Showing papers in "Essays in Biochemistry in 2017"

The mammalian ULK1 complex and autophagy initiation.

Abstract: Autophagy is a vital lysosomal degradation pathway that serves as a quality control mechanism. It rids the cell of damaged, toxic or excess cellular components, which if left to persist could be detrimental to the cell. It also serves as a recycling pathway to maintain protein synthesis under starvation conditions. A key initial event in autophagy is formation of the autophagosome, a unique double-membrane organelle that engulfs the cytosolic cargo destined for degradation. This step is mediated by the serine/threonine protein kinase ULK1 (unc-51-like kinase 1), which functions in a complex with at least three protein partners: FIP200 (focal adhesion kinase family interacting protein of 200 kDa), ATG (autophagy-related protein) 13 (ATG13), and ATG101. In this artcile, we focus on the regulation of the ULK1 complex during autophagy initiation. The complex pattern of upstream pathways that converge on ULK1 suggests that this complex acts as a node, converting multiple signals into autophagosome formation. Here, we review our current understanding of this regulation and in turn discuss what happens downstream, once the ULK1 complex becomes activated.

Regulation of selective autophagy: the p62/SQSTM1 paradigm

Abstract: In selective autophagy, cytoplasmic components are selected and tagged before being sequestered into an autophagosome by means of selective autophagy receptors such as p62/SQSTM1. In this review, we discuss how selective autophagy is regulated. An important level of regulation is the selection of proteins or organelles for degradation. Components selected for degradation are tagged, often with ubiquitin, to facilitate recognition by autophagy receptors. Another level of regulation is represented by the autophagy receptors themselves. For p62, its ability to co-aggregate with ubiquitinated substrates is strongly induced by post-translational modifications (PTMs). The transcription of p62 is also markedly increased during conditions in which selective autophagy substrates accumulate. For other autophagy receptors, the LC3-interacting region (LIR) motif is regulated by PTMs, inhibiting or stimulating the interaction with ATG8 family proteins. ATG8 proteins are also regulated by PTMs. Regulation of the capacity of the core autophagy machinery also affects selective autophagy. Importantly, autophagy receptors can induce local recruitment and activation of ULK1/2 and PI3KC3 complexes at the site of cargo sequestration.

mTORC1 as the main gateway to autophagy.

Abstract: Cells and organisms must coordinate their metabolic activity with changes in their environment to ensure their growth only when conditions are favourable. In order to maintain cellular homoeostasis, a tight regulation between the synthesis and degradation of cellular components is essential. At the epicentre of the cellular nutrient sensing is the mechanistic target of rapamycin complex 1 (mTORC1) which connects environmental cues, including nutrient and growth factor availability as well as stress, to metabolic processes in order to preserve cellular homoeostasis. Under nutrient-rich conditions mTORC1 promotes cell growth by stimulating biosynthetic pathways, including synthesis of proteins, lipids and nucleotides, and by inhibiting cellular catabolism through repression of the autophagic pathway. Its close signalling interplay with the energy sensor AMP-activated protein kinase (AMPK) dictates whether the cell actively favours anabolic or catabolic processes. Underlining the role of mTORC1 in the coordination of cellular metabolism, its deregulation is linked to numerous human diseases ranging from metabolic disorders to many cancers. Although mTORC1 can be modulated by a number of different inputs, amino acids represent primordial cues that cannot be compensated for by any other stimuli. The understanding of how amino acids signal to mTORC1 has increased considerably in the last years; however this area of research remains a hot topic in biomedical sciences. The current ideas and models proposed to explain the interrelationship between amino acid sensing, mTORC1 signalling and autophagy is the subject of the present review.

Molecular recognition of ternary complexes: a new dimension in the structure-guided design of chemical degraders.

Abstract: Molecular glues and bivalent inducers of protein degradation (also known as PROTACs) represent a fascinating new modality in pharmacotherapeutics: the potential to knockdown previously thought 'undruggable' targets at sub-stoichiometric concentrations in ways not possible using conventional inhibitors. Mounting evidence suggests these chemical agents, in concert with their target proteins, can be modelled as three-body binding equilibria that can exhibit significant cooperativity as a result of specific ligand-induced molecular recognition. Despite this, many existing drug design and optimization regimens still fixate on binary target engagement, in part due to limited structural data on ternary complexes. Recent crystal structures of protein complexes mediated by degrader molecules, including the first PROTAC ternary complex, underscore the importance of protein-protein interactions and intramolecular contacts to the mode of action of this class of compounds. These discoveries have opened the door to a new paradigm for structure-guided drug design: borrowing surface area and molecular recognition from nature to elicit cellular signalling.

ER homeostasis and autophagy

Abstract: The endoplasmic reticulum (ER) is a key site for lipid biosynthesis and folding of nascent transmembrane and secretory proteins. These processes are maintained by careful homeostatic control of the environment within the ER lumen. Signalling sensors within the ER detect perturbations within the lumen (ER stress) and employ downstream signalling cascades that engage effector mechanisms to restore homeostasis. The most studied signalling mechanism that the ER employs is the unfolded protein response (UPR), which is known to increase a number of effector mechanisms, including autophagy. In this chapter, we will discuss the emerging role of autophagy as a UPR effector pathway. We will focus on the recently discovered selective autophagy pathway for ER, ER-phagy, with particular emphasis on the structure and function of known mammalian ER-phagy receptors, namely FAM134B, SEC62, RTN3 and CCPG1. Finally, we conclude with our view of where the future of this field can lead our understanding of the involvement of ER-phagy in ER homeostasis.

Dysregulation of autophagy as a common mechanism in lysosomal storage diseases.

Abstract: The lysosome plays a pivotal role between catabolic and anabolic processes as the nexus for signalling pathways responsive to a variety of factors, such as growth, nutrient availability, energetic status and cellular stressors. Lysosomes are also the terminal degradative organelles for autophagy through which macromolecules and damaged cellular components and organelles are degraded. Autophagy acts as a cellular homeostatic pathway that is essential for organismal physiology. Decline in autophagy during ageing or in many diseases, including late-onset forms of neurodegeneration is considered a major contributing factor to the pathology. Multiple lines of evidence indicate that impairment in autophagy is also a central mechanism underlying several lysosomal storage disorders (LSDs). LSDs are a class of rare, inherited disorders whose histopathological hallmark is the accumulation of undegraded materials in the lysosomes due to abnormal lysosomal function. Inefficient degradative capability of the lysosomes has negative impact on the flux through the autophagic pathway, and therefore dysregulated autophagy in LSDs is emerging as a relevant disease mechanism. Pathology in the LSDs is generally early-onset, severe and life-limiting but current therapies are limited or absent; recognizing common autophagy defects in the LSDs raises new possibilities for therapy. In this review, we describe the mechanisms by which LSDs occur, focusing on perturbations in the autophagy pathway and present the latest data supporting the development of novel therapeutic approaches related to the modulation of autophagy.

Intrinsic, adaptive and acquired antimicrobial resistance in Gram-negative bacteria.

Abstract: Gram-negative bacteria are responsible for a large proportion of antimicrobial-resistant infections in humans and animals. Among this class of bacteria are also some of the most successful environmental organisms. Part of this success is their adaptability to a variety of different niches, their intrinsic resistance to antimicrobial drugs and their ability to rapidly acquire resistance mechanisms. These mechanisms of resistance are not exclusive and the interplay of several mechanisms causes high levels of resistance. In this review, we explore the molecular mechanisms underlying resistance in Gram-negative organisms and how these different mechanisms enable them to survive many different stress conditions.

Current perspectives in fragment-based lead discovery (FBLD)

Abstract: It is over 20 years since the first fragment-based discovery projects were disclosed. The methods are now mature for most ‘conventional’ targets in drug discovery such as enzymes (kinases and proteases) but there has also been growing success on more challenging targets, such as disruption of protein–protein interactions. The main application is to identify tractable chemical startpoints that non-covalently modulate the activity of a biological molecule. In this essay, we overview current practice in the methods and discuss how they have had an impact in lead discovery – generating a large number of fragment-derived compounds that are in clinical trials and two medicines treating patients. In addition, we discuss some of the more recent applications of the methods in chemical biology – providing chemical tools to investigate biological molecules, mechanisms and systems.

Autophagy impairment in Parkinson's disease.

Abstract: Parkinson's disease (PD) is a debilitating movement disorder typically associated with the accumulation of intracytoplasmic aggregate prone protein deposits. Over recent years, increasing evidence has led to the suggestion that the mutations underlying certain forms of PD impair autophagy. Autophagy is a degradative pathway that delivers cytoplasmic content to lysosomes for degradation and represents a major route for degradation of aggregated cellular proteins and dysfunctional organelles. Autophagy up-regulation is a promising therapeutic strategy that is being explored for its potential to protect cells against the toxicity of aggregate-prone proteins in neurodegenerative diseases. Here, we describe how the mutations in different subtypes of PD can affect different stages of autophagy.

Autophagy in health and disease: focus on the cardiovascular system.

Abstract: Autophagy is a highly conserved mechanism of lysosome-mediated protein and organelle degradation that plays a crucial role in maintaining cellular homeostasis. In the last few years, specific functions for autophagy have been identified in many tissues and organs. In the cardiovascular system, autophagy appears to be essential to heart and vessel homeostasis and function; however defective or excessive autophagy activity seems to contribute to major cardiovascular disorders including heart failure (HF) or atherosclerosis. Here, we review the current knowledge on the role of cardiovascular autophagy in physiological and pathophysiological conditions.

Metallochaperones and metalloregulation in bacteria.

Abstract: Bacterial transition metal homoeostasis or simply 'metallostasis' describes the process by which cells control the intracellular availability of functionally required metal cofactors, from manganese (Mn) to zinc (Zn), avoiding both metal deprivation and toxicity. Metallostasis is an emerging aspect of the vertebrate host-pathogen interface that is defined by a 'tug-of-war' for biologically essential metals and provides the motivation for much recent work in this area. The host employs a number of strategies to starve the microbial pathogen of essential metals, while for others attempts to limit bacterial infections by leveraging highly competitive metals. Bacteria must be capable of adapting to these efforts to remodel the transition metal landscape and employ highly specialized metal sensing transcriptional regulators, termed metalloregulatory proteins,and metallochaperones, that allocate metals to specific destinations, to mediate this adaptive response. In this essay, we discuss recent progress in our understanding of the structural mechanisms and metal specificity of this adaptive response, focusing on energy-requiring metallochaperones that play roles in the metallocofactor active site assembly in metalloenzymes and metallosensors, which govern the systems-level response to metal limitation and intoxication.

Antimicrobial resistance in healthcare, agriculture and the environment: the biochemistry behind the headlines.

Abstract: The crisis of antimicrobial resistance (AMR) is one of the most serious issues facing us today. The scale of the problem is illustrated by the recent commitment of Heads of State at the UN to coordinate efforts to curb the spread of AMR infections. In this review, we explore the biochemistry behind the headlines of a few stories that were recently published in the public media. We focus on examples from three different issues related to AMR: (i) hospital-acquired infections, (ii) the spread of resistance through animals and/or the environment and (iii) the role of antimicrobial soaps and other products containing disinfectants in the dissemination of AMR. Although these stories stem from three very different settings, the underlying message in all of them is the same: there is a direct relationship between the use of antimicrobials and the development of resistance. In addition, one type of antimicrobial could select for cross-resistance to another type and/or for multidrug resistance. Therefore, we argue the case for increased stewardship to not only cover clinical use of antibiotics, but also the use of antimicrobials in agriculture and stewardship of our crucially important biocides such as chlorhexidine.

The role played by drug efflux pumps in bacterial multidrug resistance.

Abstract: Antimicrobial resistance is a current major challenge in chemotherapy and infection control. The ability of bacterial and eukaryotic cells to recognize and pump toxic compounds from within the cell to the environment before they reach their targets is one of the important mechanisms contributing to this phenomenon. Drug efflux pumps are membrane transport proteins that require energy to export substrates and can be selective for a specific drug or poly-specific that can export multiple structurally diverse drug compounds. These proteins can be classified into seven groups based on protein sequence homology, energy source and overall structure. Extensive studies on efflux proteins have resulted in a wealth of knowledge that has made possible in-depth understanding of the structures and mechanisms of action, substrate profiles, regulation and possible inhibition of many clinically important efflux pumps. This review focuses on describing known families of drug efflux pumps using examples that are well characterized structurally and/or biochemically.

Ion channels and ion selectivity.

Abstract: Specific macromolecular transport systems, ion channels and pumps, provide the pathways to facilitate and control the passage of ions across the lipid membrane. Ion channels provide energetically favourable passage for ions to diffuse rapidly and passively according to their electrochemical potential. Selective ion channels are essential for the excitability of biological membranes: the action potential is a transient phenomenon that reflects the rapid opening and closing of voltage-dependent Na+-selective and K+-selective channels. One of the most critical functional aspects of K+ channels is their ability to remain highly selective for K+ over Na+ while allowing high-throughput ion conduction at a rate close to the diffusion limit. Permeation through the K+ channel selectivity filter is believed to proceed as a 'knockon' mechanism, in which 2-3 K+ ions interspersed by water molecules move in a single file. Permeation through the comparatively wider and less selective Na+ channels also proceeds via a loosely coupled knockon mechanism, although the ions do not need to be fully dehydrated. While simple structural concepts are often invoked to rationalize the mechanism of ion selectivity, a deeper analysis shows that subtle effects play an important role in these flexible dynamical structures.

Proteomics and metabolomics in ageing research: From biomarkers to systems biology

Abstract: Age is the single greatest risk factor for a wide range of diseases, and as the mean age of human populations grows steadily older, the impact of this risk factor grows as well. Laboratory studies on the basic biology of ageing have shed light on numerous genetic pathways that have strong effects on lifespan. However, we still do not know the degree to which the pathways that affect ageing in the lab also influence variation in rates of ageing and age-related disease in human populations. Similarly, despite considerable effort, we have yet to identify reliable and reproducible 'biomarkers', which are predictors of one's biological as opposed to chronological age. One challenge lies in the enormous mechanistic distance between genotype and downstream ageing phenotypes. Here, we consider the power of studying 'endophenotypes' in the context of ageing. Endophenotypes are the various molecular domains that exist at intermediate levels of organization between the genotype and phenotype. We focus our attention specifically on proteins and metabolites. Proteomic and metabolomic profiling has the potential to help identify the underlying causal mechanisms that link genotype to phenotype. We present a brief review of proteomics and metabolomics in ageing research with a focus on the potential of a systems biology and network-centric perspective in geroscience. While network analyses to study ageing utilizing proteomics and metabolomics are in their infancy, they may be the powerful model needed to discover underlying biological processes that influence natural variation in ageing, age-related disease, and longevity.

Structure-based drug design: aiming for a perfect fit.

Abstract: Knowledge of the three-dimensional structure of therapeutically relevant targets has informed drug discovery since the first protein structures were determined using X-ray crystallography in the 1950s and 1960s. In this editorial we provide a brief overview of the powerful impact of structure-based drug design (SBDD), which has its roots in computational and structural biology, with major contributions from both academia and industry. We describe advances in the application of SBDD for integral membrane protein targets that have traditionally proved very challenging. We emphasize the major progress made in fragment-based approaches for which success has been exemplified by over 30 clinical drug candidates and importantly three FDA-approved drugs in oncology. We summarize the articles in this issue that provide an excellent snapshot of the current state of the field of SBDD and fragment-based drug design and which offer key insights into exciting new developments, such as the X-ray free-electron laser technology, cryo-electron microscopy, open science approaches and targeted protein degradation. We stress the value of SBDD in the design of high-quality chemical tools that are used to interrogate biology and disease pathology, and to inform target validation. We emphasize the need to maintain the scientific rigour that has been traditionally associated with structural biology and extend this to other methods used in drug discovery. This is particularly important because the quality and robustness of any form of contributory data determines its usefulness in accelerating drug design, and therefore ultimately in providing patient benefit.

Fitness costs associated with the acquisition of antibiotic resistance.

Abstract: Acquisition of antibiotic resistance is a relevant problem for human health. The selection and spread of antibiotic-resistant organisms not only compromise the treatment of infectious diseases, but also the implementation of different therapeutic procedures as organ transplantation, advanced surgery or chemotherapy, all of which require proficient methods for avoiding infections. It has been generally accepted that the acquisition of antibiotic resistance will produce a general metabolic burden: in the absence of selection, the resistant organisms would be outcompeted by the susceptible ones. If that was always true, discontinuation of antibiotic use would render the disappearance of resistant microorganisms. However, several studies have shown that, once resistance emerges, the recovery of a fully susceptible population even in the absence of antibiotics is not easy. In the present study, we review updated information on the effect of the acquisition of antibiotic resistance in bacterial physiology as well as on the mechanisms that allow the compensation of the fitness costs associated with the acquisition of resistance.

Origins of mtDNA mutations in ageing.

Abstract: MtDNA mutations are one of the hallmarks of ageing and age-related diseases. It is well established that somatic point mutations accumulate in mtDNA of multiple organs and tissues with increasing age and heteroplasmy is universal in mammals. However, the origin of these mutations remains controversial. The long-lasting hypothesis stating that mtDNA mutations emanate from oxidative damage via a self-perpetuating mechanism has been extensively challenged in recent years. Contrary to this initial ascertainment, mtDNA appears to be well protected from action of reactive oxygen species (ROS) through robust protein coating and endomitochondrial microcompartmentalization. Extensive development of scrupulous high-throughput DNA sequencing methods suggests that an imperfect replication process, rather than oxidative lesions are the main sources of mtDNA point mutations, indicating that mtDNA polymerase γ (POLG) might be responsible for the majority of mtDNA mutagenic events. Here, we summarize the recent knowledge in prevention and defence of mtDNA oxidative lesions and discuss the plausible mechanisms of mtDNA point mutation generation and fixation.

Antibiotic-non-antibiotic combinations for combating extremely drug-resistant Gram-negative 'superbugs'

Abstract: The emergence of antimicrobial resistance of Gram-negative pathogens has become a worldwide crisis. The status quo for combating resistance is to employ synergistic combinations of antibiotics. Faced with this fast-approaching post-antibiotic era, it is critical that we devise strategies to prolong and maximize the clinical efficacy of existing antibiotics. Unfortunately, reports of extremely drug-resistant (XDR) Gram-negative pathogens have become more common. Combining antibiotics such as polymyxin B or the broad-spectrum tetracycline and minocycline with various FDA-approved non-antibiotic drugs have emerged as a novel combination strategy against otherwise untreatable XDR pathogens. This review surveys the available literature on the potential benefits of employing antibiotic-non-antibiotic drug combination therapy. The apex of this review highlights the clinical utility of this novel therapeutic strategy for combating infections caused by 'superbugs'.

Molecular control of chaperone-mediated autophagy.

Abstract: Chaperone-mediated autophagy (CMA) is a selective form of autophagy in which cytosolic proteins bearing a pentapeptide motif biochemically related to the KFERQ sequence, are recognized by the heat shock protein family A member 8 (HSPA8) chaperone, delivered to the lysomal membrane, and directly translocated across the lysosomal membrane by a protein complex containing lysosomal associated membrane protein 2a (Lamp2a). Since its discovery over two decades ago, the importance of this pathway in cell proteostasis has been made increasingly apparent. Deregulation of this pathway has been implicated in a variety of diseases and conditions, including lysosomal storage diseases, cancer, neurodegeneration and even aging. Here, we describe the main molecular features of the pathway, its regulation, cross-talk with other degradation pathways and importance in disease.

Fragment-based drug discovery and its application to challenging drug targets

Abstract: Fragment-based drug discovery (FBDD) is a technique for identifying low molecular weight chemical starting points for drug discovery. Since its inception 20 years ago, FBDD has grown in popularity to the point where it is now an established technique in industry and academia. The approach involves the biophysical screening of proteins against collections of low molecular weight compounds (fragments). Although fragments bind to proteins with relatively low affinity, they form efficient, high quality binding interactions with the protein architecture as they have to overcome a significant entropy barrier to bind. Of the biophysical methods available for fragment screening, X-ray protein crystallography is one of the most sensitive and least prone to false positives. It also provides detailed structural information of the protein-fragment complex at the atomic level. Fragment-based screening using X-ray crystallography is therefore an efficient method for identifying binding hotspots on proteins, which can then be exploited by chemists and biologists for the discovery of new drugs. The use of FBDD is illustrated here with a recently published case study of a drug discovery programme targeting the challenging protein-protein interaction Kelch-like ECH-associated protein 1:nuclear factor erythroid 2-related factor 2.

Antibiotic tolerance and the alternative lifestyles of Staphylococcus aureus.

Abstract: Staphylococcus aureus has an incredible ability to survive, either by adapting to environmental conditions or defending against exogenous stress. Although there are certainly important genetic traits, in part this ability is provided by the breadth of modes of growth S. aureus can adopt. It has been proposed that while within their host, S. aureus survives host-generated and therapeutic antimicrobial stress via alternative lifestyles: a persister sub-population, through biofilm growth on host tissue or by growing as small colony variants (SCVs). Key to an understanding of chronic and relapsing S. aureus infections is determining the molecular basis for its switch to these quasi-dormant lifestyles. In a multicellular biofilm, the metabolically quiescent bacterial community additionally produces a highly protective extracellular polymeric substance (EPS). Furthermore, there are bacteria within a biofilm community that have an altered physiology potentially equivalent to persister cells. Recent studies have directly linked the cellular ATP production by persister cells as their key feature and the basis for their tolerance of a range of antibiotics. In clinical settings, SCVs of S. aureus have been observed for many years; when cultured, these cells form non-pigmented colonies and are approximately ten times smaller than their counterparts. Various genotypic factors have been identified in attempts to characterize S. aureus SCVs and different environmental stresses have been implicated as important inducers.

Host–pathogen interactions and subversion of autophagy

Abstract: Macroautophagy ('autophagy'), is the process by which cells can form a double-membraned vesicle that encapsulates material to be degraded by the lysosome. This can include complex structures such as damaged mitochondria, peroxisomes, protein aggregates and large swathes of cytoplasm that can not be processed efficiently by other means of degradation. Recycling of amino acids and lipids through autophagy allows the cell to form intracellular pools that aid survival during periods of stress, including growth factor deprivation, amino acid starvation or a depleted oxygen supply. One of the major functions of autophagy that has emerged over the last decade is its importance as a safeguard against infection. The ability of autophagy to selectively target intracellular pathogens for destruction is now regarded as a key aspect of the innate immune response. However, pathogens have evolved mechanisms to either evade or reconfigure the autophagy pathway for their own survival. Understanding how pathogens interact with and manipulate the host autophagy pathway will hopefully provide a basis for combating infection and increase our understanding of the role and regulation of autophagy. Herein, we will discuss how the host cell can identify and target invading pathogens and how pathogens have adapted in order to evade destruction by the host cell. In particular, we will focus on interactions between the mammalian autophagy gene 8 (ATG8) proteins and the host and pathogen effector proteins.

Development and transmission of antimicrobial resistance among Gram-negative bacteria in animals and their public health impact.

Abstract: Gram-negative bacteria are known to cause severe infections in both humans and animals. Antimicrobial resistance (AMR) in Gram-negative bacteria is a major challenge in the treatment of clinical infections globally due to the propensity of these organisms to rapidly develop resistance against antimicrobials in use. In addition, Gram-negative bacteria possess highly efficient mechanisms through which the AMR can be disseminated between pathogenic and commensal bacteria of the same or different species. These unique traits of Gram-negative bacteria have resulted in evolution of Gram-negative bacterial strains demonstrating resistance to multiple classes of antimicrobials. The evergrowing resistance issue has not only resulted in limitation of treatment options but also led to increased treatment costs and mortality rates in humans and animals. With few or no new antimicrobials in production to combat severe life-threatening infections, AMR has been described as the one of the most severe, long-term threats to human health. Aside from overuse and misuse of antimicrobials in humans, another factor that has exacerbated the emergence of AMR in Gram-negative bacteria is the veterinary use of antimicrobials that belong to the same classes considered to be critically important for treating serious life-threatening infections in humans. Despite the fact that development of AMR dates back to before the introduction of antimicrobials, the recent surge in the resistance towards all available critically important antimicrobials has emerged as a major public health issue. This review thus focuses on discussing the development, transmission and public health impact of AMR in Gram-negative bacteria in animals.

Autophagy's secret life: secretion instead of degradation.

Abstract: Autophagy is conventionally described as a degradative, catabolic pathway and a tributary to the lysosomal system where the cytoplasmic material sequestered by autophagosomes gets degraded. However, autophagosomes or autophagosome-related organelles do not always follow this route. It has recently come to light that autophagy can terminate in cytosolic protein secretion or release of sequestered material from the cells, rather than in their degradation. In this review, we address this relatively new but growing aspect of autophagy as a complex pathway, which is far more versatile than originally anticipated.

Biological functions controlled by manganese redox changes in mononuclear Mn-dependent enzymes

Abstract: Remarkably few enzymes are known to employ a mononuclear manganese ion that undergoes changes in redox state during catalysis. Many questions remain to be answered about the role of substrate binding and/or protein environment in modulating the redox properties of enzyme-bound Mn(II), the nature of the dioxygen species involved in the catalytic mechanism, and how these enzymes acquire Mn(II) given that many other metal ions in the cell form more stable protein complexes. Here, we summarize current knowledge concerning the structure and mechanism of five mononuclear manganese-dependent enzymes: superoxide dismutase, oxalate oxidase (OxOx), oxalate decarboxylase (OxDC), homoprotocatechuate 3,4-dioxygenase, and lipoxygenase (LOX). Spectroscopic measurements and/or computational studies suggest that Mn(III)/Mn(II) are the catalytically active oxidation states of the metal, and the importance of 'second-shell' hydrogen bonding interactions with metal ligands has been demonstrated for a number of examples. The ability of these enzymes to modulate the redox properties of the Mn(III)/Mn(II) couple, thereby allowing them to generate substrate-based radicals, appears essential for accessing diverse chemistries of fundamental importance to organisms in all branches of life.

Polishing the tarnished silver bullet: the quest for new antibiotics.

Abstract: We are facing a potential catastrophe of untreatable bacterial infections, driven by the inexorable rise of extensively drug-resistant bacteria, coupled with a market failure of pharmaceutical and biotech companies to deliver new therapeutic options. While global recognition of the problem is finally apparent, solutions are still a long way from being implemented. In addition to drug stewardship programmes and better diagnostics, new antibiotics are desperately needed. The question remains as to how to achieve this goal. This review will examine the different strategies being applied to discover new antibiotics.

Impact of anthropogenic activities on the dissemination of antibiotic resistance across ecological boundaries.

Abstract: Antibiotics are considered to be one of the major medical breakthroughs in history. Nonetheless, over the past four decades, antibiotic resistance has reached alarming levels worldwide and this trend is expected to continue to increase, leading some experts to forecast the coming of a 'post-antibiotic' era. Although antibiotic resistance in pathogens is traditionally linked to clinical environments, there is a rising concern that the global propagation of antibiotic resistance is also associated with environmental reservoirs that are linked to anthropogenic activities such as animal husbandry, agronomic practices and wastewater treatment. It is hypothesized that the emergence and dissemination of antibiotic-resistant bacteria (ARB) and antibiotic-resistant genes (ARGs) within and between environmental microbial communities can ultimately contribute to the acquisition of antibiotic resistance in human pathogens. Nonetheless, the scope of this phenomenon is not clear due to the complexity of microbial communities in the environment and methodological constraints that limit comprehensive in situ evaluation of microbial genomes. This review summarizes the current state of knowledge regarding antibiotic resistance in non-clinical environments, specifically focusing on the dissemination of antibiotic resistance across ecological boundaries and the contribution of this phenomenon to global antibiotic resistance.

Genome instability: a conserved mechanism of ageing?

Abstract: DNA is the carrier of genetic information and the primary template from which all cellular information is ultimately derived Changes in the DNA information content through mutation generate diversity for evolution through natural selection but are also a source of deleterious effects It has since long been hypothesized that mutation accumulation in somatic cells of multicellular organisms could causally contribute to age-related cellular degeneration and death Assays to detect different types of mutations, from base substitutions to large chromosomal aberrations, have been developed and show unequivocally that mutations accumulate in different tissues and cell types of ageing humans and animals More recently, next-generation sequencing-based methods have been developed to accurately determine the complete landscape of base substitution mutations in single cells The first results show that the somatic mutation rate is much higher than the germline mutation rate and that base substitution loads in somatic cells are high enough to potentially affect cellular function

Interplay of autophagy, receptor tyrosine kinase signalling and endocytic trafficking.

Abstract: Vesicular trafficking events play key roles in the compartmentalization and proper sorting of cellular components. These events have crucial roles in sensing external signals, regulating protein activities and stimulating cell growth or death decisions. Although mutations in vesicle trafficking players are not direct drivers of cellular transformation, their activities are important in facilitating oncogenic pathways. One such pathway is the sensing of external stimuli and signalling through receptor tyrosine kinases (RTKs). The regulation of RTK activity by the endocytic pathway has been extensively studied. Compelling recent studies have begun to highlight the association between autophagy and RTK signalling. The influence of this interplay on cellular status and its relevance in disease settings will be discussed here.