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The Homo sapiens Na+/H+ antiporter NHA2 (SLC9B2) transports Na+ or Li+ in exchange for protons across cell membranes, and its dysfunction results in various pathologies. The activity of HsNHA2 is specifically inhibited by the flavonoid phloretin. Using bioinformatic modeling, we predicted two amino acids (R177 and S178) as being important for the binding of phloretin to the HsNHA2 molecule. Functional expression of HsNHA2 in Saccharomyces cerevisiae and its site‐directed mutagenesis revealed that while the R177T mutation resulted in an antiporter that was less sensitive to phloretin, the S178T mutation enhanced the inhibitory effect of phloretin on HsNHA2. Our data corroborate the transport properties of HsNHA2 and its interactions with an inhibitor and can be helpful for the development of new therapeutics targeting this antiporter and its pleiotropic physiological functions. [ABSTRACT FROM AUTHOR]

GOLM1, a Golgi membrane protein, is upregulated in cancers and liver diseases. Analysis of public RNAseq data from healthy human liver suggested that GOLM1 is predominantly expressed in cholangiocytes. Therefore, this study was initiated to understand the molecular functions of GOLM1 in cholangiocytes through protein interactomics. The findings reveal a number of putative GOLM1-interacting partners involved in cellular regimes such as mitochondrial and Golgi functions, ribonucleoprotein biogenesis, cell cycle, and basement membrane organization. Further, to validate select key roles, GOLM1 was silenced in MMNK-1 cholangiocytes and the effects on cell functions were studied. The silencing resulted in impaired mitochondrial function, reduced mitochondrial and P-body markers, increased apoptosis, and reduced cell adhesion, suggesting crucial roles of GOLM1 in maintaining normal cholangiocyte metabolism and function., (© 2025 Federation of European Biochemical Societies.)

In all vertebrates including mammals, the ergothioneine transporter ETT (obsolete name OCTN1; human gene symbol SLC22A4) is a powerful and highly specific transporter for the uptake of ergothioneine (ET). ETT is not expressed ubiquitously and only cells with high ETT cell-surface levels can accumulate ET to high concentration. Without ETT, there is no uptake because the plasma membrane is essentially impermeable to this hydrophilic zwitterion. Here, we review the substrate specificity and localization of ETT, which is prominently expressed in neutrophils, monocytes/macrophages, and developing erythrocytes. Most sites of strong expression are conserved across species, but there are also major differences. In particular, we critically analyze the evidence for the expression of ETT in the brain as well as recent data suggesting that the transporter SLC22A15 may also transport ET. We conclude that, to date, ETT remains the only well-defined biomarker for intracellular ET activity. In humans, the ability to take up, distribute, and retain ET depends principally on this transporter., (© 2022 Federation of European Biochemical Societies.)

Ergothioneine (ERGO) is a naturally occurring food-derived antioxidant. Despite its extremely hydrophilic properties, ERGO is easily absorbed from the gastrointestinal tract and distributed to various organs, including the brain. This is primarily because its entry into brain cells is mediated by the ERGO-specific transporter OCTN1/SLC22A4. Octn1 gene knockout mice do not have ERGO in the brain, due to the absence of OCTN1 in neurons, neural stem cells, and microglia. The existence of OCTN1 and uptake of ERGO into the brain parenchymal cells may suggest that ERGO and its transporter play a pivotal role in brain function. Oral administration of ERGO has antidepressant activities in mice. Furthermore, repeated oral administration of ERGO and ERGO-containing food extract tablets enhance memory function in mice and humans, respectively. ERGO also protects against stress-induced sleep disturbance and neuronal injury induced by amyloid β in rodents. In vitro observations suggest that ERGO benefits brain function through both its antioxidative activity and by promoting neurogenesis and neuronal maturation. This review discusses the possible involvement of ERGO in brain function and its potential therapeutic properties., (© 2022 Federation of European Biochemical Societies.)

SLC35A2 and SLC35A3 are homologous proteins with postulated nucleotide sugar transporting activities. Unlike SLC35A2, whose specificity for UDP‐Gal is well‐established, the UDP‐GlcNAc transporting activity initially attributed to SLC35A3 has recently been put into question. In this study, we constructed two hybrid proteins (SLC35A2–SLC35A3 and SLC35A3–SLC35A2) and expressed them in a previously generated SLC35A2/SLC35A3 double knockout HEK293T cell line. Our idea was to force equivalent stoichiometry of the two proteins in the cells in order to reproduce the behavior of the SLC35A2/SLC35A3 complexes in the Golgi membrane. The hybrid proteins were able to fully restore glycosylation in the double knockout. In contrast, the expression of SLC35A3 alone in these cells improved galactosylation only to a limited extent. Our study shows that the proper glycosylation requires a balanced cooperation between SLC35A2 and SLC35A3. [ABSTRACT FROM AUTHOR]

The cell membrane of Halobacterium salinarum contains a retinal‐binding photoreceptor, sensory rhodopsin II (HsSRII), coupled with its cognate transducer (HsHtrII), allowing repellent phototaxis behavior for shorter wavelength light. Previous studies on SRII from Natronomonas pharaonis (NpSRII) pointed out the importance of the hydrogen bonding interaction between Thr204NpSRII and Tyr174NpSRII in signal transfer from SRII to HtrII. Here, we investigated the effect on phototactic function by replacing residues in HsSRII corresponding to Thr204NpSRII and Tyr174NpSRII. Whereas replacement of either residue altered the photocycle kinetics, introduction of any mutations at Ser201HsSRII and Tyr171HsSRII did not eliminate negative phototaxis function. These observations imply the possibility of the presence of an unidentified molecular mechanism for photophobic signal transduction differing from NpSRII–NpHtrII. [ABSTRACT FROM AUTHOR]

Mycobacterium tuberculosis (MTB) is the etiologic agent of tuberculosis (TB), an ancient disease which causes 1.5 million deaths worldwide. Dihydroorotate dehydrogenase (DHODH) is a key enzyme of the MTB de novo pyrimidine biosynthesis pathway, and it is essential for MTB growth in vitro, hence representing a promising drug target. We present: (i) the biochemical characterization of the full‐length MTB DHODH, including the analysis of the kinetic parameters, and (ii) the previously unreleased crystal structure of the protein that allowed us to rationally screen our in‐house chemical library and identify the first selective inhibitor of mycobacterial DHODH. The inhibitor has fluorescence properties, potentially instrumental to in cellulo imaging studies, and exhibits an IC50 value of 43 μm, paving the way to hit‐to‐lead process. [ABSTRACT FROM AUTHOR]

GPRC5C is an orphan G protein‐coupled receptor (GPCR) that belongs to the class C GPCR family. Although GPRC5C is expressed in various organs, its function and ligand are still undetermined. We found that GPRC5C is expressed in mouse taste cells, enterocytes, and pancreatic α‐cells. In functional imaging assays, HEK293 cells heterologously expressing GPRC5C and the chimeric G protein α subunit Gα16‐gust44 showed robust intracellular Ca2+ increases in response to monosaccharides, disaccharides, and a sugar alcohol, but not an artificial sweetener or sweet‐tasting amino acid. Notably, Ca2+ increases occurred after washout, not during stimulation. Our findings suggest that GPRC5C has receptor properties which lead to novel 'off' responses to saccharide detachment and may work as an internal or external chemosensor specifically tuned to natural sugars. [ABSTRACT FROM AUTHOR]

Feruloyl esterases (FAEs) hydrolyze the ester bonds between hydroxycinnamic acids and arabinose residues of plant cell walls and exhibit considerable diversity in terms of substrate specificity. Here, we report the crystal structure of an FAE from Fusarium oxysporum (FoFaeC) at 1.7 Å resolution in complex with p‐coumaric acid, which is the first ligand‐bound structure of a tannase‐like FAE. Our data reveal local conformational changes around the active site upon ligand binding, suggesting alternation between an active and a resting state of the enzyme. A swinging tyrosine residue appears to be gating the substrate binding pocket, while the lid domain of the protein exerts substrate specificity by means of a well‐defined hydrophobic core that encases the phenyl moiety of the substrate. [ABSTRACT FROM AUTHOR]

Compartmentalisation of eukaryotic cells enables fundamental otherwise often incompatible cellular processes. Establishment and maintenance of distinct compartments in the cell relies not only on proteins, lipids and metabolites but also on small redox molecules. In particular, small redox molecules such as glutathione, NAD(P)H and hydrogen peroxide (H2O2) cooperate with protein partners in dedicated machineries to establish specific subcellular redox compartments with conditions that enable oxidative protein folding and redox signalling. Dysregulated redox homeostasis has been directly linked with a number of diseases including cancer, neurological disorders, cardiovascular diseases, obesity, metabolic diseases and ageing. In this review, we will summarise mechanisms regulating establishment and maintenance of redox homeostasis in the mitochondrial subcompartments of mammalian cells. [ABSTRACT FROM AUTHOR]

Metals are widely present in biological systems as simple ions or complex cofactors, and are involved in a variety of processes essential for life. Their transport inside cells and insertion into the binding sites of the proteins that need metals to function occur through complex and selective pathways involving dedicated multiprotein machineries specifically and transiently interacting with each other, often sharing the coordination of metal ions and/or cofactors. The understanding of these machineries requires integrated approaches, ranging from bioinformatics to experimental investigations, possibly in the cellular context. In this review, we report two case studies where the use of integrated in vitro and in cellulo approaches is necessary to clarify at atomic resolution essential aspects of metal trafficking in cells. [ABSTRACT FROM AUTHOR]

Previous studies have shown that amyloid‐β oligomers (AβO) bind with high affinity to cellular prion protein (PrPC). The AβO‐PrPC complex binds to cell‐surface co‐receptors, including the laminin receptor (67LR). Our current studies revealed that in Neuroscreen‐1 cells, 67LR is the major co‐receptor involved in the cellular uptake of AβO and AβΟ‐induced cell death. Both pharmacological (dibutyryl‐cAMP, forskolin and rolipram) and physiological (pituitary adenylate cyclase‐activating polypeptide) cAMP‐elevating agents decreased cell‐surface PrPC and 67LR, thereby attenuating the uptake of AβO and the resultant neuronal cell death. These cAMP protective effects are dependent on protein kinase A, but not dependent on the exchange protein directly activated by cAMP. Conceivably, cAMP protects neuronal cells from AβO‐induced cytotoxicity by decreasing cell‐surface‐associated PrPC and 67LR. [ABSTRACT FROM AUTHOR]

Ergothioneine (ERGO) is a naturally occurring food-derived antioxidant. Despite its extremely hydrophilic properties, ERGO is easily absorbed from the gastrointestinal tract and distributed to various organs, including the brain. This is primarily because its entry into brain cells is mediated by the ERGO-specific transporter OCTN1/SLC22A4. Octn1 gene knockout mice do not have ERGO in the brain, due to the absence of OCTN1 in neurons, neural stem cells, and microglia. The existence of OCTN1 and uptake of ERGO into the brain parenchymal cells may suggest that ERGO and its transporter play a pivotal role in brain function. Oral administration of ERGO has antidepressant activities in mice. Furthermore, repeated oral administration of ERGO and ERGO-containing food extract tablets enhance memory function in mice and humans, respectively. ERGO also protects against stress-induced sleep disturbance and neuronal injury induced by amyloid b in rodents. In vitro observations suggest that ERGO benefits brain function through both its antioxidative activity and by promoting neurogenesis and neuronal maturation. This review discusses the possible involvement of ERGO in brain function and its potential therapeutic properties. [ABSTRACT FROM AUTHOR]

Arylalkylamine N-acetyltransferase (aaNAT) in Aedes aegypti is primarily involved in cuticle pigmentation and formation. The reported arylalkylamine substrates are all primary amines. In this study, we report a novel substrate, a secondary amine, of Ae. aegypti aaNAT1. The recombinant aaNAT1 protein exhibited high activity to a secondary amine, epinephrine, which has not been reported for any aaNATs previously. Structure-activity relationship study demonstrated that aaNAT1 has an epinephrine-binding site, and molecular docking and dynamic simulation showed that epinephrine is quite stable in the active cavity. Further functional studies demonstrated that epinephrine affected mosquito fecundity, egg hatching and development. The new biochemical function of aaNAT1 in metabolizing epinephrine could reduce some negative effects of the compound in the mosquito. [ABSTRACT FROM AUTHOR]

Sticholysins are pore-forming toxins produced by the sea anemone Stichodactyla helianthus. When they encounter a sphingomyelin-containing membrane, these proteins bind to it and oligomerize, a process that ends in pore formation. Mounting evidence indicates that StnII can favour the activity of StnI. Previous results have shown that these two isotoxins can oligomerize together. Furthermore, StnII appeared to potentiate the activity of StnI through the membrane-binding step of the process. Hence, isotoxin interaction should occur prior to membrane encounter. Here, we have used analytical ultracentrifugation to investigate the oligomerization of Stns in solution, both separately and together. Our results indicate that while StnI seems to be more prone to oligomerize in water solution than StnII, a small percentage of StnII in StnI-StnII mixtures promotes oligomerization. [ABSTRACT FROM AUTHOR]

Cytoplasmic domains frequently promote functional assembly of multimeric ion channels. To investigate structural determinants of this process, we generated the ‘T1-chimera’ construct of the NaChBac sodium channel by truncating its C-terminal domain and splicing the T1-tetramerisation domain of the Kv1.2 channel to the N terminus. Purified T1-chimera channels were tetrameric, conducted Na+ when reconstituted into proteoliposomes, and were functionally blocked by the drug mibefradil. Both the T1-chimera and fulllength NaChBac had comparable expression levels in the membrane, whereas a NaChBac mutant lacking a cytoplasmic domain had greatly reduced membrane expression. Our findings support a model whereby bringing the transmembrane regions into close proximity enables their tetramerisation. This phenomenon is found with other channels, and thus, our findings substantiate this as a common assembly mechanism. [ABSTRACT FROM AUTHOR]

Arylalkylamine N-acetyltransferase (aaNAT) in Aedes aegypti is primarily involved in cuticle pigmentation and formation. The reported arylalkylamine substrates are all primary amines. In this study, we report a novel substrate, a secondary amine, of Ae. aegypti aaNAT1. The recombinant aaNAT1 protein exhibited high activity to a secondary amine, epinephrine, which has not been reported for any aaNATs previously. Structure-activity relationship study demonstrated that aaNAT1 has an epinephrine-binding site, and molecular docking and dynamic simulation showed that epinephrine is quite stable in the active cavity. Further functional studies demonstrated that epinephrine affected mosquito fecundity, egg hatching and development. The new biochemical function of aaNAT1 in metabolizing epinephrine could reduce some negative effects of the compound in the mosquito., (© 2022 Federation of European Biochemical Societies.)