The intricate link between membrane lipid structure and composition and membrane structural properties in bacterial membranes.

It is now evident that the cell manipulates lipid composition to regulate different processes such as membrane protein insertion, assembly and function. Moreover, changes in membrane structure and properties, lipid homeostasis during growth and differentiation with associated changes in cell size and shape, and responses to external stress have been related to drug resistance across mammalian species and a range of microorganisms. While it is well known that the biomembrane is a fluid self-assembled nanostructure, the link between the lipid components and the structural properties of the lipid bilayer are not well understood. This perspective aims to address this topic with a view to a more detailed understanding of the factors that regulate bilayer structure and flexibility. We describe a selection of recent studies that address the dynamic nature of bacterial lipid diversity and membrane properties in response to stress conditions. This emerging area has important implications for a broad range of cellular processes and may open new avenues of drug design for selective cell targeting.

Quantitative subcellular reconstruction reveals a lipid mediated inter-organelle biogenesis network.

The structures and functions of organelles in cells depend on each other but have not been systematically explored. We established stable knockout cell lines of peroxisomal, Golgi and endoplasmic reticulum genes identified in a whole-genome CRISPR knockout screen for inducers of mitochondrial biogenesis stress, showing that defects in peroxisome, Golgi and endoplasmic reticulum metabolism disrupt mitochondrial structure and function. Our quantitative total-organelle profiling approach for focussed ion beam scanning electron microscopy revealed in unprecedented detail that specific organelle dysfunctions precipitate multi-organelle biogenesis defects, impair mitochondrial morphology and reduce respiration. Multi-omics profiling showed a unified proteome response and global shifts in lipid and glycoprotein homeostasis that are elicited when organelle biogenesis is compromised, and that the resulting mitochondrial dysfunction can be rescued with precursors for ether-glycerophospholipid metabolic pathways. This work defines metabolic and morphological interactions between organelles and how their perturbation can cause disease.

Sequence differences between BAX and BAK core domains manifest as differences in their interactions with lipids.

The B-cell lymphoma 2 (BCL2) family members, BCL2-associated protein X (BAX) and BCL2 homologous antagonist killer (BAK), are required for programmed cell death via the mitochondrial pathway. When cells are stressed, damaged or redundant, the balance of power between the BCL2 family of proteins shifts towards BAX and BAK, allowing their transition from an inactive, monomeric state to a membrane-active oligomeric form that releases cytochrome c from the mitochondrial intermembrane space. That oligomeric state has an essential intermediate, a symmetric homodimer of BAX or BAK. Here we describe crystal structures of dimers of the core domain of BAX, comprising its helices α2-α5. These structures provide an atomic resolution description of the interactions that drive BAX homo-dimerisation and insights into potential interaction between core domain dimers and membrane lipids. The previously identified BAK lipid-interacting sites are not conserved with BAX and are likely to determine the differences between them in their interactions with lipids. We also describe structures of heterodimers of BAK/BAX core domains, yielding further insight into the differences in lipid binding between BAX and BAK.

Exploring the significance of lipids in Alzheimer's disease and the potential of extracellular vesicles.

Lipids play a significant role in maintaining central nervous system (CNS) structure and function, and the dysregulation of lipid metabolism is known to occur in many neurological disorders, including Alzheimer's disease. Here we review what is currently known about lipid dyshomeostasis in Alzheimer's disease. We propose that small extracellular vesicle (sEV) lipids may provide insight into the pathophysiology and progression of Alzheimer's disease. This stems from the recognition that sEV likely contributes to disease pathogenesis, but also an understanding that sEV can serve as a source of potential biomarkers. While the protein and RNA content of sEV in the CNS diseases have been studied extensively, our understanding of the lipidome of sEV in the CNS is still in its infancy.

Global lipid remodelling by hypoxia aggravates migratory potential in pancreatic cancer while maintaining plasma membrane homeostasis.

Hypoxia plays an important role in pancreatic cancer progression. It drives various metabolic reprogramming in cells including that of lipids, which in turn, can modify the structure and function of cell membranes. Homeostatic adaptation of membranes is well-recognized, but how and if it is regulated in hypoxic pancreatic cancer and its relation to aggressive phenotype and metastasis remains elusive. Here we show hypoxia-induced extensive global lipid remodelling spanning changes in lipid classes, unsaturation levels, glyceryl backbone and acyl chain lengths. No major modulation of plasma membrane biophysical properties revealed a decoupling of lipidome modulation from membrane properties under hypoxia. This was supported by observing minor changes in the lipidome of plasma membranes under hypoxia. Further, hypoxia increased migration and invasion underpinned by reduced actin volume, cell cortical stiffness and facile tether dynamics. In conclusion, we demonstrate buffering of the lipidome alterations leading to a homeostatic membrane response. These findings will help to understand the hypoxic regulation of pancreatic membrane homeostasis and identify tangible theranostic avenues.

The Proteome and Lipidome of Extracellular Vesicles from Haemonchus contortus to Underpin Explorations of Host-Parasite Cross-Talk.

Many parasitic worms have a major adverse impact on human and animal populations worldwide due to the chronicity of their infections. There is a growing body of evidence indicating that extracellular vesicles (EVs) are intimately involved in modulating (suppressing) inflammatory/immune host responses and parasitism. As one of the most pathogenic nematodes of livestock animals, Haemonchus contortus is an ideal model system for EV exploration. Here, employing a multi-step enrichment process (in vitro culture, followed by ultracentrifugation, size exclusion and filtration), we enriched EVs from H. contortus and undertook the first comprehensive (qualitative and quantitative) multi-omic investigation of EV proteins and lipids using advanced liquid chromatography-mass spectrometry and informatics methods. We identified and quantified 561 proteins and 446 lipids in EVs and compared these molecules with those of adult worms. We identified unique molecules in EVs, such as proteins linked to lipid transportation and lipid species (i.e., sphingolipids) associated with signalling, indicating the involvement of these molecules in parasite-host cross-talk. This work provides a solid starting point to explore the functional roles of EV-specific proteins and lipids in modulating parasite-host cross-talk, and the prospect of finding ways of disrupting or interrupting this relationship to suppress or eliminate parasite infection.

Phosphoinositide and redox dysregulation by the anticancer methylthioadenosine phosphorylase transition state inhibitor.

Methylthio-DADMe-immucillin-A (MTDIA) is an 86 picomolar inhibitor of 5'-methylthioadenosine phosphorylase (MTAP) with potent and specific anti-cancer efficacy. MTAP salvages S-adenosylmethionine (SAM) from 5'-methylthioadenosine (MTA), a toxic metabolite produced during polyamine biosynthesis. Changes in MTAP expression are implicated in cancer growth and development, making MTAP an appealing target for anti-cancer therapeutics. Since SAM is involved in lipid metabolism, we hypothesised that MTDIA alters the lipidomes of MTDIA-treated cells. To identify these effects, we analysed the lipid profiles of MTDIA-treated Saccharomyces cerevisiae using ultra-high resolution accurate mass spectrometry (UHRAMS). MTAP inhibition by MTDIA, and knockout of the Meu1 gene that encodes for MTAP in yeast, caused global lipidomic changes and differential abundance of lipids involved in cell signaling. The phosphoinositide kinase/phosphatase signaling network was specifically impaired upon MTDIA treatment, and was independently validated and further characterised via altered localization of proteins integral to this network. Functional consequences of dysregulated lipid metabolism included a decrease in reactive oxygen species (ROS) levels induced by MTDIA that was contemporaneous with changes in immunological response factors (nitric oxide, tumour necrosis factor-alpha and interleukin-10) in mammalian cells. These results indicate that lipid homeostasis alterations and concomitant downstream effects may be associated with MTDIA mechanistic efficacy.

Protein painting reveals pervasive remodeling of conserved proteostasis machinery in response to pharmacological stimuli.

The correct spatio-temporal organization of the proteome is essential for cellular homeostasis. However, a detailed mechanistic understanding of this organization and how it is altered in response to external stimuli in the intact cellular environment is as-yet unrealized. 'Protein painting methods provide a means to address this gap in knowledge by monitoring the conformational status of proteins within cells at the proteome-wide scale. Here, we demonstrate the ability of a protein painting method employing tetraphenylethene maleimide (TPE-MI) to reveal proteome network remodeling in whole cells in response to a cohort of commonly used pharmacological stimuli of varying specificity. We report specific, albeit heterogeneous, responses to individual stimuli that coalesce on a conserved set of core cellular machineries. This work expands our understanding of proteome conformational remodeling in response to cellular stimuli, and provides a blueprint for assessing how these conformational changes may contribute to disorders characterized by proteostasis imbalance.

Reprogrammed Lipid Metabolism and the Lipid-Associated Hallmarks of Colorectal Cancer.

Lipids have diverse structures, with multifarious regulatory functions in membrane homeostasis and bioenergetic metabolism, in mediating functional protein-lipid and protein-protein interactions, as in cell signalling and proliferation. An increasing body of evidence supports the notion that aberrant lipid metabolism involving remodelling of cellular membrane structure and changes in energy homeostasis and signalling within cancer-associated pathways play a pivotal role in the onset, progression, and maintenance of colorectal cancer (CRC) and their tumorigenic properties. Recent advances in analytical lipidome analysis technologies have enabled the comprehensive identification and structural characterization of lipids and, consequently, our understanding of the role they play in tumour progression. However, despite progress in our understanding of cancer cell metabolism and lipidomics, the key lipid-associated changes in CRC have yet not been explicitly associated with the well-established 'hallmarks of cancer' defined by Hanahan and Weinberg. In this review, we summarize recent findings that highlight the role of reprogrammed lipid metabolism in CRC and use this growing body of evidence to propose eight lipid metabolism-associated hallmarks of colorectal cancer, and to emphasize their importance and linkages to the established cancer hallmarks.

Trace residue identification, characterization, and longitudinal monitoring of the novel synthetic opioid ß-U10, from discarded drug paraphernalia.

Empirical data regarding dynamic alterations in illicit drug supply markets in response to the COVID-19 pandemic, including the potential for introduction of novel drug substances and/or increased poly-drug combination use at the "street" level, that is, directly proximal to the point of consumption, are currently lacking. Here, a high-throughput strategy employing ambient ionization-mass spectrometry is described for the trace residue identification, characterization, and longitudinal monitoring of illicit drug substances found within >6,600 discarded drug paraphernalia (DDP) samples collected during a pilot study of an early warning system for illicit drug use in Melbourne, Australia from August 2020 to February 2021, while significant COVID-19 lockdown conditions were imposed. The utility of this approach is demonstrated for the de novo identification and structural characterization of ß-U10, a previously unreported naphthamide analog within the "U-series" of synthetic opioid drugs, including differentiation from its α-U10 isomer without need for sample preparation or chromatographic separation prior to analysis. Notably, ß-U10 was observed with 23 other drug substances, most commonly in temporally distinct clusters with heroin, etizolam, and diphenhydramine, and in a total of 182 different poly-drug combinations. Longitudinal monitoring of the number and weekly "average signal intensity" (ASI) values of identified substances, developed here as a semi-quantitative proxy indicator of changes in availability, relative purity and compositions of street level drug samples, revealed that increases in the number of identifications and ASI for ß-U10 and etizolam coincided with a 50% decrease in the number of positive detections and an order of magnitude decrease in the ASI for heroin.

A Census of Hsp70-Mediated Proteome Solubility Changes upon Recovery from Heat Stress.

Eukaryotic cells respond to heat shock through several regulatory processes including upregulation of stress responsive chaperones and reversible shutdown of cellular activities through formation of protein assemblies. However, the underlying regulatory mechanisms of the recovery of these heat-induced protein assemblies remain largely elusive. Here, we measured the proteome abundance and solubility changes during recovery from heat shock in the mouse Neuro2a cell line. We found that prefoldins and translation machinery are rapidly down-regulated as the first step in the heat shock response. Analysis of proteome solubility reveals that a rapid mobilization of protein quality control machineries, along with changes in cellular energy metabolism, translational activity, and actin cytoskeleton are fundamental to the early stress responses. In contrast, longer term adaptation to stress involves renewal of core cellular components. Inhibition of the Hsp70 family, pivotal for the heat shock response, selectively and negatively affects the ribosomal machinery and delays the solubility recovery of many nuclear proteins. ProteomeXchange: PXD030069.

Hidden information on protein function in censuses of proteome foldedness.

Methods that assay protein foldedness with proteomics have generated censuses of apparent protein folding stabilities in biological milieu. However, different censuses poorly correlate with each other. Here, we show that the reason for this is that methods targeting foldedness through monitoring amino acid sidechain reactivity also detect changes in conformation and ligand binding, which can be a substantial fraction of the data. We show that the reactivity of only one quarter of cysteine or methionine sidechains in proteins in a urea denaturation curve of mammalian cell lysate can be confidently explained by a two-state unfolding isotherm. Contrary to that expected from unfolding, up to one third of the cysteines decreased reactivity. These cysteines were enriched in proteins with functions relating to unfolded protein stress. One protein, chaperone HSPA8, displayed changes arising from ligand and cofactor binding. Unmasking this hidden information using the approaches outlined here should improve efforts to understand both folding and the remodeling of protein function directly in complex biological settings.

Multi-Omic Analysis to Characterize Metabolic Adaptation of the E. coli Lipidome in Response to Environmental Stress.

As an adaptive survival response to exogenous stress, bacteria undergo dynamic remodelling of their lipid metabolism pathways to alter the composition of their cellular membranes. Here, using Escherichia coli as a well characterised model system, we report the development and application of a 'multi-omics' strategy for comprehensive quantitative analysis of the temporal changes in the lipidome and proteome profiles that occur under exponential growth phase versus stationary growth phase conditions i.e., nutrient depletion stress. Lipidome analysis performed using 'shotgun' direct infusion-based ultra-high resolution accurate mass spectrometry revealed a quantitative decrease in total lipid content under stationary growth phase conditions, along with a significant increase in the mol% composition of total cardiolipin, and an increase in 'odd-numbered' acyl-chain length containing glycerophospholipids. The inclusion of field asymmetry ion mobility spectrometry was shown to enable the enrichment and improved depth of coverage of low-abundance cardiolipins, while ultraviolet photodissociation-tandem mass spectrometry facilitated more complete lipid structural characterisation compared with conventional collision-induced dissociation, including unambiguous assignment of the odd-numbered acyl-chains as containing cyclopropyl modifications. Proteome analysis using data-dependent acquisition nano-liquid chromatography mass spectrometry and tandem mass spectrometry analysis identified 83% of the predicted E. coli lipid metabolism enzymes, which enabled the temporal dependence associated with the expression of key enzymes responsible for the observed adaptive lipid metabolism to be determined, including those involved in phospholipid metabolism (e.g., ClsB and Cfa), fatty acid synthesis (e.g., FabH) and degradation (e.g., FadA/B,D,E,I,J and M), and proteins involved in the oxidative stress response resulting from the generation of reactive oxygen species during ß-oxidation or lipid degradation.

Deleterious variants in CRLS1 lead to cardiolipin deficiency and cause an autosomal recessive multi-system mitochondrial disease.

Mitochondrial diseases are a group of inherited diseases with highly varied and complex clinical presentations. Here, we report four individuals, including two siblings, affected by a progressive mitochondrial encephalopathy with biallelic variants in the cardiolipin biosynthesis gene CRLS1. Three affected individuals had a similar infantile presentation comprising progressive encephalopathy, bull's eye maculopathy, auditory neuropathy, diabetes insipidus, autonomic instability, cardiac defects and early death. The fourth affected individual presented with chronic encephalopathy with neurodevelopmental regression, congenital nystagmus with decreased vision, sensorineural hearing loss, failure to thrive and acquired microcephaly. Using patient-derived fibroblasts, we characterized cardiolipin synthase 1 (CRLS1) dysfunction that impaired mitochondrial morphology and biogenesis, providing functional evidence that the CRLS1 variants cause mitochondrial disease. Lipid profiling in fibroblasts from two patients further confirmed the functional defect demonstrating reduced cardiolipin levels, altered acyl-chain composition and significantly increased levels of phosphatidylglycerol, the substrate of CRLS1. Proteomic profiling of patient cells and mouse Crls1 knockout cell lines identified both endoplasmic reticular and mitochondrial stress responses, and key features that distinguish between varying degrees of cardiolipin insufficiency. These findings support that deleterious variants in CRLS1 cause an autosomal recessive mitochondrial disease, presenting as a severe encephalopathy with multi-systemic involvement. Furthermore, we identify key signatures in cardiolipin and proteome profiles across various degrees of cardiolipin loss, facilitating the use of omics technologies to guide future diagnosis of mitochondrial diseases.

The impact of antibacterial peptides on bacterial lipid membranes depends on stage of growth.

The impact of maculatin 1.1 (Mac1) on the mechanical properties of supported lipid membranes derived from exponential growth phase (EGP) and stationary growth phase (SGP) E. coli lipid extracts was analysed by surface plasmon resonance and atomic force microscopy. Each membrane was analysed by quantitative nanomechanical mapping to derive measurements of the modulus, adhesion and deformation in addition to bilayer height. Image analysis revealed the presence of two domains in the EGP membrane differing in height by 0.4 nm. Three distinct domains were observed in the SGP membrane corresponding to 4.2, 4.7 and 5.4 nm in height. Using surface plasmon resonance, Mac1 was observed to bind strongly to both membranes and then disrupt the membranes as evidenced by a sharp drop in baseline. Atomic force microscopy (AFM) topographic analysis revealed the formation of domains of different height and confirmed that membrane destruction was much faster for the SGP derived bilayer. Moreover, Mac1 selectively disrupted the domain with the lowest thickness, which may correspond to a liquid ordered domain. Overall, the results provide insight into the role of lipid domains in the response of bacteria to antimicrobial peptides.

Early Warning System for Illicit Drug Use at Large Public Events: Trace Residue Analysis of Discarded Drug Packaging Samples.

Inspired by Locard's exchange principle, which states "every contact leaves a trace", a trace residue sampling strategy has been developed for the analysis of discarded drug packaging samples (DPS), as part of an early warning system for illicit drug use at large public events including music/dance festivals. Using direct analysis in real time/mass spectrometry and tandem mass spectrometry, rapid and high-throughput identification and characterization of a wide range of illicit drugs and adulterant substances was achieved, including in complex polydrug mixtures and at low relative ion abundances. A total of 1362 DPS were analyzed either off-site using laboratory-based instrumentation or on-site and in close to real time using a transportable mass spectrometer housed within a mobile analytical laboratory, with each analysis requiring less than 1 min per sample. Of the DPS analyzed, 92.2% yielded positive results for at least one of 15 different drugs and/or adulterants, including cocaine, MDMA, and ketamine, as well as numerous novel psychoactive substances (NPS). Also, 52.6% of positive DPS were found to contain polydrug mixtures, and a total of 42 different drug and polydrug combinations were observed throughout the study. For analyses performed on-site, reports to key stakeholders including event organizers, first aid and medical personnel, and peer-based harm reduction workers could be provided in as little as 5 min after sample collection. Following risk assessment of the potential harms associated with their use, drug advisories or alerts were then disseminated to event staff and patrons and subsequently to the general public when substances with particularly toxic properties were identified.

Characterization of brain-derived extracellular vesicle lipids in Alzheimer's disease.

Lipid dyshomeostasis is associated with the most common form of dementia, Alzheimer's disease (AD). Substantial progress has been made in identifying positron emission tomography and cerebrospinal fluid biomarkers for AD, but they have limited use as front-line diagnostic tools. Extracellular vesicles (EVs) are released by all cells and contain a subset of their parental cell composition, including lipids. EVs are released from the brain into the periphery, providing a potential source of tissue and disease specific lipid biomarkers. However, the EV lipidome of the central nervous system is currently unknown and the potential of brain-derived EVs (BDEVs) to inform on lipid dyshomeostasis in AD remains unclear. The aim of this study was to reveal the lipid composition of BDEVs in human frontal cortex, and to determine whether BDEVs have an altered lipid profile in AD. Using semi-quantitative mass spectrometry, we describe the BDEV lipidome, covering four lipid categories, 17 lipid classes and 692 lipid molecules. BDEVs were enriched in glycerophosphoserine (PS) lipids, a characteristic of small EVs. Here we further report that BDEVs are enriched in ether-containing PS lipids, a finding that further establishes ether lipids as a feature of EVs. BDEVs in the AD frontal cortex offered improved detection of dysregulated lipids in AD over global lipid profiling of this brain region.  AD BDEVs had significantly altered glycerophospholipid and sphingolipid levels, specifically increased plasmalogen glycerophosphoethanolamine and decreased polyunsaturated fatty acyl containing lipids, and altered amide-linked acyl chain content in sphingomyelin and ceramide lipids relative to CTL. The most prominent alteration was a two-fold decrease in lipid species containing anti-inflammatory/pro-resolving docosahexaenoic acid. The in-depth lipidome analysis provided in this study highlights the advantage of EVs over more complex tissues for improved detection of dysregulated lipids that may serve as potential biomarkers in the periphery.

Chemical Derivatization-Aided High Resolution Mass Spectrometry for Shotgun Lipidome Analysis.

Chemical derivatization coupled with nano-electrospray ionization (nESI) and ultra-high resolution accurate mass spectrometry (UHRAMS) is an established approach to overcome isobaric and isomeric mass interference limitations, and improve the analytical performance, of direct-infusion (i.e., "shotgun") lipidome analysis strategies for "sum composition" level identification and quantification of individual lipid species from within complex mixtures. Here, we describe a protocol for sequential functional group selective derivatization of aminophospholipids and O-alk-1'-enyl (i.e., plasmalogen) lipids, that when integrated into a shotgun lipidomics workflow involving deuterium-labeled internal lipid standard addition, monophasic lipid extraction, and nESI-UHRAMS analysis, enables the routine identification and quantification of >500 individual lipid species at the "sum composition" level, across four lipid categories and from >30 lipid classes and subclasses.

Helminth lipidomics: Technical aspects and future prospects.

Lipidomics is a relatively recent molecular research field, and explores lipids (fats) and their biology using advanced mass spectrometry technologies. Although this field has expanded significantly in biomedical and biotechnological disciplines, it is still in its infancy in molecular parasitology. Our goal here is to review and discuss technical aspects of MS-based lipidomics and its recent applications to parasitic worms, as well as challenges and future directions for worm lipid research. In a multi-omic paradigm, we expect that the exploration of lipidomic data for parasitic worms will yield important insights into lipid-associated biological pathways and processes, including the regulation of essential signalling pathways, parasite invasion, establishment, adaptation and development.