ABSTRACT
The huntingtin exon 1 proteins with a polyglutamine repeat in the pathological range (51 or 83 glutamines), but not with a polyglutamine tract in the normal range (20 glutamines), form aggresome-like perinuclear inclusions in human 293 Tet-Off cells. These structures contain aggregated, ubiquitinated huntingtin exon 1 protein with a characteristic fibrillar morphology. Inclusion bodies with truncated huntingtin protein are formed at centrosomes and are surrounded by vimentin filaments. Inhibition of proteasome activity resulted in a twofold increase in the amount of ubiquitinated, SDS-resistant aggregates, indicating that inclusion bodies accumulate when the capacity of the ubiquitin-proteasome system to degrade aggregation-prone huntingtin protein is exhausted. Immunofluorescence and electron microscopy with immunogold labeling revealed that the 20S, 19S, and 11S subunits of the 26S proteasome, the molecular chaperones BiP/GRP78, Hsp70, and Hsp40, as well as the RNA-binding protein TIA-1, the potential chaperone 14-3-3, and alpha-synuclein colocalize with the perinuclear inclusions. In 293 Tet-Off cells, inclusion body formation also resulted in cell toxicity and dramatic ultrastructural changes such as indentations and disruption of the nuclear envelope. Concentration of mitochondria around the inclusions and cytoplasmic vacuolation were also observed. Together these findings support the hypothesis that the ATP-dependent ubiquitin-proteasome system is a potential target for therapeutic interventions in glutamine repeat disorders.
Subject(s)
Acetylcysteine/analogs & derivatives , Heat-Shock Proteins , Inclusion Bodies/metabolism , Mutation , Nerve Tissue Proteins/metabolism , Nuclear Proteins/metabolism , Peptide Fragments/metabolism , Proteins , 14-3-3 Proteins , Acetylcysteine/pharmacology , Carrier Proteins/metabolism , Cell Line , Cysteine Endopeptidases/metabolism , Cysteine Proteinase Inhibitors/pharmacology , Endoplasmic Reticulum Chaperone BiP , Exons , Humans , Huntingtin Protein , Huntington Disease/metabolism , Immunoblotting , Inclusion Bodies/ultrastructure , Membrane Proteins/metabolism , Microscopy, Fluorescence , Models, Biological , Molecular Chaperones/metabolism , Multienzyme Complexes/antagonists & inhibitors , Multienzyme Complexes/metabolism , Nerve Tissue Proteins/genetics , Nuclear Proteins/genetics , Peptide Fragments/genetics , Poly(A)-Binding Proteins , Proteasome Endopeptidase Complex , RNA-Binding Proteins/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Synucleins , T-Cell Intracellular Antigen-1 , Transgenes , Tyrosine 3-Monooxygenase/metabolism , Vimentin/metabolism , alpha-SynucleinABSTRACT
The accumulation of insoluble protein aggregates in intra and perinuclear inclusions is a hallmark of Huntington's disease (HD) and related glutamine-repeat disorders. A central question is whether protein aggregation plays a direct role in the pathogenesis of these neurodegenerative diseases. Here we show by using a filter retardation assay that the mAb 1C2, which specifically recognizes the elongated polyglutamine (polyQ) stretch in huntingtin, and the chemical compounds Congo red, thioflavine S, chrysamine G, and Direct fast yellow inhibit HD exon 1 protein aggregation in a dose-dependent manner. On the other hand, potential inhibitors of amyloid-beta formation such as thioflavine T, gossypol, melatonin, and rifampicin had little or no inhibitory effect on huntingtin aggregation in vitro. The results obtained by the filtration assay were confirmed by electron microscopy, SDS/PAGE, and MS. Furthermore, cell culture studies revealed that the Congo red dye at micromolar concentrations reduced the extent of HD exon 1 aggregation in transiently transfected COS cells. Together, these findings contribute to a better understanding of the mechanism of huntingtin fibrillogenesis in vitro and provide the basis for the development of new huntingtin aggregation inhibitors that may be effective in treating HD.
Subject(s)
Antibodies, Monoclonal/therapeutic use , Huntington Disease/therapy , Nerve Tissue Proteins/antagonists & inhibitors , Nuclear Proteins/antagonists & inhibitors , Peptides/antagonists & inhibitors , Animals , Benzoates/pharmacology , Benzothiazoles , Biphenyl Compounds/pharmacology , COS Cells , Congo Red/pharmacology , Gossypol/pharmacology , Humans , Huntingtin Protein , Melatonin/pharmacology , Rifampin/pharmacology , Thiazoles/pharmacologyABSTRACT
To gain an insight into vertebrate genome evolution, we have analysed the organization of an approximately 40-kb genomic clone of an amphioxus (Branchiostoma floridae) cosmid library. Amphioxus is considered as being the last non-vertebrate relative to vertebrates. Sequencing and analysis of the above clone using three different exon prediction programs (Grail, GenScan, Mzef) have led to the identification of a gene of the aldo-keto reductase family as well as further exons that gave a significant database match to known genes.
Subject(s)
Alcohol Oxidoreductases/genetics , Chordata, Nonvertebrate/genetics , Aldehyde Reductase , Aldo-Keto Reductases , Amino Acid Sequence , Animals , Base Sequence , Chromosome Mapping , Cloning, Molecular , Cosmids/genetics , Molecular Sequence Data , Repetitive Sequences, Nucleic Acid , Sequence AlignmentABSTRACT
A prerequisite to understanding the evolution of the human X chromosome is the analysis of synteny of X-linked genes in different species. We have focused on the spermine synthase gene in human Xp22. 1. We show that whereas the human gene spans a genomic region of 54 kb, the Fugu rubripes gene is encompassed in a 4.7-kb region. However, we could not find conserved synteny between this region of human Xp22 and the equivalent F. rubripes region. A cosmid clone containing the F. rubripes gene does not contain other X-linked genes. Instead we identified homologs of human genes that are autosomally localized: the ryanodine receptor type I (RYRI), which is implicated in malignant hyperthermia and central core disease, and the HE6 gene. Comparison of the F. rubripes, Tetraodon fluviatilis, mouse, human, and Danio rerio 5'UTRs of spermine synthase highlights conserved sequences potentially involved in regulation. Interestingly, pseudogenes of this gene that are present in the human and mouse genomes seem to be absent in the compact F. rubripes genome. Analysis of a D. rerio PAC clone containing spermine synthase shows an intermediate genomic size in this fish. Sequence analysis of this PAC clone did not reveal other known genes: neither the RYRI gene, nor the HE6 gene, nor other human Xp22 genes were identified.
