Rachael A. Dunlop

The Non-Protein Amino Acid BMAA Is Misincorporated into Human Proteins in Place of l-Serine Causing Protein Misfolding and Aggregation

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, β-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.

Oxidized proteins: Mechanisms of removal and consequences of accumulation

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.

Recent developments in the intracellular degradation of oxidized proteins.

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.

The impact of specific oxidized amino acids on protein turnover in J774 cells

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, Alzheimers disease and Parkinsons disease.

L-DOPA is incorporated into brain proteins of patients treated for Parkinson's disease, inducing toxicity in human neuroblastoma cells in vitro

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 Parkinsons 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.

Proteins containing oxidized amino acids induce apoptosis in human monocytes.

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 Parkinsons disease.

The use of l-serine to prevent β-methylamino-l-alanine (BMAA)-induced proteotoxic stress in vitro

β-methylamino-L-alanine (BMAA), a non-protein amino acid synthesised by cyanobacteria, has been linked to a complex neurological disorder on Guam and more recently to other cases of sporadic ALS (sALS), however the mechanisms of BMAA toxicity are not completely understood. We have previously demonstrated that BMAA is misincorporated into newly synthesised proteins by human neuroblastoma cells and fibroblasts, resulting in the formation of autofluorescent material and the induction of apoptotic cell death. In the present study we show that BMAA at low levels does not cause an acute toxicity in neuroblastoma cells but increases the expression of the ER stress marker, C/EBP homologous protein (CHOP) and increases the activity of the pro-apoptotic enzyme caspase-3. We also observed an increase in the activity of the lysosomal cysteine proteases cathepsin B and L, characteristic of the accumulation of proteins in the lysosomal system. We were able to prevent these proteotoxic effects in neuroblastoma cells through co-treatment with l-serine suggesting that they resulted from incorporation of BMAA into proteins. Misincorporation provides a possible mechanism whereby BMAA could initiate misfolding, and the accumulation of aggregate-prone proteins in neurons. This build-up of misfolded proteins could explain the long latency period of the disease previously reported on Guam.

Kavalactones Yangonin and Methysticin induce apoptosis in human hepatocytes (HepG2) in vitro.

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

Using an in vitro model to study oxidised protein accumulation in ageing fibroblasts.

BACKGROUND The accumulation of oxidised proteins in ageing cells and tissues results from an increase in oxidant damage coupled with impaired degradation of the damaged proteins. Heat Shock Proteins (HSP) and other chaperones are required to recognise damaged proteins and transport them to the lysosomal and proteasomal degradation pathways. How these systems fail in ageing cells is not clear. METHODS We monitor oxidised protein accumulation, the activity of the proteasome and lysosomal proteases, and HSP levels in MRC-5 fibroblasts throughout their mitotic lifespan. We then use a novel in vitro cell culture model to experimentally generate oxidised proteins in young and old MRC-5 fibroblasts and compare their rates of degradation and changes in the key pathways involved in oxidised protein removal. RESULTS We show that the activity of the proteasome and some lysosomal enzymes decreases with ageing in MRC-5 cells as do levels of HSP70 but this is not associated with an accumulation of oxidised proteins which only occurs as cells closely approach post-mitotic senescence. Old cells are unable to degrade experimentally generated oxidised proteins as efficiently as young cells. Exposure to mild heat stress however increases the efficiency of oxidised protein degradation by young cells and increases levels of HSP70. CONCLUSIONS Our results highlight the importance of the HSP/chaperone system in oxidised protein metabolism, particularly in ageing cells. GENERAL SIGNIFICANCE These data might have implications for the development of therapies for pathologies associated with protein accumulation and suggest that the HSP/chaperone system would be an important target.

Evidence that DOPA-Derivatives are Generated After L-DOPA Incorporation into Proteins by Mammalian Cells

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.