New Methods to Study Proline-Rich Disordered Regions and Their Structural Ensembles in Protein Signaling Pathways

Ph.DDOCTOR OF PHILOSOPH

The role of PNPLA3 in the development and progression of chronic liver injury

Chronic liver disease is now of great international concern due to rapidly increasing morbidity and mortality associated with the disease. There is significant evidence that carriage of the patatin-like phospholipase domain containing protein 3 (PNPLA3) risk allele, rs738409:G, plays a key role in determining risk for the development of chronic liver disease from a variety of causes. rs738409 is a common single nucleotide polymorphism which results in substitution of an isoleucine residue for methionine at position 148 of PNPLA3 (Ile148Met; I148M). However, the physiological role of PNPLA3 and the functionality of its I148M variant, are currently largely unknown. The central aim of this thesis was to investigate the biological function of PNPLA3 and elucidate the complex role that the I148M variant plays in the development and progression of liver disease. Investigation into the primary sequence of PNPLA3 was undertaken to characterise the protein and inform latter experimental design. Phylogenetic investigation revealed human PNPLA5 to share the highest homology with PNPLA3, and revealed more distant, previously undescribed relationships with the bacterial protein ExoU. A combination of expression trials and subsequent in vitro investigation into the behaviour of PNPLA3 was attempted. Despite attempts with numerous constructs, PNPLA3 remained unstable when expressed using an E. coli expression system and was not able to be produced in sufficient quantity to facilitate structural analysis. In the latter half of the thesis, both variants of PNPLA3 are investigated through in silico structural modelling and subsequent molecular dynamic simulation. The first simulation of full-length PNPLA3 is reported, revealing a more detailed description of the domain architecture of PNPLA3 and the local impact of the I148M variation. A novel disease mechanism is proposed, in which methionine at residue 148 effects the conformational stability of the PNPLA3 active site, resulting in a loss of lipase activity

A Multiobjective Approach Applied to the Protein Structure Prediction Problem

Interest in discovering a methodology for solving the Protein Structure Prediction problem extends into many fields of study including biochemistry, medicine, biology, and numerous engineering and science disciplines. Experimental approaches, such as, x-ray crystallographic studies or solution Nuclear Magnetic Resonance Spectroscopy, to mathematical modeling, such as minimum energy models are used to solve this problem. Recently, Evolutionary Algorithm studies at the Air Force Institute of Technology include the following: Simple Genetic Algorithm (GA), messy GA, fast messy GA, and Linkage Learning GA, as approaches for potential protein energy minimization. Prepackaged software like GENOCOP, GENESIS, and mGA are in use to facilitate experimentation of these techniques. In addition to this software, a parallelized version of the fmGA, the so-called parallel fast messy GA, is found to be good at finding semi-optimal answers in reasonable wall clock time. The aim of this work is to apply a Multiobjective approach to solving this problem using a modified fast messy GA. By dividing the CHARMm energy model into separate objectives, it should be possible to find structural configurations of a protein that yield lower energy values and ultimately more correct conformations

Reclassification of serine / threonine phosphorylation sites with +1 proline (S/T-P) sites as a distinct eukaryotic post-translational modification class

+1 proline is the most frequently found sequence motif around serine/threonine phosphorylation sites. While these proline-directed serine/threonine (S/T-P) phosphorylation sites accounts for about 1/3 of known human phosphorylation sites, it is less frequently studied than other types of phosphorylation sites: partly because of its unclear sequence consensus and reduced probability of generating attestable phenotypes when modified. In this study, we propose to establish this S/T-P phosphorylation sites as a distinctive subclass of protein phosphorylation by its own. We investigated sequence preferences, biophysical fingerprints & ontological associations of known human phosphorylation sites and found there is a significant difference between S/T-P sites and other serine / threonine phosphorylation sites, which would lead to difference consequences after phosphorylation. Also, we found 'horizontal’ – sequence averaged – information plays a major role in distinguishing S/T-P sites from non-phosphorylated counterparts, while other serine/threonine phosphorylation sites and tyrosine phosphorylation sites strongly rely on ‘vertical’ – sequence specific – information to differentiate those from non-phosphorylated counterparts. These behaviors were specifically associated with +1 proline: using proline residues on other locations or other residues on +1 site as criteria were not able to reproduce these pre-stated differences. Furthermore, we identified not only +1 proline is evolutionarily conserved across phosphoprotein orthologs, but also S/T-P sites were slowly enriched within mammalian level. Interestingly, +1 proline is more likely to be in the reconstructed ancestral sequences than actually phosphorylated serine/threonine residues, which might imply about the possible origin and evolutionary advantage of S/T-P phosphorylation. These results would not only provide an insight about this ‘neglected subclass’ of phosphorylation sites, but would also suggest this particular PTM is co-evolved with eukaryotic proteome to carry out roles associated with biological complexity

The development of peptide-based inhibitors for Tau aggregation as a potential therapeutic for Alzheimer’s disease

There are currently approximately 50 million individuals worldwide with dementia resulting in predicted global societal costs of up to US $1 trillion. Approximately 60-70% of these individuals have Alzheimer’s disease, which results in a chronic and insidious decline in memory. One of the main proteins that misfolds in this disease is Tau protein, which aggregates into toxic oligomers and neurofibrillary tangles. It is these aggregates, which cause damage to the brain resulting in dementia. As a result, it is imperative to be able to prevent or suppress the pathogenic aggregation of this protein, so the onset of dementia is halted or delayed, improving quality of life. Certain amino acid sequences in Tau such as VQIINK and VQIVYK play important roles in aggregation. Targeting these sequences can potentially prevent aggregation. This project aims to produce effective peptide inhibitors based on the human Tau peptide sequences VQIINK and VQIVYK, to specifically target pathogenic Tau aggregation. Using molecular docking softrware ‘ICM-Pro’ the potential binding locations of a variety of peptide candidates were computationally investigated to determine which will be most successful in a laboratory setting. Recombinant TauΔ1-250 was incubated in the prescense of heparin and subsequently aggregated to display highly ordered parallel, in-register β-strand structures; including fibrils and paired helical filaments presenting the characteristic twist under transmission electron microscope. This aggregation was achieved using of 20μM Tau at pH 7.4 in the presence of 20mM Tris buffer, 1mM DTT, 5μM Heparin, and 15uM ThT and incubated at 37 °C for 48 hours. The first generation of peptides AG01, AG02, AG02, AG02R4, AG02R5, AGR502, AG02PR5, AG02R6, AG02R9, AG02TAT, AG02ΔI, AG02ΔV inhibited approximately 50% of Tau aggregation determined by Thioflavin-T (ThT) fluorescence assay. The next generation, AG03 was slightly more effective, however when retroinverted (RI-AG03) inhibited over 90% of Tau aggregation, confirmed by Thioflavin-T fluorescence assay, transmission electron microscopy, circular dichroism and Congo red birefringence. RI-AG03 was determined to be stable in cells at therapeutic concentrations. After determining stability of RI-AG03 using SDS-PAGE, thermal circular dichroism and mass spectrometry, it was tested in vivo in rough eye Drosophila model. Results suggested that RI-AG03 partially rescued the rough eye phenotype in this model. This research demonstrates that retro-inverted peptide RI-AG03 is a potent inhibitor of Tau aggregation and can be further developed as a novel therapeutic for Tauopathies like Alzhimer’s Disease

Self-assembly of intrinsically disordered peptide amphiphiles

Intrinsically disordered peptide amphiphiles (IDPAs) are a novel class of molecules with great potentialif incorporated into nanocarriers. IDPAs combine hydrocarbon chains that originate fromnatural lipids and polypeptide chains composed of sequences that do not fold into a static structurebut remain intrinsically disordered, fluctuating between various conformations. This amphiphilicstructure makes IDPA self-assemble into mesophases or aggregates in solution. The possible sequencevariations are vast, and their influence on the self-assembly structures has hardly beenexplored. In my Ph.D., I studied how to decode the impact on sequence composition and conformationand provide the basis for future applications and implementations of IDPAs. I worked onfour different IDPAs that differ in their amino acid sequences. We used SAXS, TEM, and turbiditymeasurements to analyze the nanoscopic self-assembled structures. We showed that permutationsand the sequence’s charge pattern remarkably alter the headgroup’s conformation. Consequently,pH-dependent phase transitions between spherical, cylindrical micelles and condensed hexagonalphases are related to the sequence variation. We demonstrated that even a single amino acid mutationcould tune the phase transition. Last, we showed that our system should phase transition forIDPAs that can be enzymatically cleaved. Altogether, we demonstrated that IDPAs enable manyapplications for lipid nanoparticle systems to add multiple functionalities by incorporating IDPAswith desired properties. For most experiments, I used small-angle X-ray scattering (SAXS). Toenable measurement under multiple conditions with ONE single probe, we developed a 3D printedsample chamber made of cyclic olefin copolymers (COC), including COC X-ray windows providingultra-low SAXS background. The chamber’s design enables both in-situ buffer exchangeand optical transmission spectroscopy. It is thus suitable for many more applications. The designconsists of a membrane insert for in-situ dialysis of the 100 μl sample volume against a reservoir.We demonstrated the chamber used by measuring our IDPA system at various pHs and polymersystems as a function of salt concentration. Our chamber’s design makes in-situ measurements atin-house sources possible. This design is proved useful and is in regular use in our lab at LMU forpH-dependent experiments. In my Ph.D. project, I studied the self-assembly of tunable IDPA andtheir properties under various environmental conditions. We developed a 3D printed chamber forin-situ dialysis to measure these conditions on one probe

Conformational design of cyclic peptides

Due to their potential importance as drug molecules as well as other applications methods to design small cyclic peptides with a rigid well-defined conformation are useful. Computational methods to predict the conformation of cyclic peptides to identify those with a well-defined conformation allow for screening of potential sequences prior to expending the resources required to make the peptides. An alternative method to produce conformationally restricted cyclic peptides would be to include structural elements that prevent the peptide changing conformation. This thesis focuses on designing well-structured cyclic peptides, either through the use of computational techniques which were developed in order to help predict the structure of cyclic peptides or, through the introduction into the cyclic peptide structure of a β-turn mimic. Chapter 1 covers current methods for the synthesis of cyclic peptides as well as methods for predicting the conformations a cyclic peptide is likely to form. β-turns, including β-turn mimics are also discussed. Chapter 2 focuses on the modification of bias-exchange metadynamics (BE META) simulations, which can be used to predict the conformation of cyclic peptides, to also predict the occurrence of cis proline within proline-containing cyclic peptides. An additional replica was added into the BEMETA to allow for cis/trans isomerisation. A series of cyclic hexapeptides was synthesised and the cis to trans ratio of the proline within the peptides obtained by NMR. These results were then used to evaluate the computational predictions. It was found faster convergence in the simulations was reached using the additional replica, but the forcefield could not always accurately model the energy difference between the cis and trans proline states. Chapter 3 presents the results of the analysis of β-turns found within a database. Cyclic hexapeptides are frequently observed to form a structure composed of two overlapping β-turns. It was hypothesised information on the β-turns extracted from the database could therefore be used to help design cyclic hexapeptides. Two peptides were designed based on the database analysis. The structure of the peptides, determined by NMR, show the amino acids in the peptides occupy the predicted positions in the major conformations. Chapter 4 explores the introduction of restraints into BE-META simulations used to predict the conformations of cyclic peptides. The restrained simulations are used to infer the lowest energy structures a cyclic hexapeptide can adopt based on the backbone conformation of the peptide when a specific β-turn type is present within the peptide. The inclusion of chiral amino acids at specific positions within the cyclic peptide structure is seen to alter the most stable conformation. In Chapter 5 a Random Forest machine learning algorithm was trained to predict the β-turn type a sequence will form based on the β-turns extracted from the database in Chapter 3. The Random Forest in combination with the lowest energy conformations determined by the restrained simulations in Chapter 4 is used to predict the conformations cyclic hexapeptides will adopt. A well-structured cyclic peptide containing the biologically active RGD motif was designed using the methods developed in this chapter. The use of the Random Forest allowed for fast filtering of potential sequences to identify those predicted to form only one major conformation. Chapter 6 focuses on the incorporation of a β-turn mimic, which forms through a chemical ligation reaction, into cyclic peptides. Conditions were found to incorporate the β-turn mimic into the cyclic peptides which allow for the cyclisation of the peptide and formation of the β-turn mimic in a single step. The reaction has a broad sequence tolerance and was found to be suitable for peptide macrocycles of varying size. Chapter 7 aims to introduce additional functionality to the β-turn mimic used in Chapter 6. The structure of the β-turn mimic was modified to include a fluorescent naphthalene group. A peptide containing the modified β-turn mimic was synthesised and circular dichroism (CD) analysis shows the modified β-turn mimic retains the β-turn structure. The fluorescent properties of the modified β-turn mimic were also analysed and found to be very similar to that of tryptophan. Chapter 8 makes use of the β-turn mimic in order to design a cyclic WW Domain mimic which retains the ability to bind proline-rich ligands. Two β-strands from a WW Domain structure were cyclised using the methods developed in Chapter 6. Molecular dynamics simulations show the β-strand structure is retained in the cyclised peptide. Binding studies also demonstrate the cyclised WW domain retains the ability to bind to a ligand known to bind to the wildtype WW Domain