Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.25.1 (proteasome)
28,817 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Huntington's disease (HD) is an inherited neurodegenerative disorder caused by polyglutamine (polyQ) expansions in the huntingtin (Ht) protein. A hallmark of HD is the proteolytic production of an N-terminal fragment of Ht, containing the polyQ repeat, that forms aggregates in the nucleus and cytoplasm of affected neurons. Proteins with longer polyQ repeats aggregate more rapidly and cause disease at an earlier age, but the mechanism of aggregation and its relationship to disease remain unclear. To provide a new, genetically tractable model system for the study of Ht, we engineered yeast cells to express an N-terminal fragment of Ht with different polyQ repeat lengths of 25, 47, 72, or 103 residues, fused to green fluorescent protein. The extent of aggregation varied with the length of the polyQ repeat: at the two extremes, most HtQ103 protein coalesced into a single large cytoplasmic aggregate, whereas HtQ25 exhibited no sign of aggregation. Mutations that inhibit the ubiquitin/proteasome pathway at three different steps had no effect on the aggregation of Ht fragments in yeast, suggesting that the ubiquitination of Ht previously noted in mammalian cells may not inherently be required for polyQ length-dependent aggregation. Changing the expression levels of a wide variety of chaperone proteins in yeast neither increased nor decreased Ht aggregation. However, Sis1, Hsp70, and Hsp104 overexpression modulated aggregation of HtQ72 and HtQ103 fragments. More dramatically, the deletion of Hsp104 virtually eliminated it. These observations establish yeast as a system for studying the causes and consequences of polyQ-dependent Ht aggregation.
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PMID:Aggregation of huntingtin in yeast varies with the length of the polyglutamine expansion and the expression of chaperone proteins. 1067 4

Huntington's disease (HD), spinocerebellar ataxias types 1 and 3 (SCA1, SCA3), and spinobulbar muscular atrophy (SBMA) are caused by CAG/polyglutamine expansion mutations. A feature of these diseases is ubiquitinated intraneuronal inclusions derived from the mutant proteins, which colocalize with heat shock proteins (HSPs) in SCA1 and SBMA and proteasomal components in SCA1, SCA3, and SBMA. Previous studies suggested that HSPs might protect against inclusion formation, because overexpression of HDJ-2/HSDJ (a human HSP40 homologue) reduced ataxin-1 (SCA1) and androgen receptor (SBMA) aggregate formation in HeLa cells. We investigated these phenomena by transiently transfecting part of huntingtin exon 1 in COS-7, PC12, and SH-SY5Y cells. Inclusion formation was not seen with constructs expressing 23 glutamines but was repeat length and time dependent for mutant constructs with 43-74 repeats. HSP70, HSP40, the 20S proteasome and ubiquitin colocalized with inclusions. Treatment with heat shock and lactacystin, a proteasome inhibitor, increased the proportion of mutant huntingtin exon 1-expressing cells with inclusions. Thus, inclusion formation may be enhanced in polyglutamine diseases, if the pathological process results in proteasome inhibition or a heat-shock response. Overexpression of HDJ-2/HSDJ did not modify inclusion formation in PC12 and SH-SY5Y cells but increased inclusion formation in COS-7 cells. To our knowledge, this is the first report of an HSP increasing aggregation of an abnormally folded protein in mammalian cells and expands the current understanding of the roles of HDJ-2/HSDJ in protein folding.
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PMID:Effects of heat shock, heat shock protein 40 (HDJ-2), and proteasome inhibition on protein aggregation in cellular models of Huntington's disease. 1071 3

Polyglutamine expansions in proteins are implicated in at least eight inherited neurodegenerative disorders, including Huntington's disease. These mutant proteins can form aggregates within the nucleus and processes of neurons possibly due to misfolding of the proteins. Polyglutamine aggregates are ubiquitinated and sequester molecular chaperone proteins and proteasome components. To investigate other protein components of polyglutamine aggregates, cerebral cortex and striata from patients with Huntington's disease and full-length cDNA transgenic mouse models for this disease were examined immunohistochemically for alpha-synuclein reactivity. Our findings demonstrate that alpha-synuclein can be used as a marker for huntingtin polyglutamine aggregates in both human and mice. Moreover in the HD transgenic mice, the intensity of immunoreactivity increases with age. The significance of recruitment of alpha-synuclein into huntingtin aggregates and its translocation away from the synapses remains to be determined. We propose that aberrant interaction of mutant huntingtin with other proteins, including alpha-synuclein, may influence disease progression.
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PMID:Alpha-synuclein immunoreactivity of huntingtin polyglutamine aggregates in striatum and cortex of Huntington's disease patients and transgenic mouse models. 1089 1

An expansion of polyglutamines in the N terminus of huntingtin causes Huntington's disease (HD) and results in the accrual of mutant protein in the nucleus and cytoplasm of affected neurons. How mutant huntingtin causes neurons to die is unclear, but some recent observations suggest that an autophagic process may occur. We showed previously that huntingtin markedly accumulates in endosomal-lysosomal organelles of affected HD neurons and, when exogenously expressed in clonal striatal neurons, huntingtin appears in cytoplasmic vacuoles causing cells to shrink. Here we show that the huntingtin-enriched cytoplasmic vacuoles formed in vitro internalized the lysosomal enzyme cathepsin D in proportion to the polyglutamine-length in huntingtin. Huntingtin-labeled vacuoles displayed the ultrastructural features of early and late autophagosomes (autolysosomes), had little or no overlap with ubiquitin, proteasome, and heat shock protein 70/heat shock cognate 70 immunoreactivities, and altered the arrangement of Golgi membranes, mitochondria, and nuclear membranes. Neurons with excess cytoplasmic huntingtin also exhibited increased tubulation of endosomal membranes. Exogenously expressed human full-length wild-type and mutant huntingtin codistributed with endogenous mouse huntingtin in soluble and membrane fractions, whereas human N-terminal huntingtin products were found only in membrane fractions that contained lysosomal organelles. We speculate that mutant huntingtin accumulation in HD activates the endosomal-lysosomal system, which contributes to huntingtin proteolysis and to an autophagic process of cell death.
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PMID:Huntingtin expression stimulates endosomal-lysosomal activity, endosome tubulation, and autophagy. 1100 84

Huntington's disease (HD) is caused by an expansion of the CAG repeat that encodes polyglutamine in huntingtin. Transient expression of an N-terminal huntingtin fragment containing an expanded polyglutamine tract induced formation of protein aggregates in cultured cells. The turnover of protein components in such aggregates has been difficult to study because of their insolubility in aqueous solutions. Here we describe a method of solubilizing the aggregates and quantifying their protein components. Insoluble pellets were collected from COS7 cells expressing an N-terminal huntingtin fragment containing an expanded polyglutamine tract and subjected to treatment with various detergent, acid, and alkaline reagents. Treatment with 100% formic acid at 37 degrees C for 30 min induced essentially complete dissociation of the aggregates to monomer. We used this solubilization technique to quantify huntingtin fusion protein in the aggregates formed in transient expression experiments. The frequency of aggregate formation increased when the proteasome inhibitor beta-lactone was added to culture media. However, the total amount of accumulated huntingtin fusion protein did not differ between cells cultured with or without beta-lactone. These results suggest that other protein components which are degraded by the proteasome, in addition to huntingtin, might be related to the dynamics of polyglutamine protein aggregates.
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PMID:Formic acid dissolves aggregates of an N-terminal huntingtin fragment containing an expanded polyglutamine tract: applying to quantification of protein components of the aggregates. 1103 34

Expansion of CAG repeats within the coding region of target genes is the cause of several autosomal dominant neurodegenerative diseases including Huntington's disease (HD). A hallmark of HD is the proteolytic production of N-terminal fragments of huntingtin containing polyglutamine repeats that form ubiquitinated aggregates in the nucleus and cytoplasm of the affected neurons. In this study, we used an ecdysone-inducible stable mouse neuro2a cell line that expresses truncated N-terminal huntingtin (tNhtt) with different polyglutamine length, along with mice transgenic for HD exon 1, to demonstrate that the ubiquitin-proteasome pathway is involved in the pathogenesis of HD. Proteasomal 20S core catalytic component was redistributed to the polyglutamine aggregates in both the cellular and transgenic mouse models. Proteasome inhibitor dramatically increased the rate of aggregate formation caused by tNhtt protein with 60 glutamine (60Q) repeats, but had very little influence on aggregate formation by tNhtt protein with 150Q repeats. Both normal and polyglutamine-expanded tNhtt proteins were degraded by proteasome, but the rate of degradation was inversely proportional to the repeat length. The shift of the proteasomal components from the total cellular environment to the aggregates, as well as the comparatively slower degradation of tNhtt with longer polyglutamine, decreased the proteasome's availability for degrading other key target proteins, such as p53. This altered proteasomal function was associated with disrupted mitochondrial membrane potential, released cytochrome c from mitochondria into the cytosol and activated caspase-9- and caspase-3-like proteases. These results suggest that the impaired proteasomal function plays an important role in polyglutamine protein-induced cell death.
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PMID:Altered proteasomal function due to the expression of polyglutamine-expanded truncated N-terminal huntingtin induces apoptosis by caspase activation through mitochondrial cytochrome c release. 1133 15

Abnormal proteins, which escape chaperone-mediated refolding or proteasome-dependent degradation, aggregate and form inclusion bodies (IBs). In several neurodegenerative diseases, such IBs can be formed by proteins with expanded polyglutamine (polyQ) domains (e.g., huntingtin). This work studies the regulation of intracellular IB formation using an NH(2)-terminal fragment of huntingtin with expanded polyQ domain. We demonstrate that the active form of MEKK1, a protein kinase that regulates several stress-activated signaling cascades, stimulates formation of the IBs. This function of MEKK1 requires kinase activity, as the kinase-dead mutant of MEKK1 cannot stimulate this process. Exposure of cells to UV irradiation or cisplatin, both of which activate MEKK1, also augmented the formation of IBs. The polyQ-containing huntingtin fragment exists in cells in two distinct forms: (a) in a discrete soluble complex, and (b) in association with insoluble fraction. MEKK1 strongly stimulated recruitment of polyQ polypeptides into the particulate fraction. Notably, a large portion of the active form of MEKK1 was associated with the insoluble fraction, concentrating in discrete sites, and polyQ-containing IBs always colocalized with them. We suggest that MEKK1 is involved in a process of IB nucleation. MEKK1 also stimulated formation of IBs with two abnormal polypeptides lacking the polyQ domain, indicating that this kinase has a general effect on protein aggregation.
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PMID:Intracellular aggregation of polypeptides with expanded polyglutamine domain is stimulated by stress-activated kinase MEKK1. 1135 44

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.
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PMID:Accumulation of mutant huntingtin fragments in aggresome-like inclusion bodies as a result of insufficient protein degradation. 1135 30

Intracellular deposition of aggregated and ubiquitylated proteins is a prominent cytopathological feature of most neurodegenerative disorders. Whether protein aggregates themselves are pathogenic or are the consequence of an underlying molecular lesion is unclear. Here, we report that protein aggregation directly impaired the function of the ubiquitin-proteasome system. Transient expression of two unrelated aggregation-prone proteins, a huntingtin fragment containing a pathogenic polyglutamine repeat and a folding mutant of cystic fibrosis transmembrane conductance regulator, caused nearly complete inhibition of the ubiquitin-proteasome system. Because of the central role of ubiquitin-dependent proteolysis in regulating fundamental cellular events such as cell division and apoptosis, our data suggest a potential mechanism linking protein aggregation to cellular disregulation and cell death.
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PMID:Impairment of the ubiquitin-proteasome system by protein aggregation. 1137 12

Neuronal intranuclear inclusions are a histopathological hallmark of Huntington's disease. Nevertheless, the precise mechanism by which they are formed and their relevance to neuronal cell death and/or dysfunction remains unclear. We recently generated a conditional mouse model of Huntington's disease (HD94) in which silencing expression of mutated huntingtin led to the disappearance of intranuclear aggregates and amelioration of the behavioral phenotype. Here, we analyze primary striatal neuronal cultures from HD94 mice to explore the dynamics of aggregate formation and reversal, the possible mechanisms involved, and the correlation between aggregates and neuronal death. In parallel, we examine symptomatic adult HD94 mice in similar studies and explored the relationship between aggregate clearance and behavioral reversal. We report that, in culture, aggregate formation and reversal were rapid processes, such that 2 d of transgene expression led to aggregate formation, and 5 d of transgene suppression led to aggregate disappearance. In mice, full reversal of aggregates and intranuclear mutant huntingtin was more rapid than reported previously and preceded the motor recovery by several weeks. Furthermore, the proteasome inhibitor lactacystin inhibited the aggregate clearance observed in culture, thus indicating that aggregate formation is a balance between the rate of huntingtin synthesis and its degradation by the proteasome. Finally, neither expression of the mutant huntingtin nor aggregates compromised the viability of HD94 cultures. This correlated with the lack of cell death in symptomatic HD94 mice, thus demonstrating that neuronal dysfunction, and not cell loss, triggered by mutant huntingtin underlies symptomatology.
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PMID:Proteasomal-dependent aggregate reversal and absence of cell death in a conditional mouse model of Huntington's disease. 1169 89


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