Lipid Protein Research Articles

Global epidemiology of Mycobacterium tuberculosis lineage 4 insights from Ecuadorian genomic data

Tuberculosis is a global public health concern, and understanding Mycobacterium tuberculosis transmission routes and genetic diversity of M. tuberculosis is crucial for outbreak control. This study aimed to explore the genomic epidemiology and genetic diversity of M. tuberculosis in Ecuador by analyzing 88 local isolates and 415 public genomes from 19 countries within the Euro-American lineage (L4). Our results revealed significant genomic diversity among the isolates, particularly in the genes related to protein processing, carbohydrate metabolism, lipid metabolism, and xenobiotic biodegradation and metabolism. The population structure analysis showed that sub-lineages 4.3.2/3 (35.4%), 4.1.2.1 (22.7%), 4.4.1 (12.7%), and 4.1.1. (10.7%) were the most prevalent. Phylogenetic and transmission network analyses suggest that these isolates circulating within Ecuador share genetic ties with isolates from other continents, implying historical and ongoing intercontinental transmission events. Our findings underscore the importance of integrating genomic data into public health strategies for tuberculosis control and suggest that enhanced genomic surveillance is essential for understanding and mitigating the global spread of M. tuberculosis. This study provides a comprehensive genomic framework for future epidemiological investigations and control measures targeting M. tuberculosis L4 in Ecuador.

Cold induces brain region-selective cell activity-dependent lipid metabolism.

It has been well documented that cold is an enhancer of lipid metabolism in peripheral tissues, yet its effect on central nervous system lipid dynamics is underexplored. It is well recognized that cold acclimations enhance adipocyte functions, including white adipose tissue lipid lipolysis and beiging, and brown adipose tissue thermogenesis in mammals. However, it remains unclear whether and how lipid metabolism in the brain is also under the control of ambient temperature. Here, we show that cold exposure predominantly increases the expressions of the lipid lipolysis genes and proteins within the paraventricular nucleus of the hypothalamus (PVH) in male mice. Mechanistically, by using innovatively combined brain-region selective pharmacology and in vivo time-lapse photometry monitoring of lipid metabolism, we find that cold activates cells within the PVH and pharmacological inactivation of cells blunts cold-induced effects on lipid peroxidation, accumulation of lipid droplets, and lipid lipolysis in the PVH. Together, these findings suggest that PVH lipid metabolism is cold sensitive and integral to cold-induced broader regulatory responses.

Cold induces brain region-selective cell activity-dependent lipid metabolism

It has been well documented that cold is an enhancer of lipid metabolism in peripheral tissues, yet its effect on central nervous system lipid dynamics is underexplored. It is well recognized that cold acclimations enhance adipocyte functions, including white adipose tissue lipid lipolysis and beiging, and brown adipose tissue thermogenesis in mammals. However, it remains unclear whether and how lipid metabolism in the brain is also under the control of ambient temperature. Here, we show that cold exposure predominantly increases the expressions of the lipid lipolysis genes and proteins within the paraventricular nucleus of the hypothalamus (PVH) in male mice. Mechanistically, by using innovatively combined brain-region selective pharmacology and in vivo time-lapse photometry monitoring of lipid metabolism, we find that cold activates cells within the PVH and pharmacological inactivation of cells blunts cold-induced effects on lipid peroxidation, accumulation of lipid droplets, and lipid lipolysis in the PVH. Together, these findings suggest that PVH lipid metabolism is cold sensitive and integral to cold-induced broader regulatory responses.

Salinity stress-induced impacts on biomass production, bioactive compounds, antioxidant activities and oxidative stress in watermeal (Wolffia globosa)

This study investigated the impact of salinity stress on the biomass production, proximate composition and bioactive contents, antioxidant activities, and oxidative stress parameters of Wolffia globosa. Wolffia was exposed to variable NaCl concentrations (0, 8.56, 17.11, 25.67, 34.22, and 42.78 mM) in nutrient-replete nutrient conditions under natural sunlight in a transparent polyhouse. The net biomass gain (NBG; 131.17 g), crude protein (33.16%) and crude lipid content (4.9%) were significantly high at 8.56 mM NaCl compared to control and most of other treatments. Total phenolic content peaked at 17.11 mM NaCl (551.39 mg GAE g−1), while total tannin content was highest at 25.67 mM NaCl (13.38 mg TAE g−1). Total flavonoid content, vitamin C, oxidative stress enzymes; superoxide dismutase, catalase, and lipid peroxidation (malondialdehyde, MDA) peaked at 42.78 mM NaCl, with values of 140.52 mg QE g−1, 218.69 mg 100 g−1, 28.18 U g−1 FW, 288.60 U min−1 g−1 FW, and 142.32 mg MDA kg−1, respectively. The control invariably had the lowest values. Total carotenoid content was significantly higher at 17.11 and 25.67 mM NaCl (1812.84 and 1749.10 μg g−1), while Chlorophyll-a (Chl-a) was highest at 17.11 mM (21.33 μg g−1). Chlorophyll-b (Chl-b) peaked at 17.11, 25.67, and 34.22 mM NaCl levels. Salinity stress generally elevated antioxidant activities, assessed via DPPH, ABTS, and FRAP assays reaching their highest values at 34.22 mM NaCl (40.52%), 17.11 mM NaCl (56.71%), and 25.67 mM NaCl (428.83 μmol Fe2⁺ g−1), respectively. FT-IR profiles indicated changes in chemical composition and functional groups due to salt stress. Overall, salinity significantly influenced the proximate composition, antioxidant activities, bioactive compounds and oxidative stress enzyme activities of wolffia.

Metallofullerenol Gd@C82(OH)22 preserves human erythrocyte plasma membrane integrity from AAPH-induced oxidative stress: molecular mechanisms and antioxidant activity.

Metallofullerenols and fullerenols have attracted attention due to their remarkable ability to interact with various biologically relevant molecules, paving the way for biomedical applications, ranging from medical imaging techniques to drug carriers, acting with increased efficiency and reduced side effects. In this work, we investigated the effects of two fullerene derivatives, Gd@C82(OH)22 and C70(OH)12, on erythrocyte membrane components under oxidative stress conditions induced by 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH) as a source of peroxyl radicals. The results demonstrated that gadolinium encapsulation within the fullerene cage enhanced the electron affinity of Gd@C82(OH)22, resulting in stronger antioxidant activity. Gd@C82(OH)22 formed a protective hydrophilic layer at the lipid-water interface, effectively preventing lipid peroxidation, oxidative protein damage, and potassium leakage, outperforming C70(OH)12. Both compounds improved membrane fluidity, but Gd@C82(OH)22 exhibited superior preservation of the erythrocyte lipid bilayer and protein function. Additionally, Gd@C82(OH)22 maintained the anion exchange function of Band 3 protein, which is critical for ionic homeostasis, by preventing oxidation-induced aggregation and preserving thiol groups. Despite its limited membrane penetration, Gd@C82(OH)22 stabilized membrane components through hydrogen bonding, which indirectly enhanced membrane stiffness and fluidity under oxidative stress. This external protection of membrane integrity reduced the risk of lipid peroxidation and oxidative damage to erythrocyte proteins. In contrast, C70(OH)12, while effective in protecting membrane lipids, was less effective in preserving protein structure. These findings highlight the unique protective properties of Gd@C82(OH)22, attributed to polarization effects induced by the encapsulated Gd atom. This study indicates that metallofullerenols, such as Gd@C82(OH)22, may have therapeutic potential in mitigating oxidative damage.

Effect of H2O2 induced oxidative stress on volatile organic compounds in differentiated 3T3-L1 cells

Oxidative stress (OS) refers to the disruption in the balance between free radical generation and antioxidant defenses, leading to potential tissue damage. Reactive oxygen species (ROS) can interact with biological components, triggering processes like protein oxidation, lipid peroxidation, or DNA damage, resulting in the generation of several volatile organic compounds (VOCs). Recently, VOCs provided new insight into cellular metabolism and can serve as potential biomarkers. The objective is to investigate the impact of OS on cell metabolism by analyzing the release or alterations of VOCs in the headspace of differentiated 3T3-L1 adipocytes. An OS model in differentiated 3T3-L1 cell lines was constructed using hydrogen peroxide (H2O2) treatment. The effect of OS on cell metabolism was analyzed by detecting VOCs in the headspace of the cells using solid phase micro extraction (SPME) and gas chromatography-mass spectrometry (GCMS). Our findings indicate that H2O2 concentrations exceeding 300 µM induce significant OS, leading to adipocyte apoptosis, as evidenced by various assays. Of the twenty VOCs identified, ten were upregulated in the cells. VOCs such as diphenyl ether, 1,3,5-trioxane, 5-methyl tridecane, 2-ethyl-1-hexanol, and 2,4-di-tert-butyl phenol emerged as potential biomarkers for OS. This study demonstrates that elevated OS alters VOC profiles in differentiated 3T3-L1 adipocytes, providing insights into the effects of OS on adipose tissue and identifying potential OS biomarkers.

Alterations in newborn metabolite patterns with preterm birth and diabetes in pregnancy.

This study examines the influence of prematurity and diabetes (DM) in pregnancy on metabolite patterns at birth, and associations with adiposity development in a prospective cohort. Term and preterm (30-36 weeks gestational age [GA]) infants were enrolled and body composition assessments completed through discharge. Targeted metabolomics was used to assess metabolites in cord or infant blood in the first 2 days. Among 91 infants, 62 were preterm and 27 were exposed to DM. In factor analysis, variation in acylcarnitines' and non-essential amino acids differed by GA and DM exposure and were associated with adiposity at term age. DM-group had 1.95-fold increase in t4-OH-pro (p = 0.003) and 2.14-fold increase in taurine (p = 0.004) compared with non-DM group. Preterm infants had 1.77-fold increase in glycerophospholipid PC aa C32:2 versus term group (p < 0.001). Pathway analysis revealed differences across DM and GA groups in pathways associated with citrulline metabolism, amino acid transport/ synthesis, and fatty acid quantity/transport. In this cohort of infants, there are unique metabolite signatures associated with DM exposure, prematurity, and adiposity development after birth. These markers may reflect early metabolism changes in the developing infant which relate to known risks of adverse growth and cardiometabolic outcomes in this group. In this study of term and preterm infants, diabetes in pregnancy was associated with unique metabolic signatures at birth, including increased expression of metabolites related to protein synthesis and lipid metabolism. Metabolites related to lipid and protein metabolism were associated with adiposity development at term age, including estimated body fat percent, skin fold thickness measures, and arm circumference measures. Unique signatures of metabolites associated with prematurity and exposure to diabetes in pregnancy may reflect early metabolism changes in the developing infant which relate to known risks of adverse growth and cardiometabolic outcomes in this group.

Cell Labeling with 15-YNE Is Useful for Tracking Protein Palmitoylation and Metabolic Lipid Flux in the Same Sample.

Protein S-palmitoylation is the process by which a palmitoyl fatty acid is attached to a cysteine residue of a protein via a thioester bond. A range of methodologies are available for the detection of protein S-palmitoylation. In this study, two methods for the S-palmitoylation of different proteins were compared after metabolic labeling of cells with 15-hexadecynoic acid (15-YNE) to ascertain their relative usefulness. It was hypothesized that labeling cells with a traceable lipid would affect lipid metabolism and the cellular lipidome. In this study, we developed a method to track 15-YNE incorporation into lipids using liquid chromatography high-resolution mass spectrometry (LC-HRMS) as well as protein palmitoylation in the same sample. We observed a time- and concentration-dependent S-palmitoylation of calnexin and succinate dehydrogenase complex flavoprotein subunit A (SDHA) depending on the cell type. The detection of S-palmitoylation with a clickable fluorophore or biotin azide followed by immunoprecipitation is shown to be equally useful. 15-YNE was observed to be incorporated into a wide array of lipid classes during the process, yet it did not appear to modify the overall lipid composition of the cells. In conclusion, we show that 15-YNE is a useful tracer to detect both protein S-palmitoylation and lipid metabolism in the same sample.

The Effects of Polyphenols on Texture and Flavor of Egg Yolk: A Molecular Docking Study.

The effect of polyphenols on the texture and flavor of egg yolk hot gel (EY) was studied. Tea polyphenols (TP), rosmarinic acid (RE), and curcumin (CC) showed significant antioxidant properties during egg yolk processing and could effectively reduce lipid oxidation products (decreased by 68.9%, 76.4%, and 58.61%, respectively) and protein oxidation products (decreased by 47.49%, 37.47%, and 52.51%, respectively) and volatile odor components (styrene, nonanal and 1-octene-3-ol). In addition, these polyphenols enhanced the hot gel properties of the yolk, but did not significantly change the taste of the yolk. This improvement could be attributed to hydrophobic interactions, hydrogen bonds and ionic bonds formed between polyphenols and egg yolk proteins. These interactions produced a more stable structure that was less likely to unfold during heat treatment. As a result, exposure to free sulfhydryl groups, free amino acids and free fatty acids was minimized, thus reducing oxidation reactions.

Evolutionary analysis of the DHHCs in Saccharinae

The DHHC domain genes are crucial for protein lipid modification, a key post-translational modification influencing membrane targeting, subcellular trafficking, and protein function. Despite their significance, the DHHC gene family in Saccharinae remains understudied. Here, we identified 32 (110 alleles), 28, 53, and 48 DHHC genes in Saccharum spontaneum Np-X, Erianthus rufipilus, Miscanthus sinensis, and Miscanthus lutarioriparius, respectively. Collinearity analysis uncovered the loss of two M. lutarioriparius genes, homologues of EruDHHC1C and EruDHHC3A. Phylogenetic and classification analyses categorized DHHC family members into six subgroups (A-F). Ka/Ks ratio analysis indicated that gene duplication in these species was primarily driven by whole-genome duplication (WGD) and dispersed duplication (DSD), with DHHC genes evolving under strong purifying selection. Gene expression and trait correlation analysis revealed a significant negative correlation between SspDHHC28A expression in S. spontaneum and sucrose content, suggesting a role in photosynthesis product transport during rapid growth. This study deepens our understanding of the DHHC gene family’s functional dynamics and evolutionary path in Saccharinae, laying a foundation for future research.

Muscle and Metabolic Genes Are Differentially Expressed During Thermal Acclimation by the Brook Trout Myotome.

Cold-water fishes, such as Brook trout (Salvelinus fontinalis), are being challenged by the consequences of climate change. The ability of these fish to acclimate to warmer environmental conditions is vital to their survival. Acclimation to warmer water may allow brook trout to reduce the metabolic costs of higher temperatures. Previous work has shown that brook trout display a significant thermal acclimation response in their myotomal muscle, with slower contractile properties observed in warm acclimated fish. In this study, gene expression was examined in hatchery brook trout acclimated to a range in temperatures (4, 10 or 20°C). Brook trout displayed variations in gene expression in their myotomal muscle in accordance with acclimation temperature. Genes important for muscle function, cellular metabolism, protein degradation, and stress response showed variation to both warm (20°C) and cold (4°C) acclimation. The warm acclimated fish also showed decreased expression of genes associated with aerobic metabolism and increased expression of genes for heat shock proteins, while the cold acclimated fish showed increased expression of genes associated with lipid metabolism and protein turnover. α-tubulin displayed a close association with thermal acclimation, increasing in expression with acclimation temperature. The patterns of muscle gene expression were the opposite of what was expected. Although warm acclimated fish have previously been shown to display slow muscle contractile properties, this study found that warm acclimation is associated with increased expression of genes for kinetically faster isoforms of important muscle proteins. Collectively, the results demonstrate a robust response to elevated temperature in the hatchery fish greater than 10,000 genes showing differential expression with temperature. These results provide a roadmap for the analysis of the acclimation response of native populations of brook trout encountering climate change.

The immune-related gene CD5 is a prognostic biomarker associated with the tumor microenvironment of breast cancer.

The occurrence and progression of breast cancer (BCa) are complex processes involving multiple factors and multiple steps. The tumor microenvironment (TME) plays an important role in this process, but the functions of immune components and stromal components in the TME require further elucidation. In this study, we obtained the RNA-seq data of 1086 patients from The Cancer Genome Atlas (TCGA) database. We calculated the proportions of tumor-infiltrating immune cells (TICs) and immune and stromal components using the CIBERSORT and ESTIMATE methods, and we screened differentially expressedgenes (DEGs). Univariate Cox regression analysis of overall survival was performed on the DEGs, and a protein-protein interaction network of their protein products was generated. Finally, the hub gene CD5 was obtained. High CD5 expression was found to be associated with longer survival than low expression. Gene set enrichment analysis showed that DEGs upregulated in the high-CD5 expression group were mainly enriched in tumor- and immune-related pathways, while those upregulated in the low-expression group were enriched in protein export and lipid synthesis. TIC analysis showed that CD5 expression was positively correlated with the infiltration of CD8+ T cells, activated memory CD4+ T cells, gamma delta T cells, and M1 macrophages and negatively correlated with the infiltration of M2 macrophages. CD5 can increase anticancer immune cell infiltration and reduce M2 macrophage infiltration. These results suggest that CD5 is likely a potential prognostic biomarker and therapeutic target, providing novel insights into the treatment and prognostic assessment of BCa.

Inhibited peroxidase activity of peroxiredoxin 1 by palmitic acid exacerbates nonalcoholic steatohepatitis in male mice

Reactive oxygen species exacerbate nonalcoholic steatohepatitis (NASH) by oxidizing macromolecules; yet how they promote NASH remains poorly understood. Here, we show that peroxidase activity of global hepatic peroxiredoxin (PRDX) is significantly decreased in NASH, and palmitic acid (PA) binds to PRDX1 and inhibits its peroxidase activity. Using three genetic models, we demonstrate that hepatic PRDX1 protects against NASH in male mice. Mechanistically, PRDX1 suppresses STAT signaling and protects mitochondrial function by scavenging hydrogen peroxide, and mitigating the oxidation of protein tyrosine phosphatases and lipid peroxidation. We further identify rosmarinic acid (RA) as a potent agonist of PRDX1. As revealed by the complex crystal structure, RA binds to PRDX1 and stabilizes its peroxidatic cysteine. RA alleviates NASH through specifically activating PRDX1’s peroxidase activity. Thus, beyond revealing the molecular mechanism underlying PA promoting oxidative stress and NASH, our study suggests that boosting PRDX1’s peroxidase activity is a promising intervention for treating NASH.

Structural Dynamics of the Slide Helix of Inactive/Closed Conformation of KirBac1.1 in Micelles and Membranes: A Fluorescence Approach

Inward rectifying potassium (Kir) channels play a critical role in maintaining the resting membrane potential and cellular homeostasis. The high-resolution crystal structure of homotetrameric KirBac1.1 in detergent micelles provides a snapshot of the closed state. Similar to micelles, KirBac1.1 is reported to be in the inactive/closed conformation in POPC membranes. The slide helix of KirBac1.1 is an important structural motif that regulates channel gating. Despite the importance of slide helix in lipid-dependent gating, conflicting models have emerged for the location of slide helix and its structural dynamics in membrane mimetics is poorly understood. Here, we monitored the structural dynamics of the slide helix (residues 46–57) of KirBac1.1 in both DM micelles and POPC membranes utilizing various site-directed fluorescence approaches. We show, using ACMA-based liposome-flux assay, the cysteine mutants of the slide helix are not functional, ensuring the inactive/closed conformation in POPC membranes similar to wild-type channel. Time-resolved fluorescence and water accessibility measurements of NBD-labeled single-cysteine mutants of slide-helix residues suggest that the location of the slide helix at the interfacial region might be shallower in membranes compared to micelles. Interestingly, the slide helix of KirBac1.1 is more dynamic in the physiologically relevant membrane environment, which is accompanied by a differential hydration dynamics throughout the slide helix. Further, REES and lifetime distribution analyses suggest significant changes in conformational heterogeneity of the slide helix in membrane mimetics. Overall, our results give an insight into how membrane mimetics affect the organization and dynamics of slide helix of the closed state of KirBac1.1, and highlight the importance of lipid–protein interactions in membranes.Graphical abstract

Bisphenol S Induces Lipid Metabolism Disorders in HepG2 and SK-Hep-1 Cells via Oxidative Stress.

Bisphenol S (BPS) is a typical endocrine disruptor associated with obesity. To observe BPS effects on lipid metabolism in HepG2 and SK-Hep-1 human HCC cells, a CCK-8 assay was used to assess cell proliferation in response to BPS, and the optimal concentration of BPS was selected. Biochemical indices such as triglyceride (TG) and total cholesterol (T-CHO), and oxidative stress indices such as malondialdehyde (MDA) and catalase (CAT) were measured. ROS and MDA levels were significantly increased after BPS treatment for 24 h and 48 h (p < 0.05), indicating an oxidative stress response. Alanine aminotransferase (ALT), T-CHO, and low-density lipoprotein cholesterol (LDL-C) levels also increased significantly after 24 or 48 h BPS treatments (p < 0.05). RT-PCR and Western blot analyses detected mRNA or protein expression levels of peroxisome proliferator-activated receptor α (PPARα) and sterol regulatory element-binding protein 1c (SREBP1C). The results indicated that BPS could inhibit the mRNA expression of PPARα and carnitine palmitoyl transferase 1B (CPT1B), reduce lipid metabolism, promote mRNA or protein expression of SREBP1C and fatty acid synthase (FASN), and increase lipid synthesis. Increased lipid droplets were observed using morphological Oil Red O staining. Our study demonstrates that BPS may cause lipid accumulation by increasing oxidative stress and perturbing cellular lipid metabolism.

Lipoprotein Signal Peptide as Adjuvants: Leveraging Lipobox-Driven TLR2 Activation in Modern Vaccine Design.

Toll-like receptor 2 (TLR2) signaling is a pivotal component of immune system activation, and it is closely linked to the lipidation of bacterial proteins. This lipidation is guided by bacterial signal peptides (SPs), which ensure the precise targeting and membrane anchoring of these proteins. The lipidation process is essential for TLR2 recognition and the activation of robust immune responses, positioning lipidated bacterial proteins as potent immunomodulators and adjuvants for vaccines against bacterial-, viral-, and cancer-related antigens. The structural diversity and cleavage pathways of bacterial SPs are critical in determining lipidation efficiency and protein localization, influencing their immunogenic potential. Recent advances in bioinformatics have significantly improved the prediction of SP structures and cleavage sites, facilitating the rational design of recombinant lipoproteins optimized for immune activation. Moreover, the use of SP-containing lipobox motifs, as adjuvants to lipidate heterologous proteins, has expanded the potential of vaccines targeting a broad range of pathogens. However, challenges persist in expressing lipidated proteins, particularly within heterologous systems. These challenges can be addressed by optimizing expression systems, such as engineering E. coli strains for enhanced lipidation. Thus, lipoprotein signal peptides (SPs) demonstrate remarkable versatility as adjuvants in vaccine development, diagnostics, and immune therapeutics, highlighting their essential role in advancing immune-based strategies to combat diverse pathogens.

Role of protein and lipid oxidation in hardening of high-protein bars during storage.

Protein bar hardening negatively impacts shelf life, quality, and consumer acceptance. Although oxidation is known to negatively affect the flavor and texture of foods, the specific roles of lipid and protein oxidation in bar hardening have not been thoroughly investigated. Furthermore, most research has concentrated on dairy proteins, with a notable lack of studies addressing the hardening of plant-based protein bars. We investigated the role of protein and lipid oxidation, Maillard reactions, moisture loss, protein aggregation, and microstructural changes in the hardening of pea, whey, and rice protein bars over a storage period of 6weeks (hardness increased 7.2×, 5.4×, and 4.4×, respectively). Changes in tryptophan fluorescence, free sulfhydryl content (e.g., loss of 57% for pea and 44% for whey), and carbonyl content demonstrated that pea and whey bars underwent protein oxidation. Lipid oxidation also occurred, demonstrated by increased peroxide and thiobarbituric acid-reactive substance values. Rice bars, however, did not undergo oxidation. Mass spectrometry indicated greater Maillard-reaction-related protein glycations formed in pea and whey bars (6.9% and 7.7%, respectively) than in rice bars (2.1%). SDS-PAGE revealed that pea and whey, but not rice, proteins aggregated during storage. Overall, this study found that moisture loss, protein and lipid oxidation, Maillard reactions, and protein aggregation correlated with bar hardening. Chemical changes may cause protein aggregation, resulting in hardening. Likely because of rice proteins' innate insolubility and disulfide linkages, rice protein bars were less susceptible to chemical changes and aggregation and hardened more slowly than whey and pea protein bars. PRACTICAL APPLICATION: This study shows that lipid and protein oxidation are correlated with protein bar hardening in both pea and whey protein bars. Additionally, this work suggests that rice protein bars may harden more slowly than pea and whey bars. These findings suggest that potential strategies to prevent bar hardening and extend shelf life include (1) adding antioxidants to prevent oxidation and (2) using rice proteins to partially or fully substitute other protein isolates.

Spectroscopic Methods for Detecting Conformational Changes During Sec18-Lipid Interactions.

Vacuole fusion is driven by SNARE proteins that require activation-or priming-by the AAA+ protein Sec18 (NSF) before they can bring membranes together and trigger the merger of two bilayers into a continuous membrane. Sec18 resides on vacuoles prior to engaging inactive cis-SNARE complexes through its interaction with the regulatory lipid phosphatidic acid (PA). Binding PA causes Sec18 to undergo large conformational changes that keeps it bound to the membrane, thus precluding its interactions with SNAREs. Such conformational changes can be measured by various biochemical and biophysical assays. The conversion of PA to diacylglycerol by the PA phosphatase Pah1 releases Sec18 from the membrane-bound pool and promotes its transfer to SNARE complexes allowing priming to occur. Here we describe four spectroscopy-based methods to distinguish conformational changes from alterations in secondary structure during PA binding. These methods only require purified protein and short chain soluble lipids, making the methods rapid and affordable ways to screen the effects of specific protein-lipid interactions. The assays described in this chapter include 1-anilino-8-naphthalenesulfonate (ANS) spectroscopy and intrinsic tryptophan fluorescence to detect exposure of hydrophobic regions; differential scanning fluorometry to measure changes in protein stability across a temperature gradient; and circular dichroism to examine changes in secondary structure.

Cooking‐Induced Lipid–Protein Oxidation in Kavurma (a Cooked Meat Product)

Optimizing the nutritional quality of cooked meat requires a better understanding of the mechanisms responsible for lipid–protein changes caused by cooking. In this study, the aim was to determine the levels of protein–lipid oxidation that could occur during the kavurma production process. For this purpose, cooking was performed at 100°C for 30, 60, 90, and 120 min, and protein oxidation was determined by measuring carbonyl content, sulfhydryl (S‐H) group levels, and Schiff base formation, while lipid oxidation was assessed by measuring thiobarbituric acid reactive substances (TBARS), peroxide, p‐anisidine, and free fatty acid (FFA) values. Also, the changes in protein structure were identified by Fourier‐transform infrared spectroscopy (FT‐IR). Carbonyl, formation of Schiff bases, peroxide, p‐anisidine, and FFA values significantly increased throughout the cooking time (p &lt; 0.05). TBARS values showed a significant increase within the first 30 min, followed by a decrease. The level of S‐H groups significantly decreased with increasing cooking time (p &lt; 0.05). FT‐IR analysis of the myofibrillar extract revealed 13 major peaks, with peak areas decreasing depending on the cooking time. Analysis of the secondary structure indicated higher relative intensities of α‐helix and random coil up to 90 min of cooking time. Results indicate that protein–lipid oxidation is dependent on the cooking time, which point out to an effect on the nutritional quality of proteins and lipids in kavurma. Also, these results not only enhance our comprehension of the complex relationship between cooking time and product quality but also present encouraging prospects for fostering healthier and safer cooking methods.

Mechanisms coupling the mTOR pathway to chronic obstructive pulmonary disease (COPD) pathogenesis.

Chronic Obstructive Pulmonary Disease (COPD) is a poorly reversible respiratory disorder distinguished by dyspnea, cough, expectoration and exacerbations due to abnormality of airways or emphysema. In this review, we consider the therapeutic potential of targeting Mammalian target of Rapamycin (mTOR) for treating COPD. The mTOR is a highly conserved serine-threonine protein kinase that integrates signals from growth factors and nutrients to control protein synthesis, lipid biogenesis and metabolism. Dysregulated mTOR pathway signaling due to genetic factors or cigarette smoking impairs autophagy, driving the buildup of abnormal cells and damaged proteins, resulting in inflammation and oxidative stress. Persistent mTOR activation also contributes to pulmonary vascular cell proliferation, facilitating the development of pulmonary resistance in COPD. Rapamycin, an inhibitor of mTOR, prevents the buildup of senescent cells in the lungs of COPD patients and inhibits the release of lung tissue-damaging proteases. mTOR also impacts the corticosteroid sensitivity in COPD patients by regulating the levels of histone deacetylases. The emerging role of gut-lung axis dysbiosis in the progression of COPD and its influence on mTOR further highlights the relevance of the mTOR pathway in COPD pathophysiology.