Showing papers on "Proteotoxicity published in 2009"

Genetic evidence linking age-dependent attenuation of the 26S proteasome with the aging process.

Abstract: The intracellular accumulation of unfolded or misfolded proteins is believed to contribute to aging and age-related neurodegenerative diseases. However, the links between age-dependent proteotoxicity and cellular protein degradation systems remain poorly understood. Here, we show that 26S proteasome activity and abundance attenuate with age, which is associated with the impaired assembly of the 26S proteasome with the 19S regulatory particle (RP) and the 20S proteasome. In a genetic gain-of-function screen, we characterized Rpn11, which encodes a subunit of the 19S RP, as a suppressor of expanded polyglutamine-induced progressive neurodegeneration. Rpn11 overexpression suppressed the age-related reduction of the 26S proteasome activity, resulting in the extension of flies' life spans with suppression of the age-dependent accumulation of ubiquitinated proteins. On the other hand, the loss of function of Rpn11 caused an early onset of reduced 26S proteasome activity and a premature age-dependent accumulation of ubiquitinated proteins. It also caused a shorter life span and an enhanced neurodegenerative phenotype. Our results suggest that maintaining the 26S proteasome with age could extend the life span and suppress the age-related progression of neurodegenerative diseases.

Proteasomal Regulation of the Hypoxic Response Modulates Aging in C.elegans

Abstract: The Caenorhabditis elegans von Hippel–Lindau tumor suppressor homolog VHL-1 is a cullin E3 ubiquitin ligase that negatively regulates the hypoxic response by promoting ubiquitination and degradation of the hypoxic response transcription factor HIF-1. Here, we report that loss of VHL-1 significantly increased life span and enhanced resistance to polyglutamine and b-amyloid toxicity. Deletion of HIF-1 was epistatic to VHL-1, indicating that HIF-1 acts downstream of VHL-1 to modulate aging and proteotoxicity. VHL-1 and HIF-1 control longevity by a mechanism distinct from both dietary restriction and insulin-like signaling. These findings define VHL-1 and the hypoxic response as an alternative longevity and protein homeostasis pathway.

Protein Homeostasis and Aging: Taking Care of Proteins From the Cradle to the Grave

Abstract: All cells count on precise mechanisms that regulate protein homeostasis to maintain a stable and functional proteome. Alterations in these fine-tuned mechanisms underlie the pathogenesis of severe human diseases including, among others, common neurodegenerative disorders such as Alzheimer's or Parkinson's disease. A progressive deterioration in the ability of cells to preserve the stability of their proteome occurs with age, even in the absence of disease, and it likely contributes to different aspects of “normal” aging. A group of experts in different aspects of the biology of aging met recently to discuss the implications of altered protein homeostasis in aging, the current gaps in our understanding of the mechanisms responsible for proteome maintenance, and future opportunities for discovery in this area. We summarize here some of the key topics and main outcomes of the discussions.

Analysis of genomic dose-response information on arsenic to inform key events in a mode of action for carcinogenicity.

Abstract: A comprehensive literature search was conducted to identify information on gene expression changes following exposures to inorganic arsenic compounds. This information was organized by compound, exposure, dose/concentration, species, tissue, and cell type. A concentration-related hierarchy of responses was observed, beginning with changes in gene/protein expression associated with adaptive responses (e.g., preinflammatory responses, delay of apoptosis). Between 0.1 and 10 microM, additional gene/protein expression changes related to oxidative stress, proteotoxicity, inflammation, and proliferative signaling occur along with those related to DNA repair, cell cycle G2/M checkpoint control, and induction of apoptosis. At higher concentrations (10-100 microM), changes in apoptotic genes dominate. Comparisons of primary cell results with those obtained from immortalized or tumor-derived cell lines were also evaluated to determine the extent to which similar responses are observed across cell lines. Although immortalized cells appear to respond similarly to primary cells, caution must be exercised in using gene expression data from tumor-derived cell lines, where inactivation or overexpression of key genes (e.g., p53, Bcl-2) may lead to altered genomic responses. Data from acute in vivo exposures are of limited value for evaluating the dose-response for gene expression, because of the transient, variable, and uncertain nature of tissue exposure in these studies. The available in vitro gene expression data, together with information on the metabolism and protein binding of arsenic compounds, provide evidence of a mode of action for inorganic arsenic carcinogenicity involving interactions with critical proteins, such as those involved in DNA repair, overlaid against a background of chemical stress, including proteotoxicity and depletion of nonprotein sulfhydryls. The inhibition of DNA repair under conditions of toxicity and proliferative pressure may compromise the ability of cells to maintain the integrity of their DNA.

Hsps and aging

Abstract: Heat-shock proteins (Hsps) are increasingly being implicated in aging phenotypes and control of life span across species. They are targets of the conserved heat-shock factor and insulin/IGF1-like signaling pathways that affect life span and aging phenotypes. Hsps are expressed in tissue-specific and disease-specific patterns during aging, and their level of expression and induction by stress correlates with and, in some instances, predicts life span. In model organisms, Hsps have been shown to increase life span and ameliorate aging-associated proteotoxicity. Finally, Hsps have emerged as key components in regulating aging-related cellular phenotypes, including cell senescence, apoptosis and cancer. The Hsps, therefore, provide a metric of individual stress and aging and are potential targets for interventions in aging and aging-related diseases.

AIP-1 ameliorates β-amyloid peptide toxicity in a Caenorhabditis elegans Alzheimer's disease model

Abstract: Multiple neurodegenerative diseases are causally linked to aggregation-prone proteins. Cellular mechanisms involving protein turnover may be key defense mechanisms against aggregating protein disorders. We have used a transgenic Caenorhabditis elegans Alzheimer's disease model to identify cellular responses to proteotoxicity resulting from expression of the human beta amyloid peptide (Aβ). We show up-regulation of aip-1 in Aβ-expressing animals. Mammalian homologues of AIP-1 have been shown to associate with, and regulate the function of, the 26S proteasome, leading us to hypothesize that induction of AIP-1 may be a protective cellular response directed toward modulating proteasomal function in response to toxic protein aggregation. Using our transgenic model, we show that overexpression of AIP-1 protected against, while RNAi knockdown of AIP-1 exacerbated, Aβ toxicity. AIP-1 overexpression also reduced accumulation of Aβ in this model, which is consistent with AIP-1 enhancing protein degradation. Transgenic expression of one of the two human aip-1 homologues (AIRAPL), but not the other (AIRAP), suppressed Aβ toxicity in C. elegans, which advocates the biological relevance of the data to human biology. Interestingly, AIRAPL and AIP-1 contain a predicted farnesylation site, which is absent from AIRAP. This farnesylation site was shown by others to be essential for an AIP-1 prolongevity function. Consistent with this, we show that an AIP-1 mutant lacking the predicted farnesylation site failed to protect against Aβ toxicity. Our results implicate AIP-1 in the regulation of protein turnover and protection against Aβ toxicity and point at AIRAPL as the functional mammalian homologue of AIP-1.

Molecular chaperones antagonize proteotoxicity by differentially modulating protein aggregation pathways.

Abstract: The self-association of misfolded or damaged proteins into ordered amyloid-like aggregates characterizes numerous neurodegenerative disorders. Insoluble amyloid plaques are diagnostic of many disease states. Yet soluble, oligomeric intermediates in the aggregation pathway appear to represent the toxic culprit. Molecular chaperones regulate the fate of misfolded proteins and thereby influence their aggregation state. Chaperones conventionally antagonize aggregation of misfolded, disease proteins and assist in refolding or degradation pathways. Recent work suggests that chaperones may also suppress neurotoxicity by converting toxic, soluble oligomers into benign aggregates. Chaperones can therefore suppress or promote aggregation of disease proteins to ameliorate the proteotoxic accumulation of soluble, assembly intermediates.

Reciprocal efficiency of RNQ1 and polyglutamine detoxification in the cytosol and nucleus.

Abstract: Onset of proteotoxicity is linked to change in the subcellular location of proteins that cause misfolding diseases. Yet, factors that drive changes in disease protein localization and the impact of residence in new surroundings on proteotoxicity are not entirely clear. To address these issues, we examined aspects of proteotoxicity caused by Rnq1-green fluorescent protein (GFP) and a huntingtin's protein exon-1 fragment with an expanded polyglutamine tract (Htt-103Q), which is dependent upon the intracellular presence of [RNQ+] prions. Increasing heat-shock protein 40 chaperone activity before Rnq1-GFP expression, shifted Rnq1-GFP aggregation from the cytosol to the nucleus. Assembly of Rnq1-GFP into benign amyloid-like aggregates was more efficient in the nucleus than cytosol and nuclear accumulation of Rnq1-GFP correlated with reduced toxicity. [RNQ+] prions were found to form stable complexes with Htt-103Q, and nuclear Rnq1-GFP aggregates were capable of sequestering Htt-103Q in the nucleus. On accumulation in the nucleus, conversion of Htt-103Q into SDS-resistant aggregates was dramatically reduced and Htt-103Q toxicity was exacerbated. Alterations in activity of molecular chaperones, the localization of intracellular interaction partners, or both can impact the cellular location of disease proteins. This, in turn, impacts proteotoxicity because the assembly of proteins to a benign state occurs with different efficiencies in the cytosol and nucleus.

A kinetic assessment of the C. elegans amyloid disaggregation activity enables uncoupling of disassembly and proteolysis

Abstract: Protein aggregation is a common feature of late onset neurodegenerative disorders, including Alzheimer's disease In Alzheimer's disease, misassembly of the Abeta peptide is genetically linked to proteotoxicity associated with disease etiology A reduction in Abeta proteotoxicity is accomplished, in part, by the previously reported Abeta disaggregation and proteolysis activities-under partial control of heat shock factor 1, a transcription factor regulating proteostasis in the cytosol and negatively regulated by insulin growth factor signaling Herein, we report an improved in vitro assay to quantify recombinant fibrillar Abeta disaggregation kinetics accomplished by the exogenous application of Celegans extracts With this assay we demonstrate that the Abeta disaggregation and proteolysis activities of Celegans are separable The disaggregation activity found in Celegans preparations is more heat resistant than the proteolytic activity Abeta disaggregation in the absence of proteolysis was found to be a reversible process Future discovery of the molecular basis of the disaggregation and proteolysis activities offers the promise of delaying the age-onset proteotoxicity that leads to neurodegeneration in a spectrum of maladies

Caenorhabditis elegans Model for Initial Screening and Mechanistic Evaluation of Potential New Drugs for Aging and Alzheimer’s Disease

Abstract: Although the worms may not be universally accepted as having a direct relevance for AD pathology, they are well suited for validation of target Aβ toxicity in vivo [12,21]. The absence of endogenous Aβ production in the worms offers an opportunity to find a direct role of the Aβ involvement in pathological behaviors [22]. In addition, predominantly intracellular expression of Aβ provides another tool to address specific roles of intracellular Aβ in relation to its toxicity. Substantial evidence implicates intracellular Aβ oligomers in early events related to AD [16]. Intracellular Aβ has also been observed in human brain neurons [23] and in triple transgenic AD mouse models, where its accumulation preceded neurofibrillary tangle formation [24]. This evidence supports the notion that Aβ toxicity assayed in the worm model reflects Aβ toxicity in mammalian neurons. A recent study indicates that the transgenic C. elegans model may be generally relevant to the proteotoxicity underlying neurodegenerative diseases [25]. Additionally, the strain has been used to investigate the role of insulin-like signaling and heat-shock factor in Aβ proteotoxicity [26,27], providing excellent examples for the relevance of the C. elegans model to AD.There are several advantages of C. elegans over the mouse model for initial drug screening and target characterization. First, there are highly conserved biochemical pathways between worms and humans. Second, established transgenic mutant linking of human Aβ expression with pathological behavioral phenotypes are easy to score. The worms have a relatively low cost of cultivation because of their small size, rapid life cycle, and short life span [28], which allow screening of thousands of animals over multiple generations on microtiter plates. The simple structure of its nervous system, consisting of only 302 neurons in an adult nematode, makes it valuable for screening drugs against age-associated neurodegeneration and the ease of genetic manipulations, which is evident in the availability of mutants and application of RNA interference (RNAi) knockdown. Several examples illustrate the power of C. elegans in screening for new drugs [29], including many known human drugs [30,31]. Some lead molecules originating from worm-based screening assays are in advanced stages of drug discovery [11].Using the C. elegans model in the past years, we have uncovered effects of natural compounds on extension of the worms’ life span; [32] on a stress response protein, the small heat-shock protein hsp-16.2; [33] on age-related behavioral declines; [34] on muscle degeneration; [35] and on Aβ-expression-induced pathological behaviors [22]. Most of those experiments would be difficult and might be impossible to perform in mice. In this chapter, we describe methods we have employed for compound screening and pharmacological evaluations of potential AD drugs using the C. elegans model.

NAD+ availability and proteotoxicity.

Abstract: It has been shown that NAD+ availability is important for neuronal survival following ischemia (Liu et al., Neuromolecular Med 11:28–42, 2009). It is proposed here that NAD+ may also control proteotoxicity by influencing both formation and catabolism of altered proteins. It is suggested that low NAD+ availability promotes synthesis of methylglyoxal (MG) which can induce formation of glycated proteins, ROS, and dysfunctional mitochondria. That glyoxalase overexpression and carnosine are both protective against MG and ischemic injury support this proposal. Recognition and elimination of altered proteins is enhanced by NAD+ through effects on stress protein expression and autophagy.

[Genetic approach for understanding of the late-onset mechanisms of polyglutamine disease].

Abstract: The intracellular accumulation of unfolded or misfolded proteins is believed to contribute to aging and age-related neurodegenerative diseases. However, the links between age-dependent proteotoxicity and cellular protein degradation systems remain poorly understood. Here, we show that 26S proteasome activity and abundance attenuate with age, which is associated with the impaired assembly of the 26S proteasome with the 19S regulatory particle (RP) and the 20S proteasome. In a genetic gain-of-function screen using Drosophila, we characterized Rpn11, which encodes a subunit of the 19S RP, as a suppressor of expanded polyglutamine-induced progressive neurodegeneration. Rpn11 overexpression suppressed the age-related reduction of the 26S proteasome activity, resulting in the extension of flies' life spans with suppression of the age-dependent accumulation of ubiquitinated proteins. On the other hand, the loss of function of Rpn11 caused an early onset of reduced 26S proteasome activity and a premature age-dependent accumulation of ubiquitinated proteins. It also caused a shorter life span and an enhanced neurodegenerative phenotype. Our results suggest that maintaining the 26S proteasome with age could extend the life span and suppress the age-related progression of polyglutamine diseases.