Mitochondrial dysfunction is an early, imminent event in neurodegenerative disorders including Parkinson disease (PD) and Alzheimer disease (AD). The enzymatic pair PINK1 and PRKN/Parkin recognize and transiently label damaged mitochondria with ubiquitin (Ub) phosphorylated at Ser65 (p-S65-Ub) as a signal for degradation via the autophagy-lysosome system (mitophagy). Despite its discovery in cell culture several years ago, robust and quantitative detection of altered mitophagy in vivo has remained challenging. Here we developed a sandwich ELISA targeting p-S65-Ub with the goal to assess mitophagy levels in mouse brain and in human clinical and pathological samples. We characterized five total Ub and four p-S65-Ub antibodies by several techniques and found significant differences in their ability to recognize phosphorylated Ub. The most sensitive antibody pair detected recombinant p-S65-Ub chains in the femtomolar to low picomolar range depending on the poly-Ub chain linkage. Importantly, this ELISA was able to assess very low baseline mitophagy levels in unstressed human cells and in brains from wild-type and prkn knockout mice as well as elevated p-S65-Ub levels in autopsied frontal cortex from AD patients vs. control cases. Moreover, the assay allowed detection of p-S65-Ub in blood plasma and was able to discriminate between PINK1 mutation carriers and controls. In summary, we developed a robust and sensitive tool to measure mitophagy levels in cells, tissue, and body fluids. Our data strongly support the idea that the stress-activated PINK1-PRKN mitophagy pathway is constitutively active in mice and humans under unstimulated, physiological and elevated in diseased, pathological conditions.
Mitochondrial genome diversity in closely related species provides an excellent platform for investigation of chromosome architecture and its evolution by means of comparative genomics. In this study, we determined the complete mitochondrial DNA sequences of eight Candida species and analyzed their molecular architectures. Our survey revealed a puzzling variability of genome architecture, including circular- and linear-mapping and multipartite linear forms. We propose that the arrangement of large inverted repeats identified in these genomes plays a crucial role in alterations of their molecular architectures. In specific arrangements, the inverted repeats appear to function as resolution elements, allowing genome conversion among different topologies, eventually leading to genome fragmentation into multiple linear DNA molecules. We suggest that molecular transactions generating linear mitochondrial DNA molecules with defined telomeric structures may parallel the evolutionary emergence of linear chromosomes and multipartite genomes in general and may provide clues for the origin of telomeres and pathways implicated in their maintenance.
Programmed translational bypassing is a process whereby ribosomes "ignore" a substantial interval of mRNA sequence. Although discovered 25 y ago, the only experimentally confirmed example of this puzzling phenomenon is expression of the bacteriophage T4 gene 60. Bypassing requires translational blockage at a "takeoff codon" immediately upstream of a stop codon followed by a hairpin, which causes peptidyl-tRNA dissociation and reassociation with a matching "landing triplet" 50 nt downstream, where translation resumes. Here, we report 81 translational bypassing elements (byps) in mitochondria of the yeast Magnusiomyces capitatus and demonstrate in three cases, by transcript analysis and proteomics, that byps are retained in mitochondrial mRNAs but not translated. Although mitochondrial byps resemble the bypass sequence in the T4 gene 60, they utilize unused codons instead of stops for translational blockage and have relaxed matching rules for takeoff/landing sites. We detected byp-like sequences also in mtDNAs of several Saccharomycetales, indicating that byps are mobile genetic elements. These byp-like sequences lack bypassing activity and are tolerated when inserted in-frame in variable protein regions. We hypothesize that byp-like elements have the potential to contribute to evolutionary diversification of proteins by adding new domains that allow exploration of new structures and functions.ribosome hopping | mitochondrial genome | proteome analysis | heterologous expression T he traditional view of translation is that mRNA is read sequentially, one codon at a time. However, low-level nonprogrammed translational bypassing (i.e., the occasional skipping of a few nucleotides) can be triggered by various factors, including tRNA paucity, unusual codons, and homo-polymer sequence tracts (1). In addition, programmed translational bypassing of 50 nt has been demonstrated for the gene 60 transcript of bacteriophage T4 (2-4). In vitro mutagenesis experiments showed that efficient translational "jumping" or "hopping" in T4 requires matching takeoff and landing codons (most effective is the wild-type GGA), a stop codon, and both a hairpin RNA secondary structure directly downstream of the takeoff site, and a Shine-Dalgarno (SD) sequence a few nucleotides upstream of the landing codon. Finally, a particular amino acid sequence in the nascent peptide encoded upstream of the takeoff site confers highest jumping efficiency. Additional cases of programmed bypassing have been postulated but currently lack supporting evidence (e.g., ref. 5), making the T4 gene 60 expression the only confirmed instance.Here, we report the massive occurrence of translational bypassing elements in mitochondria of the opportunistic human pathogen Magnusiomyces (also known as Blastoschizomyces or Geotrichum) capitatus (6), which belongs to a deeply branching lineage of Saccharomycetales (Fig. 1A). Our findings suggest that translational bypassing might be more widespread than previously thought. ResultsProtein-Coding Genes Interrupted by Dozens of In...
As a part of our initiative aimed at a large-scale comparative analysis of fungal mitochondrial genomes, we determined the complete DNA sequence of the mitochondrial genome of the yeast Candida subhashii and found that it exhibits a number of peculiar features. First, the mitochondrial genome is represented by linear dsDNA molecules of uniform length (29 795 bp), with an unusually high content of guanine and cytosine residues (52.7 %). Second, the coding sequences lack introns; thus, the genome has a relatively compact organization. Third, the termini of the linear molecules consist of long inverted repeats and seem to contain a protein covalently bound to terminal nucleotides at the 5′ ends. This architecture resembles the telomeres in a number of linear viral and plasmid DNA genomes classified as invertrons, in which the terminal proteins serve as specific primers for the initiation of DNA synthesis. Finally, although the mitochondrial genome of C. subhashii contains essentially the same set of genes as other closely related pathogenic Candida species, we identified additional ORFs encoding two homologues of the family B protein-priming DNA polymerases and an unknown protein. The terminal structures and the genes for DNA polymerases are reminiscent of linear mitochondrial plasmids, indicating that this genome architecture might have emerged from fortuitous recombination between an ancestral, presumably circular, mitochondrial genome and an invertron-like element.
Parkinson’s disease (PD) is the second most common neurodegenerative disease characterized by the progressive loss of dopaminergic neurons in the substantia nigra pars compacta and the presence of Lewy bodies, which gives rise to motor and non-motor symptoms. Unfortunately, current therapeutic strategies for PD merely treat the symptoms of the disease, only temporarily improve the patients’ quality of life, and are not sufficient for completely alleviating the symptoms. Therefore, cell-based therapies have emerged as a novel promising therapeutic approach in PD treatment. Mesenchymal stem/stromal cells (MSCs) have arisen as a leading contender for cell sources due to their regenerative and immunomodulatory capabilities, limited ethical concerns, and low risk of tumor formation. Although several studies have shown that MSCs have the potential to mitigate the neurodegenerative pathology of PD, variabilities in preclinical and clinical trials have resulted in inconsistent therapeutic outcomes. In this review, we strive to highlight the sources of variability in studies using MSCs in PD therapy, including MSC sources, the use of autologous or allogenic MSCs, dose, delivery methods, patient factors, and measures of clinical outcome. Available evidence indicates that while the use of MSCs in PD has largely been promising, conditions need to be standardized so that studies can be effectively compared with one another and experimental designs can be improved upon, such that this body of science can continue to move forward.
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