Sterol Metabolism Research Articles

Alteration of cholesterol content and oxygen level in intestinal organoids after infection with Staphylococcus aureus.

The pathogenicity elicited by Staphylococcus (S.) aureus, one of the best-studied bacteria, in the intestine is not well understood. Recently, we demonstrated that S. aureus infection induces alterations in membrane composition that are associated with concomitant impairment of intestinal function. Here, we used two organoid models, induced pluripotent stem cell (iPSC)-derived intestinal organoids and colonic intestinal stem cell-derived intestinal organoids (colonoids), to examine how sterol metabolism and oxygen levels change in response to S. aureus infection. HPLC quantification showed differences in lipid homeostasis between infected and uninfected cells, characterized by a remarkable decrease in total cellular cholesterol. As the altered sterol metabolism is often due to oxidative stress response, we next examined intracellular and extracellular oxygen levels. Three different approaches to oxygen measurement were applied: (1) cell-penetrating nanoparticles to quantify intracellular oxygen content, (2) sensor plates to quantify extracellular oxygen content in the medium, and (3) a sensor foil system for oxygen distribution in organoid cultures. The data revealed significant intracellular and extracellular oxygen drop after infection in both intestinal organoid models as well as in Caco-2 cells, which even 48 h after elimination of extracellular bacteria, did not return to preinfection oxygen levels. In summary, we show alterations in sterol metabolism and intra- and extracellular hypoxia as a result of S. aureus infection. These results will help understand the cellular stress responses during sustained bacterial infections in the intestinal epithelium.

Effects of Neonatal Hypoxic-Ischemic Injury on Brain Sterol Synthesis and Metabolism.

Neonatal hypoxic-ischemic brain injury (HIBI) results from disruptions to blood supply and oxygen in the perinatal brain. The goal of this study was to measure brain sterol metabolites and plasma oxysterols after injury in a neonatal HIBI mouse model to assess for potential therapeutic targets in the brain biochemistry as well as potential circulating diagnostic biomarkers. Postnatal day 9 CD1-IGS mouse pups were randomized to HIBI induced by carotid artery ligation followed by 30 minutes at 8% oxygen or to sham surgery and normoxia. Brain tissue was collected for sterol analysis by liquid chromatography with tandem mass spectrometry (LC-MS/MS). Plasma was collected for oxysterol analysis by LC-MS/MS. There were minimal changes in brain sterol concentrations in the first 72 hours after HIBI. In severely injured brains, there was a significant increase in desmosterol, 7-DHC, 8-DHC, and cholesterol 24 hours after injury in the ipsilateral tissue. Lanosterol, 24-dehydrolathosterol, and 14-dehydrozymostenol decreased in plasma 24 hours after injury. Severe neonatal HIBI was associated with increased cholesterol and sterol precursors in the cortex at 24 hours after injury. Differences in plasma oxysterols were seen at 24 hours but were not present at 30 minutes after injury, suggesting that these sterol intermediates would be of little value as early diagnostic biomarkers.

Structure of the ceramide-bound SPOTS complex

Sphingolipids are structural membrane components that also function in cellular stress responses. The serine palmitoyltransferase (SPT) catalyzes the rate-limiting step in sphingolipid biogenesis. Its activity is tightly regulated through multiple binding partners, including Tsc3, Orm proteins, ceramides, and the phosphatidylinositol-4-phosphate (PI4P) phosphatase Sac1. The structural organization and regulatory mechanisms of this complex are not yet understood. Here, we report the high-resolution cryo-EM structures of the yeast SPT in complex with Tsc3 and Orm1 (SPOT) as dimers and monomers and a monomeric complex further carrying Sac1 (SPOTS). In all complexes, the tight interaction of the downstream metabolite ceramide and Orm1 reveals the ceramide-dependent inhibition. Additionally, observation of ceramide and ergosterol binding suggests a co-regulation of sphingolipid biogenesis and sterol metabolism within the SPOTS complex.

Transcriptomic analysis of PADI4 target genes during multi-lineage differentiation of embryonic stem cells.

During mammalian embryo development, pluripotent epiblast cells diversify into the three primary germ layers, which will later give rise to all fetal and adult tissues. These processes involve profound transcriptional and epigenetic changes that require precise coordination. Peptidylarginine deiminase IV (PADI4) is a transcriptional regulator that is strongly associated with inflammation and carcinogenesis but whose physiological roles are less well understood. We previously found that Padi4 expression is associated with pluripotency. Here, we examined the role of PADI4 in maintaining the multi-lineage differentiation potential of mouse embryonic stem (ES) cells. Using bulk and single-cell transcriptomic analyses of embryoid bodies (EBs) derived from Padi4 knock-out (Padi4-KO) mouse ES cells, we find that PADI4 loss impairs mesoderm diversification and differentiation of cardimyocytes and endothelial cells. Additionally, Padi4 deletion leads to concerted downregulation of genes associated with polarized growth, sterol metabolism and the extracellular matrix (ECM). This study indicates a requirement for Padi4 in the specification of the mesodermal lineage and reports the Padi4 associated transcriptome, providing a platform for understanding the physiological functions of Padi4 in development and homeostasis. This article is part of the Theo Murphy meeting issue 'The virtues and vices of protein citrullination'.

Mendelian Randomization Study of Lipid Metabolites Reveals Causal Associations with Heel Bone Mineral Density.

Osteoporosis, which is a bone disease, is characterized by low bone mineral density and an increased risk of fractures. The heel bone mineral density is often used as a representative measure of overall bone mineral density. Lipid metabolism, which includes processes such as fatty acid metabolism, glycerol metabolism, inositol metabolism, bile acid metabolism, carnitine metabolism, ketone body metabolism, sterol and steroid metabolism, etc., may have an impact on changes in bone mineral density. While some studies have reported correlations between lipid metabolism and heel bone mineral density, the overall causal relationship between metabolites and heel bone mineral density remains unclear. to investigate the causal relationship between lipid metabolites and heel bone mineral density using two-sample Mendelian randomization analysis. Summary-level data from large-scale genome-wide association studies were extracted to identify genetic variants linked to lipid metabolite levels. These genetic variants were subsequently employed as instrumental variables in Mendelian randomization analysis to estimate the causal effects of each lipid metabolite on heel bone mineral density. Furthermore, metabolites that could potentially be influenced by causal relationships with bone mineral density were extracted from the KEGG and WikiPathways databases. The causal associations between these downstream metabolites and heel bone mineral density were then examined. Lastly, a sensitivity analysis was conducted to evaluate the robustness of the results and address potential sources of bias. A total of 130 lipid metabolites were analyzed, and it was found that acetylcarnitine, propionylcarnitine, hexadecanedioate, tetradecanedioate, myo-inositol, 1-arachidonoylglycerophosphorine, 1-linoleoylglycerophoethanolamine, and epiandrosterone sulfate had a causal relationship with heel bone mineral density (p < 0.05). Furthermore, our findings also indicate an absence of causal association between the downstream metabolites associated with the aforementioned metabolites identified in the KEGG and WikiPathways databases and heel bone mineral density. This work supports the hypothesis that lipid metabolites have an impact on bone health through demonstrating a causal relationship between specific lipid metabolites and heel bone mineral density. This study has significant implications for the development of new strategies to osteoporosis prevention and treatment.

Diagnostic challenges and management advances in cytochrome P450 oxidoreductase deficiency, a rare form of congenital adrenal hyperplasia, with 46, XX karyotype.

Cytochrome P450 oxidoreductase deficiency (PORD) is a rare form of congenital adrenal hyperplasia that can manifest with skeletal malformations, ambiguous genitalia, and menstrual disorders caused by cytochrome P450 oxidoreductase (POR) mutations affecting electron transfer to all microsomal cytochrome P450 and some non-P450 enzymes involved in cholesterol, sterol, and drug metabolism. With the advancement of molecular biology and medical genetics, increasing numbers of PORD cases were reported, and the clinical spectrum of PORD was extended with studies on underlying mechanisms of phenotype-genotype correlations and optimum treatment. However, diagnostic challenges and management dilemma still exists because of unawareness of the condition, the overlapping manifestations with other disorders, and no clear guidelines for treatment. Delayed diagnosis and management may result in improper sex assignment, loss of reproductive capacity because of surgical removal of ruptured ovarian macro-cysts, and life-threatening conditions such as airway obstruction and adrenal crisis. The clinical outcomes and prognosis, which are influenced by specific POR mutations, the presence of additional genetic or environmental factors, and management, include early death due to developmental malformations or adrenal crisis, bilateral oophorectomies after spontaneous rupture of ovarian macro-cysts, genital ambiguity, abnormal pubertal development, and nearly normal phenotype with successful pregnancy outcomes by assisted reproduction. Thus, timely diagnosis including prenatal diagnosis with invasive and non-invasive techniques and appropriate management is essential to improve patients' outcomes. However, even in cases with conclusive diagnosis, comprehensive assessment is needed to avoid severe complications, such as chromosomal test to help sex assignment and evaluation of adrenal function to detect partial adrenal insufficiency. In recent years, it has been noted that proper hormone replacement therapy can lead to decrease or resolve of ovarian macro-cysts, and healthy babies can be delivered by in vitro fertilization and frozen embryo transfer following adequate control of multiple hormonal imbalances. Treatment may be complicated with adverse effects on drug metabolism caused by POR mutations. Unique challenges occur in female PORD patients such as ovarian macro-cysts prone to spontaneous rupture, masculinized genitalia without progression after birth, more frequently affected pubertal development, and impaired fertility. Thus, this review focuses only on 46, XX PORD patients to summarize the potential molecular pathogenesis, differential diagnosis of classic and non-classic PORD, and tailoring therapy to maintain health, avoid severe complications, and promote fertility.

Sparassis latifolia Polysaccharide Attenuates Cholesterol in Rats Maintained on a High-Fat, High-Cholesterol Diet

Sparassis latifolia polysaccharide (SLP) has broad application prospects as a raw material of functional food. This study aimed to investigate the effects of SLP on liver cholesterol metabolism and its mechanism. Sprague–Dawley rats were fed a high-fat, high-cholesterol diet, and various assessments were conducted including hematoxylin and eosin staining, measurement of liver and metabolic indices, and antioxidant analysis. In addition, fluorescence quantitative reverse transcription-polymerase chain reaction was performed to analyze mRNA expression, and gas chromatography-mass spectrometry was employed for screening differential metabolites. The results demonstrated that SLP reduced the abnormal increase in the liver index caused by high fat and cholesterol and improved liver tissue morphology, metabolism level, and antioxidant-related enzyme activities. The polysaccharide regulated the liver sterol metabolism pathway, decreased the mRNA expression levels of 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoAR) and sterol O-acyltransferase 1 (SOAT1), upregulated the mRNA expression levels of cholesterol 7α hydroxylase (CYP7α-1) and ABCA1, and increased the contents of propionic acid and butyric acid in the liver. These findings indicated a novel pathway through which SLP prevented hepatic lipid deposition and ameliorated sterol metabolism via regulating the HMG-CoAR-CYP7α-1 axis.

Comparative physiological, antioxidant and proteomic investigation reveal robust response to cold stress in Digitalis purpurea L.

Digitalis purpurea (L) is an important medicinal plant growing at Alpine region of Himalayas and withstands low temperatures and harsh climatic conditions existing at high altitude. It serves as an ideal plant system to decipher the tolerance to cold stress (CS) in plants from high altitudes. To understand the complexity of plant response to CS, we performed a comparative physiological and biochemical study complemented with proteomics in one-month-old D. purpurea grown at 25°C (control) and 4°C (CS). We observed an enhanced accumulation of different osmo-protectants (glycine betaine, soluble sugar and proline) and higher transcription (mRNA levels) of various antioxidant enzymes with an increased antioxidant enzyme activity in D. purpurea when exposed to CS. Furthermore, higher concentrations of non-enzymatic antioxidants (flavonoids, phenolics) was also associated with the response to CS. Differential proteomic analysis revealed the role of various proteins primarily involved in redox reactions, protein stabilization, quinone and sterol metabolism involved in CS response in D. purpurea.. Our results provide a framework for better understanding the physiological and molecular mechanism of CS response in D. purpurea at high altitudes.

Identification and characterization of protein interactions with the major Niemann-Pick type C disease protein in yeast reveals pathways of therapeutic potential.

Niemann-Pick type C (NP-C) disease is a rare lysosomal storage disease caused by mutations in NPC1 (95% cases) or NPC2 (5% cases). These proteins function together in cholesterol egress from the lysosome, whereby upon mutation, cholesterol and other lipids accumulate causing major pathologies. However, it is not fully understood how cholesterol is transported from NPC1 residing at the lysosomal membrane to the endoplasmic reticulum (ER) and plasma membrane. The yeast ortholog of NPC1, Niemann-Pick type C-related protein-1 (Ncr1), functions similarly to NPC1; when transfected into a mammalian cell lacking NPC1, Ncr1 rescues the diagnostic hallmarks of cholesterol and sphingolipid accumulation. Here, we aimed to identify and characterize protein-protein interactions (PPIs) with the yeast Ncr1 protein. A genome-wide split-ubiquitin membrane yeast two-hybrid (MYTH) protein interaction screen identified 11 ER membrane-localized, full-length proteins interacting with Ncr1 at the lysosomal/vacuolar membrane. These highlight the importance of ER-vacuole membrane interface and include PPIs with the Cyb5/Cbr1 electron transfer system, the ceramide synthase complex, and the Sec61/Sbh1 protein translocation complex. These PPIs were not detected in a sterol auxotrophy condition and thus depend on normal sterol metabolism. To provide biological context for the Ncr1-Cyb5 PPI, a yeast strain lacking this PPI (via gene deletions) exhibited altered levels of sterols and sphingolipids including increased levels of glucosylceramide that mimic NP-C disease. Overall, the results herein provide new physical and genetic interaction models to further use the yeast model of NP-C disease to better understand human NP-C disease.

Cholesterol Metabolism in Aging and Age-Related Disorders

All mammalian cell membranes contain cholesterol to maintain membrane integrity. The transport of this hydrophobic lipid is mediated by lipoproteins. Cholesterol is especially enriched in the brain, particularly in synaptic and myelin membranes. Aging involves changes in sterol metabolism in peripheral organs and also in the brain. Some of those alterations have the potential to promote or to counteract the development of neurodegenerative diseases during aging. Here, we summarize the current knowledge of general principles of sterol metabolism in humans and mice, the most widely used model organism in biomedical research. We discuss changes in sterol metabolism that occur in the aged brain and highlight recent developments in cell type–specific cholesterol metabolism in the fast-growing research field of aging and age-related diseases, focusing on Alzheimer's disease. We propose that cell type–specific cholesterol handling and the interplay between cell types critically influence age-related disease processes.

Aldolase SalA dominates C24 steroidal side-chain-cleavage in the phytosterol degradation from Mycobacterium neoaurum

Understanding the mycobacteria degradation of sterol may facilitate strategies for the directed biotransformation of steroidal drug intermediates. Due to the long and complicated catabolic process, several key steps that involved in the C-C bond cleavage of steroidal side-chain remain unknown. In the present study, an aldolase SalA from Mycobacterium neoaurum was identified that was related to the synthesis of 3-oxo-4-pregnene-20-carboxyl aldehyde and 20-hydroxymethyl pregn-4-ene-3-one (4-HBC). As a member of the thiolase superfamily, the aldolase SalA-SalB is composed of a thiolase-like protein (SalA) and a DUF35 domain (SalB). As a result, the gene salA was determined that had special significance for the synthesis of 4-HBC in sterol metabolism by performing knockout and replenishment. Based on insights provided by the docking model and mutants analysis, the hypothesis of the catalytic mechanism was proposed. Furthermore, the deletion of salA absolutely eliminated the by-product 4-HBC and meanwhile the molar yield of ADD was increased by 10 %. The dominant role of SalA in the C24 steroidal side-chain cleavage as confirmed in this study provided a further clarification on the phytosterol degrading pathway in Mycobacterium neoaurum, which is of significant importance for the directed biotransformation between C19- and C22-steroids.

Effects of Dietary Chenodeoxycholic Acid Supplementation in a Low Fishmeal Diet Containing Clostridium autoethanogenum Protein on Growth, Lipid and Cholesterol Metabolism, and Hepatopancreas Health of Litopenaeus vannamei.

The study aimed to assess the impact of adding chenodeoxycholic acid (CDCA) to the diet of Litopenaeus vannamei on their growth performance, lipid and cholesterol metabolism, and hepatopancreas health while being fed a low fishmeal diet. Five diets were formulated, one of which contained 25% fishmeal (PC); fishmeal was partially replaced with Clostridium autoethanogenum protein in the remaining four diets and supplemented with 0, 0.03, 0.06, and 0.09% CDCA (NC, BA1, BA2, and BA3, respectively). In this study, four replicates of each diet were assigned and each replicate consisted of 30 shrimp with an average weight of (0.25 ± 0.03 g). The shrimp were fed four times a day for a period of 56 days. The results of this study indicate that the inclusion of CDCA in the diet had a positive impact on the growth performance of the shrimp. The final body weight (FBW), weight gain (WG), and specific growth rate (SGR) of the shrimp in the PC group were similar to those in the BA2 group, and significantly higher than those in the other three groups. The survival rate (SR) was similar among all groups. In comparison to the PC group, the low fishmeal groups exhibited a significant decrease in the crude lipid content of the whole shrimp, as well as the Total cholesterol (T-CHOL), Low-density lipoprotein (LDL-C), and High-density lipoprotein (HDL-C) levels in the hemolymph. Regarding the sterol metabolism, the dietary supplementation of CDCA up-regulated the mRNA expression of intracellular cholesterol transporter 1-like (npc1), 7-dehydrocholesterol reductase (7dhcr), Delta (24) sterol reductase (Δ24), HMG-CoA reductase membrane form (hmgcr), and sterol carrier protein 2 (scp). In the lipid metabolism, the mRNA expression of sterol-regulatory element binding protein (srebp) was significantly down-regulated in the shrimp fed the BA1 diet and the expression of AMP-activated protein kinase (ampk) was significantly up-regulated in the shrimp fed the BA1 and BA3 diets compared to the PC group. The mRNA expression of triacylglycerol lipase (tgl) was significantly up-regulated in the shrimp fed the BA2 diet compared to the NC group. Compared with the shrimp fed the PC diets, the dietary supplementation of CDCA significantly down-regulated the protein expression of SREBP1. The lumen damage in the BA1 group was significantly less severe than those in the NC group. The addition of 0.06% CDCA to low fishmeal diets can improve the growth performance, lipid and cholesterol metabolism, and hepatopancreas health of L. vannamei.

Impact of renal tubular Cpt1a overexpression on the kidney metabolome in the folic acid-induced fibrosis mouse model.

Background: Chronic kidney disease (CKD) is characterized by the progressive and irreversible deterioration of kidney function and structure with the appearance of renal fibrosis. A significant decrease in mitochondrial metabolism, specifically a reduction in fatty acid oxidation (FAO) in tubular cells, is observed in tubulointerstitial fibrosis, whereas FAO enhancement provides protection. Untargeted metabolomics offers the potential to provide a comprehensive analysis of the renal metabolome in the context of kidney injury. Methodology: Renal tissue from a carnitine palmitoyl transferase 1a (Cpt1a) overexpressing mouse model, which displays enhanced FAO in the renal tubule, subjected to folic acid nephropathy (FAN) was studied through a multiplatform untargeted metabolomics approach based on LC-MS, CE-MS and GC-MS analysis to achieve the highest coverage of the metabolome and lipidome affected by fibrosis. The expression of genes related to the biochemical routes showing significant changes was also evaluated. Results: By combining different tools for signal processing, statistical analysis and feature annotation, we were able to identify variations in 194 metabolites and lipids involved in many metabolic routes: TCA cycle, polyamines, one-carbon metabolism, amino acid metabolism, purine metabolism, FAO, glycerolipids and glycerophospholipids synthesis and degradation, glycosphingolipids interconversion, and sterol metabolism. We found several metabolites strongly altered by FAN, with no reversion induced by Cpt1a overexpression (v.g. citric acid), whereas other metabolites were influenced by CPT1A-induced FAO (v.g. glycine-betaine). Conclusion: It was implemented a successful multiplatform metabolomics approach for renal tissue analysis. Profound metabolic changes accompany CKD-associated fibrosis, some associated with tubular FAO failure. These results highlight the importance of addressing the crosstalk between metabolism and fibrosis when undertaking studies attempting to elucidate the mechanism of CKD progression.

Dietary bile acids promote sterol metabolism, bile acids enterohepatic circulation, and apoptosis in juvenile Pacific white shrimp (Litopenaeus vannamei)

Bile acids have an important role in the growth and sterol metabolism of shrimp. The aim of this study was to elucidate the effects of bile acids supplementation in the low fishmeal diets on sterol metabolism, bile acids enterohepatic circulation, bile acid profile, and apoptosis in Pacific white shrimp Litopenaeus vannamei. Six diets were formulated in this experiment, HFM diet (25% fishmeal), LFM diet (10 % fishmeal), and LFM supplemented with four levels (0.15, 0.3, 0.6, 0.9 g kg−1) of bile acids, which were LBA1, LBA2, LBA3, LBA4, respectively. Soy protein concentrate and cottonseed protein concentrate blend was used to replace fishmeal of 60 %. After 50 days of feeding trial, the shrimp fed the LBA4 diet showed significantly increased final body weight than that fed the LFM diet (P < 0.05). In the sterol metabolism, the shrimp fed the LBA1 diet was significantly increased the HMG-CoA reductase membrane form (hmgcr) and Niemann-Pick type C-2-like protein (npc2) expression compare with that fed the LFM diet, shrimp in LBA2 group was significantly increased the related genes expression [hmgcr and NPC intracellular cholesterol transporter 1-like (npc1)] than those in the LFM group of hepatopancreas (P < 0.05). In the lipid metabolism, the shrimp in LBA2 group was significantly increased the carnitine palmitoyltransferase-1 (cpt-1) and AMP-activated protein kinase (ampk) expression than that in the LFM group, shrimp in the LBA3 group was significantly increased the fatty acid binding protein fatty acid (fabp) expression than that in the LFM group (P < 0.05). In the bile acids metabolism, the multidrug resistance-associated protein 1-like (mrp2), multidrug resistance-associated protein 2-like (mrp3) and solute carrier family 22 member 7-like (oatp) expressions were significantly increased in LBA1 and LBA2 groups than those in the LFM group of hepatopancreas, organic solute transporter subunit alpha-like (ostα), mrp2, and acyl-coenzyme A thioesterase 1-like (baat) expressions were significantly up-regulated in LBA4 group than those in the LFM group of intestine (P < 0.05). Supplementary bile acids in the diet could decrease the apoptosis-related protein expression (BCL-2 and CASPASE3) compare with the shrimp fed low fishmeal diet (P < 0.05). The concentration of beta MCA in hemolymph bile acids profile increases with bile acids supplementation. The results showed that bile acids enhanced growth performance, promoted sterol metabolism, enhanced enterohepatic circulation, and reduced apoptosis in shrimp on low fishmeal diets. Data availability statementThe data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical.

Effects of 3-Hydroxy-3-methylglutaryl-CoA Reductase Inhibitors on Cholesterol Metabolism in Laying Hens.

This study aimed to investigate the effect of HMGCR inhibitors on egg yolk cholesterol content and its biological mechanisms. Four groups of 180-day-old laying hens (n = 8 cages/group, 6 laying hens/cage) were fed a corn/soybean-based diet (control) and the control diet supplemented with an HMGCR inhibitor at 60, 150, and 300 mg/kg for 4 weeks. The experimental results showed that adding HMGCR inhibitors of 150 mg/kg or more can significantly reduce the cholesterol content in the liver, yolk, serum, and pectoral muscles of laying hens. The RNA-seq results showed that compared with the control group, the addition of HMGCR inhibitors of 150 mg/kg or more to the diet significantly upregulated genes related to cholesterol synthesis in the liver, and the genes involved in steroid synthesis and metabolism, sterol synthesis and metabolism, and cholesterol synthesis and metabolism were all affected by the HMGCR inhibitors. In summary, adding HMGCR inhibitors of 150 mg/kg or more to the diet of hens can significantly reduce the cholesterol content in egg yolk. After the HMGCR inhibitors inhibited the activity of the liver HMGCR, they also altered the expression of genes related to cholesterol synthesis, bile acid synthesis, and cholesterol transport in the liver, and ultimately reduced cholesterol synthesis and cholesterol transport to the egg yolk.

Untargeted Metabolomic Analysis of Sjögren-Larsson Syndrome Reveals a Distinctive Pattern of Multiple Disrupted Biochemical Pathways.

Sjögren-Larsson syndrome (SLS) is a rare inherited neurocutaneous disease characterized by ichthyosis, spastic diplegia or tetraplegia, intellectual disability and a distinctive retinopathy. SLS is caused by bi-allelic mutations in ALDH3A2, which codes for fatty aldehyde dehydrogenase (FALDH) and results in abnormal lipid metabolism. The biochemical abnormalities in SLS are not completely known, and the pathogenic mechanisms leading to symptoms are still unclear. To search for pathways that are perturbed in SLS, we performed untargeted metabolomic screening in 20 SLS subjects along with age- and sex-matched controls. Of 823 identified metabolites in plasma, 121 (14.7%) quantitatively differed in the overall SLS cohort from controls; 77 metabolites were decreased and 44 increased. Pathway analysis pointed to disrupted metabolism of sphingolipids, sterols, bile acids, glycogen, purines and certain amino acids such as tryptophan, aspartate and phenylalanine. Random forest analysis identified a unique metabolomic profile that had a predictive accuracy of 100% for discriminating SLS from controls. These results provide new insight into the abnormal biochemical pathways that likely contribute to disease in SLS and may constitute a biomarker panel for diagnosis and future therapeutic studies.

Spatial sterol metabolism unveiled by stimulated Raman imaging.

Spatiotemporal dynamics of small-molecule metabolites have gained increasing attention for their essential roles in deciphering the fundamental machinery of life. However, subcellular-level regulatory mechanisms remain less studied, particularly due to a lack of tools to track small-molecule metabolites. To address this challenge, we developed high-resolution stimulated Raman scattering (SRS) imaging of a genetically engineered model (GEM) to map metabolites in subcellular resolution. As a result, an unexpected regulatory mechanism of a critical metabolite, sterol, was discovered in yeast by amplifying the strength of vibrational imaging by genetic modulation. Specifically, isozymes of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) were evident to promote ergosterol distribution to distinct subcellular locations, where ergosterol was enriched by a local HMGR-directed synthesis. The heterogeneity of this expression pattern thus provides new insights into sterol metabolism and related disease treatment strategies. These findings demonstrate SRS-GEM as a promising platform for new possibilities in investigating metabolic regulation, disease mechanisms, and biopharmaceutical research.

Optimization of Campesterol-Producing Yeast Strains as a Feasible Platform for the Functional Reconstitution of Plant Membrane-Bound Enzymes.

Campesterol is a major phytosterol that plays important roles in regulating membrane properties and serves as the precursor to multiple specialized metabolites, such as the phytohormone brassinosteroids. Recently, we established a campesterol-producing yeast strain and extended the bioproduction to 22-hydroxycampesterol and 22-hydroxycampest-4-en-3-one, the precursors to brassinolide. However, there is a trade-off in growth due to the disrupted sterol metabolism. In this study, we enhanced the growth of the campesterol-producing yeast by partially restoring the activity of the sterol acyltransferase and engineering upstream FPP supply. Furthermore, genome sequencing analysis also revealed a pool of genes possibly associated with the altered sterol metabolism. Retro engineering implies an essential role of ASG1, especially the C-terminal asparagine-rich domain of ASG1, in the sterol metabolism of yeast especially under stress. The performance of the campesterol-producing yeast strain was enhanced with the titer of campesterol to 18.4 mg/L, and the stationary OD600 was improved by ∼33% compared to the unoptimized strain. In addition, we examined the activity of a plant cytochrome P450 in the engineered strain, which exhibits more than 9-fold higher activity than when expressed in the wild-type yeast strain. Therefore, the engineered campesterol-producing yeast strain also serves as a robust host for the functional expression of plant membrane proteins.

Highlighting In Vitro the Role of Brain-like Endothelial Cells on the Maturation and Metabolism of Brain Pericytes by SWATH Proteomics

Within the neurovascular unit, brain pericytes (BPs) are of major importance for the induction and maintenance of the properties of the blood-brain barrier (BBB) carried by the brain microvessel endothelial cells (ECs). Throughout barriergenesis, ECs take advantage of soluble elements or contact with BPs to maintain BBB integrity and the regulation of their cellular homeostasis. However, very few studies have focused on the role of ECs in the maturation of BPs. The aim of this study is to shed light on the proteome of BPs solocultured (hBP-solo) or cocultured with ECs (hBP-coc) to model the human BBB in a non-contact manner. We first generated protein libraries for each condition and identified 2233 proteins in hBP-solo versus 2492 in hBP-coc and 2035 common proteins. We performed a quantification of the enriched proteins in each condition by sequential window acquisition of all theoretical mass spectra (SWATH) analysis. We found 51 proteins enriched in hBP-solo related to cell proliferation, contractility, adhesion and extracellular matrix element production, a protein pattern related to an immature cell. In contrast, 90 proteins are enriched in hBP-coc associated with a reduction in contractile activities as observed in vivo in 'mature' BPs, and a significant gain in different metabolic functions, particularly related to mitochondrial activities and sterol metabolism. This study highlights that BPs take advantage of ECs during barriergenesis to make a metabolic switch in favor of BBB homeostasis in vitro.

Whole-Genome Analysis of Mycobacterium neoaurum DSM 1381 and the Validation of Two Key Enzymes Affecting C22 Steroid Intermediates in Sterol Metabolism.

Mycobacterium neoaurum DSM 1381 originated from Mycobacterium neoaurum ATCC 25790 by mutagenesis screening is a strain of degrading phytosterols and accumulating important C22 steroid intermediates, including 22-hydroxy-23, 24-bisnorchola-4-en-3-one (4-HP) and 22-hydroxy-23, 24-bisnorchola-1,4-dien-3-one (HPD). However, the metabolic mechanism of these C22 products in M. neoaurum DSM 1381 remains unknown. Therefore, the whole-genome sequencing and comparative genomics analysis of M. neoaurum DSM 1381 and its parent strain M. neoaurum ATCC 25790 were performed to figure out the mechanism. As a result, 28 nonsynonymous single nucleotide variants (SNVs), 17 coding region Indels, and eight non-coding region Indels were found between the genomes of the two strains. When the wild-type 3-ketosteroid-9α-hydroxylase subunit A1 (KshA1) and β-hydroxyacyl-CoA dehydrogenase (Hsd4A) were overexpressed in M. neoaurum DSM 1381, the steroids were transformed into the 4-androstene-3, 17- dione (AD) and 1,4-androstadiene-3,17-dione (ADD) instead of C22 intermediates. This result indicated that 173N of KshA1 and 171K of Hsd4A are indispensable to maintaining their activity, respectively. Amino acid sequence alignment analysis show that both N173D in KshA1 and K171E in Hsd4A are conservative sites. The 3D models of these two enzymes were predicted by SWISS-MODEL and AlphaFold2 to understand the inactivation of the two key enzymes. These results indicate that K171E in Hsd4A may destroy the inaction between the NAD+ with the NH3+ and N173D in KshA1 and may disrupt the binding of the catalytic domain to the substrate. A C22 steroid intermediates-accumulating mechanism in M. neoaurum DSM 1381 is proposed, in which the K171E in Hsd4A leads to the enzyme's inactivation, which intercepts the C19 sub-pathways and accelerates the C22 sub-pathways, and the N173D in KshA1 leads to the enzyme's inactivation, which blocks the degradation of C22 intermediates. In conclusion, this study explained the reasons for the accumulation of C22 intermediates in M. neoaurum DSM 1381 by exploring the inactivation mechanism of the two key enzymes.