Cryo-EM structure of cytochrome bo3 quinol oxidase assembled in peptidiscs reveals an "open" conformation for potential ubiquinone-8 release.

Cytochrome bo3 quinol oxidase belongs to the heme­copper-oxidoreductase (HCO) superfamily, which is part of the respiratory chain and essential for cell survival. While the reaction mechanism of cyt bo3 has been studied extensively over the last decades, specific details about its substrate binding and product release have remained unelucidated due to the lack of structural information. Here, we report a 2.8 Å cryo-electron microscopy structure of cyt bo3 from Escherichia coli assembled in peptidiscs. Our structural model shows a conformation for amino acids 1-41 of subunit I different from all previously published structures while the remaining parts of this enzyme are similar. Our new conformation shows a "U-shape" assembly in contrast to the transmembrane helix, named "TM0", in other reported structural models. However, TM0 blocks ubiquinone-8 (reaction product) release, suggesting that other cyt bo3 conformations should exist. Our structural model presents experimental evidence for an "open" conformation to facilitate substrate/product exchange. This work helps further understand the reaction cycle of this oxidase, which could be a benefit for potential drug/antibiotic design for health science.

MALDI-MSI-LC-MS/MS Workflow for Single-Section Single Step Combined Proteomics and Quantitative Lipidomics.

We introduce a novel approach for comprehensive molecular profiling in biological samples. Our single-section methodology combines quantitative mass spectrometry imaging (Q-MSI) and a single step extraction protocol enabling lipidomic and proteomic liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis on the same tissue area. The integration of spatially correlated lipidomic and proteomic data on a single tissue section allows for a comprehensive interpretation of the molecular landscape. Comparing Q-MSI and Q-LC-MS/MS quantification results sheds new light on the effect of MSI and related sample preparation. Performing MSI before Q-LC-MS on the same tissue section led to fewer protein identifications and a lower correlation between lipid quantification results. Also, the critical role and influence of internal standards in Q-MSI for accurate quantification is highlighted. Testing various slide types and the evaluation of different workflows for single-section spatial multiomics analysis emphasized the need for critical evaluation of Q-MSI data. These findings highlight the necessity for robust quantification methods comparable to current gold-standard LC-MS/MS techniques. The spatial information from MSI allowed region-specific insights within heterogeneous tissues, as demonstrated for glioblastoma multiforme. Additionally, our workflow demonstrated the efficiency of a single step extraction for lipidomic and proteomic analyses on the same tissue area, enabling the examination of significantly altered proteins and lipids within distinct regions of a single section. The integration of these insights into a lipid-protein interaction network expands the biological information attainable from a tissue section, highlighting the potential of this comprehensive approach for advancing spatial multiomics research.

Insight into the Antioxidant Effect of Fermented and Non-Fermented Spirulina Water and Ethanol Extracts at the Proteome Level Using a Yeast Cell Model.

Spirulina is rich in various antioxidants and nutraceuticals and it has proven to be effective in the treatment of various pathological conditions. This study explores the antioxidant effect of fermented and non-fermented Spirulina extracts on the proteome level using the yeast Saccharomyces cerevisiae as a model organism. Yeast cells were treated with fermented Spirulina water extract (SV), non-fermented Spirulina water extract (NFV), fermented Spirulina ethanol extract (SE), and non-fermented Spirulina ethanol extract (NFE). Cell lysates were prepared, and label-free quantitative proteome analysis was performed. In SV, when compared to NFV samples, the levels of most differentially expressed proteins were upregulated. Alternatively, SE compared to NFE samples showed a significant downregulation for the majority of the analyzed proteins involved in different cellular processes. Additionally, a higher downregulation of stress response related proteins was observed in SE compared to NFE samples, while their abundance in SV samples increased compared to NFV. This study provided a global view, on a proteome level, of how cells cope with exogenous antioxidants and remodel their cellular processes to maintain metabolic and redox balance. Furthermore, it combined for the first time the analysis of different extract effect, including the contribution of lactic acid fermentation to the cell activity.

Inhibition of Extracellular Cathepsin D Reduces Hepatic Lipid Accumulation and Leads to Mild Changes in Inflammationin NASH Mice.

Background & Aims: The lysosomal enzyme, cathepsin D (CTSD) has been implicated in the pathogenesis of non-alcoholic steatohepatitis (NASH), a disease characterised by hepatic steatosis and inflammation. We have previously demonstrated that specific inhibition of the extracellular CTSD leads to improved metabolic features in Sprague-Dawley rats with steatosis. However, the individual roles of extracellular and intracellular CTSD in NASH are not yet known. In the current study, we evaluated the underlying mechanisms of extracellular and intracellular CTSD fractions in NASH-related metabolic inflammation using specific small-molecule inhibitors. Methods: Low-density lipoprotein receptor knock out (Ldlr-/-) mice were fed a high-fat, high cholesterol (HFC) diet for ten weeks to induce NASH. Further, to investigate the effects of CTSD inhibition, mice were injected either with an intracellular (GA-12) or extracellular (CTD-002) CTSD inhibitor or vehicle control at doses of 50 mg/kg body weight subcutaneously once in two days for ten weeks. Results: Ldlr-/- mice treated with extracellular CTSD inhibitor showed reduced hepatic lipid accumulation and an associated increase in faecal bile acid levels as compared to intracellular CTSD inhibitor-treated mice. Furthermore, in contrast to intracellular CTSD inhibition, extracellular CTSD inhibition switched the systemic immune status of the mice to an anti-inflammatory profile. In line, label-free mass spectrometry-based proteomics revealed that extra- and intracellular CTSD fractions modulate proteins belonging to distinct metabolic pathways. Conclusion: We have provided clinically translatable evidence that extracellular CTSD inhibition shows some beneficial metabolic and systemic inflammatory effects which are distinct from intracellular CTSD inhibition. Considering that intracellular CTSD inhibition is involved in essential physiological processes, specific inhibitors capable of blocking extracellular CTSD activity, can be promising and safe NASH drugs.

Proteomics analysis of human intestinal organoids during hypoxia and reoxygenation as a model to study ischemia-reperfusion injury.

Intestinal ischemia-reperfusion (IR) injury is associated with high mortality rates, which have not improved in the past decades despite advanced insight in its pathophysiology using in vivo animal and human models. The inability to translate previous findings to effective therapies emphasizes the need for a physiologically relevant in vitro model to thoroughly investigate mechanisms of IR-induced epithelial injury and test potential therapies. In this study, we demonstrate the use of human small intestinal organoids to model IR injury by exposing organoids to hypoxia and reoxygenation (HR). A mass-spectrometry-based proteomics approach was applied to characterize organoid differentiation and decipher protein dynamics and molecular mechanisms of IR injury in crypt-like and villus-like human intestinal organoids. We showed successful separation of organoids exhibiting a crypt-like proliferative phenotype, and organoids exhibiting a villus-like phenotype, enriched for enterocytes and goblet cells. Functional enrichment analysis of significantly changing proteins during HR revealed that processes related to mitochondrial metabolism and organization, other metabolic processes, and the immune response were altered in both organoid phenotypes. Changes in protein metabolism, as well as mitophagy pathway and protection against oxidative stress were more pronounced in crypt-like organoids, whereas cellular stress and cell death associated protein changes were more pronounced in villus-like organoids. Profile analysis highlighted several interesting proteins showing a consistent temporal profile during HR in organoids from different origin, such as NDRG1, SDF4 or DMBT1. This study demonstrates that the HR response in human intestinal organoids recapitulates properties of the in vivo IR response. Our findings provide a framework for further investigations to elucidate underlying mechanisms of IR injury in crypt and/or villus separately, and a model to test therapeutics to prevent IR injury.

Comparative label-free proteomic analysis of equine osteochondrotic chondrocytes.

Osteochondrosis is a developmental orthopedic disease affecting growing cartilage in young horses. In this study we compared the proteomes of equine chondrocytes obtained from healthy and osteochondrotic cartilage using a label-free mass spectrometry approach. Quantitative changes of some proteins selected for their involvement in different functional pathways highlighted by the bioinformatics analysis, were validated by western blotting, while biochemical alterations of extracellular matrix were confirmed via Raman spectroscopy analysis. In total 1637 proteins were identified, of which 59 were differentially abundant. Overall, the results highlighted differentially represented proteins involved in metabolic and functional pathways that may be related to the failure of the endochondral ossification process occurring in osteochondrosis. In particular, we identified proteins involved in extracellular matrix degradation and organization, vitamin metabolism, osteoblast differentiation, apoptosis, protein folding and localization, signalling and gene expression modulation and lysosomal activities. These results provide valuable new insights to elucidate the underlying molecular mechanisms associated with the development and progression of osteochondrosis. SIGNIFICANCE: Osteochondrosis is a common articular disorder in young horses mainly due to defects in endochondral ossification. The pathogenesis of osteochondrosis is still poorly understood and only a limited number of proteomic studies have been conducted. This study provides a comprehensive characterization of proteomic alterations occurring in equine osteochondrotic chondrocytes, the only resident cell type that modulates differentiation and maturation of articular cartilage. The results evidenced alterations in abundance of proteins involved in functional and metabolic pathways and in extracellular matrix remodelling. These findings could help clarify some molecular aspects of osteochondrosis and open new fields of research for elucidating the pathogenesis of this disease.

AntagomiR-103 and -107 Treatment Affects Cardiac Function and Metabolism.

MicroRNA-103/107 regulate systemic glucose metabolism and insulin sensitivity. For this reason, inhibitory strategies for these microRNAs are currently being tested in clinical trials. Given the high metabolic demands of the heart and the abundant cardiac expression of miR-103/107, we questioned whether antagomiR-mediated inhibition of miR-103/107 in C57BL/6J mice impacts on cardiac function. Notably, fractional shortening decreased after 6 weeks of antagomiR-103 and -107 treatment. This was paralleled by a prolonged systolic radial and circumferential time to peak and by a decreased global strain rate. Histology and electron microscopy showed reduced cardiomyocyte area and decreased mitochondrial volume and mitochondrial cristae density following antagomiR-103 and -107. In line, antagomiR-103 and -107 treatment decreased mitochondrial OXPHOS complexes' protein levels compared to scrambled, as assessed by mass spectrometry-based label-free quantitative proteomics. MiR-103/107 inhibition in primary cardiomyocytes did not affect glycolysis rates, but it decreased mitochondrial reserve capacity, reduced mitochondrial membrane potential, and altered mitochondrial network morphology, as assessed by live-cell imaging. Our data indicate that antagomiR-103 and -107 decrease cardiac function, cardiomyocyte size, and mitochondrial oxidative capacity in the absence of pathological stimuli. These data raise concern about the possible cardiac implications of the systemic use of antagomiR-103 and -107 in the clinical setting, and careful cardiac phenotyping within ongoing trials is highly recommended.

Lipid surface modifications increase mesoporous silica nanoparticle labeling properties in mesenchymal stem cells.

BACKGROUND: Nanoparticles have emerged as promising cell-labeling tools, as they can be precisely tailored in terms of chemical and physical properties. Mesoporous silica nanoparticles (MSNs), in particular, are easily tunable with regard to surface and core chemistry, and are able to confine dyes and drug molecules efficiently. PURPOSE: The aim of this study was to investigate the effect of lipid and polyethylene glycol (PEG) surface modifications on MSN stem-cell-tracking abilities. METHODS: Lipid and PEG surface functionalized MSNs were synthesized and the effect of surface functionalization on cell internalization, proliferation, differentiation and cell proteomics was investigated in patient derived mesenchymal stem cells (MSCs). RESULTS: MSNs and lipid surface-modified MSNs were internalized by >80% of the MSC population, with the exception of nanoparticles modified with short PEG chains (molecular weight 750 [MSN-PEG750]). Lipid-modified MSNs had higher labeling efficiency with maximum uptake after 2 hours of exposure and were in addition internalized 17 times higher compared to unmodified MSNs, without negatively affecting differentiation capacity. Using a mass-spectrometry-based label-free quantitative proteomics approach, we show that MSN labeling leads to the up- and downregulation of proteins that were unique for the different surface-modified MSNs. In addition, functional enrichments were found in human MSCs labeled with MSNs, MSN-PEG750, and lipid-modified MSNs. SUMMARY: Here we show that organic modifications with lipids and PEGylation can be used as a promising strategy to improve MSN labeling capabilities. In particular, we show that lipid modifications can optimize such probes in three distinct ways: significantly improved signal strength, a barrier for sustained release of additional probes, and improved stem-cell-labeling efficiency.

Adipophilin protein expression in muscle--a possible protective role against insulin resistance.

Adipophilin is a 50 kDa protein that belongs to the PAT family (perilipin, adipophilin, TIP47, S3-12 and OXPAT), which comprises proteins involved in the coating of lipid droplets. Little is known about the functional role of adipophilin in muscle. Using the C2C12 cell line as a model, we demonstrate that palmitic acid-treated cells highly express the adipophilin protein in a dose-dependent way. Next, we show that oleic acid is a more potent inducer of adipophilin protein levels than palmitic acid. Cells treated with oleic acid have a higher adipophilin protein expression and higher triglyceride levels but less impairment of insulin signaling than cells treated with palmitic acid. Additionally, we show that peroxisome proliferator-activated receptor (PPAR)alpha, PPARbeta/delta and PPARgamma agonists all increase the expression of the adipophilin protein in C2C12 cells. This effect was most pronounced for the PPARalpha agonist GW7647. Furthermore, the expression of adipophilin as a 37 kDa N-terminally truncated protein is higher in the gastrocnemius than in the quadriceps of C57BL/6J mice, especially after an 8-week high-fat diet. The expression of adipophilin was higher in the muscle of mice fed a 4-week high-fat diet based on olive oil or safflower oil than in mice fed a 4-week high-fat diet based on palm oil. After 2 weeks of intervention, plasma glucose, plasma insulin and the homeostasis model assessment of insulin resistance index were lower in mice fed a 4-week high-fat diet based on olive oil or safflower oil than in mice fed a 4-week high-fat diet based on palm oil. Taken together, the results obtained in the present study indicate that adipophilin protein expression in muscle is involved in maintaining insulin sensitivity.

An 8-week high-fat diet induces obesity and insulin resistance with small changes in the muscle transcriptome of C57BL/6J mice.

BACKGROUND: Skeletal muscle is responsible for most of the insulin-stimulated glucose uptake and metabolism. Therefore, it plays an important role in the development of insulin resistance, one of the characteristics of the metabolic syndrome (MS). As the prevalence of the MS is increasing, there is an urgent need for more effective intervention strategies. METHODS: C57BL/6J mice were fed an 8-week low-fat diet (10 kcal%; LFD) or high-fat diet (45 kcal%; HFD). Microarray analysis was performed by using two comparisons: (1) 8-week HFD transcriptome versus 8-week LFD transcriptome and (2) transcriptome of mice sacrificed at the start of the intervention versus 8-week LFD transcriptome and 8-week HFD transcriptome, respectively. RESULTS: Although an 8-week HFD induced obesity and impaired insulin sensitivity, HFD-responsive changes in the muscle transcriptome were relatively small (<1.3-fold). In fact, 8-weeks of aging induced more pronounced changes than an HFD. One comparison revealed the transcriptional downregulation of the mito- gen-activated protein kinase cascade, whereas both comparisons showed the upregulation of fatty acid oxidation, demonstrating that the two comparison strategies are confirmative as well as complementary. CONCLUSION: We suggest using complementary analysis strategies in the genome-wide search for gene expression changes induced by mild interventions, such as an HFD.

Short-term high fat-feeding results in morphological and metabolic adaptations in the skeletal muscle of C57BL/6J mice.

The prevalence of the metabolic syndrome (MS) is rapidly increasing all over the world. Consequently, there is an urgent need for more effective intervention strategies. Both animal and human studies indicate that lipid oversupply to skeletal muscle can result in insulin resistance, which is one of the characteristics of the MS. C57BL/6J mice were fed a low-fat (10 kcal%) palm oil diet or a high-fat (45 kcal%; HF) palm oil diet for 3 or 28 days. By combining transcriptomics with protein and lipid analyses we aimed to better understand the molecular events underlying the early onset of the MS. Short-term HF feeding led to altered expression levels of genes involved in a variety of biological processes including morphogenesis, energy metabolism, lipogenesis, and immune function. Protein analysis showed increased levels of the myosin heavy chain, slow fiber type protein, and the complexes I, II, III, IV, and V of the oxidative phosphorylation. Furthermore, we observed that the main mitochondrial membrane phospholipids, phosphatidylcholine and phosphatidylethanolamine, contained more saturated fatty acids. Altogether, these results point to a morphological as well as a metabolic adaptation by promoting a more oxidative fiber type. We hypothesize that after this early positive adaptation, a continued transcriptional downregulation of genes involved in oxidative phosphorylation will result in decreased oxidative capacity at a later stage. Together with increased saturation of phospholipids of the mitochondrial membrane this can result in decreased mitochondrial function, which is a hallmark observed in insulin resistance and Type 2 diabetes.