C-terminal Cytoplasmic Region Research Articles

Protective efficacy against two different viral hemorrhagic septicemia virus (VHSV) isolates belonging to genotype IVa depending on various dose-boost immunizations of rVHSV-GΔTM in olive flounder

Viral vector-based vaccines are promising candidates that deliver antigens of interest into hosts. Particularly, a single-cycle virus possesses the immunogenicity of a live virus vaccine and safety of an inactivated virus vaccine because they do not produce progeny virion after infection. The rVHSV-GΔTM single-cycle, which removes the transmembrane region and C-terminal cytoplasmic region of the G gene from the KJ2008 isolate, virus demonstrates its superiority as a vaccine in controlling the viral hemorrhagic septicemia virus (VHSV) in olive flounder, although research for establishing its suitability is still lacking. Therefore, we established optimal usage through various concentrations and booster immunizations in olive flounder and confirmed the cross-protective effect against KJ2008 and GCVP-02 belonging to different subclades of the glycoprotein genotype IVa. The olive flounder were intramuscular prime or boost immunized from 1 × 102 pfu/fish to 1 × 104 pfu/fish of rVHSV-GΔTM. After four weeks of immunization, each group was challenged with KJ2008 and GCVP-02 isolates. In the prime immunization group, protective efficacy against the GCVP-02 isolate was relatively low compared with the KJ2008 isolate, but the boost immunization group showed improved protective efficacy in both isolates. Our findings suggest that the rVHSV-GΔTM single-cycle virus vaccine based on the KJ2008 isolate can be effective in controlling VHSV in the fish culture industry because it showed a protective efficacy even against other isolates and high protective efficacy upon boosting.

Epstein–Barr virus envelope glycoprotein 110 inhibits NF-κB activation by interacting with NF-κB subunit p65

Epstein-Barr virus (EBV) is a member of the lymphotropic virus family and is highly correlated with some human malignant tumors. It has been reported that envelope glycoprotein 110 (gp110) plays an essential role in viral fusion, DNA replication, and nucleocapsid assembly of EBV. However, it has not been established whether gp110 is involved in regulating the host's innate immunity. In this study, we found that gp110 inhibits tumor necrosis factor α-mediated NF- κB promoter activity and the downstream production of NF- κB-regulated cytokines under physiological conditions. Using dual-luciferase reporter assays, we showed that gp110 might impede the NF-κB promoter activation downstream of NF-κB transactivational subunit p65. Subsequently, we used coimmunoprecipitation assays to demonstrate that gp110 interacts with p65 during EBV lytic infection, and that the C-terminal cytoplasmic region of gp110 is the key interaction domain with p65. Furthermore, we determined that gp110 can bind to the N-terminal Rel homologous and C-terminal domains of p65. Alternatively, gp110 might not disturb the association of p65 with nontransactivational subunit p50, but we showed it restrains activational phosphorylation (at Ser536) and nuclear translocation of p65, which we also found to be executed by the C-terminal cytoplasmic region of gp110. Altogether, these data suggest that the surface protein gp110 may be a vital component for EBV to antagonize the host's innate immune response, which is also helpful for revealing the infectivity and pathogenesis of EBV.

The ion channel CALHM6 controls bacterial infection-induced cellular cross-talk at the immunological synapse.

Membrane ion channels of the calcium homeostasis modulator (CALHM) family promote cell-cell crosstalk at neuronal synapses via ATP release, where ATP acts as a neurotransmitter. CALHM6, the only CALHM highly expressed in immune cells, has been linked tothe induction of natural killer (NK) cell anti-tumour activity. However, its mechanism of action and broader functions in the immune system remain unclear. Here, we generated Calhm6-/- mice and report that CALHM6 is important for theregulation of the early innate control of Listeria monocytogenes infection in vivo. We find that CALHM6 is upregulated in macrophages by pathogen-derived signals and that it relocates from the intracellular compartment to the macrophage-NK cell synapse, facilitating ATP release and controlling the kinetics of NK cell activation. Anti-inflammatory cytokines terminate CALHM6 expression. CALHM6 forms an ion channel when expressed in the plasma membrane of Xenopus oocytes, where channel opening is controlled by a conserved acidic residue, E119. In mammalian cells, CALHM6 is localised to intracellular compartments. Our results contribute to theunderstanding of neurotransmitter-like signal exchange between immune cells that fine-tunes the timing of innate immune responses.

Baculovirus Actin Rearrangement-Inducing Factor 1 Can Remodel the Mammalian Actin Cytoskeleton.

The actin rearrangement-inducing factor 1 (Arif-1) of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is an early viral protein that manipulates the actin cytoskeleton of host insect cells. Arif-1 is conserved among alphabaculoviruses and is responsible for the accumulation of F-actin at the plasma membrane during the early phase of infection. However, the molecular mechanism underlying Arif-1-induced cortical actin accumulation is still open. Recent studies have demonstrated the formation of invadosome-like structures induced by Arif-1, suggesting a function in systemic virus spread. Here, we addressed whether Arif-1 is able to manipulate the actin cytoskeleton of mammalian cells comparably to insect cells. Strikingly, transient overexpression of Arif-1 in B16-F1 mouse melanoma cells revealed pronounced F-actin remodeling. Actin assembly was increased, and intense membrane ruffling occurred at the expense of substrate-associated lamellipodia. Deletion mutagenesis studies of Arif-1 confirmed that the C-terminal cytoplasmic region was not sufficient to induce F-actin remodeling, supporting that the transmembrane region for Arif-1 function is also required in mammalian cells. The similarities between Arif-1-induced actin remodeling in insect and mammalian cells indicate that Arif-1 function relies on conserved cellular interaction partners and signal transduction pathways, thus providing an experimental tool to elucidate the underlying mechanism. IMPORTANCE Virus-induced changes of the host cell cytoskeleton play a pivotal role in the pathogenesis of viral infections. The baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is known for intervening with the regulation of the host actin cytoskeleton in a wide manner throughout the infection cycle. The actin rearrangement-inducing factor 1 (Arif-1) is a viral protein that causes actin rearrangement during the early phase of AcMNPV infection. Here, we performed overexpression studies of Arif-1 in mammalian cells to establish an experimental tool that allows elucidation of the mechanism underlying the Arif-1-induced remodeling of actin dynamics in a well-characterized and genetically accessible system.

Analysis of N-glycosylation protein of Kashin-Beck disease chondrocytes derived from induced pluripotent stem cells based on label-free strategies with LC-MS/MS.

We aimed to compare N-glycosylation proteins in Kashin-Beck disease (KBD) chondrocytes and normal chondrocytes derived from induced pluripotent stem cells (iPSCs). KBD and normal iPSCs were reprogrammed from human KBD and normal dermal fibroblasts, respectively. Subsequently, chondrocytes were differentiated from KBD and normal iPSCs separately. Immunofluorescence was utilized to assay the protein markers of iPSCs and chondrocytes. Differential N-glycosylation proteins were screened using label-free strategies with LC-MS/MS. Bioinformatics analyses were utilized to interpret the functions of differential N-glycosylation proteins. Immunofluorescence staining revealed that both KBD-iPSCs and normal-iPSCs strongly expressed pluripotency markers OCT4 and NANOG. Meanwhile, chondrocyte markers collagen II and SOX9 are presented in KBD-iPSC-chondrocytes and normal-iPSC-chondrocytes. We obtained 87 differential N-glycosylation sites which corresponded to 68 differential proteins, which were constructed into 1 cluster. We obtained collagen type I trimer and 9 other biological processes; polysaccharide binding and 9 other molecular functions; regulation of transcription by RNA polymerase II and 9 other cellular components from GO; the Pl3K-Akt signaling pathway and 9 other KEGG pathways; peroxisome and 7 other subcellular locations; and integrin alpha chain, C-terminal cytoplasmic region, conserved site and 9 other classifications of domain annotations, and 2 networks. FGFR3 and LRP1 are expressed at higher levels in KBD-iPSC-chondrocytes, while the expressions of COL2A1, TIMP1, UNC5B, NOG, LEPR, and ITGA1 were down-regulated in KBD-iPSC-chondrocytes. The differential expressions of these N-glycosylation proteins may lead to the abnormal function of KBD chondrocytes.

LapB (YciM) orchestrates protein-protein interactions at the interface of lipopolysaccharide and phospholipid biosynthesis.

The outer membrane (OM) of Gram-negative bacteria functions as an essential barrier and is characterized by an asymmetric bilayer with lipopolysaccharide (LPS) in the outer leaflet. The enzyme LpxC catalyzes the first committed step in LPS biosynthesis. It plays a critical role in maintaining the balance between LPS and phospholipids (PL), which are both derived from the same biosynthetic precursor. The essential inner membrane proteins YejM (PbgA, LapC), LapB (YciM), and the protease FtsH are known to account for optimal LpxC levels, but the mechanistic details are poorly understood. LapB is thought to be a bi-functional protein serving as an adaptor for FtsH-mediated turnover of LpxC and acting as a scaffold in the coordination of LPS biosynthesis. Here, we provide experimental evidence for the physical interaction of LapB with proteins at the biosynthetic node from where the LPS and PL biosynthesis pathways diverge. By a total of four in vivo and in vitro assays, we demonstrate protein-protein interactions between LapB and the LPS biosynthesis enzymes LpxA, LpxC, and LpxD, between LapB and YejM, the anti-adaptor protein regulating LapB activity, and between LapB and FabZ, the first PL biosynthesis enzyme. Moreover, we uncovered a new adaptor function of LapB in destabilizing not only LpxC but also LpxD. Overall, our study shows that LapB is a multi-functional protein that serves as a protein-protein interaction hub for key enzymes in LPS and PL biogenesis presumably by virtue of multiple tetratricopeptide repeat (TPR) motifs in its cytoplasmic C-terminal region.

Regulatory Roles of N- and C-Terminal Cytoplasmic Regions of P4-ATPases.

P4-ATPases, which are subfamily members of P-type ATPase superfamily, translocate membrane lipids from the exoplasmic/luminal leaflet to the cytoplasmic leaflet, thus regulating trans-bilayer lipid asymmetry. Mammalian P4-ATPases localize to the specific subcellular organelles or the plasma membrane where they translocate the specific lipids. Although recent advances in the structural analysis of P4-ATPases have improved our understanding of lipid transporting machinery, the mechanism of substrate specificity and the regulatory mechanism of the enzymes remain largely unknown. Recent studies have uncovered several specific localization and regulatory mechanisms of P4-ATPases. Here, we review the current understanding of the regulatory mechanism of P4-ATPase activity and localization in mammalian cells.

The interaction of ATP11C-b with ezrin contributes to its polarized localization

ATP11C, a member of the P4-ATPase family, translocates phosphatidylserine and phosphatidylethanolamine at the plasma membrane. We previously revealed that its C-terminal splice variant ATP11C-b exhibits polarized localization in motile cell lines, such as MDA-MB-231 and Ba/F3. In the present study, we found that the C-terminal cytoplasmic region of ATP11C-b interacts specifically with ezrin. Notably, the LLxY motif in the ATP11C-b C-terminal region is crucial for its interaction with ezrin as well as its polarized localization on the plasma membrane. A constitutively active, C-terminal phosphomimetic mutant of ezrin was colocalized with ATP11C-b in polarized motile cells. ATP11C-b was partially mislocalized in cells depleted of ezrin alone, and exhibited greater mislocalization in cells simultaneously depleted of the family members ezrin, radixin and moesin (ERM), suggesting that ERM proteins, particularly ezrin, contribute to the polarized localization of ATP11C-b. Furthermore, Atp11c knockout resulted in C-terminally phosphorylated ERM protein mislocalization, which was restored by exogenous expression of ATP11C-b but not ATP11C-a. These observations together indicate that the polarized localizations of ATP11C-b and the active form of ezrin to the plasma membrane are interdependently stabilized.

Baculovirus actin-rearrangement-inducing factor ARIF-1 induces the formation of dynamic invadosome clusters.

The baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV), a pathogen of lepidopteran insects, has a striking dependence on the host cell actin cytoskeleton. During the delayed-early stage of infection, AcMNPV was shown to induce the accumulation of actin at the cortex of infected cells. However, the dynamics and molecular mechanism of cortical actin assembly remained unknown. Here, we show that AcMNPV induces dynamic cortical clusters of dot-like actin structures that mediate degradation of the underlying extracellular matrix and therefore function similarly to clusters of invadosomes in mammalian cells. Furthermore, we find that the AcMNPV protein actin-rearrangement-inducing factor-1 (ARIF-1), which was previously shown to be necessary and sufficient for cortical actin assembly and efficient viral infection in insect hosts, is both necessary and sufficient for invadosome formation. We mapped the sequences within the C-terminal cytoplasmic region of ARIF-1 that are required for invadosome formation and identified individual tyrosine and proline residues that are required for organizing these structures. Additionally, we found that ARIF-1 and the invadosome-associated proteins cortactin and the Arp2/3 complex localize to invadosomes and Arp2/3 complex is required for their formation. These ARIF-1–induced invadosomes may be important for the function of ARIF-1 in systemic virus spread.

Role of the N- and C-terminal regions of FliF, the MS ring component in Vibrio flagellar basal body.

The MS ring is a part of the flagellar basal body and formed by 34 subunits of FliF, which consists of a large periplasmic region and two transmembrane segments connected to the N- and C-terminal regions facing the cytoplasm. A cytoplasmic protein, FlhF, which determines the position and number of the basal body, supports MS ring formation in the membrane in Vibrio species. In this study, we constructed FliF deletion mutants that lack 30 or 50 residues from the N-terminus (ΔN30 and ΔN50), and 83 (ΔC83) or 110 residues (ΔC110) at the C-terminus. The N-terminal deletions were functional and conferred motility of Vibrio cells, whereas the C-terminal deletions were nonfunctional. The mutants were expressed in Escherichia coli to determine whether an MS ring could still be assembled. When co-expressing ΔN30FliF or ΔN50FliF with FlhF, fewer MS rings were observed than with the expression of wild-type FliF, in the MS ring fraction, suggesting that the N-terminus interacts with FlhF. MS ring formation is probably inefficient without FlhF. The deletion of the C-terminal cytoplasmic region did not affect the ability of FliF to form an MS ring because a similar number of MS rings were observed for ΔC83FliF as with wild-type FliF, although further deletion of the second transmembrane segment (ΔC110FliF) abolished it. These results suggest that the terminal regions of FliF have distinct roles; the N-terminal region for efficient MS ring formation and the C-terminal region for MS ring function. The second transmembrane segment is indispensable for MS ring assembly.ImportanceThe bacterial flagellum is a supramolecular architecture involved in cell motility. At the base of the flagella, a rotary motor that begins to construct an MS ring in the cytoplasmic membrane comprises 34 transmembrane proteins (FliF). Here, we investigated the roles of the N and C terminal regions of FliF, which are MS rings. Unexpectedly, the cytoplasmic regions of FliF are not indispensable for the formation of the MS ring, but the N-terminus appears to assist in ring formation through recruitment of FlhF, which is essential for flagellar formation. The C-terminus is essential for motor formation or function.

The N- or C-terminal cytoplasmic regions of P4-ATPases determine their cellular localization.

Mammalian P4-ATPases specifically localize to the plasma membrane and the membranes of intracellular compartments. P4-ATPases contain 10 transmembrane domains, and their N- and C-terminal (NT and CT) regions face the cytoplasm. Among the ATP10 and ATP11 proteins of P4-ATPases, ATP10A, ATP10D, ATP11A, and ATP11C localize to the plasma membrane, while ATP10B and ATP11B localize to late endosomes and early/recycling endosomes, respectively. We previously showed that the NT region of ATP9B is critical for its localization to the Golgi apparatus, while the CT regions of ATP11C isoforms are critical for Ca2+-dependent endocytosis or polarized localization at the plasma membrane. Here, we conducted a comprehensive analysis of chimeric proteins and found that the NT region of ATP10 proteins and the CT region of ATP11 proteins are responsible for their specific subcellular localization. Importantly, the ATP10B NT and the ATP11B CT regions were found to harbor a trafficking and/or targeting signal that allows these P4-ATPases to localize to late endosomes and early/recycling endosomes, respectively. Moreover, dileucine residues in the NT region of ATP10B were required for its trafficking to endosomal compartments. These results suggest that the NT and CT sequences of P4-ATPases play a key role in their intracellular trafficking.

Cytoplasmic Autoinhibition in HCN Channels is Regulated by the Transmembrane Region

Hyperpolarization-activated cation-nonselective (HCN) channels regulate electrical activity in the brain and heart in a cAMP-dependent manner. The voltage-gating of these channels is mediated by a transmembrane (TM) region but is additionally regulated by direct binding of cAMP to a cyclic nucleotide-binding (CNB) fold in the cytoplasmic C-terminal region. Cyclic AMP potentiation has been explained by an autoinhibition model which views the unliganded CNB fold as an inhibitory module whose influence is disrupted by cAMP binding. However, the HCN2 subtype uses two other CNB fold-mediated mechanisms called open-state trapping and Quick-Activation to respectively slow the deactivation kinetics and speed the activation kinetics, against predictions of an autoinhibition model. To test how these multiple mechanisms are influenced by the TM region, we replaced the TM region of HCN2 with that of HCN4. This HCN4 TM-replacement preserved cAMP potentiation but augmented the magnitude of autoinhibition by the unliganded CNB fold; it moreover disrupted open-state trapping and Quick-Activation so that autoinhibition became the dominant mechanism contributed by the C-terminal region to determine kinetics. Truncation within the CNB fold partially relieved this augmented autoinhibition. This argues against the C-terminal region acting like a portable module with consistent effects on TM regions of different subtypes. Our findings provide evidence that functional interactions between the HCN2 TM region and C-terminal region govern multiple CNB fold-mediated mechanisms, implying that the molecular mechanisms of autoinhibition, open-state trapping, and Quick-Activation include participation of TM region structures.

The Role of HCN Channel Helices D and E in the Modulation of Camp Affinity

Hyperpolarization-activated cyclic-nucleotide regulated (HCN) channels are non-selective cation channels controlling key physiological functions such as cardiac pacemaking and repetitive neuronal firing. They are activated by voltage and modulated by binding of cAMP to their cytoplasmic C-terminal region named cyclic nucleotide binding domain (CNBD). Altered cAMP responses caused either by single point mutations in the protein or by non-physiological levels of the cyclic nucleotide in the cell lead to pathological conditions both in the heart and in the nervous system. The recently obtained cryo-EM structure of human HCN1 in the bound and unbound forms, confirms previous NMR studies on cAMP-induced conformational changes and reveals the presence of two additional helices at the C-terminus of the CNBD which fold in the presence of cAMP. We have previously shown, by ITC and patch clamp, that deletion of these helices (D and E) decreases the affinity for cAMP in the most studied HCN isoforms (HCN1, HCN2 and HCN4). Here we introduced single point mutations in HCN4 isoform, designed either to delete the single E helix or to selectively perturb the folding of the D helix, with the aim to dissect the role of the single D and E helices in this regulatory mechanism. Moreover, different truncations of the D helix were analyzed in order to clarify its role in the folding of the preceding C helix, known to be a key element in the transition of the CNBD from the unbound to the bound state. The patch clamp experiments were performed in whole cell configuration using a new all-in-one integrated amplifier, the ePatch.

Patch-Clamp Fluorometry Defines a Role for SUR1 in Nucleotide Inhibition of KATP Channels

ATP sensitive K+ (KATP) channels couple a cell's metabolic state to its electrical excitability. KATP is a hetero-octameric complex, with four pore-forming inward rectifier K+ channel subunits (Kir6.1 or Kir6.2), each associated with a modulatory sulfonylurea receptor (SUR1 or SUR2A/B). ATP/ADP binding to a site on the Kir6.2 subunit shuts KATP. In the presence of Mg2+, nucleotide binding to SUR1 stimulates KATP. However, SUR1 also has direct effects on gating at Kir6.2 in the absence of Mg2+. The presence of SUR1 increases channel open probability, but paradoxically also increases the apparent affinity for nucleotide inhibition at Kir6.2 by an unknown mechanism. We introduced the fluorescent non-canonical amino acid ANAP at a position (W311ANAP) in the cytoplasmic C-terminal region of Kir6.2 co-expressed with SUR1 in HEK 293T cells. This allowed us to simultaneously measure channel currents and inhibitory nucleotide binding to Kir6.2 via FRET between ANAP and a fluorescent ATP derivative (TNP-ATP). We fit models to the combined data sets to produce well-constrained estimates of open-state nucleotide affinity and the strength of coupling between nucleotide binding and channel closure. Our models suggest that “wild-type” KATP is closed when only one of its nucleotide binding sites is occupied. A Kir6.2 mutation (C166S) that profoundly increases channel activity had very little effect on nucleotide binding, but greatly perturbed the ability of bound nucleotide to inhibit KATP. In contrast, mutations at position E205 on SUR1, which is located on an intracellular loop close to the inhibitory site on Kir6.2, affected both TNP-ATP affinity and the ability of bound nucleotide to inhibit KATP. This suggests a dual role for SUR1 in KATP inhibition, both in contributing to nucleotide binding to the open state and in stabilizing the nucleotide-bound closed state.

Diacylglycerol kinase δ destabilizes serotonin transporter protein through the ubiquitin-proteasome system

Brain-specific diacylglycerol kinase (DGK) δ-knockout mice exhibited serotonin transporter (SERT) inhibitor-sensitive obsessive-compulsive disorder-like behaviors. Moreover, SERT protein levels were markedly increased in the DGKδ-deficient brain. However, its molecular mechanisms remain unclear. We found that the catalytic subdomain-a and the coiled-coil structure-containing region of DGKδ interacted with the C-terminal cytoplasmic region (CTC) of SERT. Moreover, the protein levels of full-length SERT and SERT-CTC alone were significantly decreased by DGKδ in a catalytic activity-dependent manner. A proteasome inhibitor, MG-132, inhibited DGKδ-dependent SERT degradation. Notably, DGKδ interacted with MAGE-D1 adaptor protein and Praja-1 E3 ubiquitin-protein ligase, and enhanced the ubiquitination of SERT through Praja-1. Taken together, these results indicate that DGKδ interacts with SERT and induces SERT degradation in an activity-dependent manner through the Praja-1 ubiquitin ligase-proteasome system. These new findings provide novel insights into serotonergic system regulation and the pathophysiology/therapeutics of serotonin-/SERT-related diseases such as obsessive-compulsive disorder, depression, autism and schizophrenia.

The cytoplasmic C-terminal region of the ATP11C variant determines its localization at the polarized plasma membrane.

ATP11C, a member of the P4-ATPase family, is a major phosphatidylserine (PS)-flippase located at the plasma membrane. ATP11C deficiency causes a defect in B-cell maturation, anemia and hyperbilirubinemia. Although there are several alternatively spliced variants derived from the ATP11C gene, the functional differences between them have not been considered. Here, we compared and characterized three C-terminal spliced forms (we designated as ATP11C-a, ATP11C-b and ATP11C-c), with respect to their expression patterns in cell types and tissues, and their subcellular localizations. We had previously shown that the C-terminus of ATP11C-a is critical for endocytosis upon PKC activation. Here, we found that ATP11C-b and ATP11C-c did not undergo endocytosis upon PKC activation. Importantly, we also found that ATP11C-b localized to a limited region of the plasma membrane in polarized cells, whereas ATP11C-a was distributed on the entire plasma membrane in both polarized and non-polarized cells. Moreover, we successfully identified LLXY residues within the ATP11C-b C-terminus as a critical motif for the polarized localization. These results suggest that the ATP11C-b regulates PS distribution in distinct regions of the plasma membrane in polarized cells.

Structure of the periplasmic domain of SflA involved in spatial regulation of the flagellar biogenesis of Vibrio reveals a TPR/SLR-like fold.

Bacteria have evolved various types of flagellum, an organella for bacterial motility, to adapt to their habitat environments. The number and the spatial arrangement of the flagellum are precisely controlled to optimize performance of each type of the flagellar system. Vibrio alginolyticus has a single sheathed flagellum at the cell pole for swimming. SflA is a regulator protein to prevent peritrichous formation of the sheathed flagellum, and consists of an N-terminal periplasmic region, a transmembrane helix, and a C-terminal cytoplasmic region. Whereas the cytoplasmic region has been characterized to be essential for inhibition of the peritrichous growth, the role of the N-terminal region is still unclear. We here determined the structure of the N-terminal periplasmic region of SflA (SflAN) at 1.9-Å resolution. The core of SflAN forms a domain-swapped dimer with tetratricopeptide repeat (TPR)/Sel1-like repeat (SLR) motif, which is often found in the domains responsible for protein-protein interaction in various proteins. The structural similarity and the following mutational analysis based on the structure suggest that SflA binds to unknown partner protein by SflAN and the binding signal is important for the precise control of the SflA function.

Identification of C-Terminal Binding Protein 1 as a Novel NMDA Receptor Interactor

A new N-methyl D aspartate neurotransmitter receptor interacting protein has been identified by yeast two-hybrid screening of a mouse brain cDNA library. C-terminal binding protein 1 (CtBP1) was shown to associate with the intracellular C-terminal regions of the N-methyl D aspartate receptor subunits GluN2A and GluN2D but not with GluN1-1a cytoplasmic C-terminal region. In yeast mating assays using a series of GluN2A C-terminal truncations, it was demonstrated that the CtBP1 binding domain was localized to GluN2A 1157–1382. The GluN2A binding domain was identified to lie within the CtBP1 161–224 region. CtBP1 co-immunoprecipitated with assembled GluN1/GluN2A receptors expressed in mammalian cells and also, in detergent extracts of adult mouse brain. Co-expression of CtBP1 with GluN1/GluN2A resulted in a significant decrease in receptor cell surface expression. The family of C-terminal binding proteins function primarily as transcriptional co-repressors. However, they are also known to modulate intracellular membrane trafficking mechanisms. Thus the results reported herein describe a putative role for CtBP1 in the regulation of cell surface N-methyl D aspartate receptor expression.

Two mutations at different positions in the CNBH domain of the hERG channel accelerate deactivation and impair the interaction with the EAG domain.

In the human ether-a-go-go related gene (hERG) channel, both the ether-a-go-go (EAG) domain in the N-terminal and the cyclic nucleotide (CN) binding homology (CNBH) domain in the C-terminal cytoplasmic region are known to contribute to the characteristic slow deactivation. Mutations of Phe860 in the CNBH domain, reported to fill the CN binding pocket, accelerate the deactivation and decrease the fluorescence resonance energy transfer (FRET) efficiencies between the EAG and CNBH domains. An electrostatic interaction between Arg696 and Asp727 in the C-linker domain, critical for HCN and CNG channels, is not formed in the hERG channel. Mutations of newly identified electrostatically interacting pair, Asp727 in the C-linker and Arg752 in the CNBH domains, accelerate the deactivation and decrease FRET efficiency. Voltage-dependent changes in FRET efficiency were not detected. These results suggest that the acceleration of the deactivation by mutations of C-terminal domains is a result of the lack of interaction between the EAG and CNBH domains. The human ether-a-go-go related gene (hERG) channel shows characteristic slow deactivation, and the contribution of both of the N-terminal cytoplasmic ether-a-go-go (EAG) domain and the C-terminal cytoplasmic cyclic nucleotide (CN) binding homology (CNBH) domain is well known. The interaction between these domains is known to be critical for slow deactivation. We analysed the effects of mutations in the CNBH domain and its upstream C-linker domain on slow deactivation and the interaction between the EAG and CNBH domains by electrophysiological and fluorescence resonance energy transfer (FRET) analyses using Xenopus oocyte and HEK293T cell expression systems. We first observed that mutations of Phe860 in the CNBH domain, which is reported to fill the CN binding pocket as an intrinsic ligand, accelerate deactivation and eliminate the inter-domain interaction. Next, we observed that the salt bridge between Arg696 and Asp727 in the C-linker domain, which is reported to be critical for the function of CN-regulated channels, is not formed. We newly identified an electrostatically interacting pair critical for slow deactivation: Asp727 and Arg752 in the CNBH domain. Their mutations also impaired the inter-domain interaction. Taking these results together, both mutations of the intrinsic ligand (Phe860) and a newly identified salt bridge pair (Asp727 and Arg752) in the hERG channel accelerated deactivation and also decreased the interaction between the EAG and CNBH domains. Voltage-dependent changes in FRET efficiency between the two domains were not detected. The results suggest that the CNBH domain contributes to slow deactivation of the hERG channel by a mechanism involving the EAG domain.

Assigning Function to the D and E Helices of HCN CNBD

HCN (hyperpolarization-activated cyclic-nucleotide sensitive cation nonselective) channels are activated by voltage and modulated by the direct binding of cAMP to their cytoplasmic C-terminal region named CNBD (cyclic nucleotide binding domain) (Zagotta et al., 2003). HCN channels are further regulated by TRIP8b, a brain-specific auxiliary subunit which controls channel trafficking and gating. In particular, TRIP8b interacts with the HCN channel CNBD and antagonizes the facilitatory effect of cAMP on channel opening (Hu et al., 2013 ). Recently, the cryo-EM structure of human HCN1 was solved in both the cAMP-free and cAMP-bound states (Lee and Mackinnon, 2017). A comparison of the two structures shows that, in the full-length protein, cAMP binding induces all the same changes previously highlighted in the isolated CNBD fragment using NMR (Saponaro et al., 2014), plus the unexpected folding of two additional helices at the C-terminus of the CNBD. These helices were not included in any of the previous experiments conducted using the isolated C-linker/CNBD fragment. To understand whether these two dynamic helices play a role in regulating both cAMP and TRIP8b binding, we have compared, using an in vitro binding assay, two CNBD constructs with or without the newly discovered helices. Surprisingly, their presence increases the affinity for cAMP without affecting TRIP8b binding. This result assigns to the new C-terminal helices of the HCN channel CNBD a specific role in controlling cyclic nucleotide affinity.