Recombination and replication in DNA repair of heavily irradiated Deinococcus radiodurans.

Deinococcus radiodurans' extreme resistance to ionizing radiation, desiccation, and DNA-damaging chemicals involves a robust DNA repair that reassembles its shattered genome. The repair process requires diploidy and commences with an extensive exonucleolytic erosion of DNA fragments. Liberated single-stranded overhangs prime strand elongation on overlapping fragments and the elongated complementary strands reestablish chromosomal contiguity by annealing. We explored the interdependence of the DNA recombination and replication processes in the reconstitution of the D. radiodurans genome disintegrated by ionizing radiation. The priming of extensive DNA repair synthesis involves RecA and RadA proteins. DNA polymerase III is essential for the initiation of repair synthesis, whereas efficient elongation requires DNA polymerases I and III. Inactivation of both polymerases leads to degradation of DNA fragments and rapid cell death. The present in vivo characterization of key recombination and replication processes dissects the mechanism of DNA repair in heavily irradiated D. radiodurans.

Metabolic Shifts as the Hallmark of Most Common Diseases: The Quest for the Underlying Unity.

A hyper-specialization characterizes modern medicine with the consequence of classifying the various diseases of the body into unrelated categories. Such a broad diversification of medicine goes in the opposite direction of physics, which eagerly looks for unification. We argue that unification should also apply to medicine. In accordance with the second principle of thermodynamics, the cell must release its entropy either in the form of heat (catabolism) or biomass (anabolism). There is a decreased flow of entropy outside the body due to an age-related reduction in mitochondrial entropy yield resulting in increased release of entropy in the form of biomass. This shift toward anabolism has been known in oncology as Warburg-effect. The shift toward anabolism has been reported in most diseases. This quest for a single framework is reinforced by the fact that inflammation (also called the immune response) is involved in nearly every disease. This strongly suggests that despite their apparent disparity, there is an underlying unity in the diseases. This also offers guidelines for the repurposing of old drugs.

Dysfunction and toxicity of damaged proteins in the etiology of aging and age-related degenerative and malignant diseases.

Health can be defined as a harmony, or homeostasis, of the activities of thousands of different proteins, whereas aging and diseases result from their disharmony manifested at the levels of cells and tissues. Such disharmony is caused primarily by dysfunction and toxicity of misfolded proteins damaged by oxidation. This is an overview of key data that inspired new concepts allowing interpretation and integration of the scientific literature on aging and age-related diseases. These concepts suggest strategies for prevention and attenuation of age-related degenerative and malignant diseases mimicking the life of super-centenarians.

Decreasing translation error rate in Escherichia coli increases protein function.

BACKGROUND: Over-expressed native or recombinant proteins are commonly used for industrial and pharmaceutical purposes, as well as for research. Proteins of interest need to be purified in sufficient quantity, quality and specific activity to justify their commercial price and eventual medical use. Proteome quality was previously positively correlated with ribosomal fidelity, but not on a single protein level. Here, we show that decreasing translational error rate increases the activity of single proteins. In order to decrease the amount of enzyme needed for catalysis, we propose an expression system bearing rpsL141 mutation, which confers high ribosomal fidelity. Using alpha-glucosidase (exo-alpha-1,4-glucosidase) and beta-glucanase (beta-D-glucanase) as examples, we show that proteins purified from Escherichia coli bearing rpsL141 mutation have superior activity compared to those purified from wild type E. coli, as well as some commercially available industrial enzymes. RESULTS: Our results indicate that both alpha-glucosidase and beta-glucanase isolated from E. coli bearing rpsL141 mutation have increased activity compared to those isolated from wild type E. coli. Alpha-glucosidase from rpsL141 background has a higher activity than the purchased enzymes, while beta-glucanase from the same background has a higher activity compared to the beta-glucanase purchased from Sigma, but not compared to the one purchased from Megazyme. CONCLUSION: Reduction of the error rate in protein biosynthesis via ribosomal rpsL141 mutation results in superior functionality of single proteins. We conclude that this is a viable system for expressing proteins with higher activity and that it can be easily scaled up and combined with other expression systems to meet the industrial needs.

Phenotypic and genetic consequences of protein damage.

Although the genome contains all the information necessary for maintenance and perpetuation of life, it is the proteome that repairs, duplicates and expresses the genome and actually performs most cellular functions. Here we reveal strong phenotypes of physiological oxidative proteome damage at the functional and genomic levels. Genome-wide mutations rates and biosynthetic capacity were monitored in real time, in single Escherichia coli cells with identical levels of reactive oxygen species and oxidative DNA damage, but with different levels of irreversible oxidative proteome damage (carbonylation). Increased protein carbonylation correlates with a mutator phenotype, whereas reducing it below wild type level produces an anti-mutator phenotype identifying proteome damage as the leading cause of spontaneous mutations. Proteome oxidation elevates also UV-light induced mutagenesis and impairs cellular biosynthesis. In conclusion, protein damage reduces the efficacy and precision of vital cellular processes resulting in high mutation rates and functional degeneracy akin to cellular aging.

Extreme anti-oxidant protection against ionizing radiation in bdelloid rotifers.

Bdelloid rotifers, a class of freshwater invertebrates, are extraordinarily resistant to ionizing radiation (IR). Their radioresistance is not caused by reduced susceptibility to DNA double-strand breakage for IR makes double-strand breaks (DSBs) in bdelloids with essentially the same efficiency as in other species, regardless of radiosensitivity. Instead, we find that the bdelloid Adineta vaga is far more resistant to IR-induced protein carbonylation than is the much more radiosensitive nematode Caenorhabditis elegans. In both species, the dose-response for protein carbonylation parallels that for fecundity reduction, manifested as embryonic death. We conclude that the great radioresistance of bdelloid rotifers is a consequence of an unusually effective system of anti-oxidant protection of cellular constituents, including those required for DSB repair, allowing bdelloids to recover and continue reproducing after doses of IR causing hundreds of DSBs per nucleus. Bdelloid rotifers therefore offer an advantageous system for investigation of enhanced anti-oxidant protection and its consequences in animal systems.

Stoichiometry of MutS and MutL at unrepaired mismatches in vivo suggests a mechanism of repair.

Mismatch repair (MMR) is an evolutionarily conserved DNA repair system, which corrects mismatched bases arising during DNA replication. MutS recognizes and binds base pair mismatches, while the MutL protein interacts with MutS-mismatch complex and triggers MutH endonuclease activity at a distal-strand discrimination site on the DNA. The mechanism of communication between these two distal sites on the DNA is not known. We used functional fluorescent MMR proteins, MutS and MutL, in order to investigate the formation of the fluorescent MMR protein complexes on mismatches in real-time in growing Escherichia coli cells. We found that MutS and MutL proteins co-localize on unrepaired mismatches to form fluorescent foci. MutL foci were, on average, 2.7 times more intense than the MutS foci co-localized on individual mismatches. A steric block on the DNA provided by the MutHE56A mutant protein, which binds to but does not cut the DNA at the strand discrimination site, decreased MutL foci fluorescence 3-fold. This indicates that MutL accumulates from the mismatch site toward strand discrimination site along the DNA. Our results corroborate the hypothesis postulating that MutL accumulation assures the coordination of the MMR activities between the mismatch and the strand discrimination site.

Beyond the genome: protecting the proteome may be the key to preventing skin aging.

Skin aging is associated with a progressive decline in physiological functions, skin cancers and, ultimately, death. It may be categorized as intrinsic or extrinsic, whereby intrinsic aging is attributed to chronological and genetic factors. At the molecular level, skin aging involves changes in protein conformation and function. The skin proteome changes constantly, mainly through carbonylation; an irreversible phenomenon leading to protein accumulation as toxic aggregates that impair cellular physiology and accelerate skin aging. This review details the central role of proteostasis during skin aging and why proteome protection may be a promising approach in mitigating skin aging. A comprehensive literature review of 87 articles focusing on the proteome, proteostasis, proteotoxicity, protein carbonylation, and the impact of the damaged proteome on aging, and in particular skin aging, was conducted. Skin aging is associated with deficiencies in the repair mechanisms of DNA, transcriptional control, mitochondrial function, cell cycle control, apoptosis, cellular metabolism, changes in hormonal levels secondary to toxicity of damaged proteins, and cell-to-cell communication for tissue homeostasis, which are largely controlled by proteins. In this context, a damaged proteome that leads to the loss of proteostasis may be considered as the first step in tissue aging. There is growing evidence that a healthy proteome plays a central role in skin and in maintaining healthy tissues, thus slowing down the process of skin aging. Hence, protecting the proteome against oxidative or other damage may be an appropriate strategy to prevent and delay skin aging.

Protein damage and death by radiation in Escherichia coli and Deinococcus radiodurans.

Deinococcus radiodurans is among a small number of bacterial species that are extremely resistant to ionizing radiation, UV light, toxic chemicals, and desiccation. We measured proteome oxidation (i.e., protein carbonylation, PC) in D. radiodurans as well as in standard and evolved resistant strains of Escherichia coli exposed to ionizing radiation or UVC light and found a consistent correlation with cell killing. The unique quantitative relationship between incurred PC and cell death holds over the entire range of killing for all tested bacteria and for both lethal agents, meaning that both bacterial species are equally sensitive to PC. We show that the extraordinary robustness of D. radiodurans depends on efficient proteome protection (but not DNA protection) against constitutive and radiation-induced PC consisting of low molecular weight cytosolic compounds. Remarkably, experimental evolution of resistance to ionizing radiation in E. coli coevolves with protection against PC. The decline in biosynthetic efficacy of the cellular proteome, as measured by the loss of reproduction of undamaged bacteriophage lambda in irradiated standard and evolved ionizing radiation-resistant E. coli, correlates with radiation-induced oxidative damage to host cells and their sensitivity to ionizing radiation. This correlation suggests that cell death by radiation is caused primarily by oxidative damage with consequential loss of maintenance activities including DNA repair.

A Proteome-Centric View of Ageing, including that of the Skin and Age-Related Diseases: Considerations of a Common Cause and Common Preventative and Curative Interventions.

The proteome comprises all proteins of a cell or organism. To carry their catalytic and structure-related functions, proteins must be correctly folded into their unique native three-dimensional structures. Common oxidative protein damage affects their functionality by impairing their catalytic and interactive specificities. Oxidative damage occurs preferentially to misfolded proteins and fixes the misfolded state. This review provides an overview of the mechanism and consequences of oxidative proteome damage - specifically irreversible protein carbonylation - in relation to ageing, including that of the skin as well as to age-related degeneration and diseases (ARDD) and their mitigation. A literature review of published manuscripts, available from PubMed, focusing on proteome, proteostasis, proteotoxicity, protein carbonylation, related inflammatory diseases, ARDD and the impact of the damaged proteome on ageing. During ageing, proteome damage, especially protein carbonylation, correlates with biological age. Carbonylated proteins form aggregates which can be considered as markers and accelerators of ageing and are common markers of most ARDD. Protein carbonylation leads to general ageing of the organism and organs including the skin and potentially to diseases including Alzheimer and Parkinson disease, diabetes, psoriasis, and skin cancer. Current research is promising and may open new therapeutic approaches and perspectives by targeting proteome protection as an age and ARDD management strategy.

Toxicity of the spike protein of COVID-19 is a redox shift phenomenon: A novel therapeutic approach.

We previously demonstrated that most diseases display a form of anabolism due to mitochondrial impairment: in cancer, a daughter cell is formed; in Alzheimer's disease, amyloid plaques; in inflammation cytokines and lymphokines. The infection by Covid-19 follows a similar pattern. Long-term effects include redox shift and cellular anabolism as a result of the Warburg effect and mitochondrial dysfunction. This unrelenting anabolism leads to the cytokine storm, chronic fatigue, chronic inflammation or neurodegenerative diseases. Drugs such as Lipoic acid and Methylene Blue have been shown to enhance the mitochondrial activity, relieve the Warburg effect and increase catabolism. Similarly, coMeBining Methylene Blue, Chlorine dioxide and Lipoic acid may help reduce long-term Covid-19 effects by stimulating the catabolism.

Reassembly of shattered chromosomes in Deinococcus radiodurans.

Dehydration or desiccation is one of the most frequent and severe challenges to living cells. The bacterium Deinococcus radiodurans is the best known extremophile among the few organisms that can survive extremely high exposures to desiccation and ionizing radiation, which shatter its genome into hundreds of short DNA fragments. Remarkably, these fragments are readily reassembled into a functional 3.28-megabase genome. Here we describe the relevant two-stage DNA repair process, which involves a previously unknown molecular mechanism for fragment reassembly called 'extended synthesis-dependent strand annealing' (ESDSA), followed and completed by crossovers. At least two genome copies and random DNA breakage are requirements for effective ESDSA. In ESDSA, chromosomal fragments with overlapping homologies are used both as primers and as templates for massive synthesis of complementary single strands, as occurs in a single-round multiplex polymerase chain reaction. This synthesis depends on DNA polymerase I and incorporates more nucleotides than does normal replication in intact cells. Newly synthesized complementary single-stranded extensions become 'sticky ends' that anneal with high precision, joining together contiguous DNA fragments into long, linear, double-stranded intermediates. These intermediates require RecA-dependent crossovers to mature into circular chromosomes that comprise double-stranded patchworks of numerous DNA blocks synthesized before radiation, connected by DNA blocks synthesized after radiation.

Speciation of Genes and Genomes: Conservation of DNA Polymorphism by Barriers to Recombination Raised by Mismatch Repair System.

Some basic aspects of human and animal biology and evolution involve the establishment of biological uniqueness of species and individuals within their huge variety. The discrimination among closely related species occurs in their offspring at the level of chromosomal DNA sequence homology, which is required for fertility as the hallmark of species. Biological identification of individuals, i.e., of their biological "self", occurs at the level of protein sequences presented by the MHC/HLA complex as part of the immune system that discriminates non-self from self. Here, a mechanistic molecular model is presented that can explain how DNA sequence divergence and the activity of key mismatch repair proteins, MutS and MutL, lead to 1) genetic separation of closely related species (sympatric speciation) (Fitch and Ayala, Proceedings of the National Academy of Sciences, 1994, 91, 6717-6720), 2) the stability of genomes riddled by diverged repeated sequences, and 3) conservation of highly polymorphic DNA sequence blocks that constitute the immunological self. All three phenomena involve suppression of recombination between diverged homologies, resulting in prevention of gene sharing between closely related genomes (evolution of new species) as well as sequence sharing between closely related genes within a genome (e.g., evolution of immunoglobulin, MHC, and other gene families bearing conserved polymorphisms).

The lambda red proteins promote efficient recombination between diverged sequences: implications for bacteriophage genome mosaicism.

Genome mosaicism in temperate bacterial viruses (bacteriophages) is so great that it obscures their phylogeny at the genome level. However, the precise molecular processes underlying this mosaicism are unknown. Illegitimate recombination has been proposed, but homeologous recombination could also be at play. To test this, we have measured the efficiency of homeologous recombination between diverged oxa gene pairs inserted into lambda. High yields of recombinants between 22% diverged genes have been obtained when the virus Red Gam pathway was active, and 100 fold less when the host Escherichia coli RecABCD pathway was active. The recombination editing proteins, MutS and UvrD, showed only marginal effects on lambda recombination. Thus, escape from host editing contributes to the high proficiency of virus recombination. Moreover, our bioinformatics study suggests that homeologous recombination between similar lambdoid viruses has created part of their mosaicism. We therefore propose that the remarkable propensity of the lambda-encoded Red and Gam proteins to recombine diverged DNA is effectively contributing to mosaicism, and more generally, that a correlation may exist between virus genome mosaicism and the presence of Red/Gam-like systems.

Functionalized Au15 nanoclusters as luminescent probes for protein carbonylation detection.

Atomically precise, ligand-protected gold nanoclusters (AuNCs) attract considerable attention as contrast agents in the biosensing field. However, the control of their optical properties and functionalization of surface ligands remain challenging. Here we report a strategy to tailor AuNCs for the precise detection of protein carbonylation-a causal biomarker of ageing. We produce Au15SG13 (SG for glutathione) with atomic precision and functionalize it with a thiolated aminooxy moiety to impart protein carbonyl-binding properties. Mass spectrometry and molecular modelling reveal the key structural features of Au15SG12-Aminooxy and its reactivity towards carbonyls. Finally, we demonstrate that Au15SG12-Aminooxy detects protein carbonylation in gel-based 1D electrophoresis by one- and two-photon excited fluorescence. Importantly, to our knowledge, this is the first application of an AuNC that detects a post-translational modification as a nonlinear optical probe. The significance of post-translational modifications in life sciences may open avenues for the use of Au15SG13 and other nanoclusters as contrast agents with tailored surface functionalization and optical properties.

Pancreatic Ductal Adenocarcinoma Arising in Young and Old Patients Displays Similar Molecular Features.

Pancreatic ducal adenocarcinoma is classically diagnosed in the 7th decade, but approximately 10% of patients are diagnosed under 55 years (y.o.). While the genomic and transcriptomic landscapes of late-onset tumors (LOT) have been described, little is known about early-onset tumors (EOT). Ageing is known to impact DNA methylation and proteome integrity through carbonylation-related oxidative damages. We therefore aimed to assess the global molecular features of EOT. We compared 176 EOT (≤55 y.o.) and 316 LOT (≥70 y.o.) from three distinct surgical cohorts at the clinical/genomic/epigenomic/transcriptomic level. Furthermore, we assessed oxidative stress responses and oxidative proteome damages using 2D gel electrophoresis followed by mass spectrometry protein identification. There was no consistent clinical difference between EOT and LOT across the three cohorts. The mutational landscape of key driver genes and the global methylation profile were similar in the two groups. LOT did display age-related features such as enriched DNA repair gene signatures and upregulation of oxidative stress defenses together with increased proteome carbonylation. However, these age-related differences were more preeminent in non-tumor tissues while tumor proteome and proteome damages were fairly comparable. In conclusion, this multi-omics comparison showed that EOT harbor a comparable molecular profile to that of LOT.

[Common cause and mechanism for all pathologies of aging?] / Cause commune et mécanisme commun aux maladies du vieillissement ?

Health is harmony, aging and its diseases (are) functional disharmony at the molecular, cellular and tissue levels. Our observations lead us to think that there seems to be a common cause and a common mechanism for aging and its many and diverse diseases. This common cause is the oxidative damage to particular proteins emerging from a combination of imperfect folding and oxidative stress. This common cause jointly goes with the biological clock common to various age-related diseases, whose the incidence increases exponentially over time and causes 90% of human mortality. Pharmacological interventions on the common cause could avoid and simultaneously attenuate all degenerative and malignant diseases, as it is the natural case of super-centenarians.

TITLE: Cause commune et mécanisme commun aux maladies du vieillissement ? ABSTRACT: La santé est l'harmonie, le vieillissement et ses maladies la dysharmonie fonctionnelle aux niveaux moléculaire, cellulaire et tissulaire. Nos observations semblent suggérer une cause commune et un mécanisme commun du vieillissement et de ses nombreuses et diverses maladies. Cette cause commune est le dommage oxydatif de protéines particulières, résultant à la fois de leur mauvais repliement et du stress oxydatif. La cause commune va de pair avec l'horloge biologique des diverses maladies du vieillissement, dont l'incidence augmente exponentiellement avec l'âge, responsables de 90 % de la mortalité humaine. Des interventions pharmacologiques sur la cause commune pourraient éviter et atténuer simultanément toutes les maladies dégénératives et malignes, comme c'est le cas naturellement chez les super-centenaires.

Cellular parabiosis and the latency of age-related diseases.

Cellular parabiosis is tissue-based phenotypic suppression of cellular dysfunction by intercellular molecular traffic keeping initiated age-related diseases and conditions in long latency. Interruption of cellular parabiosis (e.g. by chronic inflammation) promotes the onset of initiated pathologies. The stability of initiated latent cancers and other age-related diseases (ARD) hints to phenotypically silent genome alterations. I propose that latency in the onset of ageing and ARD is largely due to phenotypic suppression of cellular dysfunctions via molecular traffic among neighbouring cells. Intercellular trafficking ranges from the transfer of ions and metabolites (via gap junctions) to entire organelles (via tunnelling nanotubes). Any mechanism of cell-to-cell communication resulting in functional cross-complementation among the cells is called cellular parabiosis. Such 'cellular solidarity' creates tissue homeostasis by buffering defects and averaging cellular functions within the tissues. Chronic inflammation is known to (i) interrupt cellular parabiosis by the activity of extracellular proteases, (ii) activate dormant pathologies and (iii) shorten disease latency, as in tumour promotion and inflammaging. Variation in cellular parabiosis and protein oxidation can account for interspecies correlations between body mass, ARD latency and longevity. Now, prevention of ARD onset by phenotypic suppression, and healing by phenotypic reversion, become conceivable.

Ecobiological approach to research regarding ageing and diseases.

Ageing and age-related diseases (ARD) share a common biological clock that appears as their root cause: protein damage. A majority of proteins have evolved into native structures resistant to oxidative damage but any folding imperfections, including those due to "silent" amino acid substitutions, reduce oxidation resistance. Damaged proteins accumulate with age and trigger ageing-like phenotypes reversible by their turnover, while acquired genome alterations remain as stable consequences of protein malfunction. Ageing and ARD display species-specific latency in phenotypic expression. Disease latency may be proposed as to be due to phenotypic suppression of cellular defects by molecular traffic among neighbouring cells. Such cross-complementation of functional deficiencies acts as a kind of tissue-based cellular "solidarity", called cellular parabiosis. Chronic inflammation reveals dormant cell phenotypes and shortens disease latency by the breakdown of cell-cell communication, as in tumour promotion and inflammation. At the present time, predictive diagnostics, prognostics, prevention and even cure of disease by phenotypic reversion become conceivable.

Protein damage, ageing and age-related diseases.

Ageing is considered as a snowballing phenotype of the accumulation of damaged dysfunctional or toxic proteins and silent mutations (polymorphisms) that sensitize relevant proteins to oxidative damage as inborn predispositions to age-related diseases. Ageing is not a disease, but it causes (or shares common cause with) age-related diseases as suggested by similar slopes of age-related increase in the incidence of diseases and death. Studies of robust and more standard species revealed that dysfunctional oxidatively damaged proteins are the root cause of radiation-induced morbidity and mortality. Oxidized proteins accumulate with age and cause reversible ageing-like phenotypes with some irreversible consequences (e.g. mutations). Here, we observe in yeast that aggregation rate of damaged proteins follows the Gompertz law of mortality and review arguments for a causal relationship between oxidative protein damage, ageing and disease. Aerobes evolved proteomes remarkably resistant to oxidative damage, but imperfectly folded proteins become sensitive to oxidation. We show that α-synuclein mutations that predispose to early-onset Parkinson's disease bestow an increased intrinsic sensitivity of α-synuclein to in vitro oxidation. Considering how initially silent protein polymorphism becomes phenotypic while causing age-related diseases and how protein damage leads to genome alterations inspires a vision of predictive diagnostic, prognostic, prevention and treatment of degenerative diseases.