Insulin-Degrading Enzyme Efficiently Degrades polyQ Peptides but not Expanded polyQ Huntingtin Fragments.

Background: Huntington's disease is an inheritable autosomal dominant disorder caused by an expanded CAG trinucleotide repeat within the Huntingtin gene, leading to a polyglutamine (polyQ) expansion in the mutant protein. Objective: A potential therapeutic approach for delaying or preventing the onset of the disease involves enhancing the degradation of the aggregation-prone polyQ-expanded N-terminal mutant huntingtin (mHTT) exon1 fragment. A few proteases and peptidases have been identified that are able to cleave polyQ fragments with low efficiency. This study aims to identify a potent polyQ-degrading endopeptidase. Methods: Here we used quenched polyQ peptides to identify a polyQ-degrading endopeptidase. Next we investigated its role on HTT turnover, using purified polyQ-expanded HTT fragments and striatal cells expressing mHTT exon1 peptides. Results: We identified insulin-degrading enzyme (IDE) as a novel endopeptidase for degrading polyQ peptides. IDE was, however, ineffective in reducing purified polyQ-expanded HTT fragments. Similarly, in striatal cells expressing mHTT exon1 peptides, IDE did not enhance mHTT turnover. Conclusions: This study shows that despite IDE's efficiency in degrading polyQ peptides, it does not contribute to the direct degradation of polyQ-expanded mHTT fragments.

Reduction in PA28αß activation in HD mouse brain correlates to increased mHTT aggregation in cell models.

Huntington's disease is an autosomal dominant heritable disorder caused by an expanded CAG trinucleotide repeat at the N-terminus of the Huntingtin (HTT) gene. Lowering the levels of soluble mutant HTT protein prior to aggregation through increased degradation by the proteasome would be a therapeutic strategy to prevent or delay the onset of disease. Native PAGE experiments in HdhQ150 mice and R6/2 mice showed that PA28αß disassembles from the 20S proteasome during disease progression in the affected cortex, striatum and hippocampus but not in cerebellum and brainstem. Modulating PA28αß activated proteasomes in various in vitro models showed that PA28αß improved polyQ degradation, but decreased the turnover of mutant HTT. Silencing of PA28αß in cells lead to an increase in mutant HTT aggregates, suggesting that PA28αß is critical for overall proteostasis, but only indirectly affects mutant HTT aggregation.

Identification of Full-Length Wild-Type and Mutant Huntingtin Interacting Proteins by Crosslinking Immunoprecipitation in Mice Brain Cortex.

BACKGROUND: Huntington's disease is a neurodegenerative disorder caused by a CAG expansion in the huntingtin gene, resulting in a polyglutamine expansion in the ubiquitously expressed mutant huntingtin protein. OBJECTIVE: Here we set out to identify proteins interacting with the full-length wild-type and mutant huntingtin protein in the mice cortex brain region to understand affected biological processes in Huntington's disease pathology. METHODS: Full-length huntingtin with 20 and 140 polyQ repeats were formaldehyde-crosslinked and isolated via their N-terminal Flag-tag from 2-month-old mice brain cortex. Interacting proteins were identified and quantified by label-free liquid chromatography-mass spectrometry (LC-MS/MS). RESULTS: We identified 30 interactors specific for wild-type huntingtin, 14 interactors specific for mutant huntingtin and 14 shared interactors that interacted with both wild-type and mutant huntingtin, including known interactors such as F8a1/Hap40. Syt1, Ykt6, and Snap47, involved in vesicle transport and exocytosis, were among the proteins that interacted specifically with wild-type huntingtin. Various other proteins involved in energy metabolism and mitochondria were also found to associate predominantly with wild-type huntingtin, whereas mutant huntingtin interacted with proteins involved in translation including Mapk3, Eif3h and Eef1a2. CONCLUSION: Here we identified both shared and specific interactors of wild-type and mutant huntingtin, which are involved in different biological processes including exocytosis, vesicle transport, translation and metabolism. These findings contribute to the understanding of the roles that wild-type and mutant huntingtin play in a variety of cellular processes both in healthy conditions and Huntington's disease pathology.

Development of a Light-Dependent Protein Histidine Kinase.

Phosphorylation plays a critical role in facilitating signal transduction in prokaryotic and eukaryotic organisms. Our study introduces a tool for investigation of signal diffusion in a biochemical regulation network through the design and characterization of a light-stimulated histidine kinase that consists of the LOV domain from YtvA from Bacillus subtilis and the histidine kinase domain Sln1 from Saccharomyces cerevisiae. We show that blue light can be used as a trigger for modulation of the phosphorylation events in this engineered two-component signal transduction pathway in a eukaryotic cell. At the same time, we demonstrate the robustness of LOV domains and their utility for designing fusion proteins for signal transduction that can be triggered with (blue) light, providing a ready toolkit to design blue light dependent two-component signalling pathways.

Global Proteome and Ubiquitinome Changes in the Soluble and Insoluble Fractions of Q175 Huntington Mice Brains.

Huntington's disease is caused by a polyglutamine repeat expansion in the huntingtin protein which affects the function and folding of the protein, and results in intracellular protein aggregates. Here, we examined whether this mutation leads to altered ubiquitination of huntingtin and other proteins in both soluble and insoluble fractions of brain lysates of the Q175 knock-in Huntington's disease mouse model and the Q20 wild-type mouse model. Ubiquitination sites are detected by identification of Gly-Gly (diGly) remnant motifs that remain on modified lysine residues after digestion. We identified K6, K9, K132, K804, and K837 as endogenous ubiquitination sites of soluble huntingtin, with wild-type huntingtin being mainly ubiquitinated at K132, K804, and K837. Mutant huntingtin protein levels were strongly reduced in the soluble fraction whereas K6 and K9 were mainly ubiquitinated. In the insoluble fraction increased levels of huntingtin K6 and K9 diGly sites were observed for mutant huntingtin as compared with wild type. Besides huntingtin, proteins with various roles, including membrane organization, transport, mRNA processing, gene transcription, translation, catabolic processes and oxidative phosphorylation, were differently expressed or ubiquitinated in wild-type and mutant huntingtin brain tissues. Correlating protein and diGly site fold changes in the soluble fraction revealed that diGly site abundances of most of the proteins were not related to protein fold changes, indicating that these proteins were differentially ubiquitinated in the Q175 mice. In contrast, both the fold change of the protein level and diGly site level were increased for several proteins in the insoluble fraction, including ubiquitin, ubiquilin-2, sequestosome-1/p62 and myo5a. Our data sheds light on putative novel proteins involved in different cellular processes as well as their ubiquitination status in Huntington's disease, which forms the basis for further mechanistic studies to understand the role of differential ubiquitination of huntingtin and ubiquitin-regulated processes in Huntington's disease.

Design, characterization and in vivo functioning of a light-dependent histidine protein kinase in the yeast Saccharomyces cerevisiae.

Helical alignment of the α-helical linker of the LOV (light-oxygen-voltage) domain of YtvA from Bacillus subtilis with the α-helical linker of the histidine-protein kinase domain of the Sln1 kinase of the phospho-relay system for osmoregulation of Saccharomyces cerevisiae has been used to construct a light-modulatable histidine protein kinase. In vitro, illumination with blue light inhibits both the ATP-dependent phosphorylation of this hybrid kinase, as well as the phosphoryl transfer to Ypd1, the phosphoryl transfer domain of the Sln1 system. The helical alignment was carried out with conservation of the complete Jα helix of YtvA, as well as of the phosphorylatable histidine residue of the Sln1 kinase, with conservation of the hepta-helical motive of coiled-coil structures, recognizable in the helices of the two separate, constituent, proteins. Introduction of the gene encoding this hybrid histidine protein kinase into cells of S. cerevisiae in which the endogenous Sln1 kinase had been deleted, allowed us to modulate gene expression in the yeast cells with (blue) light. This was first demonstrated via the light-induced alteration of the expression level of the mannosyl-transferase OCH1, via a translational-fusion approach. As expected, illumination decreased the expression level of OCH1; the steady state decrease in saturating levels of blue light was about 40%. To visualize the in vivo functionality of this light-dependent regulation system, we fused the green fluorescent protein (GFP) to another regulatory protein, HOG1, which is also responsive to the Sln1 kinase. HOG1 is phosphorylated by the MAP-kinase-kinase Pbs2, which in turn is under control of the Sln1 kinase, via the phosphoryl transfer domain Ypd1. Fluorescence microscopy was used to show that illumination of cells that contained the combination of the hybrid kinase and the HOG1::GFP fusion protein, led to a persistent increase in the level of nuclear accumulation of HOG1, in contrast to salt stress, which-as expected-showed the well-characterized transient response. The system described in this study will be valuable in future studies on the role of cytoplasmic diffusion in signal transduction in eukaryotic cells.

On the in vivo redox state of flavin-containing photosensory receptor proteins.

Measured values of the redox midpoint potential of flavin-containing photoreceptor proteins range from physiologically very negative values, i.e., < -300 mV (compared to the calomel electrode) for some LOV domains, to slightly positive values for some cryptochromes. The actual intracellular redox potential of several key physiological electron-transfer intermediates, like the nicotinamide dinucleotides, particularly in chemoheterotrophic bacteria, may be varying beyond these two values, and are subject to physiological- and environmental regulation. The photochemical activity of photoreceptor proteins containing their flavin chromophore in the reduced, and in the fully oxidized form, is very different. We therefore have addressed the question whether or not the functioning of these flavin-containing photosensory receptors in vivo is subject to redox regulation. Here we (1) provide further evidence for the overlap of the ranges of the redox midpoint potential of the flavin in a specific photoreceptor protein and the redox potential of key intracellular redox-active metabolites, and (2) demonstrate that the redox state and photochemical activity of LOV domains can be recorded in vivo in Escherichia coli. Significantly, so far in vivo reduction of LOV domains under physiological conditions could not be detected. The implications of these observations are discussed.

Antimicrobial effect of a modified vanadium chloroperoxidase on Enterococcus faecalis biofilms at root canal pH.

INTRODUCTION: Previous research showed an antimicrobial effect of vanadium chloroperoxidase (VCPO) on in vitro Enterococcus faecalis biofilms. The current study aimed to optimize the use of this enzyme at the root canal pH using a modified VCPO (mVCPO) that was adapted to function at a higher pH and to explore the biocompatibility of mVCPO. METHODS: The activity of the original and modified VCPO was assessed using the monochlorodimedone assay. For antimicrobial assessment, 48-hour biofilms of E. faecalis OS-16 were incubated 5 or 30 minutes with mVCPO, bromide, and hydrogen peroxide, and colony-forming units were determined. A metabolic activity assay was used to evaluate the cytotoxic effect of mVCPO on oral fibroblasts. RESULTS: Reaction products generated by mVCPO at a root canal pH of 7.7 significantly inactivated the biofilm after 5 minutes and even more after 30 minutes (Mann-Whitney U test, P < .05). The mVCPO reaction products showed less cytotoxic effects than control solutions and 0.5% sodium hypochlorite (Kruskal-Wallis test, P < .05). CONCLUSIONS: The incubation of mVCPO in the presence of its substrates with in vitro E. faecalis biofilms showed a significant antimicrobial effect at the root canal pH. Also, cytotoxicity tests showed preliminary biocompatibility. Therefore, an interappointment dressing containing mVCPO could aid in improving current endodontic treatment through continuous and local generation of antimicrobials.

Effect of vanadium chloroperoxidase on Enterococcus faecalis biofilms.

INTRODUCTION: The aim of this study was to explore the antimicrobial effect of vanadium chloroperoxidase (VCPO) reaction products on Enterococcus faecalis biofilms of 4 different strains. METHODS: Twenty-four-hour biofilms of E. faecalis strains V583, ER5/1, E2, and OS-16 were incubated in mixtures with VCPO, halide (either bromide or chloride), and hydrogen peroxide. The antibacterial efficacy was assessed by colony-forming unit counts. RESULTS: The VCPO reaction products had a similar efficacy in reducing the viability of the 4 strains of E. faecalis (94%; range, 87%-100%). Bromide as the halogen of choice was more effective on E. faecalis strains E2 and OS-16, as compared with chloride (Mann-Whitney U test; P < .05). Despite different quantities of produced biofilms by the 4 strains, VCPO treatment was similarly effective toward all strains (Kruskal-Wallis test; P < .05). CONCLUSIONS: VCPO treatment results in an antimicrobial effect toward in vitro E. faecalis biofilms and might provide an addition to current endodontic treatment, possibly as an antimicrobial dressing.