Oxidation Promotes Distinct Huntingtin Aggregates in the Presence and Absence of Membranes.

Expansion of a polyglutamine (polyQ) domain within the first exon of the huntingtin (htt) protein is the underlying cause of Huntington's disease, a genetic neurodegenerative disorder. PolyQ expansion triggers htt aggregation into oligomers, fibrils, and inclusions. The 17 N-terminal amino acids (Nt17) of htt-exon1, which directly precede the polyQ domain enhances polyQ fibrillization and functions as a lipid-binding domain. A variety of post-translational modifications occur within Nt17, including oxidation of two methionine residues. Here, the impact of oxidation within Nt17 on htt aggregation both in the presence and absence of lipid membranes was investigated. Treatment with hydrogen peroxide (H2O2) reduced fibril formation in a dose-dependent manner, resulting in shorter fibrils and an increased oligomer population. With excessive H2O2 treatments, fibrils developed a unique morphological feature around their periphery. In the presence of total brain lipid vesicles, H2O2 impacted fibrillization in a similar manner. That is, oligomerization was promoted at the expense of fibril elongation. The interaction of unoxidized and oxidized htt with supported lipid bilayers was directly observed using in situ atomic force microscopy. Without oxidation, granular htt aggregates developed on the bilayer surface. However, in the presence of H2O2, distinct plateau-like regions initially developed on the bilayer surface that gave way to rougher patches containing granular aggregates. Collectively, these observations suggest that oxidation of methionine residues within Nt17 plays a crucial role in both the aggregation of htt and its ability to interact with lipid surfaces.

Mitochondrial membranes modify mutant huntingtin aggregation.

Huntington's disease (HD) is a neurodegenerative disease caused by the expansion of a polyglutamine (polyQ) tract near the N-terminus of the huntingtin (htt) protein. Expanded polyQ tracts are prone to aggregate into oligomers and insoluble fibrils. Mutant htt (mhtt) localizes to variety of organelles, including mitochondria. Specifically, mitochondrial defects, morphological alteration, and dysfunction are observed in HD. Mitochondrial lipids, cardiolipin (CL) in particular, are essential in mitochondria function and have the potential to directly interact with htt, altering its aggregation. Here, the impact of mitochondrial membranes on htt aggregation was investigated using a combination of mitochondrial membrane mimics and tissue-derived mitochondrial-enriched fractions. The impact of exposure of outer and inner mitochondrial membrane mimics (OMM and IMM respectively) to mhtt was explored. OMM and IMM reduced mhtt fibrillization, with IMM having a larger effect. The role of CL in mhtt aggregation was investigated using a simple PC system with varying molar ratios of CL. Lower molar ratios of CL (<5%) promoted fibrillization; however, increased CL content retarded fibrillization. As revealed by in situ AFM, mhtt aggregation and associated membrane morphological changes at the surface of OMM mimics was markedly different compared to IMM mimics. While globular deposits of mhtt with few fibrillar aggregates were observed on OMM, plateau-like domains were observed on IMM. A similar impact on htt aggregation was observed with exposure to purified mitochondrial-enriched fractions. Collectively, these observations suggest mitochondrial membranes heavily influence htt aggregation with implication for HD.

Synthesis and Characterization of Exopolysaccharide Encapsulated PCL/Gelatin Skin Substitute for Full-Thickness Wound Regeneration.

Loss of skin integrity can lead to serious problems and even death. In this study, for the first time, the effect of exopolysaccharide (EPS) produced by cold-adapted yeast R. mucilaginosa sp. GUMS16 on a full-thickness wound in rats was evaluated. The GUMS16 strain's EPS was precipitated by adding cold ethanol and then lyophilized. Afterward, the EPS with polycaprolactone (PCL) and gelatin was fabricated into nanofibers with two single-needle and double-needle procedures. The rats' full-thickness wounds were treated with nanofibers and Hematoxylin and eosin (H&E) and Masson's Trichrome staining was done for studying the wound healing in rats. Obtained results from SEM, DLS, FTIR, and TGA showed that EPS has a carbohydrate chemical structure with an average diameter of 40 nm. Cell viability assessments showed that the 2% EPS loaded sample exhibits the highest cell activity. Moreover, in vivo implantation of nanofiber webs on the full-thickness wound on rat models displayed a faster healing rate when EPS was loaded into a nanofiber. These results suggest that the produced EPS can be used for skin tissue engineering applications.

SUMOylation Prevents Huntingtin Fibrillization and Localization onto Lipid Membranes.

Huntington's disease (HD), a genetic neurodegenerative disease, is caused by an expanded polyglutamine (polyQ) domain in the first exon of the huntingtin protein (htt). PolyQ expansion destabilizes protein structure, resulting in aggregation into a variety of oligomers, protofibrils, and fibrils. Beyond the polyQ domain, adjacent protein sequences influence the aggregation process. Specifically, the first 17 N-terminal amino acids (Nt17) directly preceding the polyQ domain promote the formation of α-helix-rich oligomers that represent intermediate species associated with fibrillization. Due to its propensity to form an amphipathic α-helix, Nt17 also facilitates lipid binding. Three lysine residues (K6, K9, and K15) within Nt17 can be SUMOylated, which modifies htt's accumulation and toxicity within cells in a variety of HD models. The impact of SUMOylation on htt aggregation and direct interaction with lipid membranes was investigated. SUMOylation of htt-exon1 inhibited fibril formation while promoting larger, amorphous aggregate species. These amorphous aggregates were SDS soluble but nonetheless exhibited levels of ß-sheet structure similar to that of htt-exon1 fibrils. In addition, SUMOylation prevented htt binding, aggregation, and accumulation on model lipid bilayers comprised of total brain lipid extract. Collectively, these observations demonstrate that SUMOylation promotes a distinct htt aggregation pathway that may affect htt toxicity.

Lipid Membranes Influence the Ability of Small Molecules To Inhibit Huntingtin Fibrillization.

Several diseases, including Alzheimer's disease, Parkinson's disease, and Huntington's disease (HD), are associated with specific proteins aggregating and depositing within tissues and/or cellular compartments. The aggregation of these proteins is characterized by the formation of extended, ß-sheet rich fibrils, termed amyloid. In addition, a variety of other aggregate species also form, including oligomers and protofibrils. Specifically, HD is caused by the aggregation of the huntingtin (htt) protein that contains an expanded polyglutamine domain. Due to the link between protein aggregation and disease, small molecule aggregation inhibitors have been pursued as potential therapeutic agents. Two such small molecules are epigallocatechin 3-gallate (EGCG) and curcumin, both of which inhibit the fibril formation of several amyloid-forming proteins. However, amyloid formation is a complex process that is strongly influenced by the protein's environment, leading to distinct aggregation pathways. Thus, changes in the protein's environment may alter the effectiveness of aggregation inhibitors. A well-known modulator of amyloid formation is lipid membranes. Here, we investigated if the presence of lipid vesicles altered the ability of EGCG or curcumin to modulate htt aggregation and influence the interaction of htt with lipid membranes. The presence of 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine or total brain lipid extract vesicles prevented the curcumin from inhibiting htt fibril formation. In contrast, EGCG's inhibition of htt fibril formation persisted in the presence of lipids. Collectively, these results highlight the complexity of htt aggregation and demonstrate that the presence of lipid membranes is a key modifier of the ability of small molecules to inhibit htt fibril formation.

Proteins Containing Expanded Polyglutamine Tracts and Neurodegenerative Disease.

Several hereditary neurological and neuromuscular diseases are caused by an abnormal expansion of trinucleotide repeats. To date, there have been 10 of these trinucleotide repeat disorders associated with an expansion of the codon CAG encoding glutamine (Q). For these polyglutamine (polyQ) diseases, there is a critical threshold length of the CAG repeat required for disease, and further expansion beyond this threshold is correlated with age of onset and symptom severity. PolyQ expansion in the translated proteins promotes their self-assembly into a variety of oligomeric and fibrillar aggregate species that accumulate into the hallmark proteinaceous inclusion bodies associated with each disease. Here, we review aggregation mechanisms of proteins with expanded polyQ-tracts, structural consequences of expanded polyQ ranging from monomers to fibrillar aggregates, the impact of protein context and post-translational modifications on aggregation, and a potential role for lipid membranes in aggregation. As the pathogenic mechanisms that underlie these disorders are often classified as either a gain of toxic function or loss of normal protein function, some toxic mechanisms associated with mutant polyQ tracts will also be discussed.