Chemical Profiling of Astragalus membranaceus Roots (Fish.) Bunge Herbal Preparation and Evaluation of Its Bioactivity

Astragalus membranaceus (Fish.) Bunge is a perennial herb distributed in the northern part of China, and its roots, namely, Hang qi, are included as a natural ingredient in dietary supplement formulations commonly used to treat different disorders such as respiratory infections, diabetes, and heart failure. The availability of a simple method for the determination of the quality of Astragalus herbal preparations could be a challenging issue for commercial purposes. In this study, a liquid chromatography–mass spectrometry (LC–MS)/MS based approach was used to characterize specialized metabolite recovery of 3 commercial hydroalcoholic extracts of A. membranaceus (AMG1, AMG2, AMG3) in addition to a hydroalcoholic extract of A. membranaceus root (AST). The hypoglycemic effect, cholinesterase inhibition, and antioxidant activities were also evaluated. Thirty-one compounds, of which 19 polyphenols and 12 saponins, were identified. The extracts were also quantified by using a sensitive and selective Q-Trap system for their content in flavonoids and astragalosides, selecting astragaloside I and IV as chemical markers. From our results, AMG3 preparation (Axtragyl) was the most abundant in terms of both specialized classes of metabolites, showing a fingerprint similar to that of AST. Interestingly, tested enzyme inhibition ability of flavonoids, daidzein (11) and formononetin (19), reported a higher α-glucosidase inhibition in comparison with that of acarbose used as positive control. The in silico study clarified the interactions among the molecules and the importance of having a free hydroxy group. Moreover, Axtragyl was able to exert protective effects in Caco-2 cells treated with hydrogen peroxide, confirming its ability as a potential protective agent in intestinal injury

Federica Giuzio*, Maria Grazia Bonomo, Alessia Catalano*, Vittoria Infantino, Giovanni Salzano, Magnus Monné, Athina Geronikaki, Anthi Petrou, Stefano Aquaro, Maria Stefania Sinicropi#, Carmela Saturnino#

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is an RNA virus belonging to the coronavirus family responsible for coronavirus disease 2019 (COVID-19). It primarily affects the pulmonary system, which is the target of chronic obstructive pulmonary disease (COPD), for which many new compounds have been developed. In this study, phosphodiesterase 4 (PDE4) inhibitors are being investigated. The inhibition of PDE4 enzyme produces antiinflammatory and bronchodilator effects in the lung by inducing an increase in cAMP concentrations. Piclamilast and rolipram are known selective inhibitors of PDE4, which are unfortunately endowed with common side effects, such as nausea and emesis. The selective inhibition of the phosphodiesterase 4B (PDE4B) subtype may represent an intriguing technique for combating this highly contagious disease with fewer side effects. In this article, molecular docking studies for the selective inhibition of the PDE4B enzyme have been carried out on 21 in-house compounds. The compounds were docked into the pocket of the PDE4B catalytic site, and in most cases, they were almost completely superimposed onto piclamilast. Then, in order to enlarge our study, drug-likeness prediction studies were performed on the compounds under study

Antiporters of the Mitochondrial Carrier Family

The eukaryotic transport protein family SLC25 consists of mitochondrial carriers (MCs) that are recognized on the sequence level by a threefold repeated and conserved signature motif. The majority of MCs characterized so far catalyzes strict exchanges of substrates across the mitochondrial inner membrane. The substrates are nucleotides, metabolic intermediates, and cofactors that are required in cytoplasmic and matrix metabolism. This review summarizes and discusses the current knowledge of the antiport mechanism(s) of MCs that has been deduced from determining transport characteristics and by analyzing structural, sequence, and mutagenesis data. The mode of transport varies among different MCs with respect to how the substrate translocation depends on the electrical and pH gradients across the mitochondrial inner membrane, for example, the ADP/ATP carrier is electrogenic (electrophoretic), the GTP/GDP carrier is dependent on the pH gradient, the aspartate/glutamate carrier is dependent on both, and the oxoglutarate/malate carrier is independent of them. The structure of the bovine ADP/ATP carrier consists of a six-transmembrane α-helix bundle with a pseudo-threefold symmetry and a closed matrix gate. By using this structure as a template in homology modeling, residues engaged in substrate binding and the formation of a cytoplasmic gate in MCs have been proposed. The functional importance of the residues of the binding site, the matrix, and the cytoplasmic gates is supported by transport activities of different MCs with single point mutations. Cumulative evidence has been used to postulate a general transport mechanism for MCs

Discoveries, metabolic roles and diseases of mitochondrial carriers: A review

Mitochondrial carriers (MCs) are a superfamily of nuclear-encoded proteins that are mostly localized in the inner mitochondrial membrane and transport numerous metabolites, nucleotides, cofactors and inorganic anions. Their unique sequence features, i.e., a tripartite structure, six transmembrane α-helices and a three-fold repeated signature motif, allow MCs to be easily recognized. This review describes how the functions of MCs from Saccharomyces cerevisiae, Homo sapiens and Arabidopsis thaliana (listed in the first table) were discovered after the genome sequence of S. cerevisiae was determined in 1996. In the genomic era, more than 50 previously unknown MCs from these organisms have been identified and characterized biochemically using a method consisting of gene expression, purification of the recombinant proteins, their reconstitution into liposomes and transport assays (EPRA). Information derived from studies with intact mitochondria, genetic and metabolic evidence, sequence similarity, phylogenetic analysis and complementation of knockout phenotypes have guided the choice of substrates that were tested in the transport assays. In addition, the diseases associated to defects of human MCs have been briefly reviewed

Diseases Caused by Mutations in Mitochondrial Carrier Genes SLC25: A Review

In the 1980s, after the mitochondrial DNA (mtDNA) had been sequenced, several diseases resulting from mtDNA mutations emerged. Later, numerous disorders caused by mutations in the nuclear genes encoding mitochondrial proteins were found. A group of these diseases are due to defects of mitochondrial carriers, a family of proteins named solute carrier family 25 (SLC25), that transport a variety of solutes such as the reagents of ATP synthase (ATP, ADP, and phosphate), tricarboxylic acid cycle intermediates, cofactors, amino acids, and carnitine esters of fatty acids. The disease-causing mutations disclosed in mitochondrial carriers range from point mutations, which are often localized in the substrate translocation pore of the carrier, to large deletions and insertions. The biochemical consequences of deficient transport are the compartmentalized accumulation of the substrates and dysfunctional mitochondrial and cellular metabolism, which frequently develop into various forms of myopathy, encephalopathy, or neuropathy. Examples of diseases, due to mitochondrial carrier mutations are: combined D-2- and L-2-hydroxyglutaric aciduria, carnitine-acylcarnitine carrier deficiency, hyperornithinemia-hyperammonemia-homocitrillinuria (HHH) syndrome, early infantile epileptic encephalopathy type 3, Amish microcephaly, aspartate/glutamate isoform 1 deficiency, congenital sideroblastic anemia, Fontaine progeroid syndrome, and citrullinemia type II. Here, we review all the mitochondrial carrier-related diseases known until now, focusing on the connections between the molecular basis, altered metabolism, and phenotypes of these inherited disorders

Mitochondrial transport and metabolism of the vitamin B-derived cofactors thiamine pyrophosphate, coenzyme A, FAD and NAD+ , and related diseases: A review

Multiple mitochondrial matrix enzymes playing key roles in metabolism require cofactors for their action. Due to the high impermeability of the mitochondrial inner membrane, these cofactors need to be synthesized within the mitochondria or be imported, themselves or one of their precursors, into the organelles. Transporters belonging to the protein family of mitochondrial carriers have been identified to transport the coenzymes: thiamine pyrophosphate, coenzyme A, FAD and NAD(+), which are all structurally similar to nucleotides and derived from different B-vitamins. These mitochondrial cofactors bind more or less tightly to their enzymes and, after having been involved in a specific reaction step, are regenerated, spontaneously or by other enzymes, to return to their active form, ready for the next catalysis round. Disease-causing mutations in the mitochondrial cofactor carrier genes compromise not only the transport reaction but also the activity of all mitochondrial enzymes using that particular cofactor and the metabolic pathways in which the cofactor-dependent enzymes are involved. The mitochondrial transport, metabolism and diseases of the cofactors thiamine pyrophosphate, coenzyme A, FAD and NAD(+) are the focus of this review

Mitochondrial Carriers for Aspartate, Glutamate and Other Amino Acids: A Review

Members of the mitochondrial carrier (MC) protein family transport various molecules across the mitochondrial inner membrane to interlink steps of metabolic pathways and biochemical processes that take place in different compartments; i.e., are localized partly inside and outside the mitochondrial matrix. MC substrates consist of metabolites, inorganic anions (such as phosphate and sulfate), nucleotides, cofactors and amino acids. These compounds have been identified by in vitro transport assays based on the uptake of radioactively labeled substrates into liposomes reconstituted with recombinant purified MCs. By using this approach, 18 human, plant and yeast MCs for amino acids have been characterized and shown to transport aspartate, glutamate, ornithine, arginine, lysine, histidine, citrulline and glycine with varying substrate specificities, kinetics, influences of the pH gradient, and capacities for the antiport and uniport mode of transport. Aside from providing amino acids for mitochondrial translation, the transport reactions catalyzed by these MCs are crucial in energy, nitrogen, nucleotide and amino acid metabolism. In this review we dissect the transport properties, phylogeny, regulation and expression levels in different tissues of MCs for amino acids, and summarize the main structural aspects known until now about MCs. The effects of their disease-causing mutations and manipulation of their expression levels in cells are also considered as clues for understanding their physiological functions

Stop-transfer efficiency of marginally hydrophobic segments depends on the length of the carboxy-terminal tail

Hydrophobic stop-transfer sequences generally serve to halt the translocation of polypeptide chains across the endoplasmic reticulum membrane and become integrated as transmembrane α-helices. Using engineered glycosylation sites as topology reporters, we show that the length of the nascent chain between a hydrophobic segment and the carboxy terminus of the protein can affect stop-transfer efficiency. We also show that glycosylation sites located close to a protein's C terminus are modified in two distinct kinetic phases, one fast and one slow. Our findings suggest that membrane integration of a hydrophobic segment is not simply a question of thermodynamic equilibrium, but can be influenced by details of the translocation mechanism