Amyotrophic lateral sclerosis (ALS) is a debilitating, incurable, neurodegenerative disease characterized by degradation of motor neurons leading to paralysis and ultimately death in ~3-5 years. Approximately 10% of ALS cases have a dominant inheritance pattern, termed familial ALS (fALS). Mutations in the gene encoding the dimeric superoxide scavenger Cu, Zn superoxide dismutase (SOD), were found to be associated with ~20% of fALS cases. Over 110 predominantly missense SOD mutations lead to fALS by an unknown mechanism; however, it is thought that mutant SOD acquires a toxic gain of function. Mice as well as human post mortem studies have identified mutant SOD-rich aggregates in affected neurons, leading to the hypothesis that mutations in SOD increase the tendency of the protein to form toxic aggregates. SOD has a complex maturation process whereby the protein is synthesized in an apo or demetalated state, followed by formation of an intramolecular disulfide bond and binding of Zn2+ and Cu2+. Each of these post-translational modifications increases the stability of the protein. SOD has been shown to aggregate more readily from destabilized immature states, including the apo state both with and without the disulfide bond, highlighting the importance of these states. Thermal unfolding monitored by differential scanning calorimetry (DSC) and chemical denaturation monitored by optical spectroscopy were used to elucidate the folding mechanism and stability of both the apo SOD disulfide-intact and disulfide-reduced states. Chemically and structurally diverse fALS-associated mutants were investigated to gain insights into why mutant SODs may be more prone to misfold and ultimately aggregate. The mutations were introduced into a pseudo wild-type (PWT) background lacking free cysteines, resulting in highly reversible unfolding amenable to accurate thermodynamic analysis. Similarly to what was previously described for fully metallated (holo) SODs, chemical denaturation of the apo disulfide-intact SODs is well described by a 3-state dimer mechanism with native dimer, monomeric intermediate and unfolded monomer populated at equilibrium. Although removal of metals has a relatively small effect on the stability of the dimer interface, the stability of the monomer intermediate is dramatically reduced. Thermal unfolding of some disulfide-intact apo SOD mutants as well as PWT is well described by a 2-state dimer mechanism, while others unfold via a 3-state mechanism similar to chemical denaturation. All but one of the studied disulfide-intact apo mutations are destabilizing as evidenced by reductions in ΔG of unfolding. Additionally, several mutants show an increased tendency to aggregate in thermal unfolding studies through increased ratios of van’t Hoff to calorimetric enthalpy (HvH/ Hcal ). The effects of the mutations on dimer interface stability in the apo disulfide-intact form were further investigated by isothermal titration calorimetry (ITC) which provided a quantitative measure of the dissociation constant of the dimer (Kd). ITC results revealed that disulfide-intact apo SOD mutants generally have increased Kd values and hence favor dimer dissociation to the less stable monomer which has been proposed to be a precursor to toxic aggregate formation. Reduction of the disulfide bond in apo SOD leads to marked destabilization of the dimer interface, and both thermal unfolding and chemical denaturation of PWT and mutants are well described by a 2-state monomer unfolding mechanism. Most mutations destabilize the disulfide-reduced apo SOD to such an extent that the population of unfolded monomer under physiological conditions exceeds 50%. The disulfide-reduced apo mutants show increased tendency to aggregate relative to PWT in DSC experiments through increased HvH /Hcal, low or negative change in heat capacity of unfolding and/or decreased unfolding reversibility. Further evidence of enhanced aggregation tendency of disulfide-reduced apo mutants was derived from analytical ultracentrifugation sedimentation equilibrium experiments that revealed the presence of weakly associated aggregates. Overall, the results presented here provide novel insights into SOD maturation and the possible impact of stability on aggregation.

<p>Cells thrive on sustaining order and balance to maintain proper homeostatic functions. However, the primary machinery involved in protein quality control including chaperones, ubiquitin proteasome system, and autophagy all decline in function and expression with age. Failures in protein quality control lead to enhanced protein misfolding and aggregation. Efficient elimination of misfolded proteins by the proteasome system is critical for cellular proteostasis. However, inadequate proteasome capacity can lead to aberrant aggregation of misfolded proteins and inclusion body formation, which is a hallmark of numerous neurodegenerative diseases. Due to the post-mitotic nature of neurons, they are more susceptible to the collapse in proteostasis correlated with age. </p><p> </p><p>Here, we propose a cell based model of aggresome clearance using a reversible proteasome inhibitor, MG132, to identify the precise molecular machinery involved in proper processing of inclusions. It is known that once misfolded proteins are aggregated, the proteasome system can no longer degrade them. Furthermore, the continuous accumulation of aggregates often leads to aggresome formation, which results in amalgamated inclusion bodies that are simply too large for autophagosomes to engulf and degrade. Although, studies have shown that aggresomes can eventually be cleared by autophagy, the molecular mechanisms underlying this process remain unclear. </p><p>Our research reveals that regardless of impaired proteolysis, proteasomes can still stimulate autophagy-dependent aggresome clearance by producing unanchored lysine (K)63-linked ubiquitin chains via the deubiquitinating enzyme Poh1. Unanchored ubiquitin chains activate ubiquitin-binding histone deacetylase 6, which mediates actin-dependent disassembly of aggresomes. This crucial de-aggregation of aggresomes allows autophagosomes to efficiently engulf and eliminate the protein aggregates. Interestingly, the canonical function of Poh1 involves the cleavage of ubiquitin chains en bloc from proteasomal substrates prior to their degradation by the 20S core, which requires intact 26S proteasomes. In contrast, here we present evidence that during aggresome clearance, 20S proteasomes dissociate from protein aggregates, while Poh1 and selective subunits of 19S proteasomes are retained as an efficient K63 deubiquitinating enzyme complex. The dissociation of 20S proteasome components requires the molecular chaperone Hsp90. Hsp90 inhibition suppresses 26S proteasome remodeling, unanchored ubiquitin chain production, and aggresome clearance. Ultimately, we hope to apply these molecular markers of inclusion body processing to identify the underlying lesion in aggregate prone neurodegenerative disease.</p> / Dissertation

TAR DNA binding protein of 43 kDa (TDP-43) has been implicated in the pathogenesis of amyotrophic lateral sclerosis and frontotemporal lobar degeneration. Pathologically misfolded and aggregated forms of TDP-43 are found in cytoplasmic inclusion bodies of affected neurons in these diseases. The mechanism by which TDP-43 misfolding causes disease is not well understood. We postulate that the aggregation process plays a major role in pathogenesis, and we hypothesize that oligonucleotide ligands of TDP-43 can stabilize the native functional state of the protein and ameliorate aggregation of this aggregation-prone protein. Using recombinant TDP-43 we were able to examine the extent to which various oligonucleotide molecules affects its aggregation in vitro. We have found that certain natural sequence and de novo designed oligonucleotides bind TDP-43 and prevent its natural tendency to aggregate. The clinical and therapeutic implications of these findings are discussed.

TAR DNA binding protein of 43 kDa (TDP-43) has been implicated in the pathogenesis of amyotrophic lateral sclerosis and frontotemporal lobar degeneration. Pathologically misfolded and aggregated forms of TDP-43 are found in cytoplasmic inclusion bodies of affected neurons in these diseases. The mechanism by which TDP-43 misfolding causes disease is not well understood. We postulate that the aggregation process plays a major role in pathogenesis, and we hypothesize that oligonucleotide ligands of TDP-43 can stabilize the native functional state of the protein and ameliorate aggregation of this aggregation-prone protein. Using recombinant TDP-43 we were able to examine the extent to which various oligonucleotide molecules affects its aggregation in vitro. We have found that certain natural sequence and de novo designed oligonucleotides bind TDP-43 and prevent its natural tendency to aggregate. The clinical and therapeutic implications of these findings are discussed.

In this work we report a new mechanism for ageing, where passive aggregation and stochastic segregation of protein aggregates can switch cells from a non-ageing to an ageing state. This switch is activated by the increase in the total amount of protein aggregates. We established a damage reporter system by labeling Hsp104, a chaperone which binds protein aggregates, with GFP. By observing that the accumulation of Hsp104 labeled aggregates correlated with the majority of cell death in the population, and that cells which are born with a high level of aggregates are more likely to die, we validated protein aggregation as being an ageing factor in S. pombe. To identify the mechanism of damage segregation, we monitored nucleation, fusion and partition of aggregates at division. We established that aggregates are present in the cytoplasm fraction which is not occupied by vacuoles and lipid vesicles, and that they are not actively transported by the cytoskeleton. Protein aggregates were not distributed in a biased manner at division. Their position in the cytoplasm dictates to which cell they will be partitioned at division, as confirmed by studies using an asymmetrically dividing mutant, pom1Δ, in which the larger cells inherited more aggregates. This shows that aggregate segregation in S.pombe is a stochastic process. Stochastic distribution contributes to a constant dilution of damage and the maintenance of a non-ageing division, where the total levels of damage in the individuals are on average maintained constant. Together with Steven Lade and Thilo Gross, from the MPI-PKS, we designed a model in which passive aggregation and stochastic segregation reproduced our experimental results. Surprisingly, when cells are exposed to heat stress and the total levels of protein aggregates increase, aggregates are unequally segregated, i.e., ageing is turned on. The switch in segregation results from increased fusion due to a higher number of aggregates, which generates large single aggregates, which are retained by one of the cells at division. Our model reproduced the heat stress condition, showing that fusion is an essential parameter to generate clean cells quickly after the levels of damage increase. Fission yeast cells can therefore switch between non-ageing and ageing like division depending on the total amount of damage at birth. To clarify if other cellular components could be ageing factors in S. pombe, we tested if the inheritance of the old cell wall at the cell pole was associated with an increase in division time, similarly to what occurs in E. coli. We also tested if the inheritance of the new centrosome-analog, the SPB, which is segregated to the ageing mother cell in S. cerevisiae, resulted in an increase in division time. We did not find evidence for ageing associated with these structures. Finally we determined that the feature of slow division was not a transmissible trait, i.e., daughters of slow diving cells divided faster than their mothers. Another ageing hallmark, the cumulative increase in division time with the total number of divisions before death, was not present in S.pombe. Our combined results from damage segregation and pedigree analysis show that stochastic segregation of damage is a viable strategy to avoid ageing. Passive aggregation in the presence of a high number of aggregates can switch on ageing, representing an alternative to active segregation mechanisms and to the existence of pre-defined ageing lineages, as shown for other organisms. Finally, our results show that ageing is not ubiquous to life, and that it can be a facultative strategy to cope with stress.

In this thesis we present computer modelling studies that were implemented to investigate protein behavior in various environments causing their folding, unfolding and aggregation. Applications related to two important proteins - insulin and apolipoprotein C-II (ApoC-II) are presented. The use of atomistic simulation methodologies based on empirical force fields has enhanced our understanding of many physical processes governing protein structure and dynamics. However, the force fields used in classical modelling studies are often designed for a particular class of proteins and rely on continuous improvement and validation by comparison of simulations with experimental data. In Chapter 4 we present a comprehensive comparison of five popular force fields for simulation of insulin. The effect of each force field on the conformational evolution and structural properties of the protein is analysed in detail and compared with available experimental data. A fundamental phenomenon in nature is the ability of proteins to fold ab initio to their functional native conformation, also known as their biologically active state. Due to the heterogeneity and dimensionality of the systems involved, it is necessary to employ methodologies capable of accelerating rare events, specifically, configurational changes that involve the crossing of large free energy barriers. In Chapter 5, using the recently developed method BE-META we were able to identify the structural transitions and possible folding pathways of insulin. Another interesting phenomenon is the misfolding of proteins causing their aggregation, that may lead to formation of either amorphous compounds or structures of elongated-unbranched morphology known as amyloid fibrils. The deposition of amyloid fibrils in the human body may cause many debilitating diseases such as Alzheimer's and variant Creutzfeldt-Jakob diseases, thus making this field of research important and urgent. The human plasma protein apoC-II serves important roles in lipid transport, and it has been shown to form amyloid-like aggregates in solution. We have performed computational studies to investigate the effect of mutations, such as Met oxidation and the residue substitutions to hydrophobic Val and hydrophilic Gln, on dynamics of apoC-II(60-70) peptide. The conformation features relevant to the amyloidogenic propensities of the peptide were identified and presented in Chapter 6. The involvement of lipids at the various stages of development of amyloid diseases is becoming more evident in recent research efforts. In particular, micellar and sub-micellar concentrations have showed to have different effect on fibril growth and kinetics of native apoC-II and derived peptides. In Chapter 7 we investigated the influences of phospholipids at various concentrations on the structure of apoC-II(60-70) using MD and umbrella sampling methods. The molecular mechanisms of lipid effects on the peptide conformation and dynamics were identified. In Chapter 8 preliminary results on the structural stability of pre-formed oligomeric composites of apoC-II(60-70) peptide of different sizes and arrangements were also presented. The effects of mutation (oxidised Met, Met60Val and Met60Gln) on the most stable cluster was also investigated. To conclude, several ideas for continuation of research in the protein folding and aggregation field are discussed in the Future Work section of this thesis.

Epigenetics is the study of heritable biological variation not related to changes in DNA sequence. Epigenetic processes are responsible for establishing and maintaining transcriptional programs that define cell identity. Defects to epigenetic processes have been linked to a host of disorders, including mental retardation, aging, cancer and neurodegenerative diseases. The ability to control and engineer epigenetic systems would be valuable both for the basic study of these critical cellular processes as well as for synthetic biology. Indeed, while synthetic biology has made progress using bottom-up approaches to engineer transcriptional and signaling circuitry, epigenetic systems have remained largely underutilized. The predictive engineering of epigenetic systems could enable new functions to be implemented in synthetic organisms, including programmed phenotypic diversity, memory, reversibility, inheritance, and hysteresis. This thesis broadly focuses on the development of foundational tools and intellectual frameworks for applying synthetic biology to epigenetic regulation in the model eukaryote, Saccharomyces cerevisiae. Epigenetic regulation is mediated by diverse molecular mechanisms: e.g. self-sustaining feedback loops, protein structural templating, modifications to chromatin, and RNA silencing. Here we develop synthetic tools and circuits for controlling epigenetic states through (1) modifications to chromatin and (2) self-templating protein conformations. On the former, the synthetic tools we develop make it possible to study and direct how chromatin regulators operate to produce distinct gene expression programs. On the latter, we focus our studies on yeast prions, which are self-templating protein conformations that act as elements of inheritance, developing synthetic tools for detecting and controlling prion states in yeast cells. This thesis explores the application of synthetic biology to these epigenetic systems through four aims: Aim 1. Development of inducible expression systems for precise temporal expression of epigenetic regulators Aim 2. Construction of a library of chromatin regulators to study and program chromatin-based epigenetic regulation. Aim 3. Development of a genetic tool for quantifying protein aggregation and prion states in high-throughput Aim 4. Dynamics and control of prion switching Our tools and studies enable a deeper functional understanding of epigenetic regulation in cells, and the repurposing of these systems for synthetic biology toward addressing industrial and medical applications. / 2019-10-08T00:00:00Z

Amyloid $\beta$ (1-42) (A$\beta$42) is a seminal neuropathic agent in Alzheimer’s disease (AD), a multifaceted neurodegenerative disorder for which no preventative measures or disease modifying therapies currently exist. Aggregation of this peptide plays a key role in the synaptic dysfunction and neuronal death associated with the disease. Perturbing the aggregation process, therefore, represents a key strategy for the development of new AD therapeutics. A variety of issues with current screening methods, including lack of reproducibility, high reagent consumption and spectral interference from the test molecules, can limit efforts to identify new small molecule inhibitors. Furthermore, the lack of robust, time- and cost-efficient methods for screening compounds in cellular or in vivo models limits the throughput with which seemingly active small molecules can be validated and prioritised. Herein, this thesis describes efforts to overcome such limitations through the development of a unified in vitro to in vivo assay system, in which hits identified in the ‘nanoFLIM’ microfluidic-based assay can quickly be tested in cellular and whole organism disease models. The assay platform designed relies on the use of an amyloid aggregation fluorescence lifetime sensor. A$\beta$42 aggregation is monitored by changes in the fluorescence lifetime of an attached fluorophore, which is significantly quenched upon amyloid formation. To take advantage of the benefits associated with miniaturisation, an in vitro microfluidic platform was employed. A microfluidic chip capable of trapping 110 precisely ordered droplets was designed, allowing for increased sample size and greatly lowering reagent consumption relative to conventional assay formats. Optimisation of the lifetime sensor technique permitted real-time compound screening in SH-SY5Y neuroblastoma cells, as well as in disease model Caenorhabditis elegans (C. elegans). To demonstrate the potential of this assay, a selection of novel chemical libraries developed in the Spring research group was screened, resulting in the identification of a key library of interest. The inhibitory activity of the lead compound from this collection was validated using a variety of biophysical tests, and was also shown to suppress amyloid aggregation in the live cell fluorescence lifetime sensor assay, as well as in whole organism disease model C. elegans. Whilst assay development was underway, additional screening of structurally diverse chemical libraries was performed using a conventional Thioflavin T spectroscopic assay. Such work identified another molecular scaffold capable of exerting a strong inhibitory effect against A$\beta$42 aggregation. A selection of analogues was synthesised to improve the in vivo profile of this library, giving rise to a second lead inhibitory compound. The activity of this compound was subsequently validated in biophysical and cellular tests, and was also tested in disease model Drosophila melanogaster. The aggregation of A$\beta$42 lies at the root of Alzheimer’s disease. In light of the relatively few drug candidates in clinical trials for this disorder, the development of improved translational screening approaches and continued screening of novel chemical libraries is necessary to identify new potential therapeutics. In this study, an in vitro to in vivo fluorescence lifetime imaging assay has been established. Using this assay system and conventional screening approaches, two A$\beta$42 aggregation inhibitors have been identified and validated. These represent promising candidates for the development of new AD therapeutic agents, or for use as molecular probes to further dissect the mechanisms underlying this devastating disease.

In this thesis, we investigate the mechanisms driving the self-assembly of peptides and proteins using computational and theoretical tools, always validating our results with experimental measures when possible. In the first part, Chapters 2-5, we focus on the Aβ system, a peptide whose aggregation is intimately linked with the development of Alzheimer's Disease. We begin by simulating the major alloforms of the peptide, Aβ_40 and Aβ_42, demonstrating that the two populate similar disordered ensembles and matching experimental data. Next we investigate how disordered Aβ_42 monomers interact with each other, finding that oligomerisation into amorphous aggregates is driven largely by hydrophobic, non-specific forces. We then move on to probing the aggregation of Aβ_42 into amyloid structures using a native-centric coarse-grained model, and explain the results with a novel Markov state analysis from which we are able to extract structural, kinetic and thermodynamic information on elongation reactions. Finally, we probe the interactions of Aβ_42 monomers with Aβ_42 fibrillar surfaces using a specially designed enhanced sampling scheme, which allows us to obtain enthalpy-driven binding thermodynamics consistent with experiments and to propose major polar binding modes. In the second part of the thesis, Chapters 6 and 7, we model the aggregation of two other self-assembling systems, viruses and a truncated form of the molecular chaperone Hsp70. We first develop a data analysis platform to extract information on the microscopic mechanisms of viral capsid self-assembly from experimental data, synthesising the results from several different systems to draw general evolutionary conclusions about the assembly mechanism. Finally, we model the oligomerisation of Hsp70 thermodynamically and kinetically, showing that its self-assembly is a highly cooperative reaction that is under strong structural constraints.

L'exposition à la chaleur, notamment lors d'incendies est particulièrement dévastatrice et dans le cas d'objets du patrimoine elle entraine la destruction de tout ou partie de ces témoins du passé. Notre étude porte sur les effets de la chaleur sur le cuir, matériau largement présent dans les collections patrimoniales.A ce jour, aucune méthode de restauration permettant d'inverser les effets de la chaleur n'a été développée. Le premier objectif de notre étude est d'évaluer les effets d'une exposition à une chaleur sèche par une caractérisation systématique d'échantillons avant et après exposition à la chaleur. Des échantillons modèles, issus d'une même peau de veau de tannage végétal connu, ont été utilisés et caractérisés à différentes échelles structurales par un large ensemble de techniques physico-chimiques et biochimiques avant et après chauffage.Au-delà du brunissement et de la rétraction visible du cuir, la chaleur induit de nombreuses altérations au niveau de la structure du matériau, notamment, une perte de masse, une fonte des structures cristallines, une augmentation de l'hydrophobie ainsi qu'une rigidification. Une partie de ces changements sont attribués à l'agrégation protéique mise en évidence par cette recherche.Le second objectif était de développer une méthode de restauration innovante basée sur l'utilisation de molécules biologiques afin de respecter la nature de l'objet. Des enzymes de type protéase, capables de rompre les agrégats protéiques ont été utilisées. Un des défis est d'apporter suffisamment d'eau, nécessaire pour l'activité de l'enzyme, sans mouiller le cuir pour éviter tout dommage supplémentaire. Plusieurs supports d'application de la protéase ont été testés. Avec une émulsion enzymatique les résultats obtenus ne mettent en évidence ni coloration, ni rétraction et dans certains cas un gain de souplesse est observé. Des résultats encourageants ont également été obtenus dans le cas d'un cuir de veau historique (XIXe siècle). Des mesures complémentaires ont fait attribuer ces propriétés principalement à l'émulsion elle-même, cependant des mesures à plus long terme semblent mettre en évidence un effet positif de l'enzyme sur le gain de souplesse. Sous réserve de nouvelles caractérisations à des temps plus longs, le traitement élaboré pourrait constituer un nouveau support de restauration par voie biologique.Mots clefs : cuir - dénaturation thermique – agrégation protéique – bio-restauration – protéases / Heat, induced by fire, is particularly devastating for cultural heritage objects as it causes the destruction of all or part of these witnesses of the past. In this study, we focused on leather, a material largely present in heritage collections. Until now, no restoration method has been developed to treat the damaging effects of heat.The first aim of our study was to evaluate the effects of dry heat on leather samples through a systematic characterization. Model samples from a calf skin vegetable tanned in known conditions were used and the consequences of heat exposure was characterized at different structural scales using a range of physical, chemical and biochemical methods.Besides the visible browning and shrinkage of leather, heat induces many changes including a loss of mass, the melt of the crystalline regions, an increase in both hydrophobicity and rigidity. Some of these changes result from the protein aggregation induced by exposure to heat and evidenced by our research.Our second goal was to develop an innovative restoration method based on the use of biological molecules in order to respect the nature of the object. Enzymes such as proteases, able to hydrolyze protein aggregates, were used. One of the challenges was to provide the water necessary for the enzyme activity without wetting the leather surface in order to avoid further damage of the leather. Several enzyme supports were tested. The use of an enzymatic emulsion reveals neither darkening nor retraction and in some cases a flexibility gain is observed. Encouraging results were also obtained in the case of an ancient book cover made from calfskin and dated from the nineteenth century. Additional measurements lead to attribute its effect mainly to the emulsion itself, however longer-term measurements appear to show a positive effect of the enzyme on the flexibility gain. Although further characterizations on the long term are required, the treatment may constitute a new support for leather bio-restoration.Keywords: leather – thermal denaturation – protein aggregation – bio-restoration - proteases