Investigating homeostatic disruption by constitutive signals during biological ageing

PhD ThesisAgeing and disease can be understood in terms of a loss in biological homeostasis. This will often manifest as a constitutive elevation in the basal levels of biological entities. Examples include chronic inflammation, hormonal imbalances and oxidative stress. The ability of reactive oxygen species (ROS) to cause molecular damage has meant that chronic oxidative stress has been mostly studied from the point of view of being a source of toxicity to the cell. However, the known duality of ROS molecules as both damaging agents and cellular redox signals implies another perspective in the study of sustained oxidative stress. This is a perspective of studying oxidative stress as a constitutive signal within the cell. In this work a computational modelling approach is undertaken to examine how chronic oxidative stress can interfere with signal processing by redox signalling pathways in the cell. A primary outcome of this study is that constitutive signals can give rise to a ‘molecular habituation’ effect that can prime for a gradual loss of biological function. Experimental results obtained highlight the difficulties in testing for this effect in cell lines exposed to oxidative stress. However, further analysis suggests this phenomenon is likely to occur in different signalling pathways exposed to persistent signals and potentially at different levels of biological organisation.Centre for Integrated Research into Musculoskeletal Ageing (CIMA) and through them, Arthritis Research UK and the Medical Research Counc

CRYSTALLOGRAPHIC STUDY OF MUTANT INFLUENZA MATRIX (M1) PROTEIN AND AFFINITY STUDY OF SMALL MOLECULE INHIBITORS TOWARD M1 AND GROWTH FACTORS

Influenza A virus (IAV) is a seasonal infectious agent that could cause a major worldwide catastrophe. Due to its genetic properties, IAV generates new viral particles that resist the body’s immune defense and antiviral drug therapy. This occurs when a host cell has been co-infected by different IAV strains leading to the generation of hybrid viruses. This process is called reassortment. The majority of these new IAVs contain genetically altered hemagglutinin (HA) and neuraminidase (NA). Unfortunately, all current IAV drug therapies target the highly mutated proteins, HA and NA, which is not very useful. Influenza matrix protein 1 (M1) is a structural protein that accounts for a number of critical viral events. It displays a highly conserved sequence compared to other proteins, HA and NA. It is the most abundant structural viral protein. M1 has a key role in viral replication and viral assembly. During all viral steps of cellular invasion, not a single step appears to occur without the contribution of M1 in one way or another. M1 protein forms a layer underneath the lipid bilayer membrane, which contributes to vital integrity and provides an intact viral entity. Upon cellular viral entry, the M1 layer dissociates to release an RNA genome that migrates to the nucleus to utilize the host’s cellular machinery for synthesizing viral proteins. More interestingly, M1 protein exhibits different structural conformations that correlate with its physiological activity. These conformational changes come with a variety of M1-M1 interactions. Crystallographic structures have revealed a tremendous amount of information regarding the M1 mechanism in self-oligomerization and depolymerization. xi Various crystal structures of M1 are available. Our collaborator at the FDA identified an M1 mutant with G88E substitution, which is unable to form an intact M1 layer as wt-M1. In order to understand the role of a single mutated residue, M1 protein (G88E-M1) has been crystallized and its crystal structure was resolved by the groups of Desai and Safo. This crystal forms three monomers in an asymmetric unit. G88E-M1 concentration was 15 mg/mL in a buffer of 55 mM KH2PO4/K2HPO4/H3PO4, 0.2 M NaCl, pH 3.4. The condition of the reservoir was 0.1 M Tris, pH 8.5, 8% PEG (8K). The estimated pH of the crystallization drop was 6.2. In combination with the literature, significant structural manifestations were observed in different pH conditions. Under acidic conditions, this M1 mutant forms a face-to-face dimer, which is stabilized by hydrophobic interactions as well as hydrogen bond interactions. Although the monomers have less hydrophobic interactions at the monomer-monomer interface due to mutation of Gly88 into a polar amino acid, Glu88, it forms a stable dimer. That is because Glu88 generates at the interface a number of hydrogen bond interactions with Tyr100, Lys104 and Arg134. M1 is an attractive a therapeutic target protein. Recently, the Desai’s group has identified through computer-based drug design a promising anti-IAV drug candidate, called PHE that interferes with M1 layer formation leading to defects in cellular production of new viral particles. However, PHE binding affinity to M1 was unknown. Experiment of PHE-M1 binding affinity was performed using surface plasmon resonance with NeutrAvidin gold chip on which biotinylated M1 was immobilized under neutral pH. An affinity constant (Kd) of ~ 1 µM was determined. Likewise, PHE-M1 affinity was studied using microscale thermophoresis (MST), which yielded an affinity constant (Kd) of ~ 1.5 µM. Another project undertaken in this study is to evaluate the affinity of small-molecule inhibitors that bind to signaling proteins. Small-molecules that could interfere with signaling pathways are highly valued in cancer therapy. G2.2, which is a highly sulfated molecule, has previously shown anticancer activity. It seems to be safe, potent, and selective toward colorectal cancer. The mechanism of action of G2.2 mainly triggers multiple important signaling pathways of cancer stem cells including fibroblast growth factor, epidermal growth factor (EGF), bone morphogenetic protein 4, wingless-int, and transforming growth factor-β (TGFβ). EGF and TGFβ were labeled with reactive dye NT-647 on the free thiol group of cysteine residues. MST experiments were performed using phosphate buffer (pH 7.4) and serial dilution of G2.2 (1 mM as the highest concentration). MST binding studies have revealed Kd of 80 µM and 54 µM for EGF and TGFβ, respectively. xii In conclusion, this project has elucidated the crystal structure of G88E-M1 protein with valuable structural manifestations. PHE has high affinity for M1, which was confirmed using two different biophysical techniques. Moreover, G2.2 seems to be a promising drug therapy that targets cancer stem cells through inhibition of growth factors and cytokines associated with their survival and activity. However, G2.2 has low affinity for EGF and TGFβ

The physicist's guide to one of biotechnology's hottest new topics: CRISPR-Cas

Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas) constitute a multi-functional, constantly evolving immune system in bacteria and archaea cells. A heritable, molecular memory is generated of phage, plasmids, or other mobile genetic elements that attempt to attack the cell. This memory is used to recognize and interfere with subsequent invasions from the same genetic elements. This versatile prokaryotic tool has also been used to advance applications in biotechnology. Here we review a large body of CRISPR-Cas research to explore themes of evolution and selection, population dynamics, horizontal gene transfer, specific and cross-reactive interactions, cost and regulation, non-immunological CRISPR functions that boost host cell robustness, as well as applicable mechanisms for efficient and specific genetic engineering. We offer future directions that can be addressed by the physics community. Physical understanding of the CRISPR-Cas system will advance uses in biotechnology, such as developing cell lines and animal models, cell labeling and information storage, combatting antibiotic resistance, and human therapeutics.Comment: 75 pages, 15 figures, Physical Biology (2018

Structural investigation of functional nucleic acids

DNA enzymes, also known as deoxyribozymes, are synthetic single-stranded DNA molecules able to catalyze chemical reactions. There are two main reasons for studying deoxyribozymes: their practical value in various applications, and the understanding of basic properties - such as folding and catalysis - of a biopolymer that is of central importance for life. Compared to ribozymes, the DNA enzymes have a potential value as tools for industrial or therapeutic applications, owing to more cost-effective synthesis and higher stability. The first crystal structure of a deoxyribozyme demonstrated that DNA possesses the intrinsic ability to adopt complex tertiary folds that support catalysis and unveiled the active site of a DNA enzyme in the post-catalytic state (Ponce-Salvatierra, Wawrzyniak-Turek et al. 2016). The second reported crystal structure of the RNA-cleaving deoxyribozyme complements observations about the folds and catalysis of DNA enzymes although the structure was derived with DNA as a substrate mimic of RNA (Liu, Yu et al. 2017). These crystal structures represent a breakthrough in the field, but they are still insufficient to derive a clear mechanistic picture of the specific features of different RNA ligating and RNA cleaving deoxyribozymes. Therefore, ongoing efforts are devoted to structurally investigating additional deoxyribozymes. The new DNA enzymes were evolved to discriminate modified and unmodified RNA substrates and provide attractive tools for studying the natural epitranscriptomic RNA modification N6-methyladenosine (Sednev, Mykhailiuk et al. 2018). In the present study, the goal is to elucidate the structural basis for recognition of the methylated nucleobase by solving the crystal structure of the m6A sensitive RNA-cleaving deoxyribozyme in complex with an uncleavable analog of the RNA substrate, containing either methylated and unmethylated adenosine. Surprisingly, the RNA substrate dissociated from the deoxyribozyme during the crystallization process. Two structures for unmethylated and one of the methylated RNA substrate analog were solved. The next goal is to elucidate the crystal structure of the RNA-ligating deoxyribozyme in the pre-catalytic state of reaction. The previously reported crystal structure of the 9DB1 in the post-catalytic state of reaction could not explain the role of magnesium cations as cofactors for accelerating RNA ligation and properly describe the ligation mechanism. The structural investigation of the 9DB1 in the pre-catalytic state resulted in the ligation of the two RNA substrates during the crystallization process. In the future, other strategies are necessary to solve the questions on substrate recognition and catalytic mechanism of the RNA-cleaving and RNA-ligating deoxyribozymes investigated in this study. The second project deals with synthetic RNA aptamers that were identified by in vitro selection to mimic fluorescent proteins for RNA imaging and the development of biosensors. Several examples of fluorogen-activating RNA aptamers are known, and for some, the crystal structures have recently been solved e.g. of the Spinach, Mango, and Corn aptamers, that bind synthetic analogs of the GFP chromophore (Neubacher and Hennig 2019). The Chili is a new fluorogenic-RNA aptamer that mimics large Stokes shift (LSS) fluorescent proteins (FPs) by inducing highly Stokes‐shifted emission from several new green and red HBI (4-hydroxybenzylidene imidazolinone) derivatives that are non‐fluorescent when free in solution (Steinmetzger, Palanisamy et al. 2019). The new fluorophores are the first variants of fluorogenic aptamer ligands with permanently cationic sidechains that are bound by the RNA in their protonated phenol form, while emission occurs from the phenolate intermediate after excited-state proton transfer. The Chili–DMHBO+ complex is the longest wavelength-emitting (592 nm) and tightest binding (KD=12 nM) RNA fluorophore currently known in the growing family of HBI-binding aptamers. By employing X-ray crystallography, I have elucidated the three-dimensional structure of the Chili fluorophore binding site and revealed the structural basis for the large apparent Stokes shift and the promiscuity of the Chili aptamer to activate red and green-emitting chromophores2022-03-2

Mechanistics of Prothymosin alpha and Nrf2 in the Keap1-Nrf2 mediated Oxidative Stress Response

In an effort to dissect the mechanism of interaction of IDPs, in this thesis we focus on Prothymosin a (ProTa) and nuclear factor erythroid 2-related factor 2 (Nrf2), intrinsically disordered proteins, in the Nrf2 mediated oxidative stress response. Kelch-like ECH-associated protein 1 (Keap1) is an inhibitor of Nrf2, a key transcription factor of cytoprotective genes. Under unstressed conditions, Keap1 interacts with Nrf2 in the cytoplasm via its Kelch domain and suppresses Nrf2 activity. During oxidative stress, Nrf2 is released from Keap1 and is shuttled to the nucleus, where it initiates pro cell survival gene transcription. ProTa also interacts with the Kelch domain and mediates the import of Keap1 into the nucleus to inhibit Nrf2 activity. To gain a molecular basis understanding of the oxidative stress response mechanism, the interaction between ProTa and the Kelch domain of Keap1 has been delineated using nuclear magnetic resonance spectroscopy (NMR), isothermal titration calorimetry (ITC), peptide array analysis, and site-directed mutagenesis. The results revealed that ProTa retains a high level of flexibility, even in the Kelch-bound state. Mutational analysis pinpointed that the region 38NANEENGE45 of ProTa is crucial for the interaction with the Kelch domain, while the flanking residues play relatively minor roles in the affinity of binding. A high yield purification protocol with complete backbone NMR resonance assignment lays the foundation for structural and biophysical studies of the full length-Neh2 domain of the human Nrf2. In this work the full-length Neh2 domain was used to investigate binding to Kelch in the presence of cancer causing somatic mutations. To understand the mechanistic links between Keap1 mutations and cancer pathogenesis, the molecular effects of a series of mutations (G333C, G364C, G379D, G350S, R413L, R415G, A427V, G430C, and G476R on the structural and target recognition properties of Keap1 are investigated. These mutations are found to exert differential effects on the protein stability and target binding. Together with the proposed Hinge-and-Latch mechanism of Nrf2/Keap1 binding, these results provide important insight into the molecular impact of different somatic mutations on Keap1’s function as an Nrf2 repressor

Structure prediction of crystals, surfaces and nanoparticles

We review the current techniques used in the prediction of crystal structures and their surfaces and of the structures of nanoparticles. The main classes of search algorithm and energy function are summarized, and we discuss the growing role of methods based on machine learning. We illustrate the current status of the field with examples taken from metallic, inorganic and organic systems. This article is part of a discussion meeting issue 'Dynamic in situ microscopy relating structure and function'

Computational studies on fatty acid synthesis: from mechanisms to drug design

The first committed steps of the Fatty Acid synthesis pathway involves the de/carboxylation reactions of biotin. By understanding this step, potential novel antimicrobial agents could be discovered. The current tools of drug discovery can only help the research in finding and modifying potential hits. Finding a lead candidate from these programs are often equated to finding a needle in a haystack, which is due to the many assumptions used in molecular docking. The fundamental reaction kinetics can not be described by these techniques and a detailed study of the decarboxylation reaction is investigated using ab initio molecular dynamics. In this particular study, Car-Parrinello molecular dynamics is used and how the biotin model is protonated was found to play an important role in its reaction barrier. Although stable in low acidic solutions, a crucial nitrogen protonation is shown to have the lowest free energy barrier which could play a pivotal role in the enzymatic mechanism. The molecular docking knowledge of potential ligand inhibitors via a low level modeling technique connected to high level quantum mechanical reaction modeling provides a synergistic route in the search for inhibitors