Bioscience Reports最新文献

Proteins of the ATP-binding cassette (ABC) transporter superfamily are involved in diverse biological processes including multidrug resistance. As membrane proteins, they exist within a complex lipid environment, and often it is necessary to isolate them from the other membrane components to study their structure, function, and dynamics. Traditionally, detergents have been used to isolate the transporters into micelles but this can strip away lipids that may be essential for function. Polymers such as styrene maleic acid (SMA) offer attractive alternatives to detergents as they retain the protein and lipids in a nanoscale disc. However, to date, no demonstration of full ABC transporter activity in these discs has been achieved, possibly due to the inherent divalent cation sensitivity of the SMA polymers; magnesium is essential for ATP binding to ABC transporters. Novel polymers such as those based on acrylic acid styrene (AASTY) show decreased sensitivity to divalent cations and, as such, may be well placed to probe ABC transporter activity. We have demonstrated that a range of commercially available AASTY polymers solubilise biological membranes efficiently, albeit with slightly different kinetics. ABC transporters can be solubilised and purified using AASTY polymers into discs of a comparable size to those formed by SMA2000. These discs show increased magnesium tolerance but, as for SMA2000, lipids within them do not seem to undergo a full phase transition. We were unable to detect ATPase activity of ABC transporters in AASTY polymer lipid particles, suggesting that magnesium tolerance alone is not sufficient to overcome the challenges.

The nematode Caenorhabditis elegans biosynthesizes the ascarosides, a large, modular family of pheromones that are used in chemical communication. A number of carboxylesterase domain-containing (CEST) enzymes are responsible for decorating the glycolipid core of the ascarosides with a variety of modifications. However, these enzymes, which are homologous to human carboxylesterases and acetylcholinesterase, have not been characterized biochemically, and thus the mechanism whereby they attach different modifications to the ascarosides is unknown. Here, we report the expression, purification, and biochemical characterization of a soluble CEST enzyme for the first time. In this study, we focused on CEST-9.2, which is responsible for making (E)-2-methyl-2-butenoyl (MB)-modified ascarosides. We identified candidate substrates for the enzyme, and we successfully expressed a truncated version of CEST-9.2, which is lacking the transmembrane domain, in several expression systems, including Escherichia coli, Pichia pastoris, and Spodoptera frugiperda Sf9 cells. The purified CEST-9.2 from each of these systems was tested against candidate substrates, including ascarosides and either MB-coenzyme A (CoA), MB-choline, or MB-carnitine. No enzymatic activity was detected using these substrates, suggesting that either the transmembrane domain is necessary for activity or that the correct substrates have not yet been identified. We showed that the purified CEST-9.2 from Sf9 cells is well-folded and dimeric, offering a potential starting point for future structural and mechanistic studies.

Hyperhomocysteinemia is a risk factor of cardiovascular disease (CVD). High-density lipoprotein (HDL) plays an important role in anti-atherosclerosis, with its anti-atherogenic function attributed to HDL-associated proteins such as apolipoprotein A-I (apoA-I) and paraoxonase 1 (PON1). Homocysteine (Hcy) thiolactone modifies lysine residues in proteins, thereby altering their function. Although dysfunction of apoA-I and PON1 has been reported, the precise modification sites and underlying mechanisms have remained unclear. In this study, we aimed to identify Hcy-thiolactone modification sites on apoA-I and PON1. In addition, we sought to clarify the effects of Hcy-thiolactone on PON1 activity and its distribution. Modification sites were analyzed using MALDI-TOF MS. The effects of Hcy-thiolactone on various specimens, including purified proteins, reconstituted HDL (rHDL), HDL collected by ultracentrifugation, and serum samples, were characterized using enzymatic assays measuring three major PON1 activities (arylesterase, paraoxonase, and lactonase) and Western blotting. Our results demonstrated that while some Hcy-thiolactone modification sites were detected on apoA-I, PON1 itself was not directly modified by Hcy-thiolactone. Thiolactonase activity was reduced by Hcy-thiolactone in large HDL particles. Furthermore, a general reduction of PON1 activity and changes in HDL remodeling and distribution were observed in serum samples treated with Hcy-thiolactone. These findings suggest that PON1 dysfunction induced by Hcy-thiolactone is influenced by alterations in HDL remodeling and the enzyme's distribution on HDL particles. Analysis of PON1's distribution dynamics under pathological conditions may provide crucial insights into the mechanism of HDL function decline in CVD.

Mitral valve disease (MVD) is the most common valvulopathy and a frequent cause of heart failure and death. However, data regarding the molecular basis are scarce. We aimed to thoroughly explore and compare the circulating, molecular, and histopathological profiles in the main subtypes of primary chronic MVD. In total, 300 patients with chronic primary MVD undergoing mitral valve (MV) replacement were enrolled and classified in the four main etiologic subtypes: calcific mitral valve disease (CMVD, n = 81), rheumatic disease (RHVD, n = 114), Barlow's disease (BD, n = 70), and fibroelastic deficiency (FED, n = 35). Discovery studies were performed using Olink Proteomics® technology in 80 serums from MVD patients (n = 20/etiology). Histopathologic study, ELISA, and zymography were performed on resected MVs to analyze extracellular matrix (ECM) composition and remodeling, inflammation, and calcification. Serum proteomics identified markers exclusively overexpressed in each MVD etiologic subtype. Further enrichment analyses revealed specific etiology-dependent pathways involving calcification, inflammation, or ECM remodeling. Such etiology-dependent differences were mirrored in the excised MVs both architecturally and compositionally. CMVD and RHVD valves had a marked ECM disorganization with increased collagen deposition and presence of nodular and diffuse calcification, respectively. Moreover, RHVD valves exhibited high levels of tumor necrosis factor (TNF)-α, transforming growth factor (TGF)-β, and matrix degradation enzymes. BD valves showed enhanced inflammatory markers and infiltrates. Moreover, BD and FED valves presented marked proteoglycans deposition. Our study identified circulating markers involved in calcification, inflammation, and ECM remodeling, which may be associated with specific MVD etiologies. The differences in serum markers seem to mirror histopathologic and molecular alterations in the MVs with potential applications into the clinic as diagnosis biomarkers. Identification of underlying molecular mechanisms of each etiology is essential to discover new specific therapeutic targets.

The maintenance of proteostasis is essential for cellular function and organismal health. Its decline with age is a key contributor to neurodegenerative diseases, metabolic disorders, and other chronic conditions. Eukaryotic cells respond to proteotoxic stress through compartment-specific pathways, including the heat shock response (HSR), the unfolded protein response of the endoplasmic reticulum (UPRER), and the mitochondrial UPR (UPRmt). While these pathways have been extensively studied in cell-autonomous contexts, recent evidence reveals that neurons and glial cells can co-ordinate these responses across tissues through cell-non-autonomous mechanisms. Neuronal signals, including neuropeptides, biogenic amines, and possibly extracellular vesicles, can activate stress responses in distal cells, modulating lipid metabolism and impacting longevity. Emerging data also suggest a role for glial cells in systemic proteostasis regulation, though their mechanisms remain relatively uncharacterized. This review discusses both classical and emerging concepts of proteostasis stress-response pathways, their integration with neural signaling, and how their modulation influences aging and disease. Understanding how intercellular communication governs proteostasis could open new avenues for therapeutic interventions in age-related and neurodegenerative disorders.

Persistent intestinal inflammation and gut dysbiosis are key drivers of the progression from inflammatory bowel disease (IBD) to colitis-associated colorectal cancer (CAC). Eggshell membrane (ESM) has shown beneficial effects in alleviating IBD symptoms; however, its impact on inflammation-driven CAC remains unclear. This study investigated the effects of dietary ESM supplementation in an azoxymethane/dextran sulfate sodium (AOM/DSS)-induced CAC mouse model. Mice were fed either a control or ESM-supplemented diet for one month prior to CAC induction and continuing throughout the experiment. ESM supplementation significantly improved survival rates in AOM/DSS-induced CAC mice and reduced colitis severity, indicated by the down-regulation of the pro-inflammatory cytokines and M1-like macrophage markers in the colonic mucosa. In addition, ESM conferred systemic protective effects by alleviating liver inflammation and tissue damage, as evidenced by preserved hepatic structure and reduced immune cell infiltration. Importantly, ESM significantly restored cecal microbiota composition disrupted by AOM/DSS, reducing pathogenic bacteria such as Escherichia-Shigella, while enriching beneficial bacteria such as Firmicutes and Muribaculaceae. These compositional shifts were accompanied by predicted correction of dysregulated microbial metabolic pathways, reflecting a restoration of microbial functional balance. Overall, these findings demonstrate that ESM alleviates multi-organ inflammation and restores microbial balance in CAC, highlighting its potential as a dietary strategy to mitigate chronic inflammation and its systemic consequences.

Excessive hypercholesterolemia in pregnancy is a risk factor for the development of preeclampsia, but its ensuing impact on offspring cardiovascular health remains not fully understood. In the present study, Sprague Dawley rats were fed a control or high cholesterol diet (HCD) from gestational days 6-20 (term=22 days). Female and male offspring were aged to adulthood (4 months old), and in vivo and ex vivo cardiovascular function were assessed. In female offspring, body weight gain was greater in the HCD group compared with controls after 2 months of age. Blood pressure, echocardiographic parameters, cardiac capacity to recover from an ischemic insult, and endothelium-dependent vasodilation were similar between the female groups. However, vasoconstriction to a thromboxane analog (U46619) was reduced in thoracic aortas, but not in mesenteric, coronary, or carotid arteries, from the HCD females compared with controls, and was associated with lower phosphorylated myosin phosphatase target subunit 1 (MYPT1Thr855) levels. In male offspring, body weight gain, blood pressure, and echocardiographic parameters were similar between the groups, and there was no impact of HCD on vasoconstriction to U46619 or vasodilation in mesenteric arteries, carotid arteries, or thoracic aortas. However, endothelium-dependent vasodilation was reduced in coronary arteries of HCD males compared with controls and was associated with increased reactive oxygen species levels. There was also reduced cardiac capacity of the HCD males to recover from an ischemic insult. In summary, excessive hypercholesterolemia in pregnancy impaired the cardiovascular health of adult female and male offspring, but the mechanisms and vascular beds affected were specific to each sex.

Venous thromboembolism (VTE) is one of the most frequent and serious complications in cancer patients, contributing significantly to morbidity, mortality, and increased healthcare burden. Circulating tumor cells (CTCs) and circulating tumor microemboli (CTMs), components of the liquid biopsy, have emerged not only as biomarkers of disease progression and therapeutic resistance but also as potential contributors to prothrombotic states in oncologic patients. In this review, we explore the biological and clinical relationships between CTCs/CTMs and the development of VTE, highlighting mechanistic insights involving tumor-platelet interactions, immune evasion, and endothelial dysfunction. We also discuss recent findings on the prognostic value of CTCs and CTMs for thromboembolic risk stratification, as well as technological advances in their detection. Understanding the role of these circulating tumor-derived elements may open new perspectives for personalized prevention and management of thromboembolic events in cancer patients.

DExD-box (DDX) proteins are essential for RNA metabolism and are targets for treatment of cancers and neurodevelopmental disorders. The biochemical mechanisms of many DDX proteins remain unclear, including human DDX52. DDX52 is essential for cell survival and is an emerging biomarker for the onset of metastatic melanoma. In this work, we identified that human DDX52 is an ATP-dependent translocase with 3'-5' polarity, which can unwind DNA duplexes and DNA/RNA hybrids. Further, DDX52 is a nucleic acid annealase, an activity that requires an N-terminal intrinsically disordered protein region. DDX52 becomes hyperactive at DNA annealing if DDX52 helicase activity is inactivated by mutagenesis. Using CRISPR-Cas9 genetic editing, we generated U2OS cell lines heterozygous for DDX52 (DDX52+/-), which exhibit growth defects and impaired cell migration, providing direct support for previous suggestions that DDX52 may promote cancer cell metastasis and C-myc regulation.

To identify novel tyrosinase inhibitors, a series of isocryptolepine 'aza' type acyl thiourea analogs (6a-6h) were designed and synthesized using a multistep strategy. Spectroscopic methods including FTIR, UV-vis, 1H NMR, 13C NMR, and EI-MS were utilized for detailed analysis of compounds. Their tyrosinase inhibitory activities were evaluated in vitro, demonstrating superior potency compared with kojic acid (IC50 = 16.83 ± 1.162 μM). The synthesized compounds exhibited IC50 values ranging from 0.832 ± 0.03 to 7.945 ± 0.63 μM, with compound 6g emerging as the most potent inhibitor (IC50 = 0.832 ± 0.03 μM). Kinetic studies revealed competitive inhibition by compound 6g, highlighting its potential as a lead candidate for treating tyrosinase-mediated hyperpigmentation. Additional evaluations showed that these compounds also effectively inhibited other enzymes involved in cancer progression, indicating their broad therapeutic potential. Molecular modeling studies against the tyrosinase enzyme (PDB: 4OUA) confirmed strong binding interactions, while structure-activity relationship analyses provided insights into their inhibitory mechanisms. Geometry optimization of the compounds, supporting their favorable molecular properties. Drug-likeness evaluations further validated the potential of these analogs as promising anti-tyrosinase agents. Overall, this study establishes compound 6g and its analogs as compelling candidates for further development in hyperpigmentation and cancer therapeutics.