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Dr Rachael Dunlop

Biography

Rachael Dunlop PhD FSB, is a post-doctoral fellow in the School of Medical and Molecular Biosciences UTS, with an interest in motor neurone disease, in particular the role of blue green algae in triggering the sporadic form.

As a prolific science communicator, she runs her lab’s website and Twitter feed, Tellurian Toxins and blogs about all manner of science on her personal blog “The Skeptics Book of Pooh Pooh” and @DrRachie

Rachael is a reporter for The Skeptic Zone Podcast, a vice president of the Australian Skeptics, and a member of Mystery Investigators science show for children.

Rachael is the 2nd most read author on The Conversation, and her science writings have appeared in Fairfax newspapers, Mamamia, Australasian Science, Australian Doctor, and The Guardian. She is sought after as a science commentator and has appeared on Lateline, The Project, The Drum, ABC Breakfast News, Mamamia TV, Radio 2GB, 2CC, ABC and others.

Rachael is a past winner of a Twitter Shorty Award for the most interesting health and science information on Twitter. As an ex-graphic designer and copywriter, Rachael enjoys combining her love of science, art and social media as a means of communicating science to the public.

Image of Rachael Dunlop
Visiting Fellow, School of Medical and Molecular Biosciences
Copywriting & Art Direction, Photography Printmaking, Associate diploma, BSc ( Uni of Adelaide), BSc (Hon 1) (Uni of Adelaide), PhD (Uni of Sydney)
 
Phone
+61 2 9514 8384
Fax
+61 2 9514 8206
Room
CB04.06.52

Research Interests

Neurodegeneration, oxidation, protein degradation, motor neurone disease, ageing disorders, blue green algae, environmental toxins. 

Chapters

Dunlop, R.A. 2013, 'The MMR Vaccine Causes Autism' in Sykes, H. & Webb, F.J. (eds), 99 and Counting Medical Myths Debunked, Future Leaders, Melbourne Victoria, pp. 8-11.
Dunlop, R.A. 2013, 'The MMR Vaccine Causes Autism' in The Explainer: From Deja Vu to Why the Sky is Blue, and Other Conundrums, CSIRO Publishing, Australia, pp. 173-175.

Conferences

Dunlop, R.A., Cox, P.A., Banack, S.A. & Rodgers, K.J. 2013, 'The non-protein amino acid BMAA is misincorporated into human proteins in place of L-serine causing protein misfolding and aggregation.', PLoS One, p. e75376.
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Mechanisms of protein misfolding are of increasing interest in the aetiology of neurodegenerative diseases characterized by protein aggregation and tangles including Amyotrophic Lateral Sclerosis (ALS), Alzheimer's disease (AD), Parkinson's disease (PD), Lewy Body Dementia (LBD), and Progressive Supranuclear Palsy (PSP). Some forms of neurodegenerative illness are associated with mutations in genes which control assembly of disease related proteins. For example, the mouse sticky mutation sti, which results in undetected mischarging of tRNA(Ala) with serine resulting in the substitution of serine for alanine in proteins causes cerebellar Purkinje cell loss and ataxia in laboratory animals. Replacement of serine 422 with glutamic acid in tau increases the propensity of tau aggregation associated with neurodegeneration. However, the possibility that environmental factors can trigger abnormal folding in proteins remains relatively unexplored. We here report that a non-protein amino acid, ?-N-methylamino-L-alanine (BMAA), can be misincorporated in place of L-serine into human proteins. We also report that this misincorporation can be inhibited by L-serine. Misincorporation of BMAA into human neuroproteins may shed light on putative associations between human exposure to BMAA produced by cyanobacteria and an increased incidence of ALS.
Dunlop, R.A. 2011, 'Proteins containing BMAA form autofluorescent aggregates and induce cell death', Amyotrophic Lateral Sclerosis, Taylor and Francis, UK, pp. 156-156.
Dunlop, R.A. 2011, 'The cyanobacteria-derived BMAA can be incorporated into cell proteins and could thus be an environmental trigger for ALS and other neurological diseases associated with protein misfolding.', Amyotrophic Lateral Sclerosis, Informa Healthcare, UK, pp. 22-22.
Chan, P.Y., Dunlop, R.A. & Rodgers, K.J. 2006, 'Accumulation of hypochlorite modified LDL by human monocyte-derived macrophages causes a selective increase in expression of the cathepsin S gene.', FREE RADICAL RESEARCH, pp. S107-S107.
Dunlop, R.A., Dean, R.T. & Rodgers, K.J. 2006, 'Proteins containing incorporated DOPA are inefficiently degraded by proteasomes and can upregulate lysosomal proteinases.', FREE RADICAL RESEARCH, pp. S53-S53.
Zeng, J., Dunlop, R.A., Rodgers, K. & Davies, M.J. 2006, 'Evidence for inactivation of cysteine proteases by reactive carbonyls via glycation of active site thiols', FREE RADICAL RESEARCH, pp. S139-S139.
Rodgers, K.J., Dunlop, R.A. & Dean, R.T. 2005, 'A novel approach to determine the pathways involved in the cellular catabolism of oxidised proteins', FREE RADICAL RESEARCH, pp. S74-S74.

Journal articles

Dunlop, R.A., Main, B.J. & Rodgers, K.J. 2014, 'The deleterious effects of non-protein amino acids from desert plants on human and animal health', Journal of Arid Environments.
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Since plants lack the ability to remove themselves from sites of predation, they have evolved alternative defences to keep predators at bay. Mechanical defences include camouflage, the addition of thorns and spikes as well as growing in locations not easily accessed by herbivores. Chemical defences include the synthesis of compounds that are toxic to predatory species and can also inhibit or retard the growth of other plant species in a process termed "allelopathy". Amongst these chemicals is a reservoir of non-protein amino acids that mediate toxicity. These non-protein amino acids can be charged by tRNA synthetases and subsequently mis-incorporated into nascent polypeptides, resulting in aberrant, dysfunctional proteins that can be toxic to predators. Some species such as the bruchid beetle Bruchus rufimanus (Chrysomelidae), which feeds on the jack bean Canavalia ensiformis (Fabaceae) plant, have evolved advanced tRNA synthetases that are able to discriminate between protein and non-protein amino acids, thus remain unaffected. Other species including livestock and humans that do not possess such selectivity are susceptible to plant toxins. In this brief review we discuss the mechanisms of action and consequences of exposure to plant-derived non-protein amino acids with a focus on those derived from plants from arid environments. 2014 Elsevier Ltd. All rights reserved.
Dunlop, R.A. 2014, 'Trial to Assess Chelation Therapy (TACT) provides scant evidence for benefits of chelation therapy in cardiovascular disease: Commentary', Focus on Alternative and Complementary Therapies, vol. 19, no. 2, pp. 111-112.
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Dunlop, R.A., Cox, P.A., Banack, S.A. & Rodgers, K.J. 2013, 'The Non-Protein Amino Acid BMAA Is Misincorporated into Human Proteins in Place of l-Serine Causing Protein Misfolding and Aggregation', PLoS ONE, vol. 8, no. 9.
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Mechanisms of protein misfolding are of increasing interest in the aetiology of neurodegenerative diseases characterized by protein aggregation and tangles including Amyotrophic Lateral Sclerosis (ALS), Alzheimer's disease (AD), Parkinson's disease (PD), Lewy Body Dementia (LBD), and Progressive Supranuclear Palsy (PSP). Some forms of neurodegenerative illness are associated with mutations in genes which control assembly of disease related proteins. For example, the mouse sticky mutation sti, which results in undetected mischarging of tRNAAla with serine resulting in the substitution of serine for alanine in proteins causes cerebellar Purkinje cell loss and ataxia in laboratory animals. Replacement of serine 422 with glutamic acid in tau increases the propensity of tau aggregation associated with neurodegeneration. However, the possibility that environmental factors can trigger abnormal folding in proteins remains relatively unexplored. We here report that a non-protein amino acid, ?-N-methylamino-L-alanine (BMAA), can be misincorporated in place of l-serine into human proteins. We also report that this misincorporation can be inhibited by l-serine. Misincorporation of BMAA into human neuroproteins may shed light on putative associations between human exposure to BMAA produced by cyanobacteria and an increased incidence of ALS. 2013 Dunlop et al.
Chan, S.W., Dunlop, R.A., Rowe, A., Double, K.L. & Rodgers, K.J. 2012, 'L-DOPA is incorporated into brain proteins of patients treated for Parkinson's disease, inducing toxicity in human neuroblastoma cells in vitro', Experimental Neurology, vol. 238, no. 1, pp. 29-37.
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Levodopa (l-dopa), a close structural analogue of the protein amino acid l-tyrosine, can substitute for l-tyrosine in protein synthesis and be mistakenly incorporated into newly synthesised proteins in vitro. We show that l- dopa-containing proteins are present in the brain in l-DOPA-treated Parkinson's disease patients and accumulate in specific brain regions. In vitro studies demonstrate that substitution of l-tyrosine residues in proteins with l-DOPA causes protein misfolding and promotes protein aggregation in SH-SY5Y neuroblastoma cells resulting in the appearance of autofluorescent bodies. We show that the presence of l-DOPA-containing proteins causes profound changes in mitochondria and stimulates the formation of autophagic vacuoles in cells. Unlike l-DOPA, which is toxic to cells through its ability to generate radicals, proteins containing incorporated l-DOPA are toxic to SH-SY5Y cells by a mechanism independent of oxidative stress and resistant to antioxidants. These data suggest that the accumulation of l-DOPA-containing proteins in vulnerable cells might negatively impact on cell function. 2011 Elsevier Inc.
Ernst, E., Snyder, J. & Dunlop, R.A. 2012, 'National Center for Complementary and Alternative Medicine-funded randomised controlled trials of acupuncture: A systematic review', Focus on Alternative and Complementary Therapies, vol. 17, no. 1, pp. 15-21.
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The work of the National Center for Complementary and Alternative Medicine (NCCAM) has repeatedly been criticised. Much of this criticism relates to the past funding of studies of acupuncture. The aim of this article is to provide an independent, critical evaluation of the data produced by NCCAM-funded RCTs of acupuncture. Relevant studies were identified using PubMed. Studies were included if they were NCCAM-funded RCTs of acupuncture. Excluded were secondary reports of primary studies, RCTs not testing the effectiveness of acupuncture and National Institutes of Health-funded studies not mentioning NCCAM support. One author extracted the data according to predefined criteria and assessed the risk of bias. The other authors verified these tasks. Thirteen RCTs were included, with sample sizes ranging from seven to 570. Most studies reported pain as the primary outcome. Six RCTs suggested acupuncture was effective. Seven RCTs had a low risk of bias. Numerous methodological shortcomings were identified. Many NCCAM-funded RCTs of acupuncture have important limitations. These findings might improve future studies of acupuncture and could be considered in the ongoing debate regarding NCCAM-funding. 2011 The Authors. FACT 2011. Royal Pharmaceutical Society.
Tang, J., Dunlop, R.A., Rowe, A., Rodgers, K.J. & Ramzan, I. 2011, 'Kavalactones yangonin and methysticin induce apoptosis in human hepatocytes (HepG2) in vitro', Phytotherapy Research, vol. 25, no. 3, pp. 417-423.
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While cases of severe kava hepatotoxicity have been reported, studies examining the toxicity of individual kavalactones are limited. The present study examined the in vitro hepatotoxicity of kavain, methysticin and yangonin on human hepatocytes (HepG2) and the possible mechanism(s) involved. Cytotoxicity was assessed using lactate dehydrogenase (LDH) and ethidium bromide (EB) assays. The mode of cell death was analysed with acridine orange/ethidium bromide dual staining with fluorescence microscopy. Glutathione oxidation was measured using the ortho-phthalaldehyde (OPT) fluorescence assay. Kavain had minimal cytotoxicity, methysticin showed moderate concentration-dependent toxicity and yangonin displayed marked toxicity with ?40% reduction in viability in the EB assay. Acridine orange/ethidium bromide staining showed the predominant mode of cell death was apoptosis rather than necrosis. No significant changes were observed in glutathione levels, excluding this as the primary mechanism of cell death in this model. Further studies may elucidate the precise apoptotic pathways responsible and whether toxic kavalactone metabolites are involved. Copyright 2010 John Wiley & Sons, Ltd.
Dunlop, R.A., Brunk, U.T. & Rodgers, K.J. 2011, 'Proteins containing oxidized amino acids induce apoptosis in human monocytes', Biochemical Journal, vol. 435, no. 1, pp. 207-216.
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Cellular deposits of oxidized and aggregated proteins are hallmarks of a variety of age-related disorders, but whether such proteins contribute to pathology is not well understood. We previously reported that oxidized proteins form lipofuscin/ceroid-like bodies with a lysosomal-type distribution and up-regulate the transcription and translation of proteolytic lysosomal enzymes in cultured J774 mouse macrophages. Given the recently identified role of lysosomes in the induction of apoptosis, we have extended our studies to explore a role for oxidized proteins in apoptosis. Oxidized proteins were biosynthetically generated in situ by substituting oxidized analogues for parent amino acids. Apoptosis was measured with Annexin-V/PI (propidium iodide), TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP nick-end labelling), MMP (mitochondrial membrane permeabilization), caspase activation and cytochrome c release, and related to lysosomal membrane permeabilization. Synthesized proteins containing the tyrosine oxidation product L-DOPA (L-3,4- dihydroxyphenylalanine) were more potent inducers of apoptosis than proteins containing the phenylalanine oxidation product o-tyrosine. Apoptosis was dependent upon incorporation of oxidized residues, as indicated by complete abrogation in cultures incubated with the non-incorporation control D-DOPA (D-3,4-dihydroxyphenylalanine) or when incorporation was competed out by parent amino acids. The findings of the present study suggest that certain oxidized proteins could play an active role in the progression of age-related disorders by contributing to LMP (lysosomal membrane permeabilization)-initiated apoptosis and may have important implications for the long-term use of L-DOPA as a therapeutic agent in Parkinson's disease. The Authors Journal compilation 2011 Biochemical Society.
Dunlop, R.A. 2011, 'External qigong as an adjunct therapy for chronic pain - A critical analysis', Focus on Alternative and Complementary Therapies, vol. 16, no. 2, pp. 163-164.
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Dunlop, R.A. 2011, 'An inexpensive and accessible exercise regime significantly improves balance and reduces injuries in the elderly', Focus on Alternative and Complementary Therapies, vol. 16, no. 1, pp. 56-57.
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Dunlop, R.A. 2011, 'Supplementary evidence to support tai chi as an 'alternative' exercise for patients undergoing cardiac rehabilitation', Focus on Alternative and Complementary Therapies, vol. 16, no. 4, pp. 307-308.
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Dunlop, R.A. 2010, 'Critical analysis of evidence for long-term effectiveness of biofeedback intervention in hypertension', Focus on Alternative and Complementary Medicine, vol. 15, no. 4, pp. 307-308.
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Thompson, A.M., Dunlop, R.A., Dean, R.T. & Rodgers, K.J. 2009, 'Evidence that DOPA-derivatives are generated after L-DOPA incorporation into proteins by mammalian cells', Journal of Adhesion, vol. 85, no. 9, pp. 561-575.
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The adhesive and cohesive properties of the amino acid L-3,4-dihydroxyphenylalanine (DOPA) have been widely explored as a potential material for adhesion, based, among other things, on the biological system of blue mussel extracellular byssal threads and foot proteins. Proteins containing DOPA are generated within mammalian cells by oxidation of tyrosine residues during periods of oxidative stress. By generating proteins containing DOPA, in vitro, through the (mis)incorporation of DOPA during protein synthesis, we are able examine the role and fate of DOPA-containing proteins in mammalian cells. We demonstrate a decrease in catabolism of long half-life cell proteins and an increase in cellular autofluorescence when DOPA is present in cell proteins. We provide evidence for the formation of DOPA derivatives which can be detected in proteins after 14C-DOPA incorporation by HPLC analysis. Additionally, we demonstrate that the cells upregulate the expression of genes required to handle damaged proteins and protein aggregates under these conditions. Substantial evidence for DOPA derivatives and cross-linking has previously been shown in extracellular blue mussel byssal threads; here we provide evidence for cell-associated DOPA derivatives in mammalian cells.
Dunlop, R.A., Brunk, U.T. & Rodgers, K.J. 2009, 'Oxidized proteins: Mechanisms of removal and consequences of accumulation', IUBMB Life, vol. 61, no. 5, pp. 522-527.
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Elevated levels of oxidized proteins are reported in diseased tissue from age-related pathologies such as atherosclerosis, neurodegenerative disorders, and cataract. Unlike the precise mechanisms that exist for the repair of nucleic acids, lipids, and carbohydrates, the primary pathway for the repair of oxidized proteins is complete catabolism to their constitutive amino acids. This process can be inefficient as is evidenced by their accumulation. It is generally considered that damaged proteins are degraded by the proteasome; however, this is only true for mildly oxidized proteins, because substrates must be unfolded to enter the narrow catalytic core. Rather, evidence suggests that moderately or heavily oxidized proteins are endocytosed and enter the endosomal/lysosomal system, indicating co-operation between the proteasomes and the lysosomes. Heavily modified substrates are incompletely degraded and accumulate within the lysosomal compartments resulting in the formation of lipofuscin-like, autofluorescent aggregates. Accumulation eventually results in impaired turnover of large organelles such as proteasomes and mitochondria, lysosomal destablization, leakage of proteases into the cytosol and apoptosis. In this review, we summarize reports published since our last assessments of the field of oxidized protein degradation including a role for modified proteins in the induction of apoptosis. 2009 IUBMB.
Dunlop, R.A., Dean, R.T. & Rodgers, K.J. 2008, 'The impact of specific oxidized amino acids on protein turnover in J774 cells', Biochemical Journal, vol. 410, no. 1, pp. 131-140.
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Oxidized protein deposition and accumulation have been implicated in the aetiology of a wide variety of age-related pathologies. Protein oxidation in vivo commonly results in the in situ modification of amino acid side chains, generating new oxidized amino acid residues in proteins. We have demonstrated previously that certain oxidized amino acids can be (mis)incorporated into cell proteins in vitro via protein synthesis. In the present study, we show that incorporation of o- and m-tyrosine resulted in increased protein catabolism, whereas dopa incorporation generated proteins that were inefficiently degraded by cells. Incorporation of higher levels of L-dopa into proteins resulted in an increase in the activity of lysosomal cathepsins, increased autofluorescence and the generation of high-molecular-mass SDS-stable complexes, indicative of protein aggregation. These effects were due to proteins containing incorporated L-dopa, since they were not seen with the stereoisomer D-dopa, which enters the cell and generates the same reactive species as L-dopa, but cannot be incorporated into proteins. The present study highlights how the nature of the oxidative modification to the protein can determine the efficiency of its removal from the cell by proteolysis. Protection against the generation of dopa and other species that promote resistance to proteolysis might prove to be critical in preventing toxicity from oxidative stress in pathologies associated with protein deposition, such as atherosclerosis, Alzheimer's disease and Parkinson's disease. The Authors.
Zeng, J., Dunlop, R.A., Rodgers, K.J. & Davies, M.J. 2006, 'Evidence for inactivation of cysteine proteases by reactive carbonyls via glycation of active site thiols', Biochemical Journal, vol. 398, no. 2, pp. 197-206.
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Hyperglycaemia, triose phosphate decomposition and oxidation reactions generate reactive aldehydes in vivo. These compounds react non-enzymatically with protein side chains and N-terminal amino groups to give adducts and cross-links, and hence modified proteins. Previous studies have shown that free or protein-bound carbonyls inactivate glyceraldehyde-3-phosphate dehydrogenase with concomitant loss of thiol groups [Morgan, Dean and Davies (2002) Arch. Biochem. Biophys. 403, 259-269]. It was therefore hypothesized that modification of lysosomal cysteine proteases (and the structurally related enzyme papain) by free and protein-bound carbonyls may modulate the activity of these components of the cellular proteolytic machinery responsible for the removal of modified proteins and thereby contribute to a decreased removal of modified proteins from cells. It is shown that MGX (methylglyoxal), GO (glyoxal) and glycolaldehyde, but not hydroxyacetone and glucose, inhibit catB (cathepsin B), catL (cathepsin L) and catS (cathepsin S) activity in macrophage cell lysates, in a concentration-dependent manner. Protein-bound carbonyls produced similar inhibition with both cell lysates and intact macrophage cells. Inhibition was also observed with papain, with this paralleled by loss of the active site cysteine residue and formation of the adduct species S-carboxymethylcysteine, from GO, in a concentration-dependent manner. Inhibition of autolysis of papain by MGX, along with cross-link formation, was detected by SDS/PAGE. Treatment of papain and catS with the dialdehyde o-phthalaldehyde resulted in enzyme inactivation and an intramolecular active site cysteine-lysine cross-link. These results demonstrate that reactive aldehydes inhibit cysteine proteases by modification of the active site cysteine residue. This process may contribute to the accumulation of modified proteins in tissues of people with diabetes and age-related pathologies, including atherosclerosis, cataract and Al...
Rodgers, K.J., Hume, P.M., Dunlop, R.A. & Dean, R.T. 2004, 'Biosynthesis and turnover of DOPA-containing proteins by human cells', Free Radical Biology and Medicine, vol. 37, no. 11, pp. 1756-1764.
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Protein-bound 3,4-dihydroxyphenylalanine (PB-DOPA) is a major product of hydroxyl radical attack on tyrosine residues of proteins. Levels of PB-DOPA in cells and tissues have been shown to be greatly elevated in age-related diseases. We demonstrate for the first time that l-DOPA (levodopa) can be biosynthetically incorporated into cell proteins by human cells (THP-1 monocytes and monocyte-derived macrophages). The DOPA-containing proteins generated were selectively visualized on PVDF membranes using a redox-cycling staining method. Many cell proteins contained DOPA and seemed to be synthesized as their full-length forms. The cellular removal of DOPA-containing proteins by THP-1 cells was by proteolysis involving both the proteasomal and the lysosomal systems. The rate of cellular proteolysis of DOPA-containing proteins increased at lower levels of DOPA incorporation but decreased at higher levels of DOPA incorporation. The decreased rate of degradation was accompanied by an increase in the activity of cathepsins B and L but the activity of cathepsin S increased only at lower levels of DOPA incorporation. These data raise the possibility that PB-DOPA could be generated in vivo from l-DOPA, which is the most widely used treatment for Parkinson disease. 2004 Elsevier Inc. All rights reserved.
Dean, R.T., Dunlop, R., Hume, P. & Rodgers, K.J. 2003, 'Proteolytic 'defences' and the accumulation of oxidized polypeptides in cataractogenesis and atherogenesis', PROTEASES AND THE REGULATION OF BIOLOGICAL PROCESSES, vol. 70, pp. 135-146.
Dunlop, R.A., Rodgers, K.J. & Dean, R.T. 2002, 'Recent developments in the intracellular degradation of oxidized proteins', Free Radical Biology and Medicine, vol. 33, no. 7, pp. 894-906.
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The accumulation of oxidized proteins in cells and tissues is a feature of a number of age-related diseases and may also occur as a result of the aging process itself. In this article we review recent advances in our understanding of the cellular degradation of oxidized proteins directing our attention primarily to information which directly bears on the behavior of intact eukaryotic cells. We summarize new work on the key intracellular degradative machineries, proteasomes and lysosomes and examine evidence implicating an increase in protein hydrophobicity as the primary signal to the proteasome to initiate degradation. The data identifying the proteasome as the main route of degradation of oxidized proteins is examined, as well as recent data investigating changes in proteasome function after exposure of cells to oxidants and the altered catabolism of oxidized proteins in aging cells. Evidence for the cooperation between the lysosomal and proteasomal systems in the degradation of oxidized proteins is discussed. We conclude that the cellular catabolism of oxidized proteins may be a more complex process than it first appeared and suggest key issues that need to be resolved to improve our understanding of this important process. 2002 Elsevier Science Inc.
Fontaine, F.R., Dunlop, R.A., Petersen, D.R. & Burcham, P.C. 2002, 'Oxidative bioactivation of crotyl alcohol to the toxic endogenous aldehyde crotonaldehyde: Association of protein carbonylation with toxicity in mouse hepatocytes', Chemical Research in Toxicology, vol. 15, no. 8, pp. 1051-1058.
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Recent confirmation that the toxic unsaturated aldehyde crotonaldehyde (CA) contributes to protein damage during lipid peroxidation confers interest on the molecular actions of this substance. However, since a plethora of structurally related aldehydes form during membrane oxidation, clarifying the toxicological significance of individual products (e.g., CA) is challenging. To facilitate study of the mechanisms underlying CA toxicity, we explored the possibility that it can be formed enzymatically from an unsaturated precursor, crotyl alcohol. This is analogous to the way allyl alcohol is converted in vivo to its toxic oxidation product, acrolein. In kinetic studies, we found that crotyl alcohol was readily oxidized by equine liver alcohol dehydrogenase, with electrospray-mass spectrometry confirming that CA was the main product formed. Moreover, in mouse hepatocytes, crotyl alcohol produced marked time- and concentration-dependent cell killing as well as pronounced glutathione depletion. Both cytotoxicity and glutathione loss were abolished by the alcohol dehydrogenase inhibitor 4-methylpyrazole, indicating an oxidation product mediated these effects. In keeping with expectations that carbonyl-retaining Michael addition adducts would feature prominently during protein modification by CA, exposure to crotyl alcohol resulted in marked carbonylation of a wide range of cell proteins, an effect that was also abolished by 4-methylpyrazole. Damage to a subset of small proteins (e.g., 29, 32, 33 kDa) closely correlated with the severity of cell death. Collectively, these results demonstrate that crotyl alcohol is a useful tool for studying the biochemical and molecular events accompanying intracellular CA formation.