Mutant Exon1 Huntingtin Aggregation is Regulated by T3 Phosphorylation-Induced Structural Changes and Crosstalk between T3 Phosphorylation and Acetylation at K6.

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.

Reduction of cortical oxygenation in chronic kidney disease: evidence obtained with a new analysis method of blood oxygenation level-dependent magnetic resonance imaging.

BACKGROUND: Determinations of renal oxygenation by blood oxygenation level-dependent magnetic resonance imaging (BOLD-MRI) in chronic kidney disease (CKD) patients have given heterogeneous results, possibly due to the lack of a reproducible method to analyse BOLD-MRI. It therefore remains uncertain whether patients with CKD have a reduced renal tissue oxygenation. We developed a new method to analyse BOLD-MRI signals and applied it to CKD patients and controls. METHODS: MRI was performed under standardized conditions before and 15 min after IV furosemide in 104 CKD patients, 61 hypertensives and 42 controls. MR images were analysed with the new twelve-layer concentric objects method (TLCO) that divides renal parenchyma in 12 layers of equal thickness. The mean R2* value of each layer was reported, along with the change in R2* between successive layers, as measured by the slope steepness of the relevant curve. RESULTS: Inter-observer variability was 2.3 ± 0.9%, 1.9 ± 0.8% and 3.0 ± 2.3% in, respectively, controls, moderate and severe CKD. The mean R2* of the outer (more cortical) layers was significantly higher in CKD, suggesting lower cortical oxygenation as compared with controls. In CKD patients, the response to furosemide was blunted in the inner (more medullary) layers, and the R2* slope was flatter. In multivariable regression analysis, the R2* slope correlated positively with estimated glomerular filtration rate (eGFR) in patients with an eGFR <90 mL/min/1.73 m2 (P < 0.001). CONCLUSIONS: Using the new TLCO method, we confirm the hypothesis that renal cortical oxygenation is reduced in CKD in humans, and that the level of cortical oxygenation correlates with CKD severity.

Renal tissue oxygenation in children with chronic kidney disease due to vesicoureteral reflux.

BACKGROUND: Vesicoureteral reflux (VUR) is a frequent cause of chronic kidney disease (CKD) in children. Using blood oxygenation level-dependent magnetic resonance imaging (BOLD-MRI), we measured cortical and medullary oxygenation in children with CKD due to VUR and compared the results to those obtained on healthy controls. METHOD: The study population comprised 37 children (19 with CKD due to VUR and 18 healthy age-matched controls). BOLD-MRI was performed before and after furosemide treatment. MR images were analyzed with the region-of-interest (ROI) technique to assess the mean R2* values (=1/T2*) of the cortex and medulla of each kidney and with the concentric object (CO) technique that divides renal parenchyma in 12 equal layers. RESULTS: R2* values were significantly lower (corresponding to higher oxygenation) in the cortex and medulla of kidneys of children with CKD due to VUR than in those of the healthy controls (cortex 16.4 ± 1.4 vs. 17.2 ± 1.6 s(-1) , respectively; medulla 28.4 ± 3.2 vs. 30.3 ± 1.9 s(-1) , respectively; P < 0.05), and furosemide-induced changes in medullary R2* were smaller in the former than in the latter (-5.7 ± 3.0 vs. -6.9 ± 3.4 s(-1), respectively; P < 0.05). Similar results were found with the CO technique. In children with a history of unilateral reflux (n = 9), the non-affected contralateral kidneys presented similar R2* values as the diseased kidneys, but their response to furosemide was significantly larger (-7.4 ± 3.2 vs. -5.7 ± 3.0, respectively; P = 0.05). CONCLUSIONS: Chronic kidney disease due to VUR is not associated with kidney tissue hypoxia in children. The significantly larger furosemide-induced decrease in medullary R2* levels in the healthy group and unaffected contralateral kidneys of the VUR group points towards more intense renal sodium transport in these kidneys.

An Intein-based Strategy for the Production of Tag-free Huntingtin Exon 1 Proteins Enables New Insights into the Polyglutamine Dependence of Httex1 Aggregation and Fibril Formation.

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, maltose-binding protein, or thioredoxin, or released in solution upon in situ cleavage of these proteins. Herein, we report an intein-based 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.

Detection of huntingtin exon 1 phosphorylation by Phos-Tag SDS-PAGE: Predominant phosphorylation on threonine 3 and regulation by IKKß.

Expansion of a CAG triplet repeat within the first exon of the HUNTINGTIN gene encoding for a polyglutamine tract is the cause of a progressive neurodegenerative disorder known as Huntington's disease. N-terminal fragments of mutant huntingtin have a strong propensity to form oligomers and aggregates that have been linked to the Huntington's disease pathology by different mechanisms, including gain of toxic functions. The biological and biophysical properties of the polyglutamine expansion within these huntingtin fragments are influenced by neighboring domains, in particular by the first 17 amino acids of huntingtin (N17), which precede the polyglutamine expansion. It has been suggested that N17 phosphorylation modulate mutant huntingtin aggregation and toxicity, but the study of its functional and pathological relevance requires the capacity to detect this modification in biological samples in a simple, robust way, that ideally provides information on the abundance of a phosphorylated species relative to the total pool of the protein of interest. Using a modified SDS-PAGE protocol (Phos-Tag) followed by Western blotting with specific anti-HUNTINGTIN antibodies, we efficiently resolved huntingtin fragments expressed in cellular contexts based on the presence of phosphorylated residues, we defined threonine 3 as the major site of huntingtin N17 phosphorylation and, finally, we identified IKK-beta as a kinase capable of phosphorylating threonine 3 in N-terminal hungtingtin fragments.

One-pot semisynthesis of exon 1 of the Huntingtin protein: new tools for elucidating the role of posttranslational modifications in the pathogenesis of Huntington's disease.

The natural enzymes involved in regulating many of the posttranslational modifications (PTMs) within the first 17 residues (Nt17) of Huntingtin exon 1 (Httex1) remain unknown. A semisynthetic strategy that allows the site-specific introduction of PTMs within Nt17 by using expressed protein ligation (EPL) was developed. This strategy was used to produce untagged wild-type (wt) and T3-phosphorylated (pT3) Httex1 containing 23 glutamine residues (Httex1-23Q). Our studies show that pT3 significantly slows the oligomerization and fibrillization of Httex1-23Q and that Httex1 variants containing polyQ repeats below the pathogenic threshold readily aggregate and form fibrils in vitro. These findings suggest that crossing the polyQ pathogenic threshold is not essential for Httex1 aggregation. The ability to produce wt or site-specifically modified tag-free Httex1 should facilitate determining its structure and the role of N-terminal PTMs in regulating the functions of Htt in health and disease.

Novel mechanistic insight into the molecular basis of amyloid polymorphism and secondary nucleation during amyloid formation.

The formation of amyloid ß (Aß) fibrils is crucial in initiating the cascade of pathological events that culminates in Alzheimer's disease. In this study, we investigated the mechanism of Aß fibril formation from hydrodynamically well defined species under controlled aggregation conditions. We present a detailed mechanistic model that furnishes a novel insight into the process of Aß42 fibril formation and the molecular basis for the different structural transitions in the amyloid pathway. Our data reveal the structure and polymorphism of Aß fibrils to be critically influenced by the oligomeric state of the starting materials, the ratio of monomeric-to-aggregated forms of Aß42 (oligomers and protofibrils), and the occurrence of secondary nucleation. We demonstrate that monomeric Aß42 plays an important role in mediating structural transitions in the amyloid pathway, and for the first time, we provide evidences that Aß42 fibrillization occurs via a combined mechanism of nucleated polymerization and secondary nucleation. These findings will have significant implications to our understanding of the molecular basis of amyloid formation in vivo, of the heterogeneity of Aß pathology (e.g., diffuse versus amyloid plaques), and of the structural basis of Aß toxicity.

Discovery and structure activity relationship of small molecule inhibitors of toxic ß-amyloid-42 fibril formation.

Increasing evidence implicates Aß peptides self-assembly and fibril formation as crucial events in the pathogenesis of Alzheimer disease. Thus, inhibiting Aß aggregation, among others, has emerged as a potential therapeutic intervention for this disorder. Herein, we employed 3-aminopyrazole as a key fragment in our design of non-dye compounds capable of interacting with Aß42 via a donor-acceptor-donor hydrogen bond pattern complementary to that of the ß-sheet conformation of Aß42. The initial design of the compounds was based on connecting two 3-aminopyrazole moieties via a linker to identify suitable scaffold molecules. Additional aryl substitutions on the two 3-aminopyrazole moieties were also explored to enhance π-π stacking/hydrophobic interactions with amino acids of Aß42. The efficacy of these compounds on inhibiting Aß fibril formation and toxicity in vitro was assessed using a combination of biophysical techniques and viability assays. Using structure activity relationship data from the in vitro assays, we identified compounds capable of preventing pathological self-assembly of Aß42 leading to decreased cell toxicity.