Inhibition of the repair of Photosystem II by oxidative stress in cyanobacteria

The activity of Photosystem II (PS II) is severely restricted by a variety of environmental factors and, under environmental stress, is determined by the balance between the rate of damage to PS II and the rate of the repair of damaged PS II. The effects of oxidative stress on damage and repair can be examined separately, and it appears that, while light can damage PS II directly, oxidative stress acts primarily by inhibiting the repair of PS II. Studies in cyanobacteria have demonstrated that oxidative stress suppresses the de novo synthesis of proteins, in particular, the D1 protein, which is required for the repair of PS II.     

Mechanisms of clastogen-induced chromosomal aberrations: A critical review and description of a model based on failures of tethering of DNA strand ends to strand-breaking enzymes

Most theories of the mechanisms of chromosomal aberrations involve the concepts of clastogens directly acting on DNA to produce strand breaks, and subsequently, the survival of these directly caused DNA strand breaks – or misrepairs of them – through to metaphase when they appear as chromosomal ‘breaks’ or translocations. Nevertheless, various observations are inconsistent with these theories such as the fact that many chemical clastogens (e.g. caffeine, acridines) do not covalently react with DNA, while almost all of the chemical clastogens (e.g. alkylating agents) which do react covalently with DNA, do not directly cause DNA strand breaks. This paper reviews the ‘direct-clastogen damage to DNA’ theories, and the phenomenology of chromosomal aberrations which are inconsistent with them. Then the theory is considered that the breaks in chromosomes seen at metaphase and anaphase are not the survivors of DNA breaks directly induced by clastogens, but rather derive from breaks created by the enzymes which repair damaged DNA. After that, newer knowledge is reviewed that (i) strand breaks are created during normal DNA unravelling (by topoisomerases), during DNA synthesis, and during DNA repairs, and these breaks can be single- or double-stranded, (ii) breaks variously associated with unravelling, synthesis and repair can occur ‘anywhere, anytime’ (pre-synthesis, synthesis or post-synthesis) in the cell cycle, and (iii) the enzyme assemblies for DNA unravelling, synthesis and repair which make and religate the breaks must be non-covalently tethered to the ends of the DNA strands while the breaks created by the enzymes are in existence. It is then suggested that all the morphological types and other phenomena of chromosomal aberrations can be explained by aspects, mechanisms and effects of failures of this tethering function. Circumstances involving the basic mechanism (failure of DNA-end-tethering function while enzyme-created breaks are in existence) are described which might result in ‘gaps’, translocations (‘exchanges’), complex lesions such as ‘triradials’, as well as in ‘minutes’, amplifications and inversions. Predictions are made concerning likely results in various suggested studies including those involving sensitive assays for DNA-end-to-enzyme tethering functions in vitro.     

Some reassembly required

Cells invest a significant amount of their energy synthesizing proteins, and a large portion of the energy expenditure goes into making ribosomes, the RNA‐protein machines at the centre of translation. When ribosomes are damaged in a cell, i.e. during stressful conditions, cells must first recognize the damage and then mount a response. Remme et al. show that instead of having to rebuild ribosomes from scratch, bacteria can repair ribosomes by replacing damaged proteins in situ, thereby saving significant time and energy. Given the central role of translation, such repair mechanisms might be widespread in nature.     

Evolutionary couplings of amino acid residues reveal structure and function of bacterial signaling proteins

The genomic era along with major advances in high‐throughput sequencing technology has led to a rapid expansion of the genomic and consequently the protein sequence space. Bacterial extracytoplasmic function sigma factors have emerged as an important group of signaling proteins in bacteria involved in many regulatory decisions, most notably the adaptation to cell envelope stress. Their wide prevalence and amplification among bacterial genomes has led to sub‐group classification and the realization of diverse signaling mechanisms. Mathematical frameworks have been developed to utilize extensive protein sequence alignments to extract co‐evolutionary signals of interaction. This has proven useful in a number of different biological fields, including de novo structure prediction, protein–protein partner identification and the elucidation of alternative protein conformations for signal proteins, to name a few. The mathematical tools, commonly referred to under the name ‘Direct Coupling Analysis’ have now been applied to deduce molecular mechanisms of activation for sub‐groups of extracytoplasmic sigma factors adding to previous successes on bacterial two‐component signaling proteins. The amplification of signal transduction protein genes in bacterial genomes made them the first to be amenable to this approach but the sequences are available now to aid the molecular microbiologist, no matter their protein pathway of interest.     

Chromosome healing: Spontaneous and programmed de novo telomere formation by telomerase

Telomeres are protective caps for chromosome ends that are essential for genome stability. Broken chromosomes missing a telomere will not be maintained unless the chromosome is ‘healed’ with the formation of a new telomere. Chromosome healing can be a programmed event following developmentally regulated chromosome fragmentation, or it may occur spontaneously when a chromosome is accidentally broken. In this article we discuss the consequences of telomere loss and the possible mechanisms that the enzyme telomerase employs to form telomeres de novo on broken chromosome ends.     

Photoinhibition of photosystem II under environmental stress

Inhibition of the activity of photosystem II (PSII) under strong light is referred to as photoinhibition. This phenomenon is due to an imbalance between the rate of photodamage to PSII and the rate of the repair of damaged PSII. In the “classical” scheme for the mechanism of photoinhibition, strong light induces the production of reactive oxygen species (ROS), which directly inactivate the photochemical reaction center of PSII. By contrast, in a new scheme, we propose that photodamage is initiated by the direct effect of light on the oxygen-evolving complex and that ROS inhibit the repair of photodamaged PSII by suppressing primarily the synthesis of proteins de novo. The activity of PSII is restricted by a variety of environmental stresses. The effects of environmental stress on damage to and repair of PSII can be examined separately and it appears that environmental stresses, with the exception of strong light, act primarily by inhibiting the repair of PSII. Studies have demonstrated that repair-inhibitory stresses include CO₂ limitation, moderate heat, high concentrations of NaCl, and low temperature, each of which suppresses the synthesis of proteins de novo, which is required for the repair of PSII. We postulate that most types of environmental stress inhibit the fixation of CO₂ with the resultant generation of ROS, which, in turn, inhibit protein synthesis.     

Regulation and dysregulation of fibrosis in skeletal muscle

In response to skeletal muscle injury, distinct cellular pathways are activated to repair the damaged tissue. Activation and restriction of these pathways must be temporally coordinated in a precise sequence as regeneration progresses if muscle integrity and homeostasis are to be restored. However, if tissue injury persists, as in severe muscular dystrophies, the repair process becomes uncontrolled leading to the substitution of myofibers by a non-functional mass of fibrotic tissue. In this review, we provide an overview of how muscle responds to damage and aging, with special emphasis on the cellular effectors and the regulatory and inflammatory pathways that can shift normal muscle repair to fibrosis development.     

Algorithms for the de novo sequencing of peptides from tandem mass spectra

Proteomics is the study of proteins, their time- and location-dependent expression profiles, as well as their modifications and interactions. Mass spectrometry is useful to investigate many of the questions asked in proteomics. Database search methods are typically employed to identify proteins from complex mixtures. However, databases are not often available or, despite their availability, some sequences are not readily found therein. To overcome this problem, de novo sequencing can be used to directly assign a peptide sequence to a tandem mass spectrometry spectrum. Many algorithms have been proposed for de novo sequencing and a selection of them are detailed in this article. Although a standard accuracy measure has not been agreed upon in the field, relative algorithm performance is discussed. The current state of the de novo sequencing is assessed thereafter and, finally, examples are used to construct possible future perspectives of the field.     

Mediat(iz)ing Catholicism: saint,spectacle, and theopolitics in Lima,Peru

This article examines the annual public procession in Lima, Peru, of the Señor de los Milagros (Lord of Miracles) in relation to issues at the intersection of Catholic Christianity, media, and political authority. Through a theopolitical lens alert to the intermeshing of political sovereignty and authority with theological (Catholic) worldviews, I inquire into media and the Señor de los Milagros procession along three key intersecting themes that link scales of local and global Catholicism: performance, identity/belonging, and control. Key to my argument is the idea of the miraculous (lo milagroso), a culturally resonant register of embodied affective experience with compelling power, which points to how senses of belonging, authority, and ‘proper’ Catholic subjecthoods are intensified by Catholicism's diffusion through new mediatic forms, especially in church-generated productions. A consideration of media technologies, mediation, and Catholicism nuances theoretical assumptions within the anthropology of Christianity, and also suggests that the anthropology of religion should attend more closely to mediation and mediatization as newer media infrastructures – channelling flows of information, images, and affects – extend ‘the religious’ into other social spheres.     

Don't forget to be picky – selective autophagy of protein aggregates in neurodegenerative diseases

The homeostasis of cells depends on the selective degradation of damaged or superfluous cellular components. Autophagy is the major pathway that recognizes such components, sequesters them in de novo formed autophagosomes and delivers them to lysosomes for degradation. The recognition of specific cargo and the biogenesis of autophagosomes involve a dedicated machinery of autophagy related (ATG) proteins. Intense research over the past decades has revealed insights into the function of autophagy proteins and mechanisms that govern cargo recognition. Other aspects including the molecular mechanisms involved in the onset of human diseases are less well understood. However, autophagic dysfunctions, caused by age related decline in autophagy or mutations in ATG proteins, are directly related to a large number of human pathologies including neurodegenerative disorders. Here, we review most recent discoveries and breakthroughs in selective autophagy and its relationship to neurodegeneration.     

Metabolite profiling and peptidoglycan analysis of transient cell wall‐deficient bacteria in a new Escherichia coli model system

Many bacteria are able to assume a transient cell wall‐deficient (or L‐form) state under favourable osmotic conditions. Cell wall stress such as exposure to β‐lactam antibiotics can enforce the transition to and maintenance of this state. L‐forms actively proliferate and can return to the walled state upon removal of the inducing agent. We have adopted Escherichia coli as a model system for the controlled transition to and reversion from the L‐form state, and have studied these dynamics with genetics, cell biology and ‘omics’ technologies. As such, a transposon mutagenesis screen underscored the requirement for the Rcs phosphorelay and colanic acid synthesis, while proteomics show only little differences between rods and L‐forms. In contrast, metabolome comparison reveals the high abundance of lysophospholipids and phospholipids with unsaturated or cyclopropanized fatty acids in E. coli L‐forms. This increase of membrane lipids associated with increased membrane fluidity may facilitate proliferation through bud formation. Visualization of the residual peptidoglycan with a fluorescently labelled peptidoglycan binding protein indicates de novo cell wall synthesis and a role for septal peptidoglycan synthesis during bud constriction. The DD‐carboxypeptidases PBP5 and PBP6 are threefold and fourfold upregulated in L‐forms, indicating a specific role for regulation of crosslinking during L‐form proliferation.     

DNA damage-induced inhibition of rRNA synthesis by DNA-PK and PARP-1

RNA synthesis and DNA replication cease after DNA damage. We studied RNA synthesis using an in situ run-on assay and found ribosomal RNA (rRNA) synthesis was inhibited 24 h after UV light, gamma radiation or DNA cross-linking by cisplatin in human cells. Cisplatin led to accumulation of cells in S phase. Inhibition of the DNA repair proteins DNA-dependent protein kinase (DNA-PK) or poly(ADP-ribose) polymerase 1 (PARP-1) prevented the DNA damage-induced block of rRNA synthesis. However, DNA-PK and PARP-1 inhibition did not prevent the cisplatin-induced arrest of cell cycle in S phase, nor did it induce de novo BrdU incorporation. Loss of DNA-PK function prevented activation of PARP-1 and its recruitment to chromatin in damaged cells, suggesting regulation of PARP-1 by DNA-PK within a pathway of DNA repair. From these results, we propose a sequential activation of DNA-PK and PARP-1 in cells arrested in S phase by DNA damage causes the interruption of rRNA synthesis after DNA damage.     

Neutralizing Antibodies Against Allosteric Proteins: Insights From a Bacterial Adhesin

Allosteric proteins transition between ‘inactive’ and ‘active’ states. In general, such proteins assume distinct conformational states at the level of secondary, tertiary and/or quaternary structure. Different conformers of an allosteric protein can be antigenically dissimilar and induce antibodies with a highly distinctive specificities and neutralizing functional effects. Here we summarize studies on various functional types of monoclonal antibodies obtained against different allosteric conformers of the mannose-specific bacterial adhesin FimH – the most common cell attachment protein of Escherichia coli and other enterobacterial pathogens. Included are types of antibodies that activate the FimH function via interaction with ligand-induced binding sites or by wedging between domains as well as antibodies that inhibit FimH through orthosteric, parasteric, or novel dynasteric mechanisms. Understanding the molecular mechanism of antibody action against allosteric proteins provides insights on how to design antibodies with a desired functional effect, including those with neutralizing activity against bacterial and viral cell attachment proteins.     

Two-dimensional proteome reference map of Vibrio tapetis, the aetiological agent of brown ring disease in clams

Aims: Vibrio tapetis is the etiological agent of brown ring disease (BRD) in clams, one of the most threatening diseases affecting this commercially important bivalve. In this study we have constructed a proteome reference map of the V. tapetis type strain CECT 4600^T. Methods and Results: Eighty‐two proteins, consistently present in all 2D‐gels, were identified by mass spectrometry or by de novo sequencing. The majority of the proteins identified (66%) belonged to four COG categories: ‘Carbohydrate transport and metabolism’, ‘Post‐translational modification, protein turnover and chaperones’, ‘Energy production’, and ‘Amino acid transport and metabolism’. Glyceraldehyde‐3‐phosphate dehydrogenase, enolase, fructose‐bisphosphate aldolase, phosphoglycerate kinase. molecular chaperones Dnak and GroEL, alkyl hydroperoxide reductase, peptidyl‐prolyl cis‐trans isomerase B and factor Tu, were identified among the 20 most abundant proteins. A comparison of this reference map with that obtained for the V. tapetis strain GR0202RD, with different origin and pathophysiological characteristics, was performed. Conclusions: Under the culture conditions employed in this study, glucose degradation is one of the major pathways for energy production in Vibrio tapetis. In addition, the two strains studied, although with remarkable differences at genetic and pathophysiological levels, showed a high similarity under laboratory conditions. Significance and Impact of the Study: The results obtained here can be considered as a first step to gather valuable information on protein expression, related not only to diverse cellular functions and regulation but also to pathogenesis and bacterium‐host interactions in the disease process.     

Defending the mitochondria: The pathways of mitophagy and mitochondrial-derived vesicles

Mitochondria are the powerhouses for the cell, consuming oxygen to generate sufficient energy for the maintenance of normal cellular processes. However, a deleterious consequence of this process are reactive oxygen species generated as side-products of these reactions. As a means to protect mitochondria from damage, cells and mitochondria have developed a wide-range of mitochondrial quality control mechanisms that remove damaged mitochondrial cargo, enabling the mitochondria to repair the damage and ultimately restore their normal function. If the damage is extensive and mitochondria can no longer be repaired, a process termed mitophagy is initiated in which the mitochondria are directed for autophagic clearance. Canonical mitophagy is regulated by two proteins, PINK1 and Parkin, which are mutated in familial forms of Parkinson’s disease. In this review, we discuss recent work elucidating the mechanism of PINK1/Parkin-mediated mitophagy, along with recently uncovered PINK1/Parkin-independent mitophagy pathways. Moreover, we describe a novel mitochondrial quality control pathway, involving mitochondrial-derived vesicles that direct distinct and damaged mitochondrial cargo for degradation in the lysosome. Finally, we discuss the association between mitochondrial quality control, cardiac, hepatic and neurodegenerative disease and discuss the possibility of targeting these pathways for therapeutic purposes.     

Quantitative chemical proteomics profiling of de novo protein synthesis during starvation-mediated autophagy

Autophagy is an intracellular degradation mechanism in response to nutrient starvation. Via autophagy, some nonessential cellular constituents are degraded in a lysosome-dependent manner to generate biomolecules that can be utilized for maintaining the metabolic homeostasis. Although it is known that under starvation the global protein synthesis is significantly reduced mainly due to suppression of MTOR (mechanistic target of rapamycin serine/threonine kinase), emerging evidence demonstrates that de novo protein synthesis is involved in the autophagic process. However, characterizing these de novo proteins has been an issue with current techniques. Here, we developed a novel method to identify newly synthesized proteins during starvation-mediated autophagy by combining bio-orthogonal noncanonical amino acid tagging (BONCAT) and isobaric tags for relative and absolute quantitation (iTRAQ^TM). Using bio-orthogonal metabolic tagging, L-azidohomoalanine (AHA) was incorporated into newly synthesized proteins which were then enriched with avidin beads after a click reaction between alkyne-bearing biotin and AHA's bio-orthogonal azide moiety. The enriched proteins were subjected to iTRAQ labeling for protein identification and quantification using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Via the above approach, we identified and quantified a total of 1176 proteins and among them 711 proteins were found to meet our defined criteria as de novo synthesized proteins during starvation-mediated autophagy. The characterized functional profiles of the 711 newly synthesized proteins by bioinformatics analysis suggest their roles in ensuring the prosurvival outcome of autophagy. Finally, we performed validation assays for some selected proteins and found that knockdown of some genes has a significant impact on starvation-induced autophagy. Thus, we think that the BONCAT-iTRAQ approach is effective in the identification of newly synthesized proteins and provides useful insights to the molecular mechanisms and biological functions of autophagy.     

Beyond base excision repair: an evolving picture of mitochondrial DNA repair

Mitochondria are highly specialised organelles required for key cellular processes including ATP production through cellular respiration and controlling cell death via apoptosis. Unlike other organelles, mitochondria contain their own DNA genome which encodes both protein and RNA required for cellular respiration. Each cell may contain hundreds to thousands of copies of the mitochondrial genome, which is essential for normal cellular function – deviation of mitochondrial DNA (mtDNA) copy number is associated with cellular ageing and disease. Furthermore, mtDNA lesions can arise from both endogenous or exogenous sources and must either be tolerated or corrected to preserve mitochondrial function. Importantly, replication of damaged mtDNA can lead to stalling and introduction of mutations or genetic loss, mitochondria have adapted mechanisms to repair damaged DNA. These mechanisms rely on nuclear-encoded DNA repair proteins that are translocated into the mitochondria.Despite the presence of many known nuclear DNA repair proteins being found in the mitochondrial proteome, it remains to be established which DNA repair mechanisms are functional in mammalian mitochondria. Here, we summarise the existing and emerging research, alongside examining proteomic evidence, demonstrating that mtDNA damage can be repaired using Base Excision Repair (BER), Homologous Recombination (HR) and Microhomology-mediated End Joining (MMEJ). Critically, these repair mechanisms do not operate in isolation and evidence for interplay between pathways and repair associated with replication is discussed. Importantly, characterising non-canonical functions of key proteins and understanding the bespoke pathways used to tolerate, repair or bypass DNA damage will be fundamental in fully understanding the causes of mitochondrial genome mutations and mitochondrial dysfunction.