicity of this region is important for the aggregation and mutations that increase the helicity also an increase the aggregation propensity of the protein. We also identified that the residues responsible for the contact are the Gln in position 2, 28 and 36 which form a ‘spine’ of polar residues in register along the α-helix. This polar side of the helix is not the one in contact with LBD during the N/C interaction and it is possible that the two events occur in parallel. In the complex, we characterized the early oligomerization of AR in the aggregation process associated to SBMA with the perspective to provide valuable information for the development of drugs for this diseases that has currently no treatment.[cat] Les malalties neurodegeneratives són una de les malediccions de la civilització moderna i es troben estretament lligades a l’augment de l’esperança de vida de la població mundial. La majoria d’aquestes malalties estan associades a la deposició de material proteic, altrament conegut com a fibres amiloides, a les neurones i el cervell en general. Les fibres amiloides són conjunts supramoleculars lineals, composats per proteïnes disposades en fulla beta, que mostren una alta rigidesa i estabilitat termodinàmica. Exemples famosos de proteïnes amiloides són la beta amiloide (Aβ), associada a la malaltia d’Alzheimer, i l’α-­‐sinucleïna i la proteïna tau, més estretament lligades a la malaltia de Parkinson. Una altra família de desordres neurodegeneratius associats a la deposició de proteïnes és la de les malalties poliglutamines (poliQ). Aquesta família està formada per nou patologies, entre les que es troben sis atàxies espinocerebrals diferents (de les sigles en anglès, SCA 1, 2, 3, 6, 7, 17), la atròfia dentatorubral-­‐pallidoluysian (de les sigles en anglès, DRPLA) i la atròfia muscular espinal bulbar (de les sigles en anglès, SBMA), històricament la primera en ser descrita. Totes elles són hereditàries, dominants i es manifesten en edat avançada. D’altra banda, totes elles estan associades a l’adquisició de neurotoxicitat degut a l’agregació de la proteïna causant de la malaltia, que s’acumula progressivament a les neurones amb el temps. La mutació responsable de la malaltia és una expansió genètica a la regió polimòrfica de l’ADN que és comuna a totes le proteïnes associades en aquests enfermetats. Aquesta regió polimòrfica és un conjunt de repeticions CAG que codifiquen l’aminoàcid glutamina a nivell d’expressió de proteïna, és per això que es coneix com a tram de poliglutamines. Aquest tram pot tenir diverses longituds, però l’efecte tòxic només té lloc quan es supera un determinat límit d’allargada. Aquest límit fluctua entre 30 i 40 repeticions i varia de malaltia a malaltia, però en tots els casos el número de repeticions influencia la severitat i l’edat en la que s’inicia la malaltia. La raó que explica aquesta inestabilitat genètica resulta de la propensitat de les seqüències d’ADN altament repetitives (com ara els hairpins) que en determina el slippage de la cadena principal durant la replicació de l’ADN. Les expansions més llargues són causades per la reiteració d’aquesta petita mutació i s’ha observat una reducció progressiva de l’estabilitat genètica amb l’increment del número de repeticions, que en última instància determina un avançament temporal i empitjorament dels símptomes. Considerant l’estreta relació entre la presència d’agregats en els teixits dels pacients malalts i l’estadiatge de la malaltia, és fonamental entendre les propietats biofísiques dels trams de poliQ, com aquestes seqüències determinen l’agregació de la proteïna i el tipus d’estructura que presenten els agregats

Analysis of binary interactions between OTUB1 and E2 ubiquitin-conjugating enzymes

Post-translational modification of proteins via ubiquitination is mediated by three enzyme families; E1 activating enzymes, E2 conjugating enzymes and E3 ligases, all of which work in a hierarchical manner to facilitate different forms of protein ubiquitin ranging from mono-ubiquitination to the formation of different forms of ubiquitin chains (Ciechanover et al., 2000). Deubiquitinating enzymes (DUBs) act to remove ubiquitin from modified substrates. Apart from the classic interactions within the E1-E2-E3 enzymatic cascade, an unusual non-hierarchical interaction has been observed between some E2 enzymes and a DUB called OTUB1 (Markson et al., 2009). This observation raises interesting questions concerning the molecular mechanisms and specificity of this unusual E2:DUB partnership. In this study, systematic yeast two-hybrid (Y2H) screens were performed between all human E2 and DUB proteins to analyse the extent of E2:DUB interactions. Putative partnerships between OTUB1 and UBE2D1, UBE2D2, UBE2D3, UBE2D4, UBE2E1, UBE2E2, UBE2E3 and UBE2N were identified. These data correlate well with data from other independent studies, including HTP Y2H screens (Markson et al., 2009) and mass spectrometry (Sowa et al., 2009). An N-terminal truncated form of OTUB1 (ΔNOTUB1) was generated by removing a predicted 39aa N-terminal disordered region (Edelmann et al., 2009). Using this construct in combination with wild type (WT) OTUB1, complementary biophysical studies were performed to investigate the formation of complexes with UBE2D2 and UBE2E1 as these represented the strongest interactions detected in preliminary Y2H studies. Gel filtration chromatography showed convincing complex formation for both ΔNOTUB1:UBE2D2 and ΔNOTUB1:UBE2E1 in 1:1 stoichiometry. The thermodynamic profile of each complex was measured by ITC suggested a stronger affinity between ΔNOTUB1:UBE2D2 (Kd 3.89 µM) than observed for the ΔNOTUB1:UBE2E1 complex (Kd 16.55 µM). The n values for both complexes are 1.16±0.06 sites and 0.92±0.03 sites respectively, confirming that both complexes adopt a 1:1 stoichiometry. Observing the UBE2D2 (1H15N)-HSQC NMR spectral changes that occurred upon addition of unlabelled ΔNOTUB1 allowed the identification of potential residues of contact between the two proteins. From this study, we were able to predict that the 1st α-helix, the L1 loop of the 3rd and 4th β-sheet, the L2 loop connecting the 4th β-strand and the H2 α-helix within UBE2D2 were likely to be the binding surfaces for OTUB1. Point mutants corresponding to predicted contact residues in UBE2D2 were generated and tested in Y2H studies to determine their role in facilitating the formation of both E2:OTUB1 and E2:E3-RING complexes. This data suggests that in some, but not all cases, OTUB1 and E3-RINGs bind competitively to the same interface on E2 proteins. Preliminary immunofluorescence studies show that partner proteins predominantly co-localise in the cytoplasm, except UBE2E1 which is predominantly nuclear. Data from this study allowed us to propose a model of how OTUB1:UBE2D2 complex may forms and functions. Significantly, many of these predictions have now been verified by independent structural studies and subsequent live cell microscopy studies in our lab

PRINCIPLES OF COMPLEX POLYUBIQUITIN SIGNALING, THE STRUCTURAL BASIS FOR UBIQUITIN-UBISTATIN INTERACTIONS, AND NOVEL ASSAYS FOR THE CHARACTERIZATION OF DEUBIQUITINASES

Ubiquitination is the most versatile and is certainly one of the most difficult post-translation modifications to understand in eukaryotic life. In the process of ubiquitination the C-terminus of ubiquitin (Ub), a small 8.65 kDa protein is covalently attached to εNH2 groups of lysine side chains on target proteins. Once attached, additional Ubs can be added to the original Ub at eight unique linkage sites (M1, K6, K11, K27, K29, K33, K48, or K63) to form polyUb chains. This internal Ub-Ub linkage dictates the structural conformation of the polyUb chain, which in turn governs the receptors that can recognize a given chain. PolyUb chains were thought to be homogeneously linked until very recently when mixed linkage polyUb chains were detected on several cellular pathways. This observation implied that instead of having just eight distinct polyUb signals, there were now potentially quadrillions of unique chains. The results presented within represent the first in depth studies of mixed linkage polyUb chains, focusing on the structural impact of linkage mixing. For mixed K48 and K63 linked chains the findings support that their individual linkage properties are preserved regardless of linkage mixing. However, simulations for mixed linkage chains containing different linkages imply that many novel polyUb signals are possible. The ubiquitin-proteasome pathway is the primary mechanism to degrade short lived proteins in the cell and has also emerged as a top therapeutic target. Ubistatins, a class of small molecules bring about the same effects as existing proteasome inhibitor drugs by directly binding the polyUb chain. However, virtually nothing is known about the structural properties for any ubistatin/Ub complex. Here is provided the first structure of a ubistatin/Ub complex along with data that overwhelmingly validates the structure. Other important factors regarding the ubistatin/Ub interaction including the stoichiometry and dual hydrophobic / electrostatic binding mechanism are also uncovered. Proteomic analysis of polyUb conjugates has been limited to determining which linkage types are present. A novel method for K63 linked polyUb conjugates, which can measure consecutive K63 linkages is described here. This method allows the proteomics community to gain unprecedented information on cellular pathways utilizing K63 linkages