CBL/CAP Is Essential for Mitochondria Respiration Complex I Assembly and Bioenergetics Efficiency in Muscle Cells.

CBL is rapidly phosphorylated upon insulin receptor activation. Mice whole body CBL depletion improved insulin sensitivity and glucose clearance; however, the precise mechanisms remain unknown. We depleted either CBL or its associated protein SORBS1/CAP independently in myocytes and assessed mitochondrial function and metabolism compared to control cells. CBL- and CAP-depleted cells showed increased mitochondrial mass with greater proton leak. Mitochondrial respiratory complex I activity and assembly into respirasomes were reduced. Proteome profiling revealed alterations in proteins involved in glycolysis and fatty acid degradation. Our findings demonstrate CBL/CAP pathway couples insulin signaling to efficient mitochondrial respiratory function and metabolism in muscle.

Gilthead Seabream Liver Integrative Proteomics and Metabolomics Analysis Reveals Regulation by Different Prosurvival Pathways in the Metabolic Adaptation to Stress.

The study of the molecular mechanisms of stress appraisal on farmed fish is paramount to ensuring a sustainable aquaculture. Stress exposure can either culminate in the organism's adaptation or aggravate into a metabolic shutdown, characterized by irreversible cellular damage and deleterious effects on fish performance, welfare, and survival. Multiomics can improve our understanding of the complex stressed phenotype in fish and the molecular mediators that regulate the underlying processes of the molecular stress response. We profiled the stress proteome and metabolome of Sparus aurata responding to different challenges common to aquaculture production, characterizing the disturbed pathways in the fish liver, i.e., the central organ in mounting the stress response. Label-free shotgun proteomics and untargeted metabolomics analyses identified 1738 proteins and 120 metabolites, separately. Mass spectrometry data have been made fully accessible via ProteomeXchange, with the identifier PXD036392, and via MetaboLights, with the identifier MTBLS5940. Integrative multivariate statistical analysis, performed with data integration analysis for biomarker discovery using latent components (DIABLO), depicted the 10 most-relevant features. Functional analysis of these selected features revealed an intricate network of regulatory components, modulating different signaling pathways related to cellular stress, e.g., the mTORC1 pathway, the unfolded protein response, endocytosis, and autophagy to different extents according to the stress nature. These results shed light on the dynamics and extent of this species' metabolic reprogramming under chronic stress, supporting future studies on stress markers' discovery and fish welfare research.

Endoplasmic reticulum stress as a key mechanism in stunted growth of seawater rainbow trout (Oncorhynchus mykiss).

BACKGROUND: Rainbow trout (Oncorhynchus mykiss) is a salmonid species with a complex life-history. Wild populations are naturally divided into freshwater residents and sea-run migrants. Migrants undergo an energy-demanding adaptation for life in seawater, known as smoltification, while freshwater residents display these changes in an attenuated magnitude and rate. Despite this, in seawater rainbow trout farming all fish are transferred to seawater. Under these circumstances, weeks after seawater transfer, a significant portion of the fish die (around 10%) or experience growth stunting (GS; around 10%), which represents an important profitability and welfare issue. The underlying causes leading to GS in seawater-transferred rainbow trout remain unknown. In this study, we aimed at characterising the GS phenotype in seawater-transferred rainbow trout using untargeted and targeted approaches. To this end, the liver proteome (LC-MS/MS) and lipidome (LC-MS) of GS and fast-growing phenotypes were profiled to identify molecules and processes that are characteristic of the GS phenotype. Moreover, the transcription, abundance or activity of key proteins and hormones related to osmoregulation (Gill Na+, K + -ATPase activity), growth (plasma IGF-I, and liver igf1, igfbp1b, ghr1 and ctsl) and stress (plasma cortisol) were measured using targeted approaches. RESULTS: No differences in Gill Na+, K + -ATPase activity and plasma cortisol were detected between the two groups. However, a significant downregulation in plasma IGF-I and liver igf1 transcription pointed at this growth factor as an important pathomechanism for GS. Changes in the liver proteome revealed reactive-oxygen-species-mediated endoplasmic reticulum stress as a key mechanism underlying the GS phenotype. From the lipidomic analysis, key observations include a reduction in triacylglycerols and elevated amounts of cardiolipins, a characteristic lipid class associated with oxidative stress, in GS phenotype. CONCLUSION: While the triggers to the activation of endoplasmic reticulum stress are still unknown, data from this study point towards a nutritional deficiency as an underlying driver of this phenotype.

Altered ceramide metabolism is a feature in the extracellular vesicle-mediated spread of alpha-synuclein in Lewy body disorders.

Mutations in glucocerebrosidase (GBA) are the most prevalent genetic risk factor for Lewy body disorders (LBD)-collectively Parkinson's disease, Parkinson's disease dementia and dementia with Lewy bodies. Despite this genetic association, it remains unclear how GBA mutations increase susceptibility to develop LBD. We investigated relationships between LBD-specific glucocerebrosidase deficits, GBA-related pathways, and α-synuclein levels in brain tissue from LBD and controls, with and without GBA mutations. We show that LBD is characterised by altered sphingolipid metabolism with prominent elevation of ceramide species, regardless of GBA mutations. Since extracellular vesicles (EV) could be involved in LBD pathogenesis by spreading disease-linked lipids and proteins, we investigated EV derived from post-mortem cerebrospinal fluid (CSF) and brain tissue from GBA mutation carriers and non-carriers. EV purified from LBD CSF and frontal cortex were heavily loaded with ceramides and neurodegeneration-linked proteins including alpha-synuclein and tau. Our in vitro studies demonstrate that LBD EV constitute a "pathological package" capable of inducing aggregation of wild-type alpha-synuclein, mediated through a combination of alpha-synuclein-ceramide interaction and the presence of pathological forms of alpha-synuclein. Together, our findings indicate that abnormalities in ceramide metabolism are a feature of LBD, constituting a promising source of biomarkers, and that GBA mutations likely accelerate the pathological process occurring in sporadic LBD through endolysosomal deficiency.

A Metabolomics approach for the diagnosis Of SecondAry InfeCtions in COVID-19 (MOSAIC): a study protocol.

BACKGROUND: Critically ill patients with COVID-19 are at an increased risk of developing secondary bacterial infections. These are both difficult to diagnose and are associated with an increased mortality. Metabolomics may aid clinicians in diagnosing secondary bacterial infections in COVID-19 through identification and quantification of disease specific biomarkers, with the aim of identifying underlying causative microorganisms and directing antimicrobial therapy. METHODS: This is a multi-centre prospective diagnostic observational study. Patients with COVID-19 will be recruited from critical care units in three Scottish hospitals. Three serial blood samples will be taken from patients, and an additional sample taken if a patient shows clinical or microbiological evidence of secondary infection. Samples will be analysed using LC-MS and subjected to bioinformatic processing and statistical analysis to explore the metabolite changes associated with bacterial infections in COVID-19 patients. Comparisons of the data sets will be made with standard microbiological and biochemical methods of diagnosing infection. DISCUSSION: Metabolomics analyses may provide additional strategies for identifying secondary infections, which might permit faster initiation of specific tailored antimicrobial therapy to critically ill patients with COVID-19.

EMSY expression affects multiple components of the skin barrier with relevance to atopic dermatitis.

BACKGROUND: Atopic dermatitis (AD) is a common, complex, and highly heritable inflammatory skin disease. Genome-wide association studies offer opportunities to identify molecular targets for drug development. A risk locus on chromosome 11q13.5 lies between 2 candidate genes, EMSY and LRRC32 (leucine-rich repeat-containing 32) but the functional mechanisms affecting risk of AD remain unclear. OBJECTIVES: We sought to apply a combination of genomic and molecular analytic techniques to investigate which genes are responsible for genetic risk at this locus and to define mechanisms contributing to atopic skin disease. METHODS: We used interrogation of available genomic and chromosome conformation data in keratinocytes, small interfering RNA (siRNA)-mediated knockdown in skin organotypic culture and functional assessment of barrier parameters, mass spectrometric global proteomic analysis and quantitative lipid analysis, electron microscopy of organotypic skin, and immunohistochemistry of human skin samples. RESULTS: Genomic data indicate active promoters in the genome-wide association study locus and upstream of EMSY; EMSY, LRRC32, and intergenic variants all appear to be within a single topologically associating domain. siRNA-knockdown of EMSY in organotypic culture leads to enhanced development of barrier function, reflecting increased expression of structural and functional proteins, including filaggrin and filaggrin-2, as well as long-chain ceramides. Conversely, overexpression of EMSY in keratinocytes leads to a reduction in markers of barrier formation. Skin biopsy samples from patients with AD show greater EMSY staining in the nucleus, which is consistent with an increased functional effect of this transcriptional control protein. CONCLUSION: Our findings demonstrate an important role for EMSY in transcriptional regulation and skin barrier formation, supporting EMSY inhibition as a therapeutic approach.

Recreation of in-host acquired single nucleotide polymorphisms by CRISPR-Cas9 reveals an uncharacterised gene playing a role in Aspergillus fumigatus azole resistance via a non-cyp51A mediated resistance mechanism.

The human host comprises a range of specific niche environments. In order to successfully persist, pathogens such as Aspergillus fumigatus must adapt to these environments. One key example of in-host adaptation is the development of resistance to azole antifungals. Azole resistance in A. fumigatus is increasingly reported worldwide and the most commonly reported mechanisms are cyp51A mediated. Using a unique series of A. fumigatus isolates, obtained from a patient suffering from persistent and recurrent invasive aspergillosis over 2 years, this study aimed to gain insight into the genetic basis of in-host adaptation. Single nucleotide polymorphisms (SNPs) unique to a single isolate in this series, which had developed multi-azole resistance in-host, were identified. Two nonsense SNPs were recreated using CRISPR-Cas9; these were 213* in svf1 and 167* in uncharacterised gene AFUA_7G01960. Phenotypic analyses including antifungal susceptibility testing, mycelial growth rate assessment, lipidomics analysis and statin susceptibility testing were performed to associate genotypes to phenotypes. This revealed a role for svf1 in A. fumigatus oxidative stress sensitivity. In contrast, recapitulation of 167* in AFUA_7G01960 resulted in increased itraconazole resistance. Comprehensive lipidomics analysis revealed decreased ergosterol levels in strains containing this SNP, providing insight to the observed itraconazole resistance. Decreases in ergosterol levels were reflected in increased resistance to lovastatin and nystatin. Importantly, this study has identified a SNP in an uncharacterised gene playing a role in azole resistance via a non-cyp51A mediated resistance mechanism. This mechanism is of clinical importance, as this SNP was identified in a clinical isolate, which acquired azole resistance in-host.

The Glycine Lipids of Bacteroides thetaiotaomicron Are Important for Fitness during Growth In Vivo and In Vitro.

Acylated amino acids function as important components of the cellular membrane in some bacteria. Biosynthesis is initiated by the N-acylation of the amino acid, and this is followed by subsequent O-acylation of the acylated molecule, resulting in the production of the mature diacylated amino acid lipid. In this study, we use both genetics and liquid chromatography-mass spectrometry (LC-MS) to characterize the biosynthesis and function of a diacylated glycine lipid (GL) species produced in Bacteroides thetaiotaomicron We, and others, have previously reported the identification of a gene, named glsB in this study, that encodes an N-acyltransferase activity responsible for the production of a monoacylated glycine called N-acyl-3-hydroxy-palmitoyl glycine (or commendamide). In all of the Bacteroidales genomes sequenced so far, the glsB gene is located immediately downstream from a gene, named glsA, that is also predicted to encode a protein with acyltransferase activity. We use LC-MS to show that the coexpression of glsB and glsA results in the production of GL in Escherichia coli We constructed a deletion mutant of the glsB gene in B. thetaiotaomicron, and we confirm that glsB is required for the production of GL in B. thetaiotaomicron Moreover, we show that glsB is important for the ability of B. thetaiotaomicron to adapt to stress and colonize the mammalian gut. Therefore, this report describes the genetic requirements for the biosynthesis of GL, a diacylated amino acid species that contributes to fitness in the human gut bacterium B. thetaiotaomicronIMPORTANCE The gut microbiome has an important role in both health and disease of the host. The mammalian gut microbiome is often dominated by bacteria from the Bacteroidales, an order that includes Bacteroides and Prevotella In this study, we have identified an acylated amino acid, called glycine lipid, produced by Bacteroides thetaiotaomicron, a beneficial bacterium originally isolated from the human gut. In addition to identifying the genes required for the production of glycine lipids, we show that glycine lipids have an important role during the adaptation of B. thetaiotaomicron to a number of environmental stresses, including exposure to either bile or air. We also show that glycine lipids are important for the normal colonization of the murine gut by B. thetaiotaomicron This work identifies glycine lipids as an important fitness determinant in B. thetaiotaomicron and therefore increases our understanding of the molecular mechanisms underpinning colonization of the mammalian gut by beneficial bacteria.

Lipid remodeling and an altered membrane-associated proteome may drive the differential effects of EPA and DHA treatment on skeletal muscle glucose uptake and protein accretion.

In striated muscle, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have differential effects on the metabolism of glucose and differential effects on the metabolism of protein. We have shown that, despite similar incorporation, treatment of C2C12 myotubes (CM) with EPA but not DHA improves glucose uptake and protein accretion. We hypothesized that these differential effects of EPA and DHA may be due to divergent shifts in lipidomic profiles leading to altered proteomic profiles. We therefore carried out an assessment of the impact of treating CM with EPA and DHA on lipidomic and proteomic profiles. Fatty acid methyl esters (FAME) analysis revealed that both EPA and DHA led to similar but substantials changes in fatty acid profiles with the exception of arachidonic acid, which was decreased only by DHA, and docosapentanoic acid (DPA), which was increased only by EPA treatment. Global lipidomic analysis showed that EPA and DHA induced large alterations in the cellular lipid profiles and in particular, the phospholipid classes. Subsequent targeted analysis confirmed that the most differentially regulated species were phosphatidylcholines and phosphatidylethanolamines containing long-chain fatty acids with five (EPA treatment) or six (DHA treatment) double bonds. As these are typically membrane-associated lipid species we hypothesized that these treatments differentially altered the membrane-associated proteome. Stable isotope labeling by amino acids in cell culture (SILAC)-based proteomics of the membrane fraction revealed significant divergence in the effects of EPA and DHA on the membrane-associated proteome. We conclude that the EPA-specific increase in polyunsaturated long-chain fatty acids in the phospholipid fraction is associated with an altered membrane-associated proteome and these may be critical events in the metabolic remodeling induced by EPA treatment.

Autophagic lysosome reformation dysfunction in glucocerebrosidase deficient cells: relevance to Parkinson disease.

Glucocerebrosidase (GBA1) gene mutations increase the risk of Parkinson disease (PD). While the cellular mechanisms associating GBA1 mutations and PD are unknown, loss of the glucocerebrosidase enzyme (GCase) activity, inhibition of autophagy and increased α-synuclein levels have been implicated. Here we show that autophagy lysosomal reformation (ALR) is compromised in cells lacking functional GCase. ALR is a cellular process controlled by mTOR which regenerates functional lysosomes from autolysosomes formed during macroautophagy. A decrease in phopho-S6K levels, a marker of mTOR activity, was observed in models of GCase deficiency, including primary mouse neurons and the PD patient derived fibroblasts with GBA1 mutations, suggesting that ALR is compromised. Importantly Rab7, a GTPase crucial for endosome-lysosome trafficking and ALR, accumulated in GCase deficient cells, supporting the notion that lysosomal recycling is impaired. Recombinant GCase treatment reversed ALR inhibition and lysosomal dysfunction. Moreover, ALR dysfunction was accompanied by impairment of macroautophagy and chaperone-mediated autophagy, increased levels of total and phosphorylated (S129) monomeric α-synuclein, evidence of amyloid oligomers and increased α-synuclein release. Concurrently, we found increased cholesterol and altered glucosylceramide homeostasis which could compromise ALR. We propose that GCase deficiency in PD inhibits lysosomal recycling. Consequently neurons are unable to maintain the pool of mature and functional lysosomes required for the autophagic clearance of α-synuclein, leading to the accumulation and spread of pathogenic α-synuclein species in the brain. Since GCase deficiency and lysosomal dysfunction occur with ageing and sporadic PD pathology, the decrease in lysosomal reformation may be a common feature in PD.