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.
SummaryElevated 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.
Objective-Lysosomal proteinases have been implicated in a number of pathologies associated with extracellular matrix breakdown. Therefore, we investigated the possibility that the lysosomal proteinase cathepsin S may be involved in atherosclerotic plaque destabilization. Methods and Results-Atherosclerotic plaques in the brachiocephalic arteries of fat-fed apolipoprotein E/cathepsin S double knockout mice had 73% fewer acute plaque ruptures (Pϭ0.026) and were 46% smaller (Pϭ0.025) than those in age-, strain-, and sex-matched apolipoprotein E single knockout controls. When the incidence of acute plaque rupture was normalized for plaque size, the reduction in the double knockouts was 72% (Pϭ0.039). The number of buried fibrous layers, indicative of an unstable plaque phenotype, was reduced by 67% in the double knockouts (Pϭ0.008). The cysteine proteinase inhibitor, egg white cystatin, was biotinylated and used as an active-site-directed probe for cathepsins. Biotinylated cystatin selectively detected cathepsin S in extracts of human carotid atherosclerotic plaque. Active cathepsin S was detectable in extracts of human atherosclerotic plaque but not in nondiseased carotid arteries. Active cathepsins were especially prominent in macrophages in the shoulder regions of plaques, areas considered to be vulnerable to rupture. Cathepsin S protein colocalized with regions of elastin degradation in human coronary plaques. Conclusion-These data provide direct evidence that an endogenous proteinase, cathepsin S, plays an important role in atherosclerotic plaque destabilization and rupture. Key Words: atherosclerosis Ⅲ plaque Ⅲ pathology Ⅲ cathepsin T he highly thrombogenic gruel in the core of an atherosclerotic plaque is luminally covered by a fibrous cap, weakening of which leads to plaque rupture and thrombus formation. Macrophages and T cells accumulate at sites of plaque disruption. 1 Degradation of collagen by macrophagederived matrix metalloproteinases has been reported, 2 but less is known of other classes of proteinase, which may be released by activated macrophages.The lysosomal cathepsins have been implicated in the development and progression of atherosclerosis. Increased levels of cathepsins F, K, and S are present in atherosclerotic lesions, 3,4 whereas levels of the major extracellular inhibitor of cysteine proteinases, cystatin C, are decreased. 5 In humans, an association has been shown between a genetically determined decrease in cystatin C levels and the severity of coronary artery disease. 6 Cathepsin S/low-density lipoprotein (LDL) receptor double knockout mice have impaired atherogenesis when compared with LDL receptor single knockout controls. 7 Grading of atherosclerosis in the aortic arch showed that there was a delay in lesion progression in the double knockouts. For example, 12 weeks of feeding atherogenic diet to the double knockouts resulted in an average lesion severity similar to that seen in single knockout controls after just 8 weeks of feeding, and 26 weeks of atherogenic diet feeding in the ...
The incorporation of non-natural amino acids opens up the possibility to endow proteins with properties that cannot be attained with the 20 natural amino acids encoded by DNA base triplets. The incorporation of non-natural amino acids can readily be achieved with the natural protein-translational machinery, if the structure of the modified amino acid is closely related to the natural amino acid, so that it can be loaded onto tRNA by one of the natural aminoacyl-tRNA synthetases.A wide range of non-natural amino acids has been incorporated into proteins in this way [1]. In general, the efficiency of incorporation decreases with increasing K M value of the aminoacyl-tRNA synthetase for the respective amino acid. This holds, in particular, for the in vivo incorporation of non-natural amino acids, where a pool of natural amino acids is always present. This problem can be circumvented by the use of auxotrophic strains [1] or cell-free protein production systems derived from nonauxotrophic strains combined with a suitably manipulated medium for protein synthesis [2,3].Recently, high-yield, cell-free protein production systems have become available that allow the synthesis of proteins in quantities sufficient for structural genomics applications [4][5][6][7]. High-level incorporation of seleno-methionine (Se-Met) for X-ray crystallography and fluoro-tryptophan (F-Trp) for NMR has been An Escherichia coli cell-free transcription ⁄ translation system was used to explore the high-level incorporation of l-3,4-dihydroxyphenylalanine (DOPA) into proteins by replacing tyrosine with DOPA in the reaction mixtures. ESI-MS showed specific incorporation of DOPA in place of tyrosine. More than 90% DOPA incorporation at each tyrosine site was achieved, allowing the recording of clean 15 N-HSQC NMR spectra. A redox-staining method specific for DOPA was shown to provide a sensitive and generally applicable method for assessing the cell-free production of proteins. Of four proteins produced in soluble form in the presence of tyrosine, two resulted in insoluble aggregates in the presence of high levels of DOPA. DOPA has been found in human proteins, often in association with various disease states that implicate protein aggregation and ⁄ or misfolding. Our results suggest that misfolded and aggregated proteins may result, in principle, from ribosome-mediated misincorporation of intracellular DOPA accumulated due to oxidative stress. High-yield cell-free protein expression systems are uniquely suited to obtain rapid information on solubility and aggregation of nascent polypeptide chains.Abbreviations DOPA, L-3,4-dihydroxyphenylalanine; GFP, cycle 3 mutant green fluorescent protein; hCypA, human cyclophilin A; His 6 -PpiB, N-terminal His 6 -tagged PpiB; HMP, Escherichia coli flavohaemoglobin; HSQC, heteronuclear single-quantum coherence; NBT, nitroblue tetrazolium; PpiB, E. coli peptidyl-prolyl cis-trans isomerase B; RNAP, RNA polymerase; TyrRS, tyrosyl-tRNA synthetase.
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