[Show abstract][Hide abstract] ABSTRACT: Protein conformational diseases exhibit complex pathologies linked to numerous molecular defects. Aggregation of a disease-associated protein causes the misfolding and aggregation of other proteins, but how this interferes with diverse cellular pathways is unclear. Here, we show that aggregation of neurodegenerative disease-related proteins (polyglutamine, huntingtin, ataxin-1, and superoxide dismutase-1) inhibits clathrin-mediated endocytosis (CME) in mammalian cells by aggregate-driven sequestration of the major molecular chaperone heat shock cognate protein 70 (HSC70), which is required to drive multiple steps of CME. CME suppression was also phenocopied by HSC70 RNAi depletion and could be restored by conditionally increasing HSC70 abundance. Aggregation caused dysregulated AMPA receptor internalization and also inhibited CME in primary neurons expressing mutant huntingtin, showing direct relevance of our findings to the pathology in neurodegenerative diseases. We propose that aggregate-associated chaperone competition leads to both gain-of-function and loss-of-function phenotypes as chaperones become functionally depleted from multiple clients, leading to the decline of multiple cellular processes. The inherent properties of chaperones place them at risk, contributing to the complex pathologies of protein conformational diseases.
Full-text · Article · Apr 2014 · Proceedings of the National Academy of Sciences
[Show abstract][Hide abstract] ABSTRACT: Resveratrol (3,5,4'-trihydroxylstilbene) has been proposed to elicit a variety of positive health effects including protection against cancer and cardiovascular disease. The highest affinity target of resveratrol identified so far is the oxidoreductase enzyme quinone reductase 2 (QR2), which is believed to function in metabolic reduction and detoxification processes; however, evidence exists linking QR2 to the metabolic activation of quinones, which can lead to cell toxicity. Therefore, inhibition of QR2 by resveratrol may protect cells against reactive intermediates and eventually cancer. With the aim of identifying novel inhibitors of QR2, we designed, synthesized, and tested two generations of resveratrol analogue libraries for inhibition of QR2. In addition, X-ray crystal structures of six of the resveratrol analogues in the active site of QR2 were determined. Several novel inhibitors of QR2 were successfully identified as well as a compound that inhibits QR2 with a novel binding orientation.
[Show abstract][Hide abstract] ABSTRACT: The cellular heat shock response (HSR) protects cells from toxicity associated with defective protein folding, and this pathway
is widely viewed as a potential pharmacological target to treat neurodegenerative diseases linked to protein aggregation.
Here we show that the HSR is not activated by mutant huntingtin (HTT) even in cells selected for the highest expression levels
and for the presence of inclusion bodies containing aggregated protein. Surprisingly, HSR activation by HSF1 overexpression
or by administration of a small molecule activator lowers the concentration threshold at which HTT forms inclusion bodies
in cells expressing aggregation-prone, polyglutamine-expanded fragments of HTT. These data suggest that the HSR does not mitigate
inclusion body formation.
[Show abstract][Hide abstract] ABSTRACT: Huntington's disease (HD) typifies a class of inherited neurodegenerative disorders in which a CAG expansion in a single gene leads to an extended polyglutamine tract and misfolding of the expressed protein, driving cumulative neural dysfunction and degeneration. HD is invariably fatal with symptoms that include progressive neuropsychiatric and cognitive impairments, and eventual motor disability. No curative therapies yet exist for HD and related polyglutamine diseases; therefore, substantial efforts have been made in the drug discovery field to identify potential drug and drug target candidates for disease-modifying treatment. In this context, we review here a range of early-stage screening approaches based in in vitro, cellular, and invertebrate models to identify pharmacological and genetic modifiers of polyglutamine aggregation and induced neurodegeneration. In addition, emerging technologies, including high-content analysis, three-dimensional culture models, and induced pluripotent stem cells are increasingly being incorporated into drug discovery screening pipelines for protein misfolding disorders. Together, these diverse screening strategies are generating novel and exciting new probes for understanding the disease process and for furthering development of therapeutic candidates for eventual testing in the clinical setting.
No preview · Article · May 2013 · Journal of the American Society for Experimental NeuroTherapeutics
[Show abstract][Hide abstract] ABSTRACT: Protein misfolding and aggregation are widely implicated in anincreasing number of human diseases providing for new therapeutic opportunities targeting protein homeostasis (proteostasis). The cellular response to proteotoxicity is highly regulated by stress signaling pathways, molecular chaperones, transport and clearance machineriesthatfunction as a proteostasis network (PN) to protect thestability and functional properties of the proteome. Consequently, the PN is essential at the cellular and organismal level for development and lifespan. However, when challenged during aging, stress, and disease, the folding and clearance machineries can become compromised leading to both gain-of-function and loss-of-function proteinopathies. Here, we assess the role of small molecules that activate the heat shock response, the unfolded protein response, and clearance mechanismsto increase PN capacity and protect cellular proteostasis against proteotoxicity. We propose that this strategy to enhance cell stress pathways and chaperone activity establishes a cytoprotective state against misfolding and/or aggregation and represents a promising therapeutic avenue to prevent the cellular damage associated with the variety of protein conformational diseases.
Full-text · Article · Jan 2013 · Current topics in medicinal chemistry
[Show abstract][Hide abstract] ABSTRACT: Protein homeostasis (proteostasis) is essential for cellular and organismal health. Stress, aging and the chronic expression of misfolded proteins, however, challenge the proteostasis machinery and the vitality of the cell. Enhanced expression of molecular chaperones, regulated by heat shock transcription factor-1 (HSF-1), has been shown to restore proteostasis in a variety of conformational disease models, suggesting this mechanism as a promising therapeutic approach. We describe the results of a screen comprised of ∼900,000 small molecules that identified new classes of small-molecule proteostasis regulators that induce HSF-1-dependent chaperone expression and restore protein folding in multiple conformational disease models. These beneficial effects to proteome stability are mediated by HSF-1, FOXO, Nrf-2 and the chaperone machinery through mechanisms that are distinct from current known small-molecule activators of the heat shock response. We suggest that modulation of the proteostasis network by proteostasis regulators may be a promising therapeutic approach for the treatment of a variety of protein conformational diseases.
Full-text · Article · Dec 2011 · Nature Chemical Biology
[Show abstract][Hide abstract] ABSTRACT: Resveratrol has demonstrated cancer chemopreventive activity in animal models and some clinical trials are underway. In addition, resveratrol was shown to promote cell survival, increase lifespan and mimic caloric restriction, thereby improving health and survival of mice on high-calorie diet. All of these effects are potentially mediated by the pleiotropic interactions of resveratrol with different enzyme targets including COX-1 (cyclo-oxygenase-1) and COX-2, NAD+-dependent histone deacetylase SIRT1 (sirtuin 1) and QR2 (quinone reductase 2). Nonetheless, the health benefits elicited by resveratrol as a direct result of these interactions with molecular targets have been questioned, since it is rapidly and extensively metabolized to sulfate and glucuronide conjugates, resulting in low plasma concentrations. To help resolve these issues, we tested the ability of resveratrol and its metabolites to modulate the function of some known targets in vitro. In the present study, we have shown that COX-1, COX-2 and QR2 are potently inhibited by resveratrol, and that COX-1 and COX-2 are also inhibited by the resveratrol 4'-O-sulfate metabolite. We determined the X-ray structure of resveratrol bound to COX-1 and demonstrate that it occupies the COX active site similar to other NSAIDs (non-steroidal anti-inflammatory drugs). Finally, we have observed that resveratrol 3- and 4'-O-sulfate metabolites activate SIRT1 equipotently to resveratrol, but that activation is probably a substrate-dependent phenomenon with little in vivo relevance. Overall, the results of this study suggest that in vivo an interplay between resveratrol and its metabolites with different molecular targets may be responsible for the overall beneficial health effects previously attributed only to resveratrol itself.
Full-text · Article · May 2010 · Biochemical Journal
[Show abstract][Hide abstract] ABSTRACT: Melatonin exerts its biological effects through at least two transmembrane G-protein-coupled receptors, MT1 and MT2, and a lower-affinity cytosolic binding site, designated MT3. MT3 has recently been identified as QR2 (quinone reductase 2) (EC 22.214.171.124) which is of significance since it links the antioxidant effects of melatonin to a mechanism of action. Initially, QR2 was believed to function analogously to QR1 in protecting cells from highly reactive quinones. However, recent studies indicate that QR2 may actually transform certain quinone substrates into more highly reactive compounds capable of causing cellular damage. Therefore it is hypothesized that inhibition of QR2 in certain cases may lead to protection of cells against these highly reactive species. Since melatonin is known to inhibit QR2 activity, but its binding site and mode of inhibition are not known, we determined the mechanism of inhibition of QR2 by melatonin and a series of melatonin and 5-hydroxytryptamine (serotonin) analogues, and we determined the X-ray structures of melatonin and 2-iodomelatonin in complex with QR2 to between 1.5 and 1.8 A (1 A=0.1 nm) resolution. Finally, the thermodynamic binding constants for melatonin and 2-iodomelatonin were determined by ITC (isothermal titration calorimetry). The kinetic results indicate that melatonin is a competitive inhibitor against N-methyldihydronicotinamide (K(i)=7.2 microM) and uncompetitive against menadione (K(i)=92 microM), and the X-ray structures shows that melatonin binds in multiple orientations within the active sites of the QR2 dimer as opposed to an allosteric site. These results provide new insights into the binding mechanisms of melatonin and analogues to QR2.
Full-text · Article · Aug 2008 · Biochemical Journal
[Show abstract][Hide abstract] ABSTRACT: Lycopene, the red pigment of the tomato, is under investigation for the chemoprevention of prostate cancer. Because dietary lycopene has been reported to concentrate in the human prostate, its uptake and subcellular localization were investigated in the controlled environment of cell culture using the human prostate cancer cell lines LNCaP, PC-3, and DU145. After 24 hours of incubation with 1.48 micromol/L lycopene, LNCaP cells accumulated 126.6 pmol lycopene/million cells, which was 2.5 times higher than PC-3 cells and 4.5 times higher than DU145 cells. Among these cell lines, only LNCaP cells express prostate-specific antigen and fully functional androgen receptor. Levels of prostate-specific antigen secreted into the incubation medium by LNCaP cells were reduced 55% as a result of lycopene treatment at 1.48 micromol/L. The binding of lycopene to the ligand-binding domain of the human androgen receptor was carried out, but lycopene was not found to be a ligand for this receptor. Next, subcellular fractionation of LNCaP cells exposed to lycopene was carried out using centrifugation and followed by liquid chromatography-tandem mass spectrometry quantitative analysis to determine the specific cellular locations of lycopene. The majority of lycopene (55%) was localized to the nuclear membranes, followed by 26% in nuclear matrix, and then 19% in microsomes. No lycopene was detected in the cytosol. These data suggest that the rapid uptake of lycopene by LNCaP cells might be facilitated by a receptor or binding protein and that lycopene is stored selectively in the nucleus of LNCaP cells.
No preview · Article · Dec 2006 · Molecular Cancer Therapeutics
[Show abstract][Hide abstract] ABSTRACT: Organophosphorus hydrolase detoxifies a broad range of organophosphate pesticides and the chemical warfare agents (CWAs) sarin and VX. Previously, rational genetic engineering produced OPH variants with 30-fold enhancements in the hydrolysis of CWA and their analogs. One interesting variant (H254R) in which the histidine at position 254 was changed to an arginine showed a 4-fold increase in the hydrolysis of demetonS (VX analog), a 14-fold decrease with paraoxon (an insecticide), and a 183-fold decrease with DFP (sarin analog). The three-dimensional structure of this enzyme at 1.9A resolution with the inhibitor, diethyl 4-methylbenzylphosphonate (EBP), revealed that the inhibitor did not bind at the active site, but bound exclusively into a well-defined surface pocket 12 A away from the active site. This structural feature was accompanied by non-competitive inhibition of paraoxon hydrolysis by EBP with H254R, in contrast to the native enzyme, which showed competitive inhibition. These parallel structure-function characteristics identify a functional, allosteric site on the surface of this enzyme.
Full-text · Article · Nov 2005 · Archives of Biochemistry and Biophysics
[Show abstract][Hide abstract] ABSTRACT: Protein homeostasis, also called proteostasis, is critical for cellular health and its dysregulation is implicated in aging, cancer, metabolic disease, and neurodegenerative disorders. Proteostasis involves compartmentalized cellular responses (e.g. Heat Shock Response in the cytoplasm, Unfolded Protein Response in the mitochondria and endoplasmic reticulum) that limit protein misfolding and aggregation. Diseases of protein conformation are characterized by inefficient induction of these responses. As a result, identification of molecules that activate cellular stress responses and increase proteostasis may be useful for maintaining cell health. Here, we report on high throughput screening efforts that resulted in identification of a novel activator of heat shock protein 70 (Hsp70): ML346. Probe ML346 belongs to the barbituric acid scaffold. ML346 induces HSF-1-dependent chaperone expression and restores protein folding in conformational disease models. These effects are mediated by novel mechanisms involving FOXO, HSF-1, and Nfr-2.