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Open AccessReview

The Mechanotransduction Signaling Pathways in the Regulation of Osteogenesis

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Int. J. Mol. Sci. 2023, 24(18), 14326; https://doi.org/10.3390/ijms241814326 - 20 Sep 2023

Viewed by 189

Abstract

Bones are constantly exposed to mechanical forces from both muscles and Earth’s gravity to maintain bone homeostasis by stimulating bone formation. Mechanotransduction transforms external mechanical signals such as force, fluid flow shear, and gravity into intracellular responses to achieve force adaptation. However, the [...] Read more.

Bones are constantly exposed to mechanical forces from both muscles and Earth’s gravity to maintain bone homeostasis by stimulating bone formation. Mechanotransduction transforms external mechanical signals such as force, fluid flow shear, and gravity into intracellular responses to achieve force adaptation. However, the underlying molecular mechanisms on the conversion from mechanical signals into bone formation has not been completely defined yet. In the present review, we provide a comprehensive and systematic description of the mechanotransduction signaling pathways induced by mechanical stimuli during osteogenesis and address the different layers of interconnections between different signaling pathways. Further exploration of mechanotransduction would benefit patients with osteoporosis, including the aging population and postmenopausal women. Full article

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Open AccessArticle

Overexpression of KLF4 Suppresses Pulmonary Fibrosis through the HIF-1α/Endoplasmic Reticulum Stress Signaling Pathway

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Int. J. Mol. Sci. 2023, 24(18), 14008; https://doi.org/10.3390/ijms241814008 - 12 Sep 2023

Viewed by 208

Abstract

The hypoxia-inducible factor-1α/endoplasmic reticulum stress signaling pathway (HIF-1α/ERS) has a crucial role in the pathogenetic mechanism of pulmonary fibrosis (PF). However, the upstream regulatory mediators of this pathway remain unclear. In the present study, by conducting bioinformatics analysis, we found that Krüppel-like factor [...] Read more.

The hypoxia-inducible factor-1α/endoplasmic reticulum stress signaling pathway (HIF-1α/ERS) has a crucial role in the pathogenetic mechanism of pulmonary fibrosis (PF). However, the upstream regulatory mediators of this pathway remain unclear. In the present study, by conducting bioinformatics analysis, we found that Krüppel-like factor 4 (KLF4) expression was decreased in the lung tissues of patients with idiopathic pulmonary fibrosis (IPF) as compared to that in patients with non-IPF. Furthermore, KLF4 expression was significantly reduced (p = 0.0331) in bleomycin-induced fibrotic HFL-1 cells. Moreover, in mice with bleomycin-induced PF, the degree of fibrosis was significantly reduced in mice overexpressing KLF4 as compared to that in wild-type mice. In mice and HFL-1 cells, KLF4 overexpression significantly reduced bleomycin-induced protein expression of HIF-1α (p = 0.0027) and ERS markers, particularly p-IRE1α (p = 0.0255) and ATF6 (p = 0.0002). By using the JASPAR database, we predicted that KLF4 has five binding sites for the HIF-1α promoter. The results of in vitro and in vivo studies suggest that KLF4 may inhibit PF through the HIF-1α/ERS pathway. This finding could guide the development of future therapies for PF and facilitate the identification of appropriate biomarkers for routine clinical diagnosis of PF. Full article

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Open AccessArticle

Improving Geometric Validation Metrics and Ensuring Consistency with Experimental Data through TrioSA: An NMR Refinement Protocol

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Int. J. Mol. Sci. 2023, 24(17), 13337; https://doi.org/10.3390/ijms241713337 - 28 Aug 2023

Viewed by 356

Abstract

Protein model refinement a the crucial step in improving the quality of a predicted protein model. This study presents an NMR refinement protocol called TrioSA (torsion-angle and implicit-solvation-optimized simulated annealing) that improves the accuracy of backbone/side-chain conformations and the overall structural quality of [...] Read more.

Protein model refinement a the crucial step in improving the quality of a predicted protein model. This study presents an NMR refinement protocol called TrioSA (torsion-angle and implicit-solvation-optimized simulated annealing) that improves the accuracy of backbone/side-chain conformations and the overall structural quality of proteins. TrioSA was applied to a subset of 3752 solution NMR protein structures accompanied by experimental NMR data: distance and dihedral angle restraints. We compared the initial NMR structures with the TrioSA-refined structures and found significant improvements in structural quality. In particular, we observed a reduction in both the maximum and number of NOE (nuclear Overhauser effect) violations, indicating better agreement with experimental NMR data. TrioSA improved geometric validation metrics of NMR protein structure, including backbone accuracy and the secondary structure ratio. We evaluated the contribution of each refinement element and found that the torsional angle potential played a significant role in improving the geometric validation metrics. In addition, we investigated protein–ligand docking to determine if TrioSA can improve biological outcomes. TrioSA structures exhibited better binding prediction compared to the initial NMR structures. This study suggests that further development and research in computational refinement methods could improve biomolecular NMR structural determination. Full article

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Open AccessReview

Partial Reversible Inhibition of Enzymes and Its Metabolic and Pharmaco-Toxicological Implications

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Patrick Masson

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Aliya R. Mukhametgalieva

Int. J. Mol. Sci. 2023, 24(16), 12973; https://doi.org/10.3390/ijms241612973 - 19 Aug 2023

Viewed by 419

Abstract

Partial reversible inhibition of enzymes, also called hyperbolic inhibition, is an uncommon mechanism of reversible inhibition, resulting from a productive enzyme–inhibitor complex. This type of inhibition can involve competitive, mixed, non-competitive and uncompetitive inhibitors. While full reversible inhibitors show linear plots for reciprocal [...] Read more.

Partial reversible inhibition of enzymes, also called hyperbolic inhibition, is an uncommon mechanism of reversible inhibition, resulting from a productive enzyme–inhibitor complex. This type of inhibition can involve competitive, mixed, non-competitive and uncompetitive inhibitors. While full reversible inhibitors show linear plots for reciprocal enzyme initial velocity versus inhibitor concentration, partial inhibitors produce hyperbolic plots. Similarly, dose–response curves show residual fractional activity of enzymes at high doses. This article reviews the theory and methods of analysis and discusses the significance of this type of reversible enzyme inhibition in metabolic processes, and its implications in pharmacology and toxicology. Full article

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Open AccessArticle

Branched DNA-Based Electrochemical Biosensor for Sensitive Nucleic Acids Analysis with Gold Nanoparticles as Amplifier

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Int. J. Mol. Sci. 2023, 24(16), 12565; https://doi.org/10.3390/ijms241612565 - 08 Aug 2023

Viewed by 357

Abstract

A branched DNA-based electrochemical biosensor was designed to sensitively detect specific nucleic acids. On this platform, novel a branched DNA with three sticky ends could be used as a biosensor to sensitively and specifically detect nucleic acids. Meanwhile, we also employed branched DNA-modified [...] Read more.

A branched DNA-based electrochemical biosensor was designed to sensitively detect specific nucleic acids. On this platform, novel a branched DNA with three sticky ends could be used as a biosensor to sensitively and specifically detect nucleic acids. Meanwhile, we also employed branched DNA-modified AuNPs as a signal amplifier to further improve the sensitivity. Branched DNA sensors, target DNA, and DNA-modified AuNPs formed a sandwich structure to produce an electronic signal for target DNA detection. The reaction primarily involved DNA hybridization without bulky thermal cyclers and enzymes. We proved that the hybridization reaction easily occurred under different conditions, such as the NaCl concentration, reaction time, pH, and temperature, except for a pH lower than 4. The limit of detection could go as low as 0.09 pM (S/N = 3) with excellent specificity and selectivity. There was a correlation curve relationship between the peak current and the logarithm of the target DNA concentration (0.10 pM to 10 nM). The correlation coefficient reached 0.987. The electrochemical platform enables a branched DNA nanostructure to determine nucleic acids for disease diagnosis. Full article

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Open AccessArticle

Fighting Celiac Disease: Improvement of pH Stability of Cathepsin L In Vitro by Computational Design

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Valeria F. Tereshchenkova

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Irina Yu. Filippova

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Roman G. Efremov

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Elena N. Elpidina

Int. J. Mol. Sci. 2023, 24(15), 12369; https://doi.org/10.3390/ijms241512369 - 02 Aug 2023

Viewed by 404

Abstract

Roughly 1% of the global population is susceptible to celiac disease (CD)—inheritable autoimmune inflammation of the small intestine caused by intolerance to gliadin proteins present in wheat, rye, and barley grains, and called gluten in wheat. Classical treatment is a life-long gluten-free diet, [...] Read more.

Roughly 1% of the global population is susceptible to celiac disease (CD)—inheritable autoimmune inflammation of the small intestine caused by intolerance to gliadin proteins present in wheat, rye, and barley grains, and called gluten in wheat. Classical treatment is a life-long gluten-free diet, which is constraining and costly. An alternative approach is based upon the development and oral reception of effective peptidases that degrade in the stomach immunogenic proline- and glutamine-rich gliadin peptides, which are the cause of the severe reaction in the intestine. In previous research, we have established that the major digestive peptidase of an insect Tribolium castaneum—cathepsin L—hydrolyzes immunogenic prolamins after Gln residues but is unstable in the extremely acidic environment (pH 2–4) of the human stomach and cannot be used as a digestive aid. In this work, using molecular dynamics simulations, we discover the probable cause of the pH instability of cathepsin L—loss of the catalytically competent rotameric state of one of the active site residues, His 275. To “fix” the correct orientation of this residue, we designed a V277A mutant variant, which extends the range of stability of the peptidase in the acidic environment while retaining most of its activity. We suggest this protein as a lead glutenase for the development of oral medical preparation that fights CD and gluten intolerance in susceptible people. Full article

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Open AccessArticle

Oxidative Stress and Antioxidant Defense in the Brain of Bat Species with Different Feeding Habits

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Pabulo Henrique Rampelotto

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Nikolas Raphael Oliveira Giannakos

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Diego Antonio Mena Canata

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Francielly Dias Pereira

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Fernanda Schäfer Hackenhaar

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María João Ramos Pereira

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Mara Silveira Benfato

Int. J. Mol. Sci. 2023, 24(15), 12162; https://doi.org/10.3390/ijms241512162 - 29 Jul 2023

Cited by 1 | Viewed by 351

Abstract

Assessing the levels of oxidative stress markers and antioxidant enzymes in the brain is crucial in evaluating its antioxidant capacity and understanding the influence of various dietary patterns on brain well-being. This study aimed to investigate the antioxidant status and oxidative damage in [...] Read more.

Assessing the levels of oxidative stress markers and antioxidant enzymes in the brain is crucial in evaluating its antioxidant capacity and understanding the influence of various dietary patterns on brain well-being. This study aimed to investigate the antioxidant status and oxidative damage in the brain of bat species with different feeding habits to gain insights into their protective mechanisms against oxidative stress and their interspecific variation. The levels of oxidative damage markers and the activities of antioxidants were measured in the brain of four bat species with different feeding habits, namely insectivorous, frugivorous, nectarivorous, and hematophagous. Insectivorous bats showed higher levels of SOD and fumarase compared to the other groups, while hematophagous bats showed lower levels of these enzymes. On the other hand, the activities of glutathione peroxidase and glutathione S-transferase were higher in hematophagous bats and lower in insectivorous bats. The carbonyl groups and malondialdehyde levels were lower in frugivores, while they were similar in the other feeding guilds. Nitrite and nitrate levels were higher in the hematophagous group and relatively lower in all other groups. The GSSG/GSH ratio was higher in the hematophagous group and lower in frugivores. Overall, our results indicate that the levels of oxidative stress markers and the activities of antioxidant enzymes in the brain vary significantly among bat species with different feeding habitats. The findings suggest that the antioxidant status of the brain is influenced by diet and feeding habits. Full article

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Open AccessArticle

Structural Insight into the Amino Acid Environment of the Two-Domain Laccase’s Trinuclear Copper Cluster

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Ilya Kolyadenko

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Svetlana Tishchenko

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Azat Gabdulkhakov

Int. J. Mol. Sci. 2023, 24(15), 11909; https://doi.org/10.3390/ijms241511909 - 25 Jul 2023

Viewed by 455

Abstract

Laccases are industrially relevant enzymes. However, their range of applications is limited by their functioning and stability. Most of the currently known laccases function in acidic conditions at temperatures below 60 °C, but two-domain laccases (2D) oxidize some substrates in alkaline conditions and [...] Read more.

Laccases are industrially relevant enzymes. However, their range of applications is limited by their functioning and stability. Most of the currently known laccases function in acidic conditions at temperatures below 60 °C, but two-domain laccases (2D) oxidize some substrates in alkaline conditions and above 70 °C. In this study, we aim to establish the structural factors affecting the alkaline activity of the 2D laccase from Streptomyces griseoflavus (SgfSL). The range of methods used allowed us to show that the alkaline activity of SgfSL is influenced by the polar residues located close to the trinuclear center (TNC). Structural and functional studies of the SgfSL mutants Met199Ala/Asp268Asn and Met199Gly/Asp268Asn revealed that the substitution Asp268Asn (11 Å from the TNC) affects the orientation of the Asn261 (the second coordination sphere of the TNC), resulting in hydrogen-bond-network reorganization, which leads to a change in the SgfSL-activity pH profile. The combination of the Met199Gly/Arg240His and Asp268Asn substitutions increased the efficiency (k_cat/K_M) of the 2,6-DMP oxidation by 34-fold compared with the SgfSL. Our results extend the knowledge about the structure and functioning of 2D laccases’ TNC active sites and open up new possibilities for the directed engineering of laccases. Full article

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Open AccessReview

Exploring the Communication of the SASP: Dynamic, Interactive, and Adaptive Effects on the Microenvironment

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Florence Debacq-Chainiaux

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Olivier Pluquet

Int. J. Mol. Sci. 2023, 24(13), 10788; https://doi.org/10.3390/ijms241310788 - 28 Jun 2023

Viewed by 1013

Abstract

Cellular senescence is a complex cell state that can occur during physiological ageing or after exposure to stress signals, regardless of age. It is a dynamic process that continuously evolves in a context-dependent manner. Senescent cells interact with their microenvironment by producing a [...] Read more.

Cellular senescence is a complex cell state that can occur during physiological ageing or after exposure to stress signals, regardless of age. It is a dynamic process that continuously evolves in a context-dependent manner. Senescent cells interact with their microenvironment by producing a heterogenous and plastic secretome referred to as the senescence-associated secretory phenotype (SASP). Hence, understanding the cross-talk between SASP and the microenvironment can be challenging due to the complexity of signal exchanges. In this review, we first aim to update the definition of senescence and its associated biomarkers from its discovery to the present day. We detail the regulatory mechanisms involved in the expression of SASP at multiple levels and develop how SASP can orchestrate microenvironment modifications, by focusing on extracellular matrix modifications, neighboring cells’ fate, and intercellular communications. We present hypotheses on how these microenvironmental events may affect dynamic changes in SASP composition in return. Finally, we discuss the various existing approaches to targeting SASP and clarify what is currently known about the biological effects of these modified SASPs on the cellular environment. Full article

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Open AccessArticle

The Effect of Prohibitins on Mitochondrial Function during Octopus tankahkeei Spermiogenesis

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Int. J. Mol. Sci. 2023, 24(12), 10030; https://doi.org/10.3390/ijms241210030 - 12 Jun 2023

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Abstract

Mitochondria are essential for spermiogenesis. Prohibitins (PHBs; prohibitin 1, PHB1 or PHB, and prohibitin 2, PHB2) are evolutionarily conserved and ubiquitously expressed mitochondrial proteins that act as scaffolds in the inner mitochondrial membrane. In this study, we analyzed the molecular structure and dynamic [...] Read more.

Mitochondria are essential for spermiogenesis. Prohibitins (PHBs; prohibitin 1, PHB1 or PHB, and prohibitin 2, PHB2) are evolutionarily conserved and ubiquitously expressed mitochondrial proteins that act as scaffolds in the inner mitochondrial membrane. In this study, we analyzed the molecular structure and dynamic expression characteristics of Ot-PHBs, observed the colocalization of Ot-PHB1 with mitochondria and polyubiquitin, and studied the effect of phb1 knockdown on mitochondrial DNA (mtDNA) content, reactive oxygen species (ROS) levels, and apoptosis-related gene expression in spermatids. Our aim was to explore the effect of Ot-PHBs on mitochondrial function during the spermiogenesis of Octopus tankahkeei (O. tankahkeei), an economically important species in China. The predicted Ot-PHB1/PHB2 proteins contained an N-terminal transmembrane, a stomatin/prohibitin/flotillin/HflK/C (SPFH) domain (also known as the prohibitin domain), and a C-terminal coiled-coil domain. Ot-phb1/phb2 mRNA were widely expressed in the different tissues, with elevated expression in the testis. Further, Ot-PHB1 and Ot-PHB2 were highly colocalized, suggesting that they may function primarily as an Ot-PHB compiex in O. tankahkeei. Ot-PHB1 proteins were mainly expressed and localized in mitochondria during spermiogenesis, implying that their function may be localized to the mitochondria. In addition, Ot-PHB1 was colocalized with polyubiquitin during spermiogenesis, suggesting that it may be a polyubiquitin substrate that regulates mitochondrial ubiquitination during spermiogenesis to ensure mitochondrial quality. To further investigate the effect of Ot-PHBs on mitochondrial function, we knocked down Ot-phb1 and observed a decrease in mtDNA content, along with increases in ROS levels and the expressions of mitochondria-induced apoptosis-related genes bax, bcl2, and caspase-3 mRNA. These findings indicate that PHBs might influence mitochondrial function by maintaining mtDNA content and stabilizing ROS levels; in addition, PHBs might affect spermatocyte survival by regulating mitochondria-induced apoptosis during spermiogenesis in O. tankahkeei. Full article

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Open AccessArticle

Cystathionine γ-Lyase Self-Inactivates by Polysulfidation during Cystine Metabolism

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Int. J. Mol. Sci. 2023, 24(12), 9982; https://doi.org/10.3390/ijms24129982 - 10 Jun 2023

Viewed by 675

Abstract

Cystathionine γ-lyase (CSE) is an enzyme responsible for the biosynthesis of cysteine from cystathionine in the final step of the transsulfuration pathway. It also has β-lyase activity toward cystine, generating cysteine persulfide (Cys-SSH). The chemical reactivity of Cys-SSH is thought to be involved [...] Read more.

Cystathionine γ-lyase (CSE) is an enzyme responsible for the biosynthesis of cysteine from cystathionine in the final step of the transsulfuration pathway. It also has β-lyase activity toward cystine, generating cysteine persulfide (Cys-SSH). The chemical reactivity of Cys-SSH is thought to be involved in the catalytic activity of particular proteins via protein polysulfidation, the formation of -S-(S)n-H on their reactive cysteine residues. The Cys136/171 residues of CSE have been proposed to be redox-sensitive residues. Herein, we investigated whether CSE polysulfidation occurs at Cys136/171 during cystine metabolism. Transfection of wild-type CSE into COS-7 cells resulted in increased intracellular Cys-SSH production, which was significantly increased when Cys136Val or Cys136/171Val CSE mutants were transfected, instead of the wild-type enzyme. A biotin-polyethylene glycol-conjugated maleimide capture assay revealed that CSE polysulfidation occurs at Cys136 during cystine metabolism. In vitro incubation of CSE with CSE-enzymatically synthesized Cys-SSH resulted in the inhibition of Cys-SSH production. In contrast, the mutant CSEs (Cys136Val and Cys136/171Val) proved resistant to inhibition. The Cys-SSH-producing CSE activity of Cys136/171Val CSE was higher than that of the wild-type enzyme. Meanwhile, the cysteine-producing CSE activity of this mutant was equivalent to that of the wild-type enzyme. It is assumed that Cys-SSH-producing CSE activity could be auto-inactivated via the polysulfidation of the enzyme during cystine metabolism. Thus, the polysulfidation of CSE at the Cys136 residue may be an integral feature of cystine metabolism, which functions to down-regulate Cys-SSH synthesis by the enzyme. Full article

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Open AccessArticle

Endogenous TOM20 Proximity Labeling: A Swiss-Knife for the Study of Mitochondrial Proteins in Human Cells

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Int. J. Mol. Sci. 2023, 24(11), 9604; https://doi.org/10.3390/ijms24119604 - 31 May 2023

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Abstract

Biotin-based proximity labeling approaches, such as BioID, have demonstrated their use for the study of mitochondria proteomes in living cells. The use of genetically engineered BioID cell lines enables the detailed characterization of poorly characterized processes such as mitochondrial co-translational import. In this [...] Read more.

Biotin-based proximity labeling approaches, such as BioID, have demonstrated their use for the study of mitochondria proteomes in living cells. The use of genetically engineered BioID cell lines enables the detailed characterization of poorly characterized processes such as mitochondrial co-translational import. In this process, translation is coupled to the translocation of the mitochondrial proteins, alleviating the energy cost typically associated with the post-translational import relying on chaperone systems. However, the mechanisms are still unclear with only few actors identified but none that have been described in mammals yet. We thus profiled the TOM20 proxisome using BioID, assuming that some of the identified proteins could be molecular actors of the co-translational import in human cells. The obtained results showed a high enrichment of RNA binding proteins close to the TOM complex. However, for the few selected candidates, we could not demonstrate a role in the mitochondrial co-translational import process. Nonetheless, we were able to demonstrate additional uses of our BioID cell line. Indeed, the experimental approach used in this study is thus proposed for the identification of mitochondrial co-translational import effectors and for the monitoring of protein entry inside mitochondria with a potential application in the prediction of mitochondrial protein half-life. Full article

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Open AccessArticle

EAE of Mice: Enzymatic Cross Site-Specific Hydrolysis of H2A Histone by IgGs against H2A, H1, H2B, H3, and H4 Histones and Myelin Basic Protein

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Int. J. Mol. Sci. 2023, 24(10), 8636; https://doi.org/10.3390/ijms24108636 - 12 May 2023

Viewed by 885

Abstract

Histones play vital roles in chromatin function and gene transcription; however, they are very harmful in the intercellular space because they stimulate systemic inflammatory and toxic responses. Myelin basic protein (MBP) is the major protein of the axon myelin–proteolipid sheath. Antibodies–abzymes with various [...] Read more.

Histones play vital roles in chromatin function and gene transcription; however, they are very harmful in the intercellular space because they stimulate systemic inflammatory and toxic responses. Myelin basic protein (MBP) is the major protein of the axon myelin–proteolipid sheath. Antibodies–abzymes with various catalytic activities are specific features of some autoimmune diseases. IgGs against individual histones (H2A, H1, H2B, H3, and H4) and MBP were isolated from the blood of experimental-autoimmune-encephalomyelitis-prone C57BL/6 mice by several affinity chromatographies. These Abs–abzymes corresponded to various stages of EAE development: spontaneous EAE, MOG, and DNA–histones accelerated the onset, acute, and remission stages. IgGs-abzymes against MBP and five individual histones showed unusual polyreactivity in the complex formation and enzymatic cross-reactivity in the specific hydrolysis of the H2A histone. All the IgGs of 3-month-old mice (zero time) against MBP and individual histones demonstrated from 4 to 35 different H2A hydrolysis sites. The spontaneous development of EAE over 60 days led to a significant change in the type and number of H2A histone hydrolysis sites by IgGs against five histones and MBP. Mice treatment with MOG and the DNA–histone complex changed the type and number of H2A hydrolysis sites compared to zero time. The minimum number (4) of different H2A hydrolysis sites was found for IgGs against H2A (zero time), while the maximum (35) for anti-H2B IgGs (60 days after mice treatment with DNA–histone complex). Overall, it was first demonstrated that at different stages of EAE evolution, IgGs–abzymes against individual histones and MBP could significantly differ in the number and type of specific sites of H2A hydrolysis. The possible reasons for the catalytic cross-reactivity and great differences in the number and type of histone H2A cleavage sites were analyzed. Full article

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Open AccessReview

Synthesis of Bioactive Aminomethylated 8-Hydroxyquinolines via the Modified Mannich Reaction

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Oszkár Csuvik

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István Szatmári

Int. J. Mol. Sci. 2023, 24(9), 7915; https://doi.org/10.3390/ijms24097915 - 26 Apr 2023

Viewed by 1336

Abstract

8-hydroxyquinoline (oxine) is a widely known and frequently used chelating agent, and the pharmacological effects of the core molecule and its derivatives have been studied since the 19th century. There are several synthetic methods to modify this core. The Mannich reaction is one [...] Read more.

8-hydroxyquinoline (oxine) is a widely known and frequently used chelating agent, and the pharmacological effects of the core molecule and its derivatives have been studied since the 19th century. There are several synthetic methods to modify this core. The Mannich reaction is one of the most easily implementable examples, which requires mild reaction conditions and simple chemical reagents. The three components of the Mannich reaction are a primary or secondary amine, an aldehyde and a compound having a hydrogen with pronounced activity. In the modified Mannich reaction, naphthol or a nitrogen-containing naphthol analogue (e.g., 8-hydroxyquinoline) is utilised as the active hydrogen provider compound, thus affording the formation of aminoalkylated products. The amine component can be ammonia and primary or secondary amines. The aldehyde component is highly variable, including aliphatic and aromatic aldehydes. Based on the pharmacological relevance of aminomethylated 8-hydroxyquinolines, this review summarises their syntheses via the modified Mannich reaction starting from 8-hydroxyquinoline, formaldehyde and various amines. Full article

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Open AccessArticle

Smooth Muscle Cell Phenotypic Switch Induced by Traditional Cigarette Smoke Condensate: A Holistic Overview

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Int. J. Mol. Sci. 2023, 24(7), 6431; https://doi.org/10.3390/ijms24076431 - 29 Mar 2023

Cited by 1 | Viewed by 1405

Abstract

Cigarette smoke (CS) is a risk factor for inflammatory diseases, such as atherosclerosis. CS condensate (CSC) contains lipophilic components that may represent a systemic cardiac risk factor. To better understand CSC effects, we incubated mouse and human aortic smooth muscle cells (SMCs) with [...] Read more.

Cigarette smoke (CS) is a risk factor for inflammatory diseases, such as atherosclerosis. CS condensate (CSC) contains lipophilic components that may represent a systemic cardiac risk factor. To better understand CSC effects, we incubated mouse and human aortic smooth muscle cells (SMCs) with CSC. We evaluated specific markers for contractile [i.e., actin, aortic smooth muscle (ACTA2), calponin-1 (CNN1), the Kruppel-like factor 4 (KLF4), and myocardin (MYOCD) genes] and inflammatory [i.e., IL-1β, and IL-6, IL-8, and galectin-3 (LGALS-3) genes] phenotypes. CSC increased the expression of inflammatory markers and reduced the contractile ones in both cell types, with KLF4 modulating the SMC phenotypic switch. Next, we performed a mass spectrometry-based differential proteomic approach on human SMCs and could show 11 proteins were significantly affected by exposition to CSC (FC ≥ 2.7, p ≤ 0.05). These proteins are active in signaling pathways related to expression of pro-inflammatory cytokines and IFN, inflammasome assembly and activation, cytoskeleton regulation and SMC contraction, mitochondrial integrity and cellular response to oxidative stress, proteostasis control via ubiquitination, and cell proliferation and epithelial-to-mesenchymal transition. Through specific bioinformatics resources, we showed their tight functional correlation in a close interaction niche mainly orchestrated by the interferon-induced double-stranded RNA-activated protein kinase (alternative name: protein kinase RNA-activated; PKR) (EIF2AK2/PKR). Finally, by combining gene expression and protein abundance data we obtained a hybrid network showing reciprocal integration of the CSC-deregulated factors and indicating KLF4 and PKR as the most relevant factors. Full article

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Open AccessArticle

TNFα Activates the Liver X Receptor Signaling Pathway and Promotes Cholesterol Efflux from Human Brain Pericytes Independently of ABCA1

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Shiraz Dib

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Rodrigo Azevedo Loiola

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Int. J. Mol. Sci. 2023, 24(6), 5992; https://doi.org/10.3390/ijms24065992 - 22 Mar 2023

Cited by 2 | Viewed by 1480

Abstract

Neuroinflammation and brain lipid imbalances are observed in Alzheimer’s disease (AD). Tumor necrosis factor-α (TNFα) and the liver X receptor (LXR) signaling pathways are involved in both processes. However, limited information is currently available regarding their relationships in human brain pericytes [...] Read more.

Neuroinflammation and brain lipid imbalances are observed in Alzheimer’s disease (AD). Tumor necrosis factor-α (TNFα) and the liver X receptor (LXR) signaling pathways are involved in both processes. However, limited information is currently available regarding their relationships in human brain pericytes (HBP) of the neurovascular unit. In cultivated HBP, TNFα activates the LXR pathway and increases the expression of one of its target genes, the transporter ATP-binding cassette family A member 1 (ABCA1), while ABCG1 is not expressed. Apolipoprotein E (APOE) synthesis and release are diminished. The cholesterol efflux is promoted, but is not inhibited, when ABCA1 or LXR are blocked. Moreover, as for TNFα, direct LXR activation by the agonist (T0901317) increases ABCA1 expression and the associated cholesterol efflux. However, this process is abolished when LXR/ABCA1 are both inhibited. Neither the other ABC transporters nor the SR-BI are involved in this TNFα-mediated lipid efflux regulation. We also report that inflammation increases ABCB1 expression and function. In conclusion, our data suggest that inflammation increases HBP protection against xenobiotics and triggers an LXR/ABCA1 independent cholesterol release. Understanding the molecular mechanisms regulating this efflux at the level of the neurovascular unit remains fundamental to the characterization of links between neuroinflammation, cholesterol and HBP function in neurodegenerative disorders. Full article

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Open AccessArticle

A Comparative Study of the Inhibitory Action of Berberine Derivatives on the Recombinant Protein FtsZ of E. coli

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Angela Di Somma

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Carolina Canè

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Natalie Paola Rotondo

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Maria Maddalena Cavalluzzi

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Giovanni Lentini

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Angela Duilio

Int. J. Mol. Sci. 2023, 24(6), 5674; https://doi.org/10.3390/ijms24065674 - 16 Mar 2023

Viewed by 1040

Abstract

Medicinal plants belonging to the genus Berberis may be considered an interesting source of drugs to counteract the problem of antimicrobial multiresistance. The important properties associated with this genus are mainly due to the presence of berberine, an alkaloid with a benzyltetrahydroisoquinoline structure. [...] Read more.

Medicinal plants belonging to the genus Berberis may be considered an interesting source of drugs to counteract the problem of antimicrobial multiresistance. The important properties associated with this genus are mainly due to the presence of berberine, an alkaloid with a benzyltetrahydroisoquinoline structure. Berberine is active against both Gram-negative and Gram-positive bacteria, influencing DNA duplication, RNA transcription, protein synthesis, and the integrity of the cell surface structure. Countless studies have shown the enhancement of these beneficial effects following the synthesis of different berberine analogues. Recently, a possible interaction between berberine derivatives and the FtsZ protein was predicted through molecular docking simulations. FtsZ is a highly conserved protein essential for the first step of cell division in bacteria. The importance of FtsZ for the growth of numerous bacterial species and its high conservation make it a perfect candidate for the development of broad-spectrum inhibitors. In this work, we investigate the inhibition mechanisms of the recombinant FtsZ of Escherichia coli by different N-arylmethyl benzodioxolethylamines as berberine simplified analogues appropriately designed to evaluate the effect of structural changes on the interaction with the enzyme. All the compounds determine the inhibition of FtsZ GTPase activity by different mechanisms. The tertiary amine 1c proved to be the best competitive inhibitor, as it causes a remarkable increase in FtsZ K_m (at 40 μM) and a drastic reduction in its assembly capabilities. Moreover, a fluorescence spectroscopic analysis carried out on 1c demonstrated its strong interaction with FtsZ (K_d = 26.6 nM). The in vitro results were in agreement with docking simulation studies. Full article

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Open AccessArticle

Isochores and Heat Capacity of Liquid Water in Terms of the Ion–Molecular Model

by

Alexander A. Volkov

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Sergey V. Chuchupal

Int. J. Mol. Sci. 2023, 24(6), 5630; https://doi.org/10.3390/ijms24065630 - 15 Mar 2023

Viewed by 766

Abstract

Thermodynamics of liquid water in terms of a non-standard approach—the ion–molecular model—is considered. Water is represented as a dense gas of neutral H₂O molecules and single charged H₃O⁺ and OH⁻ ions. The molecules and ions perform thermal [...] Read more.

Thermodynamics of liquid water in terms of a non-standard approach—the ion–molecular model—is considered. Water is represented as a dense gas of neutral H₂O molecules and single charged H₃O⁺ and OH⁻ ions. The molecules and ions perform thermal collisional motion and interconvert due to ion exchange. The energy-rich process—vibrations of an ion in a hydration shell of molecular dipoles—well known to spectroscopists with its dielectric response at 180 cm⁻¹ (5 THz), is suggested to be key for water dynamics. Taking into account this ion–molecular oscillator, we compose an equation of state of liquid water to obtain analytical expressions for the isochores and heat capacity. Full article

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Open AccessReview

Role of Dopamine in the Heart in Health and Disease

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Int. J. Mol. Sci. 2023, 24(5), 5042; https://doi.org/10.3390/ijms24055042 - 06 Mar 2023

Cited by 3 | Viewed by 3481

Abstract

Dopamine has effects on the mammalian heart. These effects can include an increase in the force of contraction, and an elevation of the beating rate and the constriction of coronary arteries. Depending on the species studied, positive inotropic effects were strong, very modest, [...] Read more.

Dopamine has effects on the mammalian heart. These effects can include an increase in the force of contraction, and an elevation of the beating rate and the constriction of coronary arteries. Depending on the species studied, positive inotropic effects were strong, very modest, or absent, or even negative inotropic effects occurred. We can discern five dopamine receptors. In addition, the signal transduction by dopamine receptors and the regulation of the expression of cardiac dopamine receptors will be of interest to us, because this might be a tempting area of drug development. Dopamine acts in a species-dependent fashion on these cardiac dopamine receptors, but also on cardiac adrenergic receptors. We will discuss the utility of drugs that are currently available as tools to understand cardiac dopamine receptors. The molecule dopamine itself is present in the mammalian heart. Therefore, cardiac dopamine might act as an autocrine or paracrine compound in the mammalian heart. Dopamine itself might cause cardiac diseases. Moreover, the cardiac function of dopamine and the expression of dopamine receptors in the heart can be altered in diseases such as sepsis. Various drugs for cardiac and non-cardiac diseases are currently in the clinic that are, at least in part, agonists or antagonists at dopamine receptors. We define the research needs in order to understand dopamine receptors in the heart better. All in all, an update on the role of dopamine receptors in the human heart appears to be clinically relevant, and is thus presented here. Full article

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Open AccessArticle

Structural Insight into Catalysis by the Flavin-Dependent NADH Oxidase (Pden_5119) of Paracoccus denitrificans

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Martin Kryl

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Vojtěch Sedláček

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Igor Kučera

Int. J. Mol. Sci. 2023, 24(4), 3732; https://doi.org/10.3390/ijms24043732 - 13 Feb 2023

Viewed by 975

Abstract

The Pden_5119 protein oxidizes NADH with oxygen under mediation by the bound flavin mononucleotide (FMN) and may be involved in the maintenance of the cellular redox pool. In biochemical characterization, the curve of the pH-rate dependence was bell-shaped with pK_a1 = 6.6 [...] Read more.

The Pden_5119 protein oxidizes NADH with oxygen under mediation by the bound flavin mononucleotide (FMN) and may be involved in the maintenance of the cellular redox pool. In biochemical characterization, the curve of the pH-rate dependence was bell-shaped with pK_a1 = 6.6 and pK_a2 = 9.2 at 2 μM FMN while it contained only a descending limb pK_a of 9.7 at 50 μM FMN. The enzyme was found to undergo inactivation by reagents reactive with histidine, lysine, tyrosine, and arginine. In the first three cases, FMN exerted a protective effect against the inactivation. X-ray structural analysis coupled with site-directed mutagenesis identified three amino acid residues important to the catalysis. Structural and kinetic data suggest that His-117 plays a role in the binding and positioning of the isoalloxazine ring of FMN, Lys-82 fixes the nicotinamide ring of NADH to support the proS-hydride transfer, and Arg-116 with its positive charge promotes the reaction between dioxygen and reduced flavin. Full article

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Open AccessReview

Autologous Platelet and Extracellular Vesicle-Rich Plasma as Therapeutic Fluid: A Review

Int. J. Mol. Sci. 2023, 24(4), 3420; https://doi.org/10.3390/ijms24043420 - 08 Feb 2023

Cited by 3 | Viewed by 2206

Abstract

The preparation of autologous platelet and extracellular vesicle-rich plasma (PVRP) has been explored in many medical fields with the aim to benefit from its healing potential. In parallel, efforts are being invested to understand the function and dynamics of PVRP that is complex [...] Read more.

The preparation of autologous platelet and extracellular vesicle-rich plasma (PVRP) has been explored in many medical fields with the aim to benefit from its healing potential. In parallel, efforts are being invested to understand the function and dynamics of PVRP that is complex in its composition and interactions. Some clinical evidence reveals beneficial effects of PVRP, while some report that there were no effects. To optimize the preparation methods, functions and mechanisms of PVRP, its constituents should be better understood. With the intention to promote further studies of autologous therapeutic PVRP, we performed a review on some topics regarding PVRP composition, harvesting, assessment and preservation, and also on clinical experience following PVRP application in humans and animals. Besides the acknowledged actions of platelets, leukocytes and different molecules, we focus on extracellular vesicles that were found abundant in PVRP. Full article

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Open AccessCommunication

Domain Swapping between AtACS7 and PpACL1 Results in Chimeric ACS-like Proteins with ACS or C_β-S Lyase Single Enzymatic Activity

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Int. J. Mol. Sci. 2023, 24(3), 2956; https://doi.org/10.3390/ijms24032956 - 03 Feb 2023

Viewed by 839

Abstract

The gaseous hormone ethylene plays a pivotal role in plant growth and development. In seed plants, the key rate-limiting enzyme that controls ethylene biosynthesis is ACC synthase (ACS). ACS has, for a long time, been believed to be a single-activity enzyme until we [...] Read more.

The gaseous hormone ethylene plays a pivotal role in plant growth and development. In seed plants, the key rate-limiting enzyme that controls ethylene biosynthesis is ACC synthase (ACS). ACS has, for a long time, been believed to be a single-activity enzyme until we recently discovered that it also possesses C_β-S lyase (CSL) activity. This discovery raises fundamental questions regarding the biological significance of the dual enzymatic activities of ACS. To address these issues, it is highly necessary to obtain ACS mutants with either ACS or CSL single activity. Here, domain swapping between Arabidopsis AtACS7 and moss CSL PpACL1 were performed. Enzymatic activity assays of the constructed chimeras revealed that, R10, which was produced by replacing AtACS7 box 6 with that of PpACL1, lost ACS but retained CSL activity, whereas R12 generated by box 4 substitution lost CSL and only had ACS activity. The activities of both chimeric proteins were compared with previously obtained single-activity mutants including R6, AtACS7^Q98A, and AtACS7^D245N. All the results provided new insights into the key residues required for ACS and CSL activities of AtACS7 and laid an important foundation for further in-depth study of the biological functions of its dual enzymatic activities. Full article

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Open AccessArticle

Moderate Constraint Facilitates Association and Force-Dependent Dissociation of HA-CD44 Complex

by

Ziyang Yao

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Jianhua Wu

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Ying Fang

Int. J. Mol. Sci. 2023, 24(3), 2243; https://doi.org/10.3390/ijms24032243 - 23 Jan 2023

Cited by 2 | Viewed by 1097

Abstract

Binding of cell surface glycoprotein CD44 to hyaluronic acid (HA) is a key event for mediating cell adhesion, motility, metastasis, inflammatory responses and tumor development, but the regulation mechanism and its molecular basis under diverse mechanical constraints remain unclear. We herein investigated interaction [...] Read more.

Binding of cell surface glycoprotein CD44 to hyaluronic acid (HA) is a key event for mediating cell adhesion, motility, metastasis, inflammatory responses and tumor development, but the regulation mechanism and its molecular basis under diverse mechanical constraints remain unclear. We herein investigated interaction of CD44 HABD (HA binding site domain) to HA through free and steered molecular dynamics (MD) simulations as well as atomic force microscope (AFM) measurement using different constraints on HA. The middle, two ends or both of the constrained HA chains were fixed for MD simulations, while one and two biotin–avidin linkage or physical absorption were used to immobilize HA on substrates for AFM experiments, to model HA chains with low, moderate and high HA flexibilities, respectively. We found that binding of CD44 to moderate fixed HA was possessed of a better thermo-stability, a lower mechanical strength and a higher dissociation probability, while higher adhesive frequency, smaller rupture force and shorter lifetime were assigned to CD44 on the two biotin-immobilized HA rather than one biotin-immobilized or physically absorbed HA on substrates, suggesting a moderate HA flexibility requirement in favor of association and force-induced dissociation of CD44-HA complex. Tensile-induced convex conformation of HA chain was responsible for reduction of complex mechano-stability and did inversely a shrunken CD44 HABD under stretching; transition from catch bond to slip bond governed CD44-HA interaction. This study uncovered the regulation mechanism and its molecular basis for CD44-HA affinity under diverse mechano-microenvironments and provided a new insight into CD44-HA interaction-mediated cell inflammatory responses and tumor development. Full article

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Open AccessArticle

New Semisynthetic Penicillins Obtained by Coupling of the 6-Aminopenicillanic Acid with 5-Mercapto-1,2,4-triazoles-3,4-disubstituted

by

Corina Cheptea

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Alexandru Zara

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Dan Gheorghe Dimitriu

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Valeriu Sunel

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Dana Ortansa Dorohoi

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Toni Andor Cigu

Int. J. Mol. Sci. 2023, 24(2), 1497; https://doi.org/10.3390/ijms24021497 - 12 Jan 2023

Viewed by 1185

Abstract

In a basic medium, 5-Mercapto-1,2,4-triazoles pass into the thiol form, allowing their transformation into sodium salts, which, in reaction with sodium monochloroacetate, lead to sodium 5-thioacetates of 1,2,4-triazoles-3,4-disubstituted. Sulfur derivatives converted to pivalic mixed anhydrides were used as active forms in the acylation [...] Read more.

In a basic medium, 5-Mercapto-1,2,4-triazoles pass into the thiol form, allowing their transformation into sodium salts, which, in reaction with sodium monochloroacetate, lead to sodium 5-thioacetates of 1,2,4-triazoles-3,4-disubstituted. Sulfur derivatives converted to pivalic mixed anhydrides were used as active forms in the acylation of 6-amino penicillanic acid (6-AP) to obtain new semisynthetic penicillins. They contain in the molecule, together with the β-lactam ring, the nucleus 3-[(5-nitroindazol-1′-yl-methyl)]-4-aryl-5-mercapto-1,2,4-triazole, both contributing to an important antibacterial effect. The structure of the new antibiotics was confirmed by the results of elemental and spectral analysis (FT-IR, ¹H- and ¹³C-NMR). The synthetic penicillins were tested for toxicological action and antibacterial activity and the obtained results were close to those for amoxicillin, the reference drug. Full article

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Open AccessReview

Assessing the Role of Aquaporin 4 in Skeletal Muscle Function

by

Tejal Aslesh

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Ammar Al-aghbari

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Toshifumi Yokota

Int. J. Mol. Sci. 2023, 24(2), 1489; https://doi.org/10.3390/ijms24021489 - 12 Jan 2023

Viewed by 1835

Abstract

Water transport across the biological membranes is mediated by aquaporins (AQPs). AQP4 and AQP1 are the predominantly expressed AQPs in the skeletal muscle. Since the discovery of AQP4, several studies have highlighted reduced AQP4 levels in Duchenne muscular dystrophy (DMD) patients and mouse [...] Read more.

Water transport across the biological membranes is mediated by aquaporins (AQPs). AQP4 and AQP1 are the predominantly expressed AQPs in the skeletal muscle. Since the discovery of AQP4, several studies have highlighted reduced AQP4 levels in Duchenne muscular dystrophy (DMD) patients and mouse models, and other neuromuscular disorders (NMDs) such as sarcoglycanopathies and dysferlinopathies. AQP4 loss is attributed to the destabilizing dystrophin-associated protein complex (DAPC) in DMD leading to compromised water permeability in the skeletal muscle fibers. However, AQP4 knockout (KO) mice appear phenotypically normal. AQP4 ablation does not impair physical activity in mice but limits them from achieving the performance demonstrated by wild-type mice. AQP1 levels were found to be upregulated in DMD models and are thought to compensate for AQP4 loss. Several groups investigated the expression of other AQPs in the skeletal muscle; however, these findings remain controversial. In this review, we summarize the role of AQP4 with respect to skeletal muscle function and findings in NMDs as well as the implications from a clinical perspective Full article

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Open AccessArticle

Açai Berry Administration Promotes Wound Healing through Wnt/β-Catenin Pathway

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Livia Interdonato

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Ylenia Marino

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Gianluca Antonio Franco

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Int. J. Mol. Sci. 2023, 24(1), 834; https://doi.org/10.3390/ijms24010834 - 03 Jan 2023

Cited by 2 | Viewed by 1055

Abstract

Recently, wound healing has received increased attention from both a scientific and clinical point of view. It is characterized by an organized series of processes: angiogenesis, cell migration and proliferation, extracellular matrix production, and remodeling. Many of these processes are controlled by the [...] Read more.

Recently, wound healing has received increased attention from both a scientific and clinical point of view. It is characterized by an organized series of processes: angiogenesis, cell migration and proliferation, extracellular matrix production, and remodeling. Many of these processes are controlled by the Wnt pathway, which activates them. The aim of the study was to evaluate the molecular mechanism of açai berry administration in a mouse model of wound healing. CD1 male mice were used in this research. Two full-thickness excisional wounds (5 mm) were performed with a sterile biopsy punch on the dorsum to create two circular, full-thickness skin wounds on either side of the median line on the dorsum. Açai berry was administered by oral administration (500 mg/kg dissolved in saline) for 6 days after induction of the wound. Our study demonstrated that açai berry can modulate the Wnt pathway, reducing the expression of Wnt3a, the cysteine-rich domain of frizzled (FZ)8, and the accumulation of cytosolic and nuclear β-catenin. Moreover, açai berry reduced the levels of TNF-α and IL-18, which are target genes strictly downstream of the Wnt/β-catenin pathway. It also showed important anti-inflammatory activities by reducing the activation of the NF-κB pathway. Furthermore, Wnt can modulate the activity of growth factors, such as TGF-β, and VEGF, which are the basis of the wound-healing process. In conclusion, we can confirm that açai berry can modulate the activity of the Wnt/β-catenin pathway, as it is involved in the inflammatory process and in the activity of the growth factor implicated in wound healing. Full article

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2022

Jump to: 2023

Open AccessArticle

Elucidating the Interaction between Pyridoxine 5′-Phosphate Oxidase and Dopa Decarboxylase: Activation of B6-Dependent Enzyme

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Mohammed H. AL Mughram

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Mohini S. Ghatge

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Glen E. Kellogg

and

Martin K. Safo

Int. J. Mol. Sci. 2023, 24(1), 642; https://doi.org/10.3390/ijms24010642 - 30 Dec 2022

Cited by 1 | Viewed by 1687

Abstract

Pyridoxal 5′-phosphate (PLP), the active form of vitamin B6, serves as a cofactor for scores of B6-dependent (PLP-dependent) enzymes involved in many cellular processes. One such B6 enzyme is dopa decarboxylase (DDC), which is required for the biosynthesis of key neurotransmitters, e.g., dopamine [...] Read more.

Pyridoxal 5′-phosphate (PLP), the active form of vitamin B6, serves as a cofactor for scores of B6-dependent (PLP-dependent) enzymes involved in many cellular processes. One such B6 enzyme is dopa decarboxylase (DDC), which is required for the biosynthesis of key neurotransmitters, e.g., dopamine and serotonin. PLP-dependent enzymes are biosynthesized as apo-B6 enzymes and then converted to the catalytically active holo-B6 enzymes by Schiff base formation between the aldehyde of PLP and an active site lysine of the protein. In eukaryotes, PLP is made available to the B6 enzymes through the activity of the B6-salvage enzymes, pyridoxine 5′-phosphate oxidase (PNPO) and pyridoxal kinase (PLK). To minimize toxicity, the cell keeps the content of free PLP (unbound) very low through dephosphorylation and PLP feedback inhibition of PNPO and PLK. This has led to a proposed mechanism of complex formation between the B6-salvage enzymes and apo-B6 enzymes prior to the transfer of PLP, although such complexes are yet to be characterized at the atomic level, presumably due to their transient nature. A computational study, for the first time, was used to predict a likely PNPO and DDC complex, which suggested contact between the allosteric PLP tight-binding site on PNPO and the active site of DDC. Using isothermal calorimetry and/or surface plasmon resonance, we also show that PNPO binds both apoDDC and holoDDC with dissociation constants of 0.93 ± 0.07 μM and 2.59 ± 0.11 μM, respectively. Finally, in the presence of apoDDC, the tightly bound PLP on PNPO is transferred to apoDDC, resulting in the formation of about 35% holoDDC. Full article

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Open AccessArticle

Protein Kinase CK2 Contributes to Glucose Homeostasis by Targeting Fructose-1,6-Bisphosphatase 1

by

Mandy Pack

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Tim Nikolai Gulde

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Michelle Victoria Völcker

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Int. J. Mol. Sci. 2023, 24(1), 428; https://doi.org/10.3390/ijms24010428 - 27 Dec 2022

Viewed by 1242

Abstract

Glucose homeostasis is of critical importance for the survival of organisms. It is under hormonal control and often coordinated by the action of kinases and phosphatases. We have previously shown that CK2 regulates insulin production and secretion in pancreatic β-cells. In order to [...] Read more.

Glucose homeostasis is of critical importance for the survival of organisms. It is under hormonal control and often coordinated by the action of kinases and phosphatases. We have previously shown that CK2 regulates insulin production and secretion in pancreatic β-cells. In order to shed more light on the CK2-regulated network of glucose homeostasis, in the present study, a qRT-PCR array was carried out with 84 diabetes-associated genes. After inhibition of CK2, fructose-1,6-bisphosphatase 1 (FBP1) showed a significant lower gene expression. Moreover, FBP1 activity was down-regulated. Being a central enzyme of gluconeogenesis, the secretion of glucose was decreased as well. Thus, FBP1 is a new factor in the CK2-regulated network implicated in carbohydrate metabolism control. Full article

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Open AccessArticle

Coprinopsis cinerea Galectin CGL1 Induces Apoptosis and Inhibits Tumor Growth in Colorectal Cancer Cells

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Int. J. Mol. Sci. 2023, 24(1), 235; https://doi.org/10.3390/ijms24010235 - 23 Dec 2022

Viewed by 1220

Abstract

Mushroom galectins are promising anticancer agents for their low IC₅₀ values against cancer cells in vitro. In this study, two Coprinopsis cinerea galectins, CGL1 and CGL2, were heterologously expressed, and their biochemistry properties and anticancer effects were evaluated. The purified galectins were [...] Read more.

Mushroom galectins are promising anticancer agents for their low IC₅₀ values against cancer cells in vitro. In this study, two Coprinopsis cinerea galectins, CGL1 and CGL2, were heterologously expressed, and their biochemistry properties and anticancer effects were evaluated. The purified galectins were thermostable at neutral pH conditions. They both existed as tetramers and shared a high affinity towards lactose. CGL1 and CGL2 strongly inhibited the cell viability of many cancer cell lines, including three colorectal cancer cells, in a dose-dependent manner by inducing mitochondria-mediated caspase-dependent apoptosis. Furthermore, CGL1 exhibited higher apoptosis-inducing ability and cytotoxicity than CGL2. In vivo cell viability experiments based on two xenograft mouse models showed that CGL1 had a more substantial inhibitory effect than CGL2 on HCT116 tumor growth (p < 0.0001), whereas only CGL1 inhibited DLD1 tumor growth (p < 0.01). This is the first study to evaluate the anti-colorectal cancer effect of mushroom lectins in vivo, and our results showed that CGL1 is a potent agent for colorectal cancer treatment. Full article

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Open AccessReview

The Mutual Relationship between Glycosylation and Non-Coding RNAs in Cancer and Other Physio-Pathological Conditions

by

Martina Duca

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Nadia Malagolini

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Fabio Dall’Olio

Int. J. Mol. Sci. 2022, 23(24), 15804; https://doi.org/10.3390/ijms232415804 - 13 Dec 2022

Cited by 2 | Viewed by 1633

Abstract

Glycosylation, which consists of the enzymatic addition of sugars to proteins and lipids, is one of the most important post-co-synthetic modifications of these molecules, profoundly affecting their activity. Although the presence of carbohydrate chains is crucial for fine-tuning the interactions between cells and [...] Read more.

Glycosylation, which consists of the enzymatic addition of sugars to proteins and lipids, is one of the most important post-co-synthetic modifications of these molecules, profoundly affecting their activity. Although the presence of carbohydrate chains is crucial for fine-tuning the interactions between cells and molecules, glycosylation is an intrinsically stochastic process regulated by the relative abundance of biosynthetic (glycosyltransferases) and catabolic (glycosidases) enzymes, as well as sugar carriers and other molecules. Non-coding RNAs, which include microRNAs, long non-coding RNAs and circRNAs, establish a complex network of reciprocally interacting molecules whose final goal is the regulation of mRNA expression. Likewise, these interactions are stochastically regulated by ncRNA abundance. Thus, while protein sequence is deterministically dictated by the DNA/RNA/protein axis, protein abundance and activity are regulated by two stochastic processes acting, respectively, before and after the biosynthesis of the protein axis. Consequently, the worlds of glycosylation and ncRNA are closely interconnected and mutually interacting. In this paper, we will extensively review the many faces of the ncRNA–glycosylation interplay in cancer and other physio-pathological conditions. Full article

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Open AccessArticle

Mathematical Modeling of ROS Production and Diode-like Behavior in the SDHA/SDHB Subcomplex of Succinate Dehydrogenases in Reverse Quinol-Fumarate Reductase Direction

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Nikolay I. Markevich

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Lubov N. Markevich

Int. J. Mol. Sci. 2022, 23(24), 15596; https://doi.org/10.3390/ijms232415596 - 09 Dec 2022

Cited by 1 | Viewed by 996

Abstract

Succinate dehydrogenase (SDH) plays an important role in reverse electron transfer during hypoxia/anoxia, in particular, in ischemia, when blood supply to an organ is disrupted, and oxygen is not available. It was detected in the voltammetry studies about three decades ago that the [...] Read more.

Succinate dehydrogenase (SDH) plays an important role in reverse electron transfer during hypoxia/anoxia, in particular, in ischemia, when blood supply to an organ is disrupted, and oxygen is not available. It was detected in the voltammetry studies about three decades ago that the SDHA/SDHB subcomplex of SDH can have such a strong nonlinear property as a “tunnel-diode” behavior in reverse quinol-fumarate reductase direction. The molecular and kinetic mechanisms of this phenomenon, that is, a strong drop in the rate of fumarate reduction as the driving force is increased, are still unclear. In order to account for this property of SDH, we developed and analyzed a mechanistic computational model of reverse electron transfer in the SDHA/SDHB subcomplex of SDH. It was shown that a decrease in the rate of succinate release from the active center during fumarate reduction quantitatively explains the experimentally observed tunnel-diode behavior in SDH and threshold values of the electrode potential of about −80 mV. Computational analysis of ROS production in the SDHA/SDHB subcomplex of SDH during reverse electron transfer predicts that the rate of ROS production decreases when the tunnel-diode behavior appears. These results predict a low rate of ROS production by the SDHA/SDHB subcomplex of SDH during ischemia. Full article

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Open AccessReview

Insights into Molecular Structure of Pterins Suitable for Biomedical Applications

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Int. J. Mol. Sci. 2022, 23(23), 15222; https://doi.org/10.3390/ijms232315222 - 03 Dec 2022

Cited by 2 | Viewed by 1849

Abstract

Pterins are an inseparable part of living organisms. Pterins participate in metabolic reactions mostly as tetrahydropterins. Dihydropterins are usually intermediates of these reactions, whereas oxidized pterins can be biomarkers of diseases. In this review, we analyze the available data on the quantum chemistry [...] Read more.

Pterins are an inseparable part of living organisms. Pterins participate in metabolic reactions mostly as tetrahydropterins. Dihydropterins are usually intermediates of these reactions, whereas oxidized pterins can be biomarkers of diseases. In this review, we analyze the available data on the quantum chemistry of unconjugated pterins as well as their photonics. This gives a comprehensive overview about the electronic structure of pterins and offers some benefits for biomedicine applications: (1) one can affect the enzymatic reactions of aromatic amino acid hydroxylases, NO synthases, and alkylglycerol monooxygenase through UV irradiation of H₄pterins since UV provokes electron donor reactions of H₄pterins; (2) the emission properties of H₂pterins and oxidized pterins can be used in fluorescence diagnostics; (3) two-photon absorption (TPA) should be used in such pterin-related infrared therapy because single-photon absorption in the UV range is inefficient and scatters in vivo; (4) one can affect pathogen organisms through TPA excitation of H₄pterin cofactors, such as the molybdenum cofactor, leading to its detachment from proteins and subsequent oxidation; (5) metal nanostructures can be used for the UV-vis, fluorescence, and Raman spectroscopy detection of pterin biomarkers. Therefore, we investigated both the biochemistry and physical chemistry of pterins and suggested some potential prospects for pterin-related biomedicine. Full article

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Open AccessArticle

Resveratrol-like Compounds as SIRT1 Activators

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Int. J. Mol. Sci. 2022, 23(23), 15105; https://doi.org/10.3390/ijms232315105 - 01 Dec 2022

Cited by 9 | Viewed by 1531

Abstract

The sirtuin 1 (SIRT1) activator resveratrol has emerged as a promising candidate for the prevention of vascular oxidative stress, which is a trigger for endothelial dysfunction. However, its clinical use is limited by low oral bioavailability. In this work, we have applied a [...] Read more.

The sirtuin 1 (SIRT1) activator resveratrol has emerged as a promising candidate for the prevention of vascular oxidative stress, which is a trigger for endothelial dysfunction. However, its clinical use is limited by low oral bioavailability. In this work, we have applied a previously developed computational protocol to identify the most promising derivatives from our in-house chemical library of resveratrol derivatives. The most promising compounds in terms of SIRT1 activation and oral bioavailability, predicted in silico, were evaluated for their ability to activate the isolated SIRT1 enzyme. Then, we assessed the antioxidant effects of the most effective derivative, compound 3d, in human umbilical vein endothelial cells (HUVECs) injured with H₂O₂ 100 µM. The SIRT1 activator 3d significantly preserved cell viability and prevented an intracellular reactive oxygen species increase in HUVECs exposed to the oxidative stimulus. Such effects were partially reduced in the presence of a sirtuin inhibitor, sirtinol, confirming the potential role of sirtuins in the activity of resveratrol and its derivatives. Although 3d appeared less effective than resveratrol in activating the isolated enzyme, the effects exhibited by both compounds in HUVECs were almost superimposable, suggesting a higher ability of 3d to cross cell membranes and activate the intracellular target SIRT1. Full article

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Open AccessReview

The Role of L-Arginine-NO System in Female Reproduction: A Narrative Review

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Int. J. Mol. Sci. 2022, 23(23), 14908; https://doi.org/10.3390/ijms232314908 - 28 Nov 2022

Cited by 2 | Viewed by 2623

Abstract

Accumulating evidence are available on the involvement of l-arginine-nitric oxide (NO) system in complex biological processes and numerous clinical conditions. Particular attention was made to reveal the association of l-arginine and methylarginines to outcome measures of women undergoing in vitro fertilization (IVF). This [...] Read more.

Accumulating evidence are available on the involvement of l-arginine-nitric oxide (NO) system in complex biological processes and numerous clinical conditions. Particular attention was made to reveal the association of l-arginine and methylarginines to outcome measures of women undergoing in vitro fertilization (IVF). This review attempts to summarize the expression and function of the essential elements of this system with particular reference to the different stages of female reproduction. A literature search was performed on the PubMed and Google Scholar systems. Publications were selected for evaluation according to the results presented in the Abstract. The regulatory role of NO during the period of folliculogenesis, oocyte maturation, fertilization, embryogenesis, implantation, placentation, pregnancy, and delivery was surveyed. The major aspects of cellular l-arginine uptake via cationic amino acid transporters (CATs), arginine catabolism by nitric oxide synthases (NOSs) to NO and l-citrulline and by arginase to ornithine, and polyamines are presented. The importance of NOS inhibition by methylated arginines and the redox-sensitive elements of the process of NO generation are also shown. The l-arginine-NO system plays a crucial role in all stages of female reproduction. Insufficiently low or excessively high rates of NO generation may have adverse influences on IVF outcome. Full article

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Open AccessArticle

Testing Serum Albumins and Cyclodextrins as Potential Binders of the Mycotoxin Metabolites Alternariol-3-Sulfate, Alternariol-9-Monomethylether and Alternariol-9-Monomethylether-3-Sulfate

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Int. J. Mol. Sci. 2022, 23(22), 14353; https://doi.org/10.3390/ijms232214353 - 18 Nov 2022

Cited by 2 | Viewed by 1128

Abstract

Alternaria mycotoxins, including alternariol (AOH), alternariol-9-monomethylether (AME), and their masked/modified derivatives (e.g., sulfates or glycosides), are common food contaminants. Their acute toxicity is relatively low, while chronic exposure can lead to the development of adverse health effects. Masked/modified metabolites can probably release the [...] Read more.

Alternaria mycotoxins, including alternariol (AOH), alternariol-9-monomethylether (AME), and their masked/modified derivatives (e.g., sulfates or glycosides), are common food contaminants. Their acute toxicity is relatively low, while chronic exposure can lead to the development of adverse health effects. Masked/modified metabolites can probably release the more toxic parent mycotoxin due to their enzymatic hydrolysis in the intestines. Previously, we demonstrated the complex formation of AOH with serum albumins and cyclodextrins; these interactions were successfully applied for the extraction of AOH from aqueous matrices (including beverages). Therefore, in this study, the interactions of AME, alternariol-3-sulfate (AS), and alternariol-9-monomethylether-3-sulfate (AMS) were investigated with albumins (human, bovine, porcine, and rat) and with cyclodextrins (sulfobutylether-β-cyclodextrin, sugammadex, and cyclodextrin bead polymers). Our major results/conclusions are the following: (1) The stability of mycotoxin–albumin complexes showed only minor species dependent variations. (2) AS and AMS formed highly stable complexes with albumins in a wide pH range, while AME–albumin interactions preferred alkaline conditions. (3) AME formed more stable complexes with the cyclodextrins examined than AS and AMS. (4) Beta-cyclodextrin bead polymer proved to be highly suitable for the extraction of AME, AS, and AMS from aqueous solution. (5) Albumins and cyclodextrins are promising binders of the mycotoxins tested. Full article

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Open AccessArticle

Benzenesulfonamides Incorporating Hydantoin Moieties Effectively Inhibit Eukaryoticand Human Carbonic Anhydrases

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Int. J. Mol. Sci. 2022, 23(22), 14115; https://doi.org/10.3390/ijms232214115 - 15 Nov 2022

Cited by 8 | Viewed by 1105

Abstract

A series of novel 1-(4-benzenesulfonamide)-3-alkyl/benzyl-hydantoin derivatives were synthesized and evaluated for the inhibition of eukaryotic and human carbonic anhydrases (CAs, EC 4.2.1.1). The prepared compounds were screened for their hCA inhibitory activities against three cytosolic isoforms as well as two β-CAs from fungal [...] Read more.

A series of novel 1-(4-benzenesulfonamide)-3-alkyl/benzyl-hydantoin derivatives were synthesized and evaluated for the inhibition of eukaryotic and human carbonic anhydrases (CAs, EC 4.2.1.1). The prepared compounds were screened for their hCA inhibitory activities against three cytosolic isoforms as well as two β-CAs from fungal pathogens. The best inhibition was observed against hCA II and VII as well as Candida glabrata enzyme CgNce103. hCA I and Malassezia globosa MgCA enzymes were, on the other hand, less effectively inhibited by these compounds. The inhibitory potency of these compounds against CAs was found to be dependent on the electronic and steric effects of substituent groups on the N3-position of the hydantoin ring, which included alkyl, alkenyl and substituted benzyl moieties. The interesting results against CgNce103 make the compounds of interest for investigations in vivo as potential antifungals. Full article

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Open AccessReview

The bHLH Transcription Factors in Neural Development and Therapeutic Applications for Neurodegenerative Diseases

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Dong Gi Lee

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Young-Kwang Kim

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Kwang-Hyun Baek

Int. J. Mol. Sci. 2022, 23(22), 13936; https://doi.org/10.3390/ijms232213936 - 11 Nov 2022

Cited by 2 | Viewed by 2779

Abstract

The development of functional neural circuits in the central nervous system (CNS) requires the production of sufficient numbers of various types of neurons and glial cells, such as astrocytes and oligodendrocytes, at the appropriate periods and regions. Hence, severe neuronal loss of the [...] Read more.

The development of functional neural circuits in the central nervous system (CNS) requires the production of sufficient numbers of various types of neurons and glial cells, such as astrocytes and oligodendrocytes, at the appropriate periods and regions. Hence, severe neuronal loss of the circuits can cause neurodegenerative diseases such as Huntington’s disease (HD), Parkinson’s disease (PD), Alzheimer’s disease (AD), and Amyotrophic Lateral Sclerosis (ALS). Treatment of such neurodegenerative diseases caused by neuronal loss includes some strategies of cell therapy employing stem cells (such as neural progenitor cells (NPCs)) and gene therapy through cell fate conversion. In this report, we review how bHLH acts as a regulator in neuronal differentiation, reprogramming, and cell fate determination. Moreover, several different researchers are conducting studies to determine the importance of bHLH factors to direct neuronal and glial cell fate specification and differentiation. Therefore, we also investigated the limitations and future directions of conversion or transdifferentiation using bHLH factors. Full article

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Open AccessArticle

A Note on the Effects of Linear Topology Preservation in Monte Carlo Simulations of Knotted Proteins

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João N. C. Especial

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Antonio Rey

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Patrícia F. N. Faísca

Int. J. Mol. Sci. 2022, 23(22), 13871; https://doi.org/10.3390/ijms232213871 - 10 Nov 2022

Cited by 1 | Viewed by 939

Abstract

Monte Carlo simulations are a powerful technique and are widely used in different fields. When applied to complex molecular systems with long chains, such as those in synthetic polymers and proteins, they have the advantage of providing a fast and computationally efficient way [...] Read more.

Monte Carlo simulations are a powerful technique and are widely used in different fields. When applied to complex molecular systems with long chains, such as those in synthetic polymers and proteins, they have the advantage of providing a fast and computationally efficient way to sample equilibrium ensembles and calculate thermodynamic and structural properties under desired conditions. Conformational Monte Carlo techniques employ a move set to perform the transitions in the simulation Markov chain. While accepted conformations must preserve the sequential bonding of the protein chain model and excluded volume among its units, the moves themselves may take the chain across itself. We call this a break in linear topology preservation. In this manuscript, we show, using simple protein models, that there is no difference in equilibrium properties calculated with a move set that preserves linear topology and one that does not. However, for complex structures, such as those of deeply knotted proteins, the preservation of linear topology provides correct equilibrium results but only after long relaxation. In any case, to analyze folding pathways, knotting mechanisms and folding kinetics, the preservation of linear topology may be an unavoidable requirement. Full article

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Open AccessReview

Biomolecular Profiling by MALDI-TOF Mass Spectrometry in Food and Beverage Analyses

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Marek Šebela

Int. J. Mol. Sci. 2022, 23(21), 13631; https://doi.org/10.3390/ijms232113631 - 07 Nov 2022

Cited by 4 | Viewed by 2250

Abstract

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has frequently been applied to the analysis of biomolecules. Its strength resides not only in compound identification but particularly in acquiring molecular profiles providing a high discriminating power. The main advantages include its speed, simplicity, [...] Read more.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has frequently been applied to the analysis of biomolecules. Its strength resides not only in compound identification but particularly in acquiring molecular profiles providing a high discriminating power. The main advantages include its speed, simplicity, versatility, minimum sample preparation needs, and a relatively high tolerance to salts. Other benefits are represented by the possibility of automation, high throughput, sensitivity, accuracy, and good reproducibility, allowing quantitative studies. This review deals with the prominent use of MALDI-TOF MS profiling in food and beverage analysis ranging from the simple detection of sample constituents to quantifications of marker compounds, quality control, and assessment of product authenticity. This review summarizes relevant discoveries that have been obtained with milk and milk products, edible oils, wine, beer, flour, meat, honey, and other alimentary products. Marker molecules are specified: proteins and peptides for milk, cheeses, flour, meat, wine and beer; triacylglycerols and phospholipids for oils; and low-molecular-weight metabolites for wine, beer and chocolate. Special attention is paid to sample preparation techniques and the combination of spectral profiling and statistical evaluation methods, which is powerful for the differentiation of samples and the sensitive detection of frauds and adulterations. Full article

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Graphical abstract

Open AccessArticle

Potential of Vitamin B6 Dioxime Analogues to Act as Cholinesterase Ligands

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Int. J. Mol. Sci. 2022, 23(21), 13388; https://doi.org/10.3390/ijms232113388 - 02 Nov 2022

Viewed by 1250

Abstract

Seven pyridoxal dioxime quaternary salts (1–7) were synthesized with the aim of studying their interactions with human acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). The synthesis was achieved by the quaternization of pyridoxal monooxime with substituted 2-bromoacetophenone oximes (phenacyl bromide oximes). [...] Read more.

Seven pyridoxal dioxime quaternary salts (1–7) were synthesized with the aim of studying their interactions with human acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). The synthesis was achieved by the quaternization of pyridoxal monooxime with substituted 2-bromoacetophenone oximes (phenacyl bromide oximes). All compounds, prepared in good yields (43–76%) and characterized by 1D and 2D NMR spectroscopy, were evaluated as reversible inhibitors of cholinesterase and/or reactivators of enzymes inhibited by toxic organophosphorus compounds. Their potency was compared with that of their monooxime analogues and medically approved oxime HI-6. The obtained pyridoxal dioximes were relatively weak inhibitors for both enzymes (K_i = 100–400 µM). The second oxime group in the structure did not improve the binding compared to the monooxime analogues. The same was observed for reactivation of VX-, tabun-, and paraoxon-inhibited AChE and BChE, where no significant efficiency burst was noted. In silico analysis and molecular docking studies connected the kinetic data to the structural features of the tested compound, showing that the low binding affinity and reactivation efficacy may be a consequence of a bulk structure hindering important reactive groups. The tested dioximes were non-toxic to human neuroblastoma cells (SH-SY5Y) and human embryonal kidney cells (HEK293). Full article

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Open AccessArticle

Polysaccharides of Salsola passerina: Extraction, Structural Characterization and Antioxidant Activity

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Alexander S. Shashkov

Int. J. Mol. Sci. 2022, 23(21), 13175; https://doi.org/10.3390/ijms232113175 - 29 Oct 2022

Cited by 4 | Viewed by 1210

Abstract

The above-ground part of the Salsola passerine was found to contain ~13% (w/w) of polysaccharides extractable with water and aqueous solutions of ammonium oxalate and sodium carbonate. The fractions extracted with aqueous sodium carbonate solutions had the highest yield. [...] Read more.

The above-ground part of the Salsola passerine was found to contain ~13% (w/w) of polysaccharides extractable with water and aqueous solutions of ammonium oxalate and sodium carbonate. The fractions extracted with aqueous sodium carbonate solutions had the highest yield. The polysaccharides of majority fractions are characterized by similar monosaccharide composition; namely, galacturonic acid and arabinose residues are the principal components of their carbohydrate chains. The present study focused on the determination of antioxidant activity of the extracted polysaccharide fractions and elucidation of the structure of polysaccharides using nuclear magnetic resonance (NMR) spectroscopy. Homogalacturonan (HG), consisting of 1,4-linked residues of α-D-galactopyranosyluronic acid (GalpA), rhamnogalacturonan-I (RG-I), which contains a diglycosyl repeating unit with a strictly alternating sequence of 1,4-linked D-GalpA and 1,2-linked L-rhamnopyranose (Rhap) residues in the backbone, and arabinan, were identified as the structural units of the obtained polysaccharides. HMBC spectra showed that arabinan consisted of alternating regions formed by 3,5-substituted and 1,5-linked arabinofuranose residues, but there was no alternation of these residues in the arabinan structure. Polysaccharide fractions scavenged the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical at 0.2–1.8 mg/mL. The correlation analysis showed that the DPPH scavenging activity of polysaccharide fractions was associated with the content of phenolic compounds (PCs). Full article

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Open AccessArticle

Analysis of the Site-Specific Myoglobin Modifications in the Melibiose-Derived Novel Advanced Glycation End-Product

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Kinga Gostomska-Pampuch

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Magdalena Staniszewska

Int. J. Mol. Sci. 2022, 23(21), 13036; https://doi.org/10.3390/ijms232113036 - 27 Oct 2022

Viewed by 975

Abstract

MAGE (melibiose-derived advanced glycation end-product) is the glycation product generated in the reaction of a model protein with melibiose. The in vivo analog accumulates in several tissues; however, its origin still needs explanation. In vitro MAGE is efficiently generated under dry conditions in [...] Read more.

MAGE (melibiose-derived advanced glycation end-product) is the glycation product generated in the reaction of a model protein with melibiose. The in vivo analog accumulates in several tissues; however, its origin still needs explanation. In vitro MAGE is efficiently generated under dry conditions in contrast to the reaction carried in an aqueous solvent. Using liquid chromatography coupled with mass spectrometry, we analyzed the physicochemical properties and structures of myoglobin glycated with melibiose under different conditions. The targeted peptide analysis identified structurally different AGEs, including crosslinking and non-crosslinking modifications associated with lysine, arginine, and histidine residues. Glycation in a dry state was more efficient in the formation of structures containing an intact melibiose moiety (21.9%) compared to glycation under aqueous conditions (15.6%). The difference was reflected in characteristic fluorescence that results from protein structural changes and impact on a heme group of the model myoglobin protein. Finally, our results suggest that the formation of in vitro MAGE adduct is initiated by coupling melibiose to a model myoglobin protein. It is confirmed by the identification of intact melibiose moieties. The intermediate glycation product can further rearrange towards more advanced structures, including cross-links. This process can contribute to a pool of AGEs accumulating locally in vivo and affecting tissue biology. Full article

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Open AccessArticle

Identification of Molecular Determinants in iRhoms1 and 2 That Contribute to the Substrate Selectivity of Stimulated ADAM17

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Ariana I. Rabinowitsch

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Jose Manuel Perez-Aguilar

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Carl P. Blobel

Int. J. Mol. Sci. 2022, 23(21), 12796; https://doi.org/10.3390/ijms232112796 - 24 Oct 2022

Cited by 3 | Viewed by 1427

Abstract

The metalloprotease ADAM17 is a key regulator of the TNFα, IL-6R and EGFR signaling pathways. The maturation and function of ADAM17 is controlled by the seven-membrane-spanning proteins iRhoms1 and 2. The functional properties of the ADAM17/iRhom1 and ADAM17/iRhom2 complexes differ, in that stimulated [...] Read more.

The metalloprotease ADAM17 is a key regulator of the TNFα, IL-6R and EGFR signaling pathways. The maturation and function of ADAM17 is controlled by the seven-membrane-spanning proteins iRhoms1 and 2. The functional properties of the ADAM17/iRhom1 and ADAM17/iRhom2 complexes differ, in that stimulated shedding of most ADAM17 substrates tested to date can be supported by iRhom2, whereas iRhom1 can only support stimulated shedding of very few ADAM17 substrates, such as TGFα. The first transmembrane domain (TMD1) of iRhom2 and the sole TMD of ADAM17 are important for the stimulated shedding of ADAM17 substrates by iRhom2. However, little is currently known about how the iRhoms interact with different substrates to control their stimulated shedding by ADAM17. To provide new insights into this topic, we tested how various chimeras between iRhom1 and iRhom2 affect the stimulated processing of the EGFR-ligands TGFα (iRhom1- or 2-dependent) and EREG (iRhom2-selective) by ADAM17. This uncovered an important role for the TMD7 of the iRhoms in determining their substrate selectivity. Computational methods utilized to characterize the iRhom1/2/substrate interactions suggest that the substrate selectivity is determined, at least in part, by a distinct accessibility of the substrate cleavage site to stimulated ADAM17. These studies not only provide new insights into why the substrate selectivity of stimulated iRhom2/ADAM17 differs from that of iRhom1/ADAM17, but also suggest new approaches for targeting the release of specific ADAM17 substrates. Full article

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Open AccessArticle

Lack of NPR1 Increases Vascular Endothelial Adhesion through Induction of Integrin Beta 4

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Int. J. Mol. Sci. 2022, 23(20), 12627; https://doi.org/10.3390/ijms232012627 - 20 Oct 2022

Viewed by 1424

Abstract

Natriuretic peptide receptor 1 (NPR1) serves as a modulator of vascular endothelial homeostasis. Interactions between monocytes and endothelial cells may initiate endothelium dysfunction, which is known as an early hallmark of atherosclerosis. In this study, we performed RNA-sequencing analysis for the aorta of [...] Read more.

Natriuretic peptide receptor 1 (NPR1) serves as a modulator of vascular endothelial homeostasis. Interactions between monocytes and endothelial cells may initiate endothelium dysfunction, which is known as an early hallmark of atherosclerosis. In this study, we performed RNA-sequencing analysis for the aorta of Npr1 knockout (Npr1^+/−) mice and found that differentially expressed genes were significantly related to cell adhesion. This result was supported by an increased expression of intercellular adhesion molecule 1 (ICAM-1) in the aortic endothelium of Npr1^+/− mice. Moreover, we observed that the knockdown of NPR1 increased ICAM-1 expression and promoted THP-1 monocyte adhesion to human umbilical vein endothelial cells (HUVECs). NPR1 overexpression decreased ICAM-1 expression and inhibited the adhesion of monocytes to HUVECs treated by TNF-α (a cell adhesion inducer). Further analysis showed that adhesion-related genes were enriched in the focal adhesion signaling pathway, in which integrin beta 4 (Itgb4) was determined as a key gene. Notably, ITGB4 expression increased in vascular endothelium of Npr1^+/− mice and in NPR1-knockdown HUVECs. The deficiency of ITGB4 decreased ICAM-1 expression and attenuated monocyte adhesion to NPR1-knockdown endothelial cells. Additionally, a reduced NPR1 and an increased ITGB4 expression level were found in an atherosclerosis mouse model. In conclusion, our findings demonstrate that NPR1 deficiency increases vascular endothelial cell adhesion by stimulating ITGB4 expression, which may contribute to the development of atherosclerosis. Full article

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Open AccessArticle

Mutation Hotspot for Changing the Substrate Specificity of β-N-Acetylhexosaminidase: A Library of GlcNAcases

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Int. J. Mol. Sci. 2022, 23(20), 12456; https://doi.org/10.3390/ijms232012456 - 18 Oct 2022

Viewed by 1139

Abstract

β-N-Acetylhexosaminidase from Talaromyces flavus (TfHex; EC 3.2.1.52) is an exo-glycosidase with dual activity for cleaving N-acetylglucosamine (GlcNAc) and N-acetylgalactosamine (GalNAc) units from carbohydrates. By targeting a mutation hotspot of the active site residue Glu332, we prepared [...] Read more.

β-N-Acetylhexosaminidase from Talaromyces flavus (TfHex; EC 3.2.1.52) is an exo-glycosidase with dual activity for cleaving N-acetylglucosamine (GlcNAc) and N-acetylgalactosamine (GalNAc) units from carbohydrates. By targeting a mutation hotspot of the active site residue Glu332, we prepared a library of ten mutant variants with their substrate specificity significantly shifted towards GlcNAcase activity. Suitable mutations were identified by in silico methods. We optimized a microtiter plate screening method in the yeast Pichia pastoris expression system, which is required for the correct folding of tetrameric fungal β-N-acetylhexosaminidases. While the wild-type TfHex is promiscuous with its GalNAcase/GlcNAcase activity ratio of 1.2, the best single mutant variant Glu332His featured an 8-fold increase in selectivity toward GlcNAc compared with the wild-type. Several prepared variants, in particular Glu332Thr TfHex, had significantly stronger transglycosylation capabilities than the wild-type, affording longer chitooligomers – they behaved like transglycosidases. This study demonstrates the potential of mutagenesis to alter the substrate specificity of glycosidases. Full article

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