Protein aggregation is a hallmark of a number of neurodegenerative disorders including Alzheimer???s Disease, Huntington???s Disease, and Amyotrophic Lateral Sclerosis. Despite the common occurrence of protein aggregation in disease, the fundamental mechanisms controlling the propensity of a protein to aggregate are not well understood. Over the past decade, one of the most significant advancements in the field of understanding protein aggregation has been the development of several aggregation prediction algorithms. In this study, two separate approaches were used to investigate the detailed molecular mechanisms of protein aggregation. First, a thorough investigation that compared nine protein aggregation prediction techniques was performed. Protein aggregation propensity calculations were performed on wild type and mutant sequences of three diverse proteins including Superoxide Dismutase (SOD), human Acylphosphatase (AcP), and the amyloid beta peptide (A??42). This study presents the first wide-scale comparison of such a large number of prediction algorithms, and additionally provides new information on the ability of the algorithms to successfully predict the experimentally observed aggregation of several mutations of diverse proteins. The algorithms were predominantly developed based on a set of known amyloid-forming proteins and peptides, however, are quite diverse in the way they were designed and the proteins on which they were tested. Interestingly, significant variation was observed when predicting the aggregation propensity of identical sequences by multiple techniques, indicating that the algorithms do not possess a consensus on the primary factors that govern aggregation. Further analyses compared predicted and observed aggregation data for several mutants of the test proteins. The aggregation prediction algorithms predominantly demonstrated poor to moderate correlations with observed aggregation, and the strongest correlations occurred in instances where the test data was used in the development of the algorithms. The general lack of ability of the algorithms to predict the aggregation patterns of more than one test protein suggests that aggregation may be a much more specific process that it is generally attributed to be in that there may be inherently different properties modulating the aggregation mechanisms of different proteins towards varying aggregate structures. The second component of this project was to experimentally examine the role of salt in influencing protein aggregation as a method to elucidate the specific molecular mechanisms controlling protein aggregation pathways. The ALS-causing SOD1 mutation, A4V, in both the oxidized and reduced apo form, was used as a model protein. The role of NaCl and Na2SO4 in mediating protein aggregation was studied using several techniques. While oxidized apo A4V showed very little evidence of aggregation even in the presence of salt, for reduced apo, aggregates readily formed and were promoted by the addition of salt. This finding correlated with the increasing kosmotropic nature of the salt as described by the Hofmeister series. The aggregates formed in the presence of salt contained intermolecular disulphide bonds and demonstrated ANS and ThT binding, indicating aggregates are likely to be largely hydrophobic and possess beta-sheet morphology. Salt promotes protein aggregation in two ways: 1) electrostatic interactions shield protein charges and reduce repulsion between proteins, and 2) specific interactions stabilize various aggregation-prone conformations of the protein. This work is evidence of the important role of salt in influencing protein aggregation and provides a framework for future studies into the complex effects of solution conditions in modulating protein aggregation pathways. Both aspects of this study contribute greatly to furthering the understanding of the molecular mechanisms governing protein aggregation. This is of particular importance to neurodegenerative diseases, where uncovering the factors that modulate the formation of toxic aggregate species is important for disease treatment and prevention. The potential aggregation mechanisms of SOD1, and the contributions it may play in ALS pathogenesis, will be discussed throughout this study.

No description available.

Neuronal pentraxin with chromo domain (NPCD) belongs to a family of neuronally-expressed pentraxin proteins thought to be involved in synaptic refinement and plasticity. One isoform of Npcd, neuronal pentraxin receptor (NPR), is a type-II transmembrane protein responsible for the clustering of related neuronal pentraxins 1 and 2. However, recently identified cytosolic NPCD isoforms with no known function were discovered through their interaction with the intracellular domain of a receptor protein tyrosine phosphatase PTPRO. PTPRO is a signaling molecule known to be involved in the development of the nervous system. Additionally, upregulated expression of neuronal pentraxins has been implicated in neuronal cytotoxicity and associated with neurodegenerative diseases. Here, we demonstrate that a novel cytosolic NPCD isoform interacts with the BTB-Kelch protein Mayven/KLHL2. This interaction was identified through a yeast two-hybrid screen using the C-terminal pentraxin domain region of NPCD and confirmed through mammalian cell colocalization and co-immunoprecipitation studies. Domain truncation analysis suggests that the kelch domains of Mayven/KLHL2 are responsible for this interaction with NPCD. We also show that Mayven/KLHL2 is capable of interacting with Cullin 3, an integral protein in the Cullin-RING ubiquitin ligase complex. An in-vivo ubiquitylation assay demonstrates that overexpression of Mayven/KLHL2 increases NPCD ubiquitylation, and suggests a novel E3 ubiquitin ligase function of Mayven/KLHL2 with NPCD as its substrate. Furthermore, we observed an increased propensity of overexpressed NPCD to form aggresomes with coexpression of Mayven/KLHL2. As the formation of aggresomes is often associated with protein aggregation and deposition diseases, including a multitude of neurodegenerative diseases, we tested NPCD overexpression and the effects of Mayven/KLHL2 coexpression on neuronal cytotoxicity and apoptosis. Overexpressed NPCD in hippocampal neuron cultures resulted in increased cytotoxicity and apoptosis, further exacerbated by Mayven/KLHL2 coexpression. Our findings report an interaction between NPCD and Mayven/KLHL2, demonstrate a novel role of Mayven/KLHL2 as an E3 ubiquitin ligase, and explore a possible intersection between the ubiquitin-proteasome degradation pathway, neuronal pentraxins, and neurodegenerative disease.

Many neuro-degenerative and metabolic diseases like Parkinson’s and Alzheimer’s are attributed to the effect of mis-folded and aggregated state of proteins in cells. This suggests that the phenomenon of in vivo protein aggregation may be relatively common, perhaps more than currently appreciated. In this study, we aimed to decipher the cause behind an intriguing and potentially related phenomenon observed in yeast cells - a widespread reorganization of hundreds of cytosolic proteins into punctate foci under starvation conditions. The key question that emerges is whether this phenomenon represents organization of proteins into functional assemblies or catastrophic aggregation. This thesis supports the aggregation hypothesis and provides evidence of its role in shaping the dynamics of cellular proteomes. We have been able to demonstrate that the proteins forming foci share a high propensity to aggregate and that these foci may represent sites of homogenous protein aggregation, structures which are typically associated with chaperones. A link between the formation of foci to the yeast aging process has also been established. With evidence correlating protein aggregation propensities to the cellular energy state, we have extended the current "living on the edge" hypothesis (which demonstrates an inverse correlation between protein expression levels and their aggregation propensities). For a specific case of the "purinosome", which is inferred to be a functional enzyme complex responsible for purine biosynthesis, we have shown that the observations may be explained alternatively as a generic protein aggregation phenomenon. This study highlights a systems approach to studying cellular proteins, which can corroborate or provide an alternative explanation to inferences drawn from traditional reductionistic analysis. / text

Submitted to the faculty of Indiana University in partial fulfillment of the requirements for the degree Master of Science in the Department of Bioinformatics in the School of Informatics of, Indiana University December, 2004 / Electrostatic modeling was done with Delphi of insight II to explain and predict protein aggregation, measured here for β-lactoglobulin and insulin using turbidimetry and stopped flow spectrophotometry. The initial rate of aggregation of β-Lactoglobulin was studied between pH 3.8 and 5.2 in 4.5mM NaCl; and for ionic strengths from 4.5 to 500mM NaCl at pH 5.0. The initial slope of the turbidity vs. time curve was used to define the initial rate of aggregation. The highest initial rate was observed near pH < pI i.e., 4.6 (< 5.2). The decrease in aggregation rate when the pH was increased from 4.8 to 5.0 was large compared to its decrease when the pH was reduced from 4.4 to 4.2; i.e., the dependence of initial rate on pH was highly asymmetric. The initial rate of aggregation at pH 5.0 increased linearly with the reciprocal of ionic strength in the range I = 0.5 to 0.0045M. Protein electrostatic potential distributions are used to understand the pH and ionic strength dependence of the initial rate of aggregation. Similar studies were done with insulin. In contrast to BLG, the highest initial aggregation rate for insulin was observed at pH = pI. Electrostatic computer modeling shows that these differences arise from the distinctly different surface charge distributions of insulin and BLG.

A common maxim of protein biochemistry states, “structure is function.” This is generally just as true for an individual polypeptide chains as for multi-protein complexes. The advent of yeast tagged-protein libraries has allowed systematic screening of a protein’s local interaction partners as well as a roughly mapping its cellular location. Recently our group and others discovered hundreds proteins forming new structures in stationary phase yeast cells using the yeast GFP-tag library. That equates to well over a quarter of normally diffuse cytoplasmic proteins assembled into discrete structures that appear as foci or fibers, all of unknown function. This study provides evidence that many of these foci are formed by protein aggregation- that contrary the maxim, structure can be dysfunction. Furthermore, this study uses yeast to demonstrate the generality of cytoplasmic protein aggregation in response to a variety of stresses, provides evidence that increasing aggregation of particular cytoplasmic proteins correlates with aging even across organisms, and proposes a theoretical framework for how cellular energy levels affect protein aggregation propensity. / text

The aim of this thesis is to investigate and better understand the mechanisms of protein self-assembly. Specifically, I study three protein systems which form morphologically and structurally distinct brillar protein aggregates. The first of these studies is concerned with the self-assembly of amyloid brils formed from bovine insulin. Amyloid brils are associated with human diseases such as Alzheimers Disease and type-2 diabetes, and are also garnering interest in biomaterial applications. Fragmentation-dominated models for the self-assembly of amyloid brils have had important successes in explaining the kinetics of amyloid bril formation but predict bril length distributions that do not match experimental observations. Here I resolve this inconsistency using a combination of experimental kinetic measurements and computer simulations. I provide evidence for a structural transition demarcated by a critical bril mass concentration, or CFC, above which fragmentation of the brils is suppressed. Our simulations predict the formation of distinct bril length distributions above and below the CFC, which I confirm by electron microscopy. These results point to a new picture of amyloid bril growth in which structural transitions that occur during self-assembly have strong effects on the final population of aggregate species with small, and potentially cytotoxic, oligomers dominating for long periods of time at protein concentrations below the CFC. I further show that the CFC can be modulated by environmental conditions, pointing to possible in vivo strategies for controlling cytotoxicity. I probe the structural nature of the transition by performing small angle neutron scattering. Secondly, I study the formation of amyloid-like brils from the protein ovalbumin. I undertake kinetic experiments of self-assembly and find two key features emerge: the lack of a lag time and the existence of a slow growth regime in the long-time limit. I observe, using TEM, that these brils are worm-like in nature and form closed-loops. I find the growth kinetics are intimately connected to this particular morphology. I present a simple kinetic model which captures the features of the kinetics found in experiments by incorporating end-to-end association of brils. I comment on the ramifications this type of amyloid bril assembly may have on oligomeric toxicity. Thirdly, the DNA-mimic protein ocr is highly charged (-56e at pH 8) and forms non-amyloid brillar assemblies at very high ammonium sulphate concentrations (3.2M). The fact that ocr forms translucent brillar gels at such high salt concentrations is extremely unique. Typically under such high salt conditions, non-specific amorphous aggregates are formed. In order to better understand the mechanism of why ocr forms specific bril aggregates, I used variants of the wile-type protein in which extensive regions of surface have been removed or modified. The structural characteristics of gels formed from the variants were probed using microrheological techniques. I find that non-specific electrostatic charge screening plays an important role in ocr aggregation. However, I also locate a potentially important α-helical region which may play a part in establishing specific interactions so that ocr may form ordered brillar assemblies.

Protein deposition in the form of amyloid fibrils is the hallmark of more than 40 human pathologies, including Alzheimer's disease (AD) and Parkinson's disease (PD). Misfolded protein oligomers formed as intermediates during the aggregation process have been strongly implicated in the onset and progression of these diseases. In this thesis, I describe our efforts to uncover molecular agents that can reduce the toxicity caused by protein aggregation via targeting the generation, the physiochemical properties or the membrane affinity of oligomeric species. We employed an integrative approach combining in vitro techniques, including chemical kinetics, atomic force microscopy, and biophysical measurements, and in vivo methods, including neuroblastoma cells and C. elegans models of AD and PD, to identify a range of small molecules and antibodies that can suppress the toxicity related to protein aggregation through a variety of mechanisms. In Chapter 3, we show that the deleterious effects of protein aggregation can be suppressed in AD and PD worms by interfering with the aggregation rates of the amyloid-β peptide (Aβ) and the α-synuclein protein (αS). In Chapter 4, we resolve the mechanism of action for a molecule that enhances the rate of Aβ42 aggregation in AD worms with the result that toxicity is reduced, and find that it potentiates the secondary nucleation microscopic step in vitro. In Chapter 5, we characterize molecules and antibodies that modify the physiochemical properties and self-association of oligomers comprised of several proteins into clusters with reduced diffusibility. In Chapter 6, we classify a family of molecules that protect the cell by displacing several types of oligomeric species from the membrane through a generic mechanism. These results demonstrate strategies by which one can target the aggregation process to alter its resulting toxicity, provide insight into modifying the properties of the most deleterious species associated with protein aggregation and suggest that the protection of the cell from the oligomer-induced cytotoxicity associated with numerous protein misfolding diseases is a promising strategy to combat protein misfolding diseases.

Peroxiredoxins are ubiquitous, thiol-specific proteins that have multiple functions in stress protection, including oxidative stress. Tsa1 is the major yeast peroxiredoxin and we show that it functions as a specific antioxidant to protect against oxidative stress caused by nascent protein misfolding and aggregation. Yeast mutants lacking TSA1 are sensitive to misfolding caused by exposure to the proline analogue azetidine-2-carboxylic acid (AZC). AZC promotes protein aggregation and its toxicity to a tsa1 mutant is caused by reactive oxygen species (ROS). Generation of [rho0] cells lacking mitochondrial DNA rescues the tsa1 mutant AZC sensitivity indicating that mitochondria are the source of ROS. Inhibition of nascent protein synthesis with cycloheximide prevents AZC-induced protein aggregation and abrogates ROS generation confirming that aggregate formation causes ROS production. Protein aggregation is accompanied by mitochondrial fragmentation and we show that Tsa1 localizes to the sites of protein aggregation, which are formed adjacent to mitochondria. Further investigation reveals that AZC-induced protein aggregation leads to an inhibition of mitochondrial respiration and the depolarisation of the mitochondrial membrane. Remarkably, this was entirely dependent on the presence of Tsa1. We show that the effects of protein aggregation on mitochondrial function are mediated by the Ras/PKA pathway and that Tsa1 appears to influence the activity of this pathway through its effects on the yeast phosphodiesterase, Pde2. Together, these data indicate a new role for peroxiredoxins in the response to ROS, generated as a result of protein misfolding and aggregate formation. Finally, we analysed the characteristics of proteins found within protein aggregates, isolated from different conditions during the course of the study. Our results highlight the differences between proteins that aggregate under normal, mid-exponential growth conditions (physiological aggregates) and those which aggregate during cellular stress. We were able to establish the characteristics of an archetypical physiological aggregate, through an assessment of a range of properties, identifying factors that significantly differed from genomic expectations. Furthermore, our observations indicate that, in general, cellular stress reduces the threshold of metrics associated with protein aggregation propensity. We also found that different stresses result in the aggregation of proteins that are, largely, physicochemically indistinct from one another, regardless of the mode of toxicity. Finally we show that a significant number of proteins, identified in our protein aggregates, were also present in protein aggregates isolated from aged C. elegans. This suggests that the factors and components of protein aggregates are conserved.

Protein aggregation is a phenomenon that plays a major role in protein crystallization and in protein formulation. In protein crystallization, aggregation is the prerequisite step; however, in protein formulation it has to be suppressed to assure therapeutic efficiency of the product. Light scattering techniques are the most promising methods to study the hydrodynamic properties of macromolecular solutions, which directly measures protein aggregation. Unfortunately, the normal dynamic light scattering technique is regarded as expensive because of the amount of protein used for these experiments. In order to address this problem, a scale down dynamic light scattering device has been designed. The osmotic second virial coefficient, a dilute solution parameter helps in identifying solution conditions for protein crystal growth. The second part of this thesis involves comparison of osmotic second virial coefficient (B) measurements of lysozyme using laser light scattering techniques with B measurements of lysozyme performed using self-interaction chromatography (SIC).