Biochemical and Biophysical Research Communications

The mucosal chemoattractant C-X-C motif chemokine ligand 17 (CXCL17) was recently identified as a ligand for the orphan G protein-coupled receptor 25 (GPR25). Although CXCL17 orthologs have been identified in fishes, amphibians, and mammals, their presence in reptiles and birds remains unclear. In this study, we employed bioinformatic searches based on gene synteny and sequence features to identify CXCL17 orthologs in public databases. We identified functional CXCL17 orthologs in 46 reptilian species, including lizards, snakes, turtles, and alligators. In contrast, we found only non-functional gene relics in 22 bird species, suggesting the avian lineage lost functional CXCL17 during evolution. A recombinant reptilian CXCL17 from the loggerhead turtle (Caretta caretta), termed Cc-CXCL17, directly bound to and efficiently activated its corresponding receptor, Cc-GPR25, in a C-terminal fragment-dependent manner. Activation of Cc-GPR25 by Cc-CXCL17 also induced chemotactic movement of transfected human embryonic kidney (HEK) 293T cells. In cross-species activity assays, CXCL17s from human and tropical clawed frog could activate Cc-GPR25 albeit with lower potency, but fish orthologs lacked this activity; all tested CXCL17s had no detectable activity towards chicken GPR25, but Cc-CXCL17 had low activity towards mallard GPR25. Our findings demonstrate the presence of functional CXCL17 orthologs in extant reptiles and provide evidence for their evolutionary loss in birds, offering new insights into the phylogenetic distribution of the newly identified CXCL17-GPR25 signaling system.

The gastric peptide ghrelin and its receptor GHSR have important functions in energy metabolism. Recently, liver-expressed antimicrobial peptide 2 (LEAP2) was identified as an endogenous antagonist for GHSR. Ghrelin, LEAP2, and GHSR are ubiquitously present from fishes to mammals and are highly conserved in evolution. However, our recent study suggested that GHSRs from the Actinopterygii fish Danio rerio (zebrafish) and Larimichthys crocea (large yellow croaker) have lost their binding to ghrelin, despite binding normally to LEAP2. Do these fish GHSRs use another peptide as their agonist? To answer this question, in the present study, we tested to two fish motilins that are closely related to ghrelin. In ligand binding and activation assays, the fish GHSRs from D. rerio and L. crocea displayed no detectable or very low binding to all tested motilins; however, the GHSR from the Sarcopterygii fish Latimeria chalumnae (coelacanth) bound to its motilin with high affinity and was efficiently activated by it. Therefore, it seemed that motilin is not a ligand for GHSR in D. rerio and L. crocea, but is an efficient agonist for GHSR in L. chalumnae, which is known as a living fossil and is believed to be one of the closest fish ancestors of tetrapods. The results of present study suggested that in ancient fishes, GHSR had two efficient agonists, ghrelin and motilin; however, this feature might be only preserved in some extant fishes with ancient evolutionary origins. Our present work shed new light on the ligand usage of GHSR in different fish species and in evolution.

Taxonomic status of house mice in the Korean Peninsula remains poorly understood. Here, we analyze genetic and morphological characteristics of mice from Korea and evaluate their phylogenetic relationships to the well-known primary subspecies. Using a comprehensive set of publicly available genetic data (mtDNA cytb gene), Korean mice including our specimens from islands, mountains, and agricultural fields were identified to Mus mus musculus. External morphology, such as tail ratios of our specimens, resembled previously assigned subspecies (e.g., M. m. molossinus, M. m. utsuryonis, and M. m. yamashinai), suggesting a single subspecific group within M. m. musculus. Korean mice displayed a distinctive landmark configuration around the snout, with a relatively short and slender premaxillary tooth-patch width (PMXW) and a larger maxillary tooth-row length (MXTL) compared to laboratory strains derived from M. m. domesticus. Our investigation provides insights into the phylogenetic relationships and taxonomic status of Korean mice relative to the primary lineages of M. musculus subspecies. Understanding the evolutionary history of Korean M. m. musculus sheds new light on how their spatiotemporal dynamics led to diversification, with the Korean Peninsula serving as an ecological bridge between East Eurasia and neighboring regions.

Zinc plays a critical role in cellular functions, but its excess may lead to detrimental effects, including cardiac abnormalities. The impact of zinc overload on cardiomyocytes is investigated in this study. For the first time, we report the regulatory relationship between zinc and histone acetylation. Excessive zinc downregulates the transcription of Bmp4 in HL-1 cell line and primary cardiomyocytes. This downregulation is linked to reduced histone acetylation (H3K9ac), mediated by the suppression of histone acetyltransferases (HATs), rather than changes in histone deacetylases (HDACs). When zinc is introduced directly into the nucleus, Bmp4 expression is upregulated, suggesting that the reduction of histone acetylation by zinc is in an indirect way. This study underscores the importance of zinc homeostasis in maintaining cardiac health and provides insights into the molecular basis of zinc-induced cardiac dysfunction.

Cell growth is a crucial biological property of cells, which plays key roles in several major biological processes including organ development, tissue repair and cancer development. It is of both scientific and medical significance to discover new strategies to modulate cell growth. Koisio technology is a novel technology that modulates the properties of water solely by physical approaches without additions of any external substances. In our current study we obtained the following findings regarding the effects of Koisio technology-modulated solutions on cell growth: First, compared with the lung fibroblast (L929) cell cultures cultured in normal media, the L929 cells cultured in Koisio technology-modulated media grew at approximately 20% higher speed; second, compared with the lung cancer cell cultures (LLC cells) cultured in normal media, the LLC cells cultured in Koisio technology-modulated media grew at approximately 9% lower speed; and third, compared with the telomere lengths of the L929 cells cultured in normal media, the L929 cells cultured in Koisio technology-modulated media had approximately 14% longer telomere length. Collectively, our study has provided the first evidence indicating that Koisio technology-modulated solutions affect differentially the growth of lung fibroblast cell cultures and that of lung cancer cell cultures. The capacity of the Koisio technology-modulated solutions to promote the growth of lung fibroblast cell cultures may result at least partially from its capacity to protect the telomere length.

Serotonin regulates egg-laying by activating vulva muscle contraction. Its oxidization metabolite 5-HIAA inhibits vulva muscle contraction. The loss-of-function mutants of grk-2 have strong egg-laying defects because they have serotonin depletion and high level of 5-HIAA. The experiment results show that 5-HIAAs function depends on 5-HT1/HT2 receptor SER-1. The egg-laying inhibition can be completely reverted by loss-of-function goa-1, the Go homolog in C. elegans. Serotonin and 5-HIAA competes for SER-1 binding and they activate different G protein complexes. The tricyclic antidepression drug cyproheptadine can block 5-HIAA function. The experiment results propose a plausible mechanism of cyproheptadine function. On the postsynaptic membrane, SER-1 binding with cyproheptadine can only recruit serotonin, thus, it biased activates Gq instead of Go coupled signaling.

The omicron variant of SARS-CoV-2 is responsible for the COVID-19 pandemic, serving as a significant origin for the variants still being detected today. It affects the spike protein that most vaccines used to target when the Omicron strain was discovered. Here, we demonstrate that the receptor binding domain (RBD) of the Omicron variant of SARS-CoV-2 exhibits an increased affinity for human angiotensin-converting enzyme type 2 (hACE2) as a viral cell receptor compared to the prototype RBD. We also identified that {beta}- and {gamma}-actin are Omicron-specific binding partners of RBD. Protein complex predictions suggested that many of the Omicron-specific amino acid substitutions might be involved in the affinity of RBD and actin. Accordingly, we highlight the intriguing observation that proteins expected to localize to different cellular compartments exhibit strong binding.

The RAG complex (RAG1 and RAG2) can bind to recombination signal sequences of antigen receptor loci gene segments and coordinate V(D)J recombination which is the primary method of generating antigen receptor diversity. Previous biochemistry studies discovered RAG1 D600, D708 and E962 residues as essential for catalytic DNA nicking and hairpin forming activity of the RAG complex. Neutralization of each of the acidic residues does not impair DNA binding to recombination signal sequence containing DNA substrates, but cleavage of the substrates is severely compromised. These three acidic residues are thought to comprise a DDE motif that is responsible for binding to a divalent cation that is necessary for cleavage activity. Although a Rag1-/-; RAG1-D708A transgenic mouse model system has been used to study dynamics of RAG activity, transgenic expression may not precisely mimic expression from the endogenous locus. In order to improve upon this model, we created Rag1D600A mice that lack B and T cells and demonstrate a developmental block at the pro-B and DN stages, respectively. Thus, Rag1D600A mice provide a novel mouse model system for studying the poorly understood noncanonical functions of RAG1.

The newly identified signaling system comprising C-X-C motif chemokine ligand 17 (CXCL17) and G protein-coupled receptor 25 (GPR25) is involved in immune regulation and tumor development. However, the evolutionary origin of this pair has remained unclear because CXCL17 orthologs in lower vertebrates exhibit extreme sequence variation and cannot be identified through conventional homology-based searches. In this study, we identified seven possible CXCL17 orthologs in primitive cartilaginous fishes, including sharks and rays, using an integrated approach based on key amino acid sequence features as well as gene synteny, architecture, and RNA sequencing data in the NCBI gene database. To validate these candidates, a representative ortholog from the cloudy catshark (Scyliorhinus torazame), termed St-CXCL17, was prepared via bacterial overexpression and in vitro refolding. In cell-based functional assays, St-CXCL17 demonstrated high binding affinity and activation potency toward its corresponding receptor, St-GPR25. Further analysis revealed that removing three conserved C-terminal residues almost completely abolished this activity. While these cartilaginous fish CXCL17s share considerable homology with one another, they lack significant overall similarity to orthologs in mammals, amphibians, or bony fishes. These findings identify functional CXCL17 orthologs in cartilaginous fishes for the first time, implying that the CXCL17-GPR25 signaling pair likely originated in ancient cartilaginous fish ancestors or earlier and has been conserved throughout the evolution of jawed vertebrates.

The chemokine-like peptide GPR15LG is a known agonist of G protein-coupled receptor 15 (GPR15), a ligand-receptor pair primarily implicated in mammalian mucosal immunity and lymphocyte homing. However, the evolutionary origin and phylogenetic distribution of this signaling system remain poorly understood due to the extreme sequence diversity of GPR15LG orthologs. In this study, we identified GPR15LG orthologs in several fish species for the first time according to their conserved gene synteny, genomic organization, and amino acid sequence features. A representative ortholog from the spotted gar (Lepisosteus oculatus), termed Lo-GPR15LG, was recombinantly prepared and functionally characterized using NanoLuc Binary Technology (NanoBiT)-based {beta}-arrestin recruitment assay and homogenous ligand-receptor binding assay. Our results demonstrated that Lo-GPR15LG directly binds to and efficiently activates its cognate receptor, Lo-GPR15, with a dissociation constant (Kd) of approximately 60 nM and an EC50 value of approximately 10 nM. Functional assays further revealed that receptor activation is critically dependent on the conserved C-terminal residues. Notably, human and fish orthologs exhibited no cross-species activity, consistent with their high sequence divergence. These findings reveal that the GPR15LG-GPR15 signaling system originated in ancient fish ancestors and has remained a conserved signaling axis throughout vertebrate evolution, suggesting a fundamental role in immunity across all vertebrate lineages.

The COVID-19 (coronavirus disease 2019) pandemic is caused by SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2). SARS-CoV-2 produces a small hydrophobic envelope (E) protein which shares high homology with SARS-CoV E protein. By patch-clamp recording, the E protein is demonstrated to be a cation-selective ion channel. Furthermore, the SARS-CoV-2 E protein can be blocked by a SARS-CoV E protein inhibitor hexamethylene amiloride. Using structural model and virtual screening, another E protein inhibitor AZD5153 is discovered. AZD5153 is a bromodomain protein 4 inhibitor against hematologic malignancies in clinical trial. The E protein amino acids Phe23 and Val29 are key determinants for AZD5153 sensitivity. This study provides two promising lead compounds and a functional assay of SARS-CoV-2 E protein for the future drug candidate discovery.

The nicotinic acetylcholine receptors (nAChR) are complexes that are composed of subunits evolved from common ancestor. Although the subunits are similar in sequences and structures, their molecular function varies significantly. Therefore, detecting the molecular sites specific to each subunit is important to understand the property of the subunits and the receptors formed by them. As we know, the molecular sites critical to the structure and molecular function of a protein family usually are conserved in evolution, and those specific to each member of the family are often closely related to its structural and functional specificity. In this study, we analyzed the sequence specific sites in nAChR subunits by adopting the evolutionary trace method and the two-state model, and explored the relationship between structure and function in combination with the spatial location of the sites. The results showed that the detected sites in 7 subunit were closely related to ligand binding and conformational changes of the energetic coupling pathway. The conserved sites tended to be distributed in the interior of the spatial structure of protein molecules, and the sites potentially related to new functions were distributed on the surface of the spatial structure. In summary, our results could be helpful to understanding the molecular features related to the function specificity and diversity of the nAChR subunits.

ABSTRACTGlucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common inherited enzymopathy in humans and is associated with a predisposition to hemolysis. However, there are few animal models to that adequately mimic associated human disease states that could be used to evaluate strategies to address clinical syndromes attributable to G6PD deficiency. In the present study, we aimed to establish a stable transgenic zebrafish model of G6PD deficiency that recapitulates the clinical manifestations of G6PD deficiency. We incorporated a stable transgene of G6PD lacking nucleotides from 1315 to 1443 denoted Tg(zgata1:g6pdM1315-1443-egfp). Functional analysis showed that Tg(zgata1:g6pdM1315-1443-egfp) transgenic zebrafish demonstrate a decrease in g6pd activity, reduced GSH levels and hemoglobin content, and increases in pericardial edema in response to α-naphthol exposure, similar to human subjects with G6PD deficiency. We detected no other significant phenotypic abnormalities compared to controls. Taken together, these observations indicate that the Tg(zgata1:g6pdM1315-1443-egfp) zebrafish line mirrors key clinical manifestations of G6PD deficiency in humans. This model may facilitate mechanistic studies and promote translational research related to G6PD deficiency.Competing Interest StatementThe authors have declared no competing interest.View Full Text

BacMam system utilizes baculovirus to deliver exogenous genes into mammalian cells and is extensively used for recombinant production of eukaryotic proteins. Here, we described an improved BacMam vector (pBMCL1, Addgene#178203) which allows convenient tracing of virus production, provides higher infection efficiency towards mammalian cells, minimizes unwanted transcription of toxic genes in insect cells, and provides the capability for co-expression of multiple proteins via a single virus. We demonstrate the successful application of the pBMCL1 vector for the expression of not only the human TRPC3 channel but also the heteromeric KATP channel.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causing agent of the COVID-19 pandemic, has spread globally. Angiotensin-converting enzyme 2 (ACE2) has been identified as the host cell receptor that binds to receptor-binding domain (RBD) of the SARS-COV-2 spike protein and mediates cell entry. Because the ACE2 proteins are widely available in mammals, it is important to investigate the interactions between the RBD and the ACE2 of other mammals. Here we analyzed the sequences of ACE2 proteins from 16 mammals and predicted the structures of ACE2-RBD complexes. Analyses on sequence, structure, and dynamics synergistically provide valuable insights into the interactions between ACE2 and RBD. The comparison results suggest that the ACE2 of bovine, cat and panda form strong binding with RBD, while in the cases of rat, least horseshoe bat, horse, pig, mouse and civet, the ACE2 proteins interact weakly with RBD.

Every known life form senses and reacts to mechanical forces. These mechanical stimuli can be converted into electrical signals by mechanically gated ion channels, a transduction cascade pivotal to numerous physiological functions including touch, hearing, mechanical pain, circulation, gastrointestinal function, and mechanical loading in various tissues. Despite continuous efforts, numerous mechanically gated ion channels with the mechanotransduction process underlying these physiological functions remain unidentified. Here, we focused on the transmembrane channel-like (TMC) protein family expressed in the cultured cells to identify those with potential mechanosensitive activity. Remarkably, in contrast to human TMC1/2 (HsTMC1/2), human TMC3-8 (HsTMC3-8) proteins are localized to the plasma membrane when heterologously expressed in the cultured cells. Further experiments revealed that mechanical poking stimuli can effectively activate HsTMC3-8. In addition, HsTMC3-8 induced stretch-activated currents and elicited well-resolved single-channel activities in response to negative pressure stimulation. The mutants near the putative pore region altered reversal potentials (Erev) of HsTMC3-8, suggesting that TMC3-8 are likely pore-forming subunits of ion channels. In summary, we proposed that TMC proteins are the largest mammalian mechanically gated ion channel family.

The serotonin type 3A (5-HT3A) receptor is a homopentameric cation-selective member of the pentameric ligand-gated ion channel (pLGIC) superfamily. Members of this superfamily assemble from five subunits, each of which consists of three domains, extracellular (ECD), transmembrane (TMD), and intracellular domain (ICD). Previously, we have also demonstrated that 5-HT3A-ICD is required and sufficient for the interaction between 5-HT3A and RIC-3. Additionally, we have shown that 5-HT3A-ICD fused to maltose binding protein (MBP) directly interacts with the chaperone protein resistance to inhibitors of choline esterase (RIC-3), without the involvement of other protein(s). To elucidate the molecular determinants of this interaction we developed different MBP-fused 5-HT3A-ICD constructs by deletion of large portions of its amino acid sequence. We have expressed seven mutants in Escherichia coli and purified them to homogeneity. Using a RIC-3 affinity pull-down assay, the interaction of MBP-5HT3A-ICD constructs and RIC-3 is investigated. Furthermore, we co-expressed 5-HT3A and 5-HT3AB, a heteromeric form of 5-HT3Rs, with RIC-3 in Xenopus oocytes to compare their interaction with RIC-3 in-vivo by two electrode voltage clamp (TEVC) recordings. Full-length 5-HT3A-and 5-HT3AB mediated currents are significantly reduced when RIC-3 is co-expressed in either condition. In summary, we identify a 24-amino acid long segment of the 5-HT3A-ICD as a molecular determinant for the interaction between the 5-HT3A-ICD and RIC-3.\n\nStatement of SignificanceThe chaperone protein RIC-3 is known to modulate the functional surface expression of cation-conducting pentameric ligand-gated ion channels. Previously we have demonstrated that the intracellular domain of serotonin channels mediates this effect. Here we provide experimental evidence for a 24-amino acid long segment within the 115-amino acid long intracellular domain as a determinant for RIC-3 interaction. Recently it was found experimentally that the identified segment contains an alpha helix that has been observed or predicted to be present in other cation-conducting channels. The present work provides novel insights into protein-protein interactions that are likely also relevant for other cation-conducting members of this large ion channel family that includes nACh and 5-HT3 receptors.

DBC1 (deleted in breast cancer 1) is a human nuclear protein that modulates the activities of various proteins. NAD+ (oxidized form of nicotinamide adenine dinucleotide) is thought to potentially bind to the Nudix homology domains (NHDs) of DBC1, thereby regulating DBC1-PARP1 [poly (adenosine diphosphate-ribose) polymerase] interactions, the modulation of which may restore DNA repair to protect against cancer, radiation, and aging. Therefore, our study comprehensively employed methods including NMR (Nuclear Magnetic Resonance), ITC (isothermal titration calorimetry), genetic mutation, and computer biology to thoroughly investigate the molecular mechanism of the binding interaction between NAD+ and its precursor NMN with the NHD domain of DBC1 (DBC1354-396). The results from NMR and ITC indicate that NAD+ likely interacts with DBC1354-396 through hydrogen bonding, with a binding affinity nearly twice that of NMN. The key binding sites are primarily E363 and D372. Molecular Docking further revealed the importance of conventional hydrogen bonds and carbon-hydrogen bonds in the binding process. These findings may lead to a better understanding of how NAD+ regulates the physiological functions of DBC1, thereby offering guiding principles for the development of targeted therapies and drug research focused on tumor diseases associated with DBC1.

Many drugs have the potential to induce the expression of drug-metabolizing enzymes, particularly cytochrome P450 3A4 (CYP3A4), in hepatocytes. Hepatocytes can accurately evaluate drug-mediated CYP3A4 induction as the gold standard for in vitro hepatic toxicology test, but their lot variation is an issue to be solved. Only a limited number of immortalized hepatocyte cells have been reported. In this study, we generated an immortalized cell expressing CYP3A4 from a patient with drug-induced liver injury (DILI). To generate DILI-derived cells with a high expression of CYP3A4, we employed a three-step approach: 1. Differentiation of DILI-induced pluripotent stem cells (DILI-iPSCs); 2. Immortalization of the differentiated cells; 3. Selection of the cells with puromycin. We hypothesize that cells with a high expression of cytochrome P450 genes can survive even after exposure to cytotoxic antibiotics because of high drug-metabolism activity. Puromycin, one of the cytotoxic antibiotics, was used in this study because of its rapid cytocidal effect at a low concentration. Phenotypic studies in vitro revealed that the puromycin-selected cells (HepaSM or SI cells) constitutively expressed the CYP3A4 gene at an extremely high level, and continued to proliferate at least up to 34 population doublings for more than 250 days. The expression profiles were independent of population doublings. Drug-mediated induction test revealed that the cells significantly increased CYP3A4 after exposure to rifampicin, suggesting that the immortalized cells would serve as another useful source for in vitro examination of drug metabolism and CYP3A4 induction.

Endo-beta-N-acetylglucosaminidase (ENGASE) is one of the key enzymes involved in the regulation of structure and function of glycoproteins. It is conserved from prokaryotic to eukaryotic cells. Although their activities in vitro and applications have been well studied, the biological function of ENGASE remains to be illustrated. In this study, we analyzed the molecular and physiological function of Engase from Caenorhabditis elegans homolog eng-1(CeEngase). We found that CeEngase knockout or knockdown increased the environmental stresses adaptability, such as heat stress and osmotic stress. Preliminary glycomics analysis showed that the basement membrane proteins of extracellular matrix may be the main targets of CeENGASE. In addition, CeENGASE may selectively prefer to N2H7 glycans on glycoproteins. In conclusion, our data illustrated that the defection and/or down regulation of CeEngase may provide a beneficially adaptation for stresses.