Structural Basis of Huntingtin Fibril Polymorphism Revealed by Cryogenic Electron Microscopy of Exon 1 HTT Fibrils.

The lack of detailed insight into the structure of aggregates formed by the huntingtin protein (HTT) has hampered the efforts to develop therapeutics and diagnostics targeting pathology formation in the brain of patients with Huntington's disease. To address this knowledge gap, we investigated the structural properties of in vitro-generated fibrils from exon1 of the huntingtin protein by cryogenic electron microscopy and single-particle analyses. We show that wildtype and mutant exon1 of the huntingtin protein form nonhelical fibrils with a polyglutamine amyloid core composed of β-hairpins with unique characteristics that have not been previously observed with other amyloid filaments.

Pharmacological characterization of mutant huntingtin aggregate-directed PET imaging tracer candidates.

Huntington's disease (HD) is caused by a CAG trinucleotide repeat expansion in the first exon of the huntingtin (HTT) gene coding for the huntingtin (HTT) protein. The misfolding and consequential aggregation of CAG-expanded mutant HTT (mHTT) underpin HD pathology. Our interest in the life cycle of HTT led us to consider the development of high-affinity small-molecule binders of HTT oligomerized/amyloid-containing species that could serve as either cellular and in vivo imaging tools or potential therapeutic agents.

Investigating Crosstalk Among PTMs Provides Novel Insight Into the Structural Basis Underlying the Differential Effects of Nt17 PTMs on Mutant Httex1 Aggregation.

Post-translational modifications (PTMs) within the first 17 amino acids (Nt17) of the Huntingtin protein (Htt) have been shown to inhibit the aggregation and attenuate the toxicity of mutant Htt proteins and in various models of Huntington's disease. Here, we expand on these studies by investigating the effect of methionine eight oxidation (oxM8) and its crosstalk with lysine 6 acetylation (AcK6) or threonine 3 phosphorylation (pT3) on the aggregation of mutant Httex1 (mHttex1). We show that M8 oxidation delays but does not inhibit the aggregation and has no effect on the final morphologies of mHttex1aggregates.

Site-Specific Phosphorylation of Huntingtin Exon 1 Recombinant Proteins Enabled by the Discovery of Novel Kinases.

Post-translational modifications (PTMs) within the first 17 amino acids (Nt17) of exon 1 of the Huntingtin protein (Httex1) play important roles in modulating its cellular properties and functions in health and disease. In particular, phosphorylation of threonine and serine residues (T3, S13, and/or S16) has been shown to inhibit Htt aggregation in vitro and inclusion formation in cellular and animal models of Huntington's disease (HD). In this paper, we describe a new and simple methodology for producing milligram quantities of highly pure wild-type or mutant Httex1 proteins that are site-specifically phosphorylated at T3 or at both S13 and S16.

Extent of N-terminus exposure of monomeric alpha-synuclein determines its aggregation propensity.

As an intrinsically disordered protein, monomeric alpha-synuclein (aSyn) occupies a large conformational space. Certain conformations lead to aggregation prone and non-aggregation prone intermediates, but identifying these within the dynamic ensemble of monomeric conformations is difficult. Herein, we used the biologically relevant calcium ion to investigate the conformation of monomeric aSyn in relation to its aggregation propensity.

Phosphorylation of the overlooked tyrosine 310 regulates the structure, aggregation, and microtubule- and lipid-binding properties of Tau.

The microtubule-associated protein Tau is implicated in the pathogenesis of several neurodegenerative disorders, including Alzheimer's disease. Increasing evidence suggests that post-translational modifications play critical roles in regulating Tau's normal functions and its pathogenic properties in tauopathies. Very little is known about how phosphorylation of tyrosine residues influences the structure, aggregation, and microtubule- and lipid-binding properties of Tau.

A simple, versatile and robust centrifugation-based filtration protocol for the isolation and quantification of α-synuclein monomers, oligomers and fibrils: Towards improving experimental reproducibility in α-synuclein research.

Increasing evidence suggests that the process of alpha-synuclein (α-syn) aggregation from monomers into amyloid fibrils and Lewy bodies, via oligomeric intermediates plays an essential role in the pathogenesis of different synucleinopathies, including Parkinson's disease (PD), multiple system atrophy and dementia with Lewy bodies (DLB). However, the nature of the toxic species and the mechanisms by which they contribute to neurotoxicity and disease progression remain elusive. Over the past two decades, significant efforts and resources have been invested in studies aimed at identifying and targeting toxic species along the pathway of α-syn fibrillization.

Chronic corticosterone aggravates behavioral and neuronal symptomatology in a mouse model of alpha-synuclein pathology.

Debilitating, yet underinvestigated nonmotor symptoms related to mood/emotion, such as depression, are common in Parkinson's disease. Here, we explore the role of depression and of the amygdala, a brain region robustly linked to mood/emotion, in synucleinopathy. We hypothesized that mood/emotional deficits might accelerate Parkinson's disease-linked symptomatology, including the formation of α-synuclein pathology.

Generation of Native, Untagged Huntingtin Exon1 Monomer and Fibrils Using a SUMO Fusion Strategy.

Huntington's Disease (HD) is an inherited fatal neurodegenerative disease caused by a CAG expansion (≥36) in the first exon of the HD gene, resulting in the expression of the Huntingtin protein (Htt) or N-terminal fragments thereof with an expanded polyglutamine (polyQ) stretch. The exon1 of the Huntingtin protein (Httex1) is the smallest Htt fragment that recapitulates many of the features of HD in cellular and animal models and is one of the most widely studied fragments of Htt. The small size of Httex1 makes it experimentally more amenable to biophysical characterization using standard and high-resolution techniques in comparison to longer fragments or full-length Htt.

Exploring the role of post-translational modifications in regulating α-synuclein interactions by studying the effects of phosphorylation on nanobody binding.

Intracellular deposits of α-synuclein in the form of Lewy bodies are major hallmarks of Parkinson's disease (PD) and a range of related neurodegenerative disorders. Post-translational modifications (PTMs) of α-synuclein are increasingly thought to be major modulators of its structure, function, degradation and toxicity. Among these PTMs, phosphorylation near the C-terminus at S129 has emerged as a dominant pathogenic modification as it is consistently observed to occur within the brain and cerebrospinal fluid (CSF) of post-mortem PD patients, and its level appears to correlate with disease progression.

Phosphorylation of huntingtin at residue T3 is decreased in Huntington's disease and modulates mutant huntingtin protein conformation.

Posttranslational modifications can have profound effects on the biological and biophysical properties of proteins associated with misfolding and aggregation. However, their detection and quantification in clinical samples and an understanding of the mechanisms underlying the pathological properties of misfolding- and aggregation-prone proteins remain a challenge for diagnostics and therapeutics development. We have applied an ultrasensitive immunoassay platform to develop and validate a quantitative assay for detecting a posttranslational modification (phosphorylation at residue T3) of a protein associated with polyglutamine repeat expansion, namely Huntingtin, and characterized its presence in a variety of preclinical and clinical samples.

Elucidating the Role of Site-Specific Nitration of α-Synuclein in the Pathogenesis of Parkinson's Disease via Protein Semisynthesis and Mutagenesis.

Parkinson's disease (PD) is characterized by the loss of dopaminergic neurons in the substantia nigra and the presence of intraneuronal inclusions consisting of aggregated and post-translationally modified α-synuclein (α-syn). Despite advances in the chemical synthesis of α-syn and other proteins, the generation of site-specifically nitrated synthetic proteins has not been reported. Consequently, it has not been possible to determine the roles of nitration at specific residues in regulating the physiological and pathogenic properties of α-syn.