The receptor binding domain of SARS-CoV-2 Omicron subvariants targets Siglec-9 to decrease its immunogenicity by preventing macrophage phagocytosis.

The development of a vaccine specific to severe acute respiratory syndrome coronavirus 2 Omicron has been hampered due to its low immunogenicity. Here, using reverse mutagenesis, we found that a phenylalanine-to-serine mutation at position 375 (F375S) in the spike protein of Omicron to revert it to the sequence found in Delta and other ancestral strains significantly enhanced the immunogenicity of Omicron vaccines. Sequence FAPFFAF at position 371-377 in Omicron spike had a potent inhibitory effect on macrophage uptake of receptor-binding domain (RBD) nanoparticles or spike-pseudovirus particles containing this sequence. Omicron RBD enhanced binding to Siglec-9 on macrophages to impair phagocytosis and antigen presentation and promote immune evasion, which could be abrogated by the F375S mutation. A bivalent F375S Omicron RBD and Delta-RBD nanoparticle vaccine elicited potent and broad nAbs in mice, rabbits and rhesus macaques. Our research suggested that manipulation of the Siglec-9 pathway could be a promising approach to enhance vaccine response.

A novel NhaD-type Na+/H+ antiporter from the moderate halophile and alkaliphile Halomonas alkaliphila.

In this study, a NhaD-type Na+/H+ antiporter gene designated Ha-nhaD was obtained by selection of genomic DNA from the moderate halophile and alkaliphile Halomonas alkaliphila in Escherichia coli KNabc lacking 3 major Na+/H+ antiporters. The presence of Ha-NhaD conferred tolerance of E. coli KNabc to NaCl up to 0.6 mol·L-1 and to LiCl up to 0.2 mol·L-1 and to an alkaline pH. pH-dependent Na+(Li+)/H+ antiport activity was detected from everted membrane vesicles prepared from E. coli KNabc/pUC-nhaD but not those of KNabc/pUC18. Ha-NhaD exhibited Na+(Li+)/H+ antiport activity over a wide pH range from 7.0 to 9.5, with the highest activity at pH 9.0. Protein sequence alignment and phylogenetic analysis revealed that Ha-NhaD is significantly different from the 7 known NhaD-type Na+/H+ antiporters, including Dw-NhaD, Dl-NhaD, Vp-NhaD, Vc-NhaD, Aa-NhaD, He-NhaD, and Ha-NhaD1. Although Ha-NhaD showed a closer phylogenetic relationship with Ha-NhaD2, a significant difference in pH-dependent activity profile exists between Ha-NhaD and Ha-NhaD2. Taken together, Ha-nhaD encodes a novel pH-dependent NhaD-type Na+/H+ antiporter.

The histamine receptor H1 acts as an alternative receptor for SARS-CoV-2.

Numerous host factors, in addition to human angiotensin-converting enzyme 2 (hACE2), have been identified as coreceptors of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), demonstrating broad viral tropism and diversified druggable potential. We and others have found that antihistamine drugs, particularly histamine receptor H1 (HRH1) antagonists, potently inhibit SARS-CoV-2 infection. In this study, we provided compelling evidence that HRH1 acts as an alternative receptor for SARS-CoV-2 by directly binding to the viral spike protein. HRH1 also synergistically enhanced hACE2-dependent viral entry by interacting with hACE2. Antihistamine drugs effectively prevent viral infection by competitively binding to HRH1, thereby disrupting the interaction between the spike protein and its receptor. Multiple inhibition assays revealed that antihistamine drugs broadly inhibited the infection of various SARS-CoV-2 mutants with an average IC50 of 2.4 µM. The prophylactic function of these drugs was further confirmed by authentic SARS-CoV-2 infection assays and humanized mouse challenge experiments, demonstrating the therapeutic potential of antihistamine drugs for combating coronavirus disease 19.IMPORTANCEIn addition to human angiotensin-converting enzyme 2, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can utilize alternative cofactors to facilitate viral entry. In this study, we discovered that histamine receptor H1 (HRH1) not only functions as an independent receptor for SARS-CoV-2 but also synergistically enhances ACE2-dependent viral entry by directly interacting with ACE2. Further studies have demonstrated that HRH1 facilitates the entry of SARS-CoV-2 by directly binding to the N-terminal domain of the spike protein. Conversely, antihistamine drugs, primarily HRH1 antagonists, can competitively bind to HRH1 and thereby prevent viral entry. These findings revealed that the administration of repurposable antihistamine drugs could be a therapeutic intervention to combat coronavirus disease 19.

Structural basis of ligand binding modes of human EAAT2.

In the central nervous system (CNS), excitatory amino acid transporters (EAATs) mediate the uptake of excitatory neurotransmitter glutamate and maintain its low concentrations in the synaptic cleft for avoiding neuronal cytotoxicity. Dysfunction of EAATs can lead to many psychiatric diseases. Here we report cryo-EM structures of human EAAT2 in an inward-facing conformation, in the presence of substrate glutamate or selective inhibitor WAY-213613. The glutamate is coordinated by extensive hydrogen bonds and further stabilized by HP2. The inhibitor WAY-213613 occupies a similar binding pocket to that of the substrate glutamate. Upon association with the WAY-213613, the HP2 undergoes a substantial conformational change, and in turn stabilizes the inhibitor binding by forming hydrophobic interactions. Electrophysiological experiments elucidate that the unique S441 plays pivotal roles in the binding of hEAAT2 with glutamate or WAY-213613, and the I464-L467-V468 cluster acts as a key structural determinant for the selective inhibition of this transporter by WAY-213613.

A novel three-TMH Na+/H+ antiporter and the functional role of its oligomerization.

Na+/H+antiportersare a category of ubiquitous transmembrane proteins with various important physiological roles in almost all living organisms ranging from bacteria to humans. However, the knowledge of novel Na+/H+antiporters remains to be broadened, and the functional roles ofoligomerization in theseantiportershave not yet been thoroughly understood. Here, we reported functional analysis of an unknown transmembrane protein composed of 103 amino acid residues. This protein was found to function as a Na+(Li+, K+)/H+ antiporter. To the best of our knowledge, this antiporter is the minimal one of known Na+/H+antiporters and thus designated as NhaM to represent the minimal Na+/H+antiporter. NhaM and its homologs have not yet been classified into any protein family. Based on phylogenetic analysis and protein alignment, we propose NhaM and its homologs to constitute a novel transporter family designated as NhaM family. More importantly, we found that NhaM is assembled with parallel protomers into a homo-oligomer and oligomerization is vital for the function of this antiporter. This implies that NhaM may adopt and require an oligomer structure for its normal function to create a similar X-shaped structure to that of the NhaA fold. Taken together, current findings not only present the proposal of a novel transporter family but also positively contribute to the functional roles of oligomerization in Na+/H+antiporters.

A Novel MFS-MDR Transporter, MdrP, Employs D223 as a Key Determinant in the Na+ Translocation Coupled to Norfloxacin Efflux.

Multidrug resistance (MDR) transporters of the major facilitator superfamily (MFS) were previously believed to drive the extrusion of multiple antimicrobial drugs through the coupling to proton translocation. Here, we present the identification of the first Na+-coupled MFS-MDR transporter, MdrP, which also can achieve H+-coupled drug efflux independently of Na+. Importantly, we propose that MdrP can extrude norfloxacin in a mode of drug/Na+ antiport, which has not yet been reported in any MFS member. On this basis, we further provide the insights into a novel Na+ and H+ coupling mechanism of MFS-MDR transporters, even for all secondary transporters. The most important finding lies in that D223 should mainly act as a key determinant in the Na+ translocation coupled to norfloxacin efflux. Furthermore, our results partially modify the knowledge of the conformational stability-related residues in the motif A of MFS transporters and imply the importance of a new positively charged residue, R361, for the stabilization of outward-facing conformation of MFS transporters. These novel findings positively contribute to the knowledge of MFS-MDR transporters, especially about Na+ and H+ coupling mechanism. This study is based mainly on measurements in intact cells or everted membranes, and a biochemical assay with a reconstituted MdrP protein should be necessary to come to conclusion to be assured.

Characterization of a Functionally Unknown Arginine-Aspartate-Aspartate Family Protein From Halobacillus andaensis and Functional Analysis of Its Conserved Arginine/Aspartate Residues.

Arginine-aspartate-aspartate (RDD) family, representing a category of transmembrane proteins containing one highly conserved arginine and two highly conserved aspartates, has been functionally uncharacterized as yet. Here we present the characterization of a member of this family designated RDD from the moderate halophile Halobacillus andaensis NEAU-ST10-40T and report for the first time that RDD should function as a novel Na+(Li+, K+)/H+ antiporter. It's more interesting whether the highly conserved arginine/aspartate residues among the whole family or between RDD and its selected homologs are related to the protein function. Therefore, we analyzed their roles in the cation-transporting activity through site-directed mutagenesis and found that D154, R124, R129, and D158 are indispensable for Na+(Li+, K+)/H+ antiport activity whereas neither R35 nor D42 is involved in Na+(Li+, K+)/H+ antiport activity. As a dual representative of Na+(Li+, K+)/H+ antiporters and RDD family proteins, the characterization of RDD and the analysis of its important residues will positively contribute to the knowledge of the cation-transporting mechanisms of this novel antiporter and the roles of highly conserved arginine/aspartate residues in the functions of RDD family proteins.

An Uncharacterized Major Facilitator Superfamily Transporter From Planococcus maritimus Exhibits Dual Functions as a Na+(Li+, K+)/H+ Antiporter and a Multidrug Efflux Pump.

Within major facilitator superfamily (MFS), up to 27 unknown major facilitator families and many members of 60 well-characterized families have been functionally unknown as yet, due to their sharing no or significantly low sequence identity with characterized MFS members. Here we present the first report on the characterization of one functionally unknown MFS transporter designated MdrP with the accession version No. ANU18183.1 from the slight halophile Planococcus maritimus DS 17275T. During the screening of Na+/H+ antiporter genes, we found at first that MdrP exhibits Na+(Li+, K+)/H+ antiport activity, and propose that it should represent a novel class of Na+(Li+, K+)/H+ antiporters. However, we speculate that MdrP may possess an additional protein function. The existence of the signature Motif A of drug/H+antiporter (DHA) family members and phylogenetic analysis suggest that MdrP may also function as a drug efflux pump, which was established by minimum inhibitory concentration tests and drug efflux activity assays. Taken together, this novel MFS transporter exhibits dual functions as a Na+(Li+, K+)/H+ antiporter and a multidrug efflux pump, which will be very helpful to not only positively contribute to the function prediction of uncharacterized MFS members especially DHA1 family ones, but also broaden the knowledge of Na+/H+ antiporters.

Characterization of a novel two-component Na+(Li+, K+)/H+ antiporter from Halomonas zhaodongensis.

In this study, genomic DNA was screened for novel Na+/H+ antiporter genes from Halomonas zhaodongensis by selection in Escherichia coli KNabc lacking three major Na+/H+ antiporters. Co-expression of two genes designated umpAB, encoding paired homologous unknown membrane proteins belonging to DUF1538 (domain of unknown function with No. 1538) family, were found to confer E. coli KNabc the tolerance to 0.4 M NaCl and 30 mM LiCl, and an alkaline pH resistance at 8.0. Western blot and co-immunoprecipitation establish that UmpAB localize as a hetero-dimer in the cytoplasmic membranes. Functional analysis reveals that UmpAB exhibit pH-dependent Na+(Li+, K+)/H+ antiport activity at a wide pH range of 6.5 to 9.5 with an optimal pH at 9.0. Neither UmpA nor UmpB showed homology with known single-gene or multi-gene Na+/H+ antiporters, or such proteins as ChaA, MdfA, TetA(L), Nap and PsmrAB with Na+/H+ antiport activity. Phylogenetic analysis confirms that UmpAB should belong to DUF1538 family, which are significantly distant with the above-mentioned proteins with Na+/H+ antiport activity. Taken together, we propose that UmpAB represent a novel two-component Na+(Li+, K+)/H+ antiporter. To the best of our knowledge, this is the first report on the functional analysis of unknown membrane proteins belonging to DUF1538 family.

Identification of dicalcium phosphate dihydrate deposited during osteoblast mineralization in vitro.

The hydroxyapatite (HAP) with variable chemical substitutions has been considered as the major component in the mineralized part of bones. Various metastable crystalline phases have been suggested as transitory precursors of HAP in bone, but there are no consensuses as to the nature of these phases and their temporal evolution. In the present study, we cultured rat calvarial osteoblasts with ascorbate and ß-glycerophosphate to explore which calcium phosphate precursor phases comprise the initial mineral in the process of osteoblast mineralization in vitro. At the indicated time points, the deposited calcium phosphate was analyzed after removing organic substances from the extracellular matrix with hydrazine. The features comparable to dicalcium phosphate dihydrate (DCPD) and octacalcium phosphate (OCP), in addition to HAP, were detected in the mineral phases by high resolution transmission electron microscopy. And there was a trend of conversion from DCPD- and OCP-like phases to HAP in the course of mineralization, as indicated by Fourier-transform infrared microspectroscopy, energy-dispersive X-ray spectroscopy and synchrotron X-ray powder diffraction analyses. Besides, biochemical assay showed a progressive decrease in the ratio of mineral-associated proteins to calcium with time. These findings suggest that DCPD- and OCP-like phases are likely to occur on the course of osteoblast mineralization, and the mineral-associated proteins might be involved in modulating the mineral phase transformation.