polyQ Region Research Articles

Predicting the involvement of polyQ- and polyA in protein-protein interactions by their amino acid context

Homorepeats, specifically polyglutamine (polyQ) and polyalanine (polyA), are often implicated in protein-protein interactions (PPIs). So far, a method to predict the participation of homorepeats in protein interactions is lacking. We propose a machine learning approach to identify PPI-involved polyQ and polyA regions within the human proteome based on known interacting regions. Using the dataset of human homorepeats, we identified 157 polyQ and 745 polyA regions potentially involved in PPIs. Machine learning models, trained on amino acid context and homorepeat length, demonstrated high precision (0.90–0.98) but variable recall (0.42–0.85). Random forest outperformed other models (AUC polyQ = 0.686, AUC polyA = 0.732) using the positions surrounding the homorepeat −10 to +10. Integrating paralog information marginally improved predictions but was excluded for model simplicity. Further optimization revealed that for polyQ, using amino acid surrounding positions from −6 to +6 increased AUC to 0.715. For polyA, no improvement was found. Incorporating coiled coil overlap information enhanced polyA predictions (AUC = 0.745) but not polyQ. Finally, we applied these models to predict PPI involvement across all polyQ and polyA regions, identifying potential interactions. Case studies illustrated the method's predictive capacity, highlighting known interacting regions with high scores and elucidating potential false negatives.

Drosophila in the study of hTBP protein interactions in the development and modeling of SCA17.

Protein interactions participate in many molecular mechanisms involved in cellular processes. The human TATA box binding protein (hTBP) interacts with Antennapedia (Antp) through its N-terminal region, specifically via its glutamine homopeptides. This PolyQ region acts as a binding site for other transcription factors under normal conditions, but when it expands, it generates spinocerebellar ataxia 17 (SCA17), whose protein aggregates in the brain prevent its correct functioning. To determine whether the hTBP glutamine-rich region is involved in its interaction with homeoproteins and the role it plays in the formation of protein aggregates in SCA17. We characterized hTBP interaction with other homeoproteins using BiFC, and modeled SCA17 in Drosophila melanogaster by targeting hTBPQ80 to the fly brain using UAS/GAL4. There was hTBP interaction with homeoproteins through its glutamine-rich region, and hTBP protein aggregates with expanded glutamines were found to affect the locomotor capacity of flies. The study of hTBP interactions opens the possibility for the search for new therapeutic strategies in neurodegenerative pathologies such as SCA17.

Phase separation and molecular ordering of the prion-like domain of the Arabidopsis thermosensory protein EARLY FLOWERING 3

Liquid-liquid phase separation (LLPS) is an important mechanism enabling the dynamic compartmentalization of macromolecules, including complex polymers such as proteins and nucleic acids, and occurs as a function of the physicochemical environment. In the model plant, Arabidopsis thaliana, LLPS by the protein EARLY FLOWERING3 (ELF3) occurs in a temperature-sensitive manner and controls thermoresponsive growth. ELF3 contains a largely unstructured prion-like domain (PrLD) that acts as a driver of LLPS in vivo and in vitro. The PrLD contains a poly-glutamine (polyQ) tract, whose length varies across natural Arabidopsis accessions. Here, we use a combination of biochemical, biophysical, and structural techniques to investigate the dilute and condensed phases of the ELF3 PrLD with varying polyQ lengths. We demonstrate that the dilute phase of the ELF3 PrLD forms a monodisperse higher-order oligomer that does not depend on the presence of the polyQ sequence. This species undergoes LLPS in a pH- and temperature-sensitive manner and the polyQ region of the protein tunes the initial stages of phase separation. The liquid phase rapidly undergoes aging and forms a hydrogel as shown by fluorescence and atomic force microscopies. Furthermore, we demonstrate that the hydrogel assumes a semiordered structure as determined by small-angle X-ray scattering, electron microscopy, and X-ray diffraction. These experiments demonstrate a rich structural landscape for a PrLD protein and provide a framework to describe the structural and biophysical properties of biomolecular condensates.

Comparative molecular dynamics simulations of pathogenic and non-pathogenic huntingtin protein monomers and dimers.

Polyglutamine expansion at the N-terminus of the huntingtin protein exon 1 (Htt-ex1) is closely associated with a number of neurodegenerative diseases, which result from the aggregation of the increased polyQ repeat. However, the underlying structures and aggregation mechanism are still poorly understood. We performed microsecond-long all-atom molecular dynamics simulations to study the folding and dimerization of Htt-ex1 (about 100 residues) with non-pathogenic and pathogenic polyQ lengths, and uncovered substantial differences. The non-pathogenic monomer adopts a long α-helix that includes most of the polyQ residues, which forms the interaction interface for dimerization, and a PPII-turn-PPII motif in the proline-rich region. In the pathogenic monomer, the polyQ region is disordered, leading to compact structures with many intra-protein interactions and the formation of short β-sheets. Dimerization can proceed via different modes, where those involving the N-terminal headpiece bury more hydrophobic residues and are thus more stable. Moreover, in the pathogenic Htt-ex1 dimers the proline-rich region interacts with the polyQ region, which slows the formation of β-sheets.

ATXN3 controls DNA replication and transcription by regulating chromatin structure.

The deubiquitinating enzyme Ataxin-3 (ATXN3) contains a polyglutamine (PolyQ) region, the expansion of which causes spinocerebellar ataxia type-3 (SCA3). ATXN3 has multiple functions, such as regulating transcription or controlling genomic stability after DNA damage. Here we report the role of ATXN3 in chromatin organization during unperturbed conditions, in a catalytic-independent manner. The lack of ATXN3 leads to abnormalities in nuclear and nucleolar morphology, alters DNA replication timing and increases transcription. Additionally, indicators of more open chromatin, such as increased mobility of histone H1, changes in epigenetic marks and higher sensitivity to micrococcal nuclease digestion were detected in the absence of ATXN3. Interestingly, the effects observed in cells lacking ATXN3 are epistatic to the inhibition or lack of the histone deacetylase 3 (HDAC3), an interaction partner of ATXN3. The absence of ATXN3 decreases the recruitment of endogenous HDAC3 to the chromatin, as well as the HDAC3 nuclear/cytoplasm ratio after HDAC3 overexpression, suggesting that ATXN3 controls the subcellular localization of HDAC3. Importantly, the overexpression of a PolyQ-expanded version of ATXN3 behaves as a null mutant, altering DNA replication parameters, epigenetic marks and the subcellular distribution of HDAC3, giving new insights into the molecular basis of the disease.

Structural Elements of ‘Anti‐prion J protein 1’ (Apj1) Required for Efficient Curing of the [PSI+] Prion by Hsp104 Overexpression

Prions are self‐propagating aggregates of misfolded protein that often form ordered amyloids. Amyloids are associated with a variety of human neurodegenerative diseases, including but not limited to Alzheimer’s disease, Huntington’s disease, and Parkinson’s disease. Amyloid‐forming prions are also present in Saccharomyces cerevisiae, where they are propagated through populations by a fragmentation mechanism that requires three chaperone proteins: the disaggregase Hsp104, the Hsp70 Ssa, and the J‐protein Sis1. When Hsp104 is ectopically overexpressed, however, the prion [PSI+] is specifically eliminated from cell populations in a Sis1‐dependent manner. We previously determined that the J‐protein Apj1 is also required for efficient elimination, or curing, of the prion variants [PSI+]STR and [PSI+]Sc4 by Hsp104 overexpression. Additionally, we showed that Apj1 and Sis1 have overlapping functions in the Hsp104‐mediated curing process, as overexpressing one in the absence of a functional version of the other restores curing. A construct bearing the first 161 residues of Apj1 is sufficient to restore efficient curing in Δapj1cells. Residues 1‐161 can be subdivided based on sequence features and homology into: a J‐domain, a polyglutamine track with an imperfect QA repeat region, a region bearing homology to the G/F region of Sis1, and finally a region rich in glycine, serine, phenylalanine, and asparagine which resembles the amino acid content of many prion‐forming domains. Various constructs produced by mutating and truncating regions of Apj1‐161 were ectopically expressed in Δapj1 cells to determine which regions, if any, were sufficient to replace Apj1 in Hsp104‐mediated curing. We found that constructs containing the polyQ region restored efficient curing and those lacking the polyQ region did not support efficient curing, indicating the importance of the polyQ region in the curing process. The Sis1 homologous region and the SFN rich region proved nonessential for Hsp104‐mediated curing. Ongoing experiments are being conducted to determine which regions of Apj1‐161 are required to replace the function of Sis1 in Hsp104‐mediated curing.

Effects of charge on the interaction of the huntingtin N-terminal sequence with model cell membranes

Huntington’s Disease (HD) is a genetic neurodegenerative disorder caused by an expansion in the polyglutamine (poly-Q) region near the N-terminus of the huntingtin (htt) protein. When the poly-Q expansion surpasses a threshold of 35-40 glutamine repeats, the protein misfolds into a beta sheet rich structure that forms insoluble neuronal aggregates. The poly-Q region of htt is flanked by a 17 amino acid sequence (Nt17) that facilitates insertion of htt into lipid membranes via an amphipathic alpha helical structure, a function that is proposed to anchor the protein and may affect the aggregation process.

The conformational dynamics of the flanking polyQ regions in the membrane-bound state of huntingtin exon 1

Huntington's disease (HD) is an inherited neurodegenerative disorder caused by an abnormal expansion of the polyglutamine (polyQ) repeat within the first exon of huntingtin protein (httex1). The membrane interaction of httex1 is critical for its self-assembly and toxicity. While initial studies were essentially focused on polyQ peptide-lipid interaction, recent evidence supports that both flanking polyQ regions [the N17 segment and proline-rich domain (PRD)] strongly modulate the httex1 aggregation at membrane surfaces.

Between Interactions and Aggregates: The PolyQ Balance.

Polyglutamine (polyQ) regions are highly abundant consecutive runs of glutamine residues. They have been generally studied in relation to the so-called polyQ-associated diseases, characterized by protein aggregation caused by the expansion of the polyQ tract via a CAG-slippage mechanism. However, more than 4,800 human proteins contain a polyQ, and only nine of these regions are known to be associated with disease. Computational sequence studies and experimental structure determinations are completing a more interesting picture in which polyQ emerge as a motif for modulation of protein–protein interactions. But long polyQ regions may lead to an excess of interactions, and produce aggregates. Within this mechanistic perspective of polyQ function and malfunction, we discuss polyQ definition and properties such as variable codon usage, sequence and context structure imposition, functional relevance, evolutionary patterns in species-centered analyses, and open resources.

An Expanded Polyproline Domain Maintains Mutant Huntingtin Soluble in vivo and During Aging.

Huntington’s disease is a dominantly inherited neurodegenerative disorder caused by the expansion of a CAG repeat, encoding for the amino acid glutamine (Q), present in the first exon of the protein huntingtin. Over the threshold of Q39 HTT exon 1 (HTTEx1) tends to misfold and aggregate into large intracellular structures, but whether these end-stage aggregates or their on-pathway intermediates are responsible for cytotoxicity is still debated. HTTEx1 can be separated into three domains: an N-terminal 17 amino acid region, the polyglutamine (polyQ) expansion and a C-terminal proline rich domain (PRD). Alongside the expanded polyQ, these flanking domains influence the aggregation propensity of HTTEx1: with the N17 initiating and promoting aggregation, and the PRD modulating it. In this study we focus on the first 11 amino acids of the PRD, a stretch of pure prolines, which are an evolutionary recent addition to the expanding polyQ region. We hypothesize that this proline region is expanding alongside the polyQ to counteract its ability to misfold and cause toxicity, and that expanding this proline region would be overall beneficial. We generated HTTEx1 mutants lacking both flanking domains singularly, missing the first 11 prolines of the PRD, or with this stretch of prolines expanded. We then followed their aggregation landscape in vitro with a battery of biochemical assays, and in vivo in novel models of C. elegans expressing the HTTEx1 mutants pan-neuronally. Employing fluorescence lifetime imaging we could observe the aggregation propensity of all HTTEx1 mutants during aging and correlate this with toxicity via various phenotypic assays. We found that the presence of an expanded proline stretch is beneficial in maintaining HTTEx1 soluble over time, regardless of polyQ length. However, the expanded prolines were only advantageous in promoting the survival and fitness of an organism carrying a pathogenic stretch of Q48 but were extremely deleterious to the nematode expressing a physiological stretch of Q23. Our results reveal the unique importance of the prolines which have and still are evolving alongside expanding glutamines to promote the function of HTTEx1 and avoid pathology.

Inhibition of huntingtin aggregation by its N-terminal 17-residue peptide and its analogs

The N-terminal 17-residue stretch of huntingtin (httN17) folds into an amphipathic α-helix. The httN17–harboring polyQ peptides form oligomers that are mediated via the assembly of the httN17 α-helices. The oligomerization results in higher local concentration of the polyglutamine (polyQ) region, thereby facilitating amyloid formation. The httN17 co-assembles with the httN17-harbouring polyQ peptides, thereby reducing the local polyQ concentration, and consequently inhibiting aggregation. This study presents the aggregation inhibition of the exon I region of pathogenic huntingtin by httN17 and its analogs. The C-terminal amidation of httN17 is found to be essential for activity. The httN17 peptides with free amino terminus and the acetylated amino terminus possess comparable activity. The httN17 analog, wherein the Leu7 and Ala10 are substituted with 2-aminoisobutyric acid residues, exhibits significantly higher activity than the native httN17.

Effects of the Flanking polyQ Regions and Membrane Physical Properties on Huntingtin Binding to Lipid Vesicles

The pathological expansion of the polyglutamine (polyQ) repeat within the first exon of huntingtin (HTTex1) protein is a hallmark of Huntington’s disease (HD). Multiple evidences support that the membrane interaction of huntingtin is critical for its misfolding and aggregation in HD. Here, we focus on obtaining a detailed understanding of the initial steps of HTTex1-lipid interaction. This characterization has been exceptionally challenging using traditional ensemble methods due to its heterogeneous nature and the high propensity of HTTex1 to aggregate. To overcome these limitations, we used single-molecule approaches based mainly on fluorescence correlation spectroscopy (FCS) to monitor the binding of HTTex1 to synthetic lipid vesicles. The dependence on membrane curvature and lipid composition was studied, considering as models anionic lipids, raft-mimicking mixtures, and total brain lipid extract. The FCS results show a preferential binding of HTTex1 towards POPS (anionic lipid)-containing vesicles of small curvature. Moreover, time-resolved anisotropy measurements reveal distinct conformational dynamics of the adjacent polyQ regions in the HTTex1 membrane-bound state. Notably, the proline-rich domain remains highly flexible and solvent exposed even upon membrane binding. instead, the N17 segment converts into a less dynamic state. Our findings provide unique insight into how membrane curvature/composition and flanking polyQ sequences modulate the early stages of HTTex1 aggregation at the membrane surface. Work supported by FCT-Portugal (PTDC/BIA-BFS/30959/2017 grant and CEECIND/00884/2017 contract to AMM, and UIDB/04565/2020 funding to iBB-IST).

Excess Rab4 rescues synaptic and behavioral dysfunction caused by defective HTT-Rab4 axonal transport in Huntington\u2019s disease

Huntington’s disease (HD) is characterized by protein inclusions and loss of striatal neurons which result from expanded CAG repeats in the poly-glutamine (polyQ) region of the huntingtin (HTT) gene. Both polyQ expansion and loss of HTT have been shown to cause axonal transport defects. While studies show that HTT is important for vesicular transport within axons, the cargo that HTT transports to/from synapses remain elusive. Here, we show that HTT is present with a class of Rab4-containing vesicles within axons in vivo. Reduction of HTT perturbs the bi-directional motility of Rab4, causing axonal and synaptic accumulations. In-vivo dual-color imaging reveal that HTT and Rab4 move together on a unique putative vesicle that may also contain synaptotagmin, synaptobrevin, and Rab11. The moving HTT-Rab4 vesicle uses kinesin-1 and dynein motors for its bi-directional movement within axons, as well as the accessory protein HIP1 (HTT-interacting protein 1). Pathogenic HTT disrupts the motility of HTT-Rab4 and results in larval locomotion defects, aberrant synaptic morphology, and decreased lifespan, which are rescued by excess Rab4. Consistent with these observations, Rab4 motility is perturbed in iNeurons derived from human Huntington’s Disease (HD) patients, likely due to disrupted associations between the polyQ-HTT-Rab4 vesicle complex, accessory proteins, and molecular motors. Together, our observations suggest the existence of a putative moving HTT-Rab4 vesicle, and that the axonal motility of this vesicle is disrupted in HD causing synaptic and behavioral dysfunction. These data highlight Rab4 as a potential novel therapeutic target that could be explored for early intervention prior to neuronal loss and behavioral defects observed in HD.

When Less Is More: Combining Site-Specific Isotope Labeling and NMR Unravels Structural Details of Huntingtin Repeats

In this issue of Structure, Urbanek etal. (2020a) combine site-specific isotope labeling and NMR spectroscopy to investigate opposing effects of flanking regions onto the conformation of the poly-Q region in Huntingtin. Poly-Q interactions with preceding residues promote an α-helical conformation while a following proline-rich region favors extended conformations.

The features of polyglutamine regions depend on their evolutionary stability

BackgroundPolyglutamine regions (polyQ) are one of the most studied and prevalent homorepeats in eukaryotes. They have a particular length-dependent codon usage, which relates to a characteristic CAG-slippage mechanism. Pathologically expanded tracts of polyQ are known to form aggregates and are involved in the development of several human neurodegenerative diseases. The non-pathogenic function of polyQ is to mediate protein-protein interactions via a coiled-coil pairing with an interactor. They are usually located in a helical context.ResultsHere we study the stability of polyQ regions in evolution, using a set of 60 proteomes from four distinct taxonomic groups (Insecta, Teleostei, Sauria and Mammalia). The polyQ regions can be distinctly grouped in three categories based on their evolutionary stability: stable, unstable by length variation (inserted), and unstable by mutations (mutated). PolyQ regions in these categories can be significantly distinguished by their glutamine codon usage, and we show that the CAG-slippage mechanism is predominant in inserted polyQ of Sauria and Mammalia. The polyQ amino acid context is also influenced by the polyQ stability, with a higher proportion of proline residues around inserted polyQ. By studying the secondary structure of the sequences surrounding polyQ regions, we found that regarding the structural conformation around a polyQ, its stability category is more relevant than its taxonomic information. The protein-protein interaction capacity of a polyQ is also affected by its stability, as stable polyQ have more interactors than unstable polyQ.ConclusionsOur results show that apart from the sequence of a polyQ, information about its orthologous sequences is needed to assess its function. Codon usage, amino acid context, structural conformation and the protein-protein interaction capacity of polyQ from all studied taxa critically depend on the region stability. There are however some taxa-specific polyQ features that override this importance. We conclude that a taxa-driven evolutionary analysis is of the highest importance for the comprehensive study of any feature of polyglutamine regions.

The importance of definitions in the study of polyQ regions: A tale of thresholds, impurities and sequence context

Polyglutamine (polyQ) regions are one of the most prevalent homorepeats in eukaryotes. It is however difficult to evaluate their prevalence because various studies claim different results. The reason is the lack of a consensus to define what is indeed a polyQ region. We have tackled this issue by studying how the use of different thresholds (i.e., minimum number of glutamines required in a protein region of a given size), to detect polyQ regions in the human proteome influences not only their prevalence but also their general features and sequence context. Threshold definition shapes the length distribution of the polyQ dataset, and changes the observed number and position of impurities (amino acids other than glutamine) within polyQ regions. Irrespective of the chosen threshold, leucine and proline residues are enriched both within and around polyQ. While leucine is enriched at the N-terminus of polyQ and specially at position −1 (amino acid preceding the polyQ), proline is prevalent in the C-terminus (positions +1 to +5, that is, the first five amino acids after the polyQ). We also checked the suitability of these thresholds for other species, and compared their polyQ features with those found in humans. As the sequence context and features of polyQ regions are threshold-dependent, we propose a method to quickly scan the polyQ landscape of a proteome. We complement our results with a summarized overview about which biases are to be expected per threshold when studying polyQ regions.

ATXN1 N-terminal region explains the binding differences of wild-type and expanded forms

BackgroundWild-type (wt) polyglutamine (polyQ) regions are implicated in stabilization of protein-protein interactions (PPI). Pathological polyQ expansion, such as that in human Ataxin-1 (ATXN1), that causes spinocerebellar ataxia type 1 (SCA1), results in abnormal PPI. For ATXN1 a larger number of interactors has been reported for the expanded (82Q) than the wt (29Q) protein.MethodsTo understand how the expanded polyQ affects PPI, protein structures were predicted for wt and expanded ATXN1, as well as, for 71 ATXN1 interactors. Then, the binding surfaces of wt and expanded ATXN1 with the reported interactors were inferred.ResultsOur data supports that the polyQ expansion alters the ATXN1 conformation and that it enhances the strength of interaction with ATXN1 partners. For both ATXN1 variants, the number of residues at the predicted binding interface are greater after the polyQ, mainly due to the AXH domain. Moreover, the difference in the interaction strength of the ATXN1 variants was due to an increase in the number of interactions at the N-terminal region, before the polyQ, for the expanded form.ConclusionsThere are three regions at the AXH domain that are essential for ATXN1 PPI. The N-terminal region is responsible for the strength of the PPI with the ATXN1 variants. How the predicted motifs in this region affect PPI is discussed, in the context of ATXN1 post-transcriptional modifications.

Structure of Membrane-Bound Huntingtin Exon 1 Reveals Membrane Interaction and Aggregation Mechanisms

Huntington's disease is caused by a polyQ expansion in the first exon of huntingtin (Httex1). Membrane interaction of huntingtin is of physiological and pathological relevance. Using electron paramagnetic resonance and Overhauser dynamic nuclear polarization, we find that the N-terminal residues 3-13 of wild-type Httex1(Q25) form a membrane-bound, amphipathic α helix. This helix is positioned in the interfacial region, where it is sensitive to membrane curvature and electrostatic interactions with head-group charges. Residues 14-22, which contain the first five residues of the polyQ region, are in a transition region that remains in the interfacial region without taking up a stable, α-helical structure. The remaining C-terminal portion is solvent exposed. The phosphomimetic S13D/S16D mutations, which are known to protect from toxicity, inhibit membrane binding and attenuate membrane-mediated aggregation of mutant Httex1(Q46) due to electrostatic repulsion. Targeting the N-terminal membrane anchor using post-translational modifications or specific binders could be a potential means to reduce aggregation and toxicity invivo.

Inter-generational resemblance of methylation levels at circadian genes and associations with phenology in the barn swallow

Regulation of gene expression can occur via epigenetic effects as mediated by DNA methylation. The potential for epigenetic effects to be transmitted across generations, thus modulating phenotypic variation and affecting ecological and evolutionary processes, is increasingly appreciated. However, the study of variation in epigenomes and inter-generational transmission of epigenetic alterations in wild populations is at its very infancy. We studied sex- and age-related variation in DNA methylation and parent-offspring resemblance in methylation profiles in the barn swallows. We focused on a class of highly conserved ‘clock’ genes (clock, cry1, per2, per3, timeless) relevant in the timing of activities of major ecological importance. In addition, we considerably expanded previous analyses on the relationship between methylation at clock genes and breeding date, a key fitness trait in barn swallows. We found positive assortative mating for methylation at one clock locus. Methylation varied between the nestling and the adult stage, and according to sex. Individuals with relatively high methylation as nestlings also had high methylation levels when adults. Extensive parent-nestling resemblance in methylation levels was observed. Occurrence of extra-pair fertilizations allowed to disclose evidence hinting at a prevalence of paternal germline or sperm quality effects over common environment effects in generating father-offspring resemblance in methylation. Finally, we found an association between methylation at the clock poly-Q region, but not at other loci, and breeding date. We thus provided evidence for sex-dependent variation and the first account of parent-offspring resemblance in methylation in any wild vertebrate. We also showed that epigenetics may influence phenotypic plasticity of timing of life cycle events, thus having a major impact on fitness.

Huntingtin Aggregation Is Modified in the Presence of a Variety of Lipid Membranes

Huntington's Disease is a fatal neurodegenerative disorder characterized by an expanded polyglutamine (polyQ) region of the huntingtin protein (htt) that leads to the formation of toxic aggregates. The first 17 amino-terminal residues of htt (Nt17) have been shown to promote this oligomer formation, and also comprise a lipid binding domain. The subcellular localization and interaction of htt containing expanded polyQ domains with membranous surfaces has been well documented and suggests that these interactions play a role in HD pathogenesis. While protein/lipid membrane interactions play an important role in a number of amyloid-related diseases, the protein represents only one of the participants. For example, the size and charge of the lipid headgroups likely influence the interaction between Nt17 and lipid membranes. Any effect on lipid binding would also affect the aggregation and trafficking of htt. Determining factors that regulate the affinity of htt for membranes could not only help understand the normal functions of htt, but could lead to a better understanding of how to modify protein-lipid interaction for therapeutic purposes. As a result, we undertook a series of experiments to determine the role of specific lipids in modulating the htt/lipid interaction and resulting aggregation. Htt aggregation in the presence of POPC, DOPC, DMPC, POPG, POPE, POPS, and total brain lipid extract were investigated via thioflavin T aggregation assays and other biochemical assays. In addition, the modulating effect of cholesterol was also explored.