The presence of expanded poly-glutamine (polyQ) repeats in proteins is directly linked to the pathogenesis of several neurodegenerative diseases, including Huntington’s disease. However, the molecular and structural basis underlying the increased toxicity of aggregates formed by proteins containing expanded polyQ repeats remain poorly understood, in part due to the size and morphological heterogeneity of the aggregates they form in vitro. To address this knowledge gap and technical limitations, we investigated the structural, mechanical and morphological properties of fibrillar aggregates at the single molecule and nanometer scale using the first exon of the Huntingtin protein as a model system (Exon1). Our findings demonstrate a direct correlation of the morphological and mechanical properties of Exon1 aggregates with their structural organization at the single aggregate and nanometric scale and provide novel insights into the molecular and structural basis of Huntingtin Exon1 aggregation and toxicity.

Herein, we used protein semisynthesis to investigate, for the first time, the effect of lysine acetylation and phosphorylation, as well as the crosstalk between these modifications on the structure and aggregation of mutant huntingtin exon1 (Httex1). Our results demonstrate that phosphorylation at T3 stabilizes the α-helical conformation of the N-terminal 17 amino acids (Nt17) and significantly inhibits the aggregation of mutant Httex1. Acetylation of single lysine residues, K6, K9 or K15, had no effect on Httex1 aggregation. Interestingly, acetylation at K6, but not at K9 or K15, reversed the inhibitory effect of T3 phosphorylation. Together, our results provide novel insight into the role of Nt17 post-translational modifications in regulating the structure and aggregation of Httex1 and suggest that its aggregation and possibly its function(s) are controlled by regulatory mechanisms involving crosstalk between different PTMs.

Increasing evidence suggests that amyloid polymorphism gives rise to different strains of amyloids with distinct toxicities and pathology-spreading properties. Validating this hypothesis is challenging due to a lack of tools and methods that allow for the direct characterization of amyloid polymorphism in hydrated and complex biological samples. Here, we report on the development of 11-mercapto-1-undecanesulfonate-coated gold nanoparticles (NPs) that efficiently label the edges of synthetic, recombinant, and native amyloid fibrils derived from different amyloidogenic proteins. We demonstrate that these NPs represent powerful tools for assessing amyloid morphological polymorphism, using cryogenic transmission electron microscopy (cryo-EM). The NPs allowed for the visualization of morphological features that are not directly observed using standard imaging techniques, including transmission electron microscopy with use of the negative stain or cryo-EM imaging. The use of these NPs to label native paired helical filaments (PHFs) from the postmortem brain of a patient with Alzheimer’s disease, as well as amyloid fibrils extracted from the heart tissue of a patient suffering from systemic amyloid light-chain amyloidosis, revealed a high degree of homogeneity across the fibrils derived from human tissue in comparison with fibrils aggregated in vitro. These findings are consistent with, and strongly support, the emerging view that the physiologic milieu is a key determinant of amyloid fibril strains. Together, these advances should not only facilitate the profiling and characterization of amyloids for structural studies by cryo-EM, but also pave the way to elucidate the structural basis of amyloid strains and toxicity, and possibly the correlation between the pathological and clinical heterogeneity of amyloid diseases.

The first exon of the Huntingtin protein (Httex1) is one of the most actively studied Htt fragments because its overexpression in R6/2 transgenic mice has been shown to recapitulate several key features of Huntington disease. However, the majority of biophysical studies of Httex1 are based on assessing the structure and aggregation of fusion constructs where Httex1 is fused to large proteins, such as glutathione S-transferase, maltosebinding protein, or thioredoxin, or released in solution upon in situ cleavage of these proteins. Herein, we report an inteinbased strategy that allows, for the first time, the rapid and efficient production of native tag-free Httex1 with polyQ repeats ranging from 7Q to 49Q. Aggregation studies on these proteins enabled us to identify interesting polyQ-length-dependent effects on Httex1 oligomer and fibril formation that were previously not observed using Httex1 fusion proteins or Httex1 proteins produced by in situ cleavage of fusion proteins. Our studies revealed the inability of Httex1-7Q/15Q to undergo amyloid fibril formation and an inverse correlation between fibril length and polyQ repeat length, suggesting possible polyQ length-dependent differences in the structural properties of the Httex1 aggregates. Altogether, our findings underscore the importance of working with tag-free Httex1 proteins and indicate that model systems based on non-native Httex1 sequences may not accurately reproduce the effect of polyQ repeat length and solution conditions on Httex1 aggregation kinetics and structural properties. Huntington disease (HD)2 is a fatal neurodegenerative disorder caused by a CAG expansion within the first exon (Exon 1) of the huntingtin gene, IT15 (1). The CAG repeat length ranges between 6 and 35 in healthy subjects, whereas patients with HD exhibit lengths of 36 or greater resulting in the synthesis of a mutant Huntingtin protein (Htt) with an expanded polyglutamine (polyQ) domain (2). The length of the polyQ tract directly correlates with disease severity and is inversely correlated with disease age of onset (3). HD patients suffer from motor impairments, cognitive decline, and depression due to neurodegeneration in the striatum and cortex (4 -8).The accumulation of N-terminal fragments of mutant Htt in the nucleus and cytoplasm of striatal neurons led to the hypothesis that these fragments play causative roles in the neurodegeneration and pathology observed in HD (9 -13). This hypothesis is supported by the observation that the overexpression of the expanded Exon 1 in R6/2 transgenic mice recapitulates several key symptoms of HD (14). Subsequent studies demonstrated that aberrant splicing in HttQ150 knock-in mice gave rise to a short mRNA, which translates into the Huntingtin Exon 1 protein (Httex1), thus linking the genetic cause of HD to the generation of a highly toxic N-terminal Htt fragment (15). Together, these findings highlight the importance of this region in HD pathogenesis and underscore the critical importance of investigating the structure, aggregation, a...

Loss of function mutations in the PINK1 kinase are causal for autosomal recessive Parkinson disease (PD) whilst gain of function mutations in the LRRK2 kinase cause autosomal dominant PD. PINK1 indirectly regulates the phosphorylation of a subset of Rab GTPases at a conserved Serine111 (Ser111) residue within the SF3 motif. Using genetic code expansion technologies we have produced stoichiometric Ser111-phosphorylated Rab8A revealing impaired interactions with its cognate guanine nucleotide exchange factor (GEF) and GTPase activating protein (GAP). In a screen for Rab8A kinases we identify TAK1 and MST3 kinases that can efficiently phosphorylate the Switch II residue Threonine72 (Thr72) in a similar manner as LRRK2. Strikingly we demonstrate that Ser111 phosphorylation negatively regulates the ability of LRRK2 but not MST3 or TAK1 to phosphorylate Thr72 in vitro and demonstrate an interplay of PINK1-and LRRK2-mediated phosphorylation of Rab8A in cells. Finally, we present the crystal structure of Ser111-phosphorylated Rab8A and NMR structure of Ser111phosphorylated Rab1B that does not demonstrate any major changes suggesting that the phosphorylated SF3 motif may disrupt effector-Switch II interactions. Overall, we demonstrate antagonistic regulation between PINK1-dependent Ser111 phosphorylation and LRRK2-mediated Thr72 phosphorylation of Rab8A suggesting that small molecule activators of PINK1 may have therapeutic potential in patients harbouring LRRK2 mutations. Materials and MethodsReagents [γ-32 P] ATP was from PerkinElmer, MLi-2 was obtained from Merck [30]. LRRK2 (G2019S) Recombinant Human Protein (residues 970-2527 #10499963) was purchased from Invitrogen and all additional kinases used in the in vitro screen were from MRC PPU Reagents and Services -including TAK1 (1-303), TAB1 (437-504) fusion DU753 and MST3 (1-431) DU number: 30889 N-terminal GST. All mutagenesis was carried out using the QuikChange site-directed mutagenesis method (Stratagene) with KOD polymerase (Novagen). All DNA constructs were verified by DNA sequencing, which was performed by The Sequencing Service, School of Life Sciences, University of Dundee, using DYEnamic ET terminator chemistry (Amersham Biosciences) on Applied Biosystems automated DNA sequencers. DNA for bacterial protein expression was transformed into E. coli BL21 DE3 RIL (codon plus) cells (Stratagene). All cDNA plasmids, antibodies and recombinant proteins generated for this study are available to request through our reagents website https://mrcppureagents.dundee.ac.uk/ or from the Itzen laboratory. Expression of non-phosphorylated (WT) Rab proteinsWT-Rab1B(3-174) was expressed in fusion with a N-terminal His6-MBP tag (pMAL) or a C-terminal His6 tag (pNHD), while WT-Rab8A(6-176) was fused to a N-terminal His6 tag or a C-terminal His6 tag (pET19). The N-terminal Rab1B and Rab8A fusion constructs contained a TEV cleavage site to remove the His6-MBP tag or His6 tag from the N-terminus of the Rab proteins. The C-terminal His6 tags remained on the Rab proteins. All WT-Rab pro...

GTPases act as key regulators of many cellular processes by switching between active (GTP-bound) and inactive (GDP-bound) states. In many cases, understanding their mode of action has been aided by artificially stabilizing one of these states either by designing mutant proteins or by complexation with non-hydrolysable GTP analogues. Because of inherent disadvantages in these approaches, we have developed acryl-bearing GTP and GDP derivatives that can be covalently linked with strategically placed cysteines within the GTPase of interest. Binding studies with GTPase-interacting proteins and X-ray crystallography analysis demonstrate that the molecular properties of the covalent GTPase–acryl–nucleotide adducts are a faithful reflection of those of the corresponding native states and are advantageously permanently locked in a defined nucleotide (that is active or inactive) state. In a first application, in vivo experiments using covalently locked Rab5 variants provide new insights into the mechanism of correct intracellular localization of Rab proteins.

Loss of function mutations in the PTEN-induced kinase 1 (PINK1) kinase are causal for autosomal recessive Parkinson's disease (PD) whilst gain of function mutations in the LRRK2 kinase cause autosomal dominant PD. PINK1 indirectly regulates the phosphorylation of a subset of Rab GTPases at a conserved Serine111 (Ser111) residue within the SF3 motif. Using genetic code expansion technologies, we have produced stoichiometric Ser111-phosphorylated Rab8A revealing impaired interactions with its cognate guanine nucleotide exchange factor and GTPase activating protein. In a screen for Rab8A kinases we identify TAK1 and MST3 kinases that can efficiently phosphorylate the Switch II residue Threonine72 (Thr72) in a similar manner as LRRK2 in vitro. Strikingly, we demonstrate that Ser111 phosphorylation negatively regulates the ability of LRRK2 but not MST3 or TAK1 to phosphorylate Thr72 of recombinant nucleotide-bound Rab8A in vitro and demonstrate an interplay of PINK1- and LRRK2-mediated phosphorylation of Rab8A in transfected HEK293 cells. Finally, we present the crystal structure of Ser111-phosphorylated Rab8A and nuclear magnetic resonance structure of Ser111-phosphorylated Rab1B. The structures reveal that the phosphorylated SF3 motif does not induce any major changes, but may interfere with effector-Switch II interactions through intramolecular H-bond formation and/or charge effects with Arg79. Overall, we demonstrate antagonistic regulation between PINK1-dependent Ser111 phosphorylation and LRRK2-mediated Thr72 phosphorylation of Rab8A indicating a potential cross-talk between PINK1-regulated mitochondrial homeostasis and LRRK2 signalling that requires further investigation in vivo.

Herein, we used protein semisynthesis to investigate, for the first time, the effect of lysine acetylation and phosphorylation, as well as the crosstalk between these modifications on the structure and aggregation of mutant huntingtin exon1 (Httex1). Our results demonstrate that phosphorylation at T3 stabilizes the α-helical conformation of the N-terminal 17 amino acids (Nt17) and significantly inhibits the aggregation of mutant Httex1. Acetylation of single lysine residues, K6, K9 or K15, had no effect on Httex1 aggregation. Interestingly, acetylation at K6, but not at K9 or K15, reversed the inhibitory effect of T3 phosphorylation. Together, our results provide novel insight into the role of Nt17 posttranslational modifications in regulating the structure and aggregation of Httex1 and suggest that its aggregation and possibly its function(s) are controlled by regulatory mechanisms involving crosstalk between different PTMs. Main textSeveral lines of evidence suggest that the first N-terminal 17 amino acids of the Huntingtin protein (Htt) play important roles in modulating Htt structure, oligomerization, fibrils formation, membrane binding, subcellular localization and interactions with other proteins. [1] This, combined with the fact that this short N-terminal sequence harbors several residues that are subjected to diverse posttranslational modifications (PTMs), including phosphorylation, acetylation, ubiquitination and sumoylation (Scheme S1-A in the Supporting Information), suggests that PTMs may serve as reversible molecular switches for regulating Htt structure, interactome, cellular properties and toxicity. [2] However, the lack of knowledge about the enzymes involved in regulating these modifications has made it difficult to decipher their role in regulating the function(s) of Htt in health and disease.To address these limitations and enable deciphering of the Nt17 PTM code, our group has pursued the development of semisynthetic strategies that permit site-specific introduction of single or multiple PTMs within Nt17 of Huntingtin exon 1 (Httex1). Recently, we reported a semisynthetic strategy that permits site-specific phosphorylation at T3.[3] However, this strategy suffered from several limitations and was only suitable for introducing PTMs in the first N-terminal 9 residues of wild-type Httex1 (≤23 glutamine residues) (Scheme S2). [3] To allow investigations of all Nt17 PTMs, we developed and optimized two new modular semisynthetic protocols that permit the generation of wild-type and for the first time mutant Httex1 (mHttex1) proteins containing single or multiple PTMs (Scheme S3). The first strategy (Scheme S3-A) is only suitable for introducing PTMs in the N-terminal 9 residues of Httex1, whereas the second strategy allows the investigation of all the PTMs within the Nt17 domain. Additionally, the optimized conditions enabled the semisynthesis of mutant Httex1 (full details about these semisynthetic strategies are given in the Supporting Information). This optimized strategy enabled us, for...