Essays in biochemistry最新文献

Cyanobacteria, also known as blue-green algae, are ubiquitous organisms on the planet. They contain tremendous protein machineries that are of interest to the biotechnology industry and beyond. Recently, the number of annotated cyanobacterial genomes has expanded, enabling structural studies on known gene-coded proteins to accelerate. This review focuses on the advances in mass spectrometry (MS) that have enabled structural proteomics studies to be performed on the proteins and protein complexes within cyanobacteria. The review also showcases examples whereby MS has revealed critical mechanistic information behind how these remarkable machines within cyanobacteria function.

Cross-linking mass spectrometry has become an established technology to provide structural information on the topology and dynamics of protein complexes. Readily accessible workflows can provide detailed data on simplified systems, such as purified complexes. However, using this technology to study the structure of protein complexes in situ, such as in organelles, cells, and even tissues, is still a technological frontier. The complexity of these systems remains a considerable challenge, but there have been dramatic improvements in sample handling, data acquisition, and data processing. Here, we summarise these developments and describe the paths towards comprehensive and comparative structural interactomes by cross-linking mass spectrometry.

Proteins and RNAs are fundamental parts of biological systems, and their interactions affect many essential cellular processes. Therefore, it is crucial to understand at a molecular and at a systems level how proteins and RNAs form complexes and mutually affect their functions. In the present mini-review, we will first provide an overview of different mass spectrometry (MS)-based methods to study the RNA-binding proteome (RBPome), most of which are based on photochemical cross-linking. As we will show, some of these methods are also able to provide higher-resolution information about binding sites, which are important for the structural characterisation of protein-RNA interactions. In addition, classical structural biology techniques such as nuclear magnetic resonance (NMR) spectroscopy and biophysical methods such as electron paramagnetic resonance (EPR) spectroscopy and fluorescence-based methods contribute to a detailed understanding of the interactions between these two classes of biomolecules. We will discuss the relevance of such interactions in the context of the formation of membrane-less organelles (MLOs) by liquid-liquid phase separation (LLPS) processes and their emerging importance as targets for drug discovery.

Integral membrane proteins (IMPs) perform a range of diverse functions and their dysfunction underlies numerous pathological conditions. Consequently, IMPs constitute most drug targets, and the elucidation of their mechanism of action has become an intense field of research. Historically, IMP studies have relied on their extraction from membranes using detergents, which have the potential to perturbate their structure and dynamics. To circumnavigate this issue, an array of membrane mimetics has been developed that aim to reconstitute IMPs into native-like lipid environments that more accurately represent the biological membrane. Hydrogen/deuterium exchange-mass spectrometry (HDX-MS) has emerged as a versatile tool for probing protein dynamics in solution. The continued development of HDX-MS methodology has allowed practitioners to investigate IMPs using increasingly native-like membrane mimetics, and even pushing the study of IMPs into the in vivo cellular environment. Consequently, HDX-MS has come of age and is playing an ever-increasingly important role in the IMP structural biologist toolkit. In the present mini-review, we discuss the evolution of membrane mimetics in the HDX-MS context, focusing on seminal publications and recent innovations that have led to this point. We also discuss state-of-the-art methodological and instrumental advancements that are likely to play a significant role in the generation of high-quality HDX-MS data of IMPs in the future.

Multidrug efflux pumps are ubiquitous across both eukaryotes and prokaryotes, and have major implications in antimicrobial and multidrug resistance. They reside within cellular membranes and have proven difficult to study owing to their hydrophobic character and relationship with their compositionally complex lipid environment. Advances in structural mass spectrometry (MS) techniques have made it possible to study these systems to elucidate critical information on their structure-function relationships. For example, MS techniques can report on protein structural dynamics, stoichiometry, connectivity, solvent accessibility, and binding interactions with ligands, lipids, and other proteins. This information proving powerful when used in conjunction with complementary structural biology methods and molecular dynamics (MD) simulations. In the present review, aimed at those not experts in MS techniques, we report on the current uses of MS in studying multidrug efflux systems, practical considerations to consider, and the future direction of the field. In the first section, we highlight the importance of studying multidrug efflux proteins, and introduce a range of different MS techniques and explain what information they yield. In the second section, we review recent studies that have utilised MS techniques to study and characterise a range of different multidrug efflux systems.

This review aims to summarise the current capabilities of surface mass spectrometry (MS) approaches that offer intact protein analysis, and that of non-covalent complexes. Protein analysis is largely achieved via matrix-assisted laser desorption/ionisation (MALDI), which is in itself a surface analysis approach or solvent-based electrospray ionisation (ESI). Several surface sampling approaches have been developed based on ESI, and those that have been used for intact protein analysis will be discussed below. The extent of protein coverage, top-down elucidation, and probing of protein structure for native proteins and non-covalent complexes will be discussed for each approach. Strategies for improving protein analysis, ranging from sample preparation, and sampling methods to instrument modifications and the inclusion of ion mobility separation in the workflow will also be discussed. The relative benefits and drawbacks of each approach will be summarised, providing an overview of current capabilities.

The discovery of oxidative cleavage of glycosidic bonds by enzymes currently known as lytic polysaccharide monooxygenases (LPMOs) has profoundly changed our current understanding of enzymatic processes underlying the conversion of polysaccharides in the biosphere. LPMOs are truly unique enzymes, harboring a single copper atom in a solvent-exposed active site, allowing them to oxidize C-H bonds at the C1 and/or C4 carbon of glycosidic linkages found in recalcitrant, often crystalline polysaccharides such as cellulose and chitin. To catalyze this challenging reaction, LPMOs harness and control a powerful oxidative reaction that involves Fenton-like chemistry. In this essay, we first draw a brief portrait of the LPMO field, notably explaining the shift from the monooxygenase paradigm (i.e., using O2 as cosubstrate) to that of a peroxygenase (i.e., using H2O2). Then, we briefly review current understanding of how LPMOs generate and control a hydroxyl radical (HO•) generated through Cu(I)-catalyzed H2O2 homolysis, and how this radical is used to create the proposed Cu(II)-oxyl species, abstracting hydrogen atom of the C-H bond. We also point at the complexity of analyzing redox reactions involving reactive oxygen species and address potential deficiencies in the interpretation of existing LPMO data. Being the first copper enzymes shown to enable site-specific Fenton-like chemistry, and maybe not the only ones, LPMOs may serve as a blueprint for future research on monocopper peroxygenases.

Astrocytes show unique anatomical, morphological, and metabolic features to take up substrates from the blood and metabolize them for local delivery to active synapses to sustain neuron function. In the present review, we specifically focus on key molecular aspects of energy and redox metabolism that facilitate this astrocyte-neuronal coupling in a controlled manner. Basal glycolysis is co-ordinated by the anaphase-promoting complex/cyclosome (APC/C)-Cdh1, a ubiquitin ligase that targets the proglycolytic enzyme 6-phosphofructokinase-2,6-bisphosphastate-3 (PFKFB3) for degradation. APC/C-Cdh1 activity is more robust in neurons than in astrocytes, which determine that PFKFB3 abundance and glycolytic rate are weaker in neurons. The low PFKFB3 activity in neurons facilitates glucose-6-phosphate oxidation via the pentose-phosphate pathway, which promotes antioxidant protection. Conversely, the high PFKFB3 activity in astrocytes allows the production and release of glycolytic lactate, which is taken up by neurons that use it as an oxidizable substrate. Importantly, the mitochondrial respiratory chain is tighter assembled in neurons than in astrocytes, thus the bioenergetic efficiency of mitochondria is higher in neurons. Because of this, the production of reactive oxygen species (mROS) by mitochondrial complex I is very low in neurons and very high in astrocytes. Such a naturally occurring high abundance of mROS in astrocytes physiologically determines a specific transcriptional fingerprint that contributes to sustaining cognitive performance. We conclude that the energy and redox metabolism of astrocytes must complementarily match that of neurons to regulate brain function and animal welfare.

Astrocytes are a type of non-neuronal, glial cells, anatomically placed in the intersection between the brain blood vessels and other neural cells-including neurons. Such a strategic situation confers these cells a unique opportunity to sense circulating molecules and adapt according to different organismal conditions. By acting as sentinel cells, astrocytes thus co-ordinate gene expression profiles, immune responses, signal transduction pathways, and metabolic programs that play essential roles in the formation of brain circuits to modulate neurotransmission and higher-order organismal functions.

Synaptic regulation of the primary inhibitory neurotransmitter γ-aminobutyric acid (GABA) is essential for brain function. Cerebral GABA homeostasis is tightly regulated through multiple mechanisms and is directly coupled to the metabolic collaboration between neurons and astrocytes. In this essay, we outline and discuss the fundamental roles of astrocytes in regulating synaptic GABA signaling. A major fraction of synaptic GABA is removed from the synapse by astrocytic uptake. Astrocytes utilize GABA as a metabolic substrate to support glutamine synthesis. The astrocyte-derived glutamine is subsequently transferred to neurons where it serves as the primary precursor of neuronal GABA synthesis. The flow of GABA and glutamine between neurons and astrocytes is collectively termed the GABA-glutamine cycle and is essential to sustain GABA synthesis and inhibitory signaling. In certain brain areas, astrocytes are even capable of synthesizing and releasing GABA to modulate inhibitory transmission. The majority of oxidative GABA metabolism in the brain takes place in astrocytes, which also leads to synthesis of the GABA-related metabolite γ-hydroxybutyric acid (GHB). The physiological roles of endogenous GHB remain unclear, but may be related to regulation of tonic inhibition and synaptic plasticity. Disrupted inhibitory signaling and dysfunctional astrocyte GABA handling are implicated in several diseases including epilepsy and Alzheimer's disease. Synaptic GABA homeostasis is under astrocytic control and astrocyte GABA uptake, metabolism, and recycling may therefore serve as relevant targets to ameliorate pathological inhibitory signaling.