PDZ Motif Research Articles

CFTR anion channel modulates expression of human transmembrane mucin MUC3 through the PDZ protein GOPC

The transmembrane mucins in the enterocyte are type 1 transmembrane proteins with long and rigid mucin domains, rich in proline, threonine and serine residues that carry numerous O-glycans. Three of these mucins, MUC3, MUC12 and MUC17 are unique in harboring C-terminal class I PDZ motifs, making them suitable ligands for PDZ proteins. A screening of 123 different human PDZ domains for binding to MUC3 identified a strong interaction with the PDZ protein GOPC (Golgi-associated PDZ and coiled-coil motif-containing protein). This interaction was mediated by the C-terminal PDZ motif of MUC3, binding to the single GOPC PDZ domain. GOPC is also a binding partner for cystic fibrosis transmembrane conductance regulator (CFTR) that directs CFTR for degradation. Overexpression of GOPC downregulated the total levels of MUC3, an effect that was reversed by introducing CFTR. The results suggest that CFTR and MUC3 compete for binding to GOPC, which in turn can regulate levels of these two proteins. For the first time a direct coupling between mucins and the CFTR channel is demonstrated, a finding that will shed further light on the still poorly understood relationship between cystic fibrosis and the mucus phenotype of this disease.

Targeting the regulation of CFTR channels

In this issue of the Biochemical Journal, Zhang et al. reveal a new strategy for modifying the regulated function of CFTR (cystic fibrosis transmembrane conductance regulator) on the apical surface of epithelial cells. Simply stated, these authors tested the idea that the cAMP-dependent channel activity of CFTR could be effectively enhanced by disruption of a protein-protein interaction which is normally inhibitory for the production of cAMP. This particular protein-protein interaction [between the PDZ motif of LPA2 (type 2 lysophosphatidic acid receptor) and the scaffold protein Nherf2 (Na+/H+ exchanger regulatory factor 2)] is localized in the CFTR interactome on the apical membrane of epithelial cells. Hence disruption of the LPA2-Nherf2 interaction should lead to a localized elevation in cAMP and, consequently, increased cAMP-dependent CFTR activity on the surface of epithelial cells. Zhang et al. confirmed these expectations for a small-molecule compound targeting the LPA2-Nherf2 interaction using relevant cultures and tissues thought to model the human respiratory epithelium. The success of this strategy depended on previous knowledge regarding the role for multiple PDZ-motif-mediated interactions in signalling (directly or indirectly) to CFTR. Given the number and diversity of such PDZ-mediated interactions, future structural and computational studies will be essential for guiding the design of specific pharmacological interventions.

Genetic variation on internal protein matric (M1) and non structural protein (NS1) of Indonesian avian influenza virus H5N1 subtype

The mutation and genetic variation of avian influenza virus ussually associated with Hemmaglutinin (HA) and Neuraminidase (NA). The HA and NA protein are surface glycoproteins which have role for receptor binding site of the virus, determine virus subtype and genetic variation occured in those proteins. On the other site, the virus have the internal protein that posses function for virus replication. This study analyzed the mutation on the the internal protein virus especially the Matrix (M1) and non structural (NS1) protein and its three dimensioanal structure of proteins. The methods used in this study were virus propagation, (Reverse Transcriptase Polymerase Chain Reaction) RT-PCR sequencing of M1 and NS1 and using DS Modeller dan DS Standalone from Discovery Studio for Modeling and Simulation to predict the three dimentional structure of the proteins. The result of this study showed that Indonesian AI H5N1 subtype had genetic variation internal protein and have no change on conserved of condition such as putative zinc finger and nuclear localization signal (NLS). Genetic variation that occured on PDZ motif of NS1 especially have human origin motif that might be correlated with virus adaptation on human. Key Words: Avian Influenza, Matrix (M1), Non Structural (NS1), Genetic Variation

Serotonin transporter interacts with the PDGFβ receptor in PDGF-BB-induced signaling and mitogenesis in pulmonary artery smooth muscle cells

The serotonin transporter (SERT) and the platelet-derived growth factor receptor (PDGFR) have been implicated in both clinical and experimental pulmonary hypertension (PH) and the facilitation of pulmonary artery smooth muscle cell (PASMC) growth. To gain a better understanding of the possible relationship of these two cell surface molecules we have explored interactions between SERT and PDGFR. We have previously demonstrated that SERT transactivates PDGFRβ in serotonin-stimulated PASMC proliferation. We now provide evidence for a role for SERT in PDGF-BB signaling and PASMC proliferation by using pharmacological inhibitors, genetic ablation, and construct overexpression of SERT. The results show that four tested SERT blockers dose dependently inhibit PDGF-stimulated human and bovine PASMC proliferation with comparable efficacy to that of PDGFR inhibitors, whereas 5-HT1B or 5-HT2A receptor inhibitors had no effect. Combinations of the SERT and PDGFR inhibitors led to synergistic/additive inhibition. Similarly, PDGF-induced PASMC proliferation was attenuated by small interfering RNA downregulation of SERT. Inhibition of SERT in PASMCs attenuated PDGF-induced phosphorylation of PDGFRβ, Akt, and p38 but not Erk. Overexpression of SERT in HEK293 cells led to enhanced Akt phosphorylation by PDGF, which was blunted by a SERT PDZ motif mutant, indicating the mechanistic need for the PDZ motif of SERT in PDGF signaling. Furthermore, coimmunoprecipitation experiments showed that SERT and PDGFRβ become physically associated upon PDGF stimulation. In total, the data show for the first time an important interactive relationship between SERT and the PDGFRβ in the production of PASMC proliferation triggered by PDGF that may be important in PH.

RGS19 enhances cell proliferation through its C-terminal PDZ motif

Regulator of G protein signaling 19 (RGS19), also known as Gα-interacting protein (GAIP), is a GTPase activating protein (GAP) for Gα i subunits. Apart from its GAP function, RGS19 has been implicated in growth factor signaling through binding to GAIP-interacting protein C-terminus (GIPC) via its C-terminal PDZ-binding motif. To gain additional insight on its function, we have stably expressed RGS19 in a number of mammalian cell lines and examined its effect on cell proliferation. Interestingly, overexpression of RGS19 stimulated the growth of HEK293, PC12, Caco2, and NIH3T3 cells. This growth promoting effect was not shared by other RGS proteins including RGS4, RGS10 and RGS20. Despite its ability to stimulate cell proliferation, RGS19 failed to induce neoplastic transformation in NIH3T3 cells as determined by focus formation and soft-agar assays, and it did not induce tumor growth in athymic nude mice. Deletion mutants of RGS19 lacking the PDZ-binding motif failed to complex with GIPC and did not exhibit any growth promoting effect. Overexpression of GIPC alone in HEK293 cells stimulated cell proliferation whereas its knockdown in H1299 non-small cell lung carcinomas suppressed cell proliferation. This study demonstrates that RGS19, in addition to acting as a GAP, is able to stimulate cell proliferation in a GIPC-dependent manner.

A newly discovered mutation in PICK1 in a human with globozoospermia

Globozoospermia is a human infertility syndrome caused by spermatogenesis defects (OMIM 102530). Acrosome plays an important role at the site of sperm-zonapellucida binding during the fertilization process. Thus, malformation of the acrosome is the most prominent feature seen in globozoospermia. Disruption of several mouse genes, including Gopc (Golgi-associated PDZ and coiled-coil motif containing protein), Hrb (HIV-1 Rev binding protein), Csnk2a2 (casein kinase 2, alpha prime polypeptide) and Pick1 (protein interacting with C kinase 1), results in a phenotype similar to globozoospermia in humans, which suggests their potential role in the disease. However, no mutations with a clear link to globozoospermia have been identified in these genes in humans. In this study, we screened the candidate genes mentioned above in three globozoospermia type I patients and discovered a homozygous missense mutation (G198A) in exon 13 of the PICK1 gene in a Chinese family. The family member affected by this homozygous missense mutation showed a complete lack of acrosome. Using the candidate gene screening strategy, our study is the first to identify an autosomal recessive genetic mutation in PICK1 that was responsible for globozoospermia in humans.

Binding of PDZ-RhoGEF to ATP-binding Cassette Transporter A1 (ABCA1) Induces Cholesterol Efflux through RhoA Activation and Prevention of Transporter Degradation

ATP-binding cassette transporter A1 (ABCA1)-mediated lipid efflux to apolipoprotein A1 (apoA-I) initiates the biogenesis of high density lipoprotein. Here we show that the Rho guanine nucleotide exchange factors PDZ-RhoGEF and LARG bind to the C terminus of ABCA1 by a PDZ-PDZ interaction and prevent ABCA1 protein degradation by activating RhoA. ABCA1 is a protein with a short half-life, and apoA-I stabilizes ABCA1 protein; however, depletion of PDZ-RhoGEF/LARG by RNA interference suppressed the apoA-I stabilization of ABCA1 protein in human primary fibroblasts. Exogenous PDZ-RhoGEF expression activated RhoA and increased ABCA1 protein levels and cholesterol efflux activity. Likewise, forced expression of a constitutively active RhoA mutant significantly increased ABCA1 protein levels, whereas a dominant negative RhoA mutant decreased them. The constitutively active RhoA retarded ABCA1 degradation, thus accounting for its ability to increase ABCA1 protein. Moreover, stimulation with apoA-I transiently activated RhoA, and the pharmacological inhibition of RhoA or the dominant negative RhoA blocked the ability of apoA-I to stabilize ABCA1. Finally, depletion of RhoA or RhoGEFs/RhoA reduces the cholesterol efflux when transcriptional regulation via PPARgamma is eliminated. Taken together, our results have identified a novel physical and functional interaction between ABCA1 and PDZ-RhoGEF/LARG, which activates RhoA, resulting in ABCA1 stabilization and cholesterol efflux activity.

Ephrin-B2 regulates endothelial cell morphology and motility independently of Eph-receptor binding

The transmembrane protein ephrin-B2 regulates angiogenesis, i.e. the formation of new blood vessels through endothelial sprouting, proliferation and remodeling processes. In addition to essential roles in the embryonic vasculature, ephrin-B2 expression is upregulated in the adult at sites of neovascularization, such as tumors and wounds. Ephrins are known to bind Eph receptor family tyrosine kinases on neighboring cells and trigger bidirectional signal transduction downstream of both interacting molecules. Here we show that ephrin-B2 dynamically modulates the motility and cellular morphology of isolated endothelial cells. Even in the absence of Eph-receptor binding, ephrin-B2 stimulates repeated cycling between actomyosin-dependent cell contraction and spreading episodes, which requires the presence of the C-terminal PDZ motif. Our results show that ephrin-B2 is a potent regulator of endothelial cell behavior, and indicate that the control of cell migration and angiogenesis by ephrins might involve both receptor-dependent and receptor-independent activities.

Ephrin-B2 regulates endothelial cell morphology and motility independently of Eph-receptor binding

The transmembrane protein ephrin-B2 regulates angiogenesis, i.e. the formation of new blood vessels through endothelial sprouting, proliferation and remodeling processes. In addition to essential roles in the embryonic vasculature, ephrin-B2 expression is upregulated in the adult at sites of neovascularization, such as tumors and wounds. Ephrins are known to bind Eph receptor family tyrosine kinases on neighboring cells and trigger bidirectional signal transduction downstream of both interacting molecules. Here we show that ephrin-B2 dynamically modulates the motility and cellular morphology of isolated endothelial cells. Even in the absence of Eph-receptor binding, ephrin-B2 stimulates repeated cycling between actomyosin-dependent cell contraction and spreading episodes, which requires the presence of the C-terminal PDZ motif. Our results show that ephrin-B2 is a potent regulator of endothelial cell behavior, and indicate that the control of cell migration and angiogenesis by ephrins might involve both receptor-dependent and receptor-independent activities.

ZASP1-D117N ON Z-LINE DECREASES HNAV1.5 FUNCTION IN A DCM SUBJECT WITH CONDUCTION DISTURBANCES

Abstract Category: Electrophysiology--BasicPresentation Number: 1078-125Authors: Yutao Xi, Tomohiko AI, Zhaohui Li, Geru Wu, Enkhsaikhan Purevjav, Shahrzad Abbasi, Jie Cheng, Matteo Vatta, Electrophysiology Research Laboratory, Texas Heart Institute/St. Luke’s Episcopal Hospital, Hoston, TX, Pediatric Cardiology, Texas Children’s Hospital/Baylor College of Medicine, Houston, TX Background: Dilated cardiomyopathy (DCM) is often associated with conduction system diseases. However, the underlying molecular mechanisms remain unclear. We have previously identified the D117N mutation in the Z-band Alternatively Spliced PDZ motif (ZASP1), in a DCM patient with conduction disturbances, while the ZASP1-K136M was found in a DCM subject with no conduction defects. This study sought to elucidate the role of ZASP1-D117N in regulating the cardiac sodium channel (hNav1.5), which is critically involved in conduction system diseases. Methods: Patch-clamp techniques were used to measure INa in either HEK293 cells stably expressing hNav1.5 or neonatal rat cardiomyocytes (NRCM), both transiently transfected with WT or mutant ZASP1. Pull-down assay and immunohistochemistry (IHC) analysis were employed to study the interaction and localization between hNav1.5, WT and mutant ZASP and other cytoskeletal proteins.Results: Macroscopic INa were significantly decreased in cells expressing D117N compared to WT and K136M (WT, -252.0 ± 24.5 pA/pF, n = 20; K136M, -259.7 ± 37.1 pA/pF, n = 17; D117N, -182.2 ± 18.5 pA/pF, p<0.05). ZASP1-D117N, not K136M, rightward shifted steady-state activation (Vh: WT, -44.3 ± 1.6 mV, n = 23; K136M, -44.0 ± 2.5 mV, n = 17; D117N, -36.9 ± 1.6 mV, n = 18, p<0.05) and steady-state inactivation (Vh: WT, -100.3 ± 1.4 mV, n = 16; K136M, -100.9 ± 2.6, n = 17; D117N, -92.9 ± 1.4 mV, n = 19, p<0.005). Same INa reduction occurred in NRCM expressing D117N, but not ZASP1-WT. Furthermore, pull-down and IHC experiments demonstrated that both WT and D117N ZASP1 co-localized with the complex including hNav1.5, Telethonin and ACTN2, although NRCM expressing ZASP1-D117N demonstrate less organized Z-line. Treatment using ML-7, a cytoskeleton disruption agent, normalized INa parameters, rescuing hNav1.5 function.Conclusions: ZASP1-D117N decreases hNav1.5 function via a hNav1.5/Teletholnin/ZASP1/ACTN2 complex, and might account for conduction disturbances associated with DCM. Our study suggests that disrupted cytoarchitectural proteins are the primary cause of cardiomyopathies, and may predispose to secondary ion channel dysfunction and cardiac arrhythmias.

Deletion of the distal COOH-terminus of the A2B adenosine receptor switches internalization to an arrestin- and clathrin-independent pathway and inhibits recycling.

We have investigated the effect of deletions of a postsynaptic density, disc large and zo-1 protein (PDZ) motif at the end of the COOH-terminus of the rat A(2B) adenosine receptor on intracellular trafficking following long-term exposure to the agonist 5'-(N-ethylcarboxamido)-adenosine. The trafficking of the wild type A(2B) adenosine receptor and deletion mutants expressed in Chinese hamster ovary cells was studied using an enzyme-linked immunosorbent assay in combination with immunofluorescence microscopy. The wild type A(2B) adenosine receptor and deletion mutants were all extensively internalized following prolonged treatment with NECA. The intracellular compartment through which the Gln(325)-stop receptor mutant, which lacks the Type II PDZ motif found in the wild type receptor initially trafficked was not the same as the wild type receptor. Expression of dominant negative mutants of arrestin-2, dynamin or Eps-15 inhibited internalization of wild type and Leu(330)-stop receptors, whereas only dominant negative mutant dynamin inhibited agonist-induced internalization of Gln(325)-stop, Ser(326)-stop and Phe(328)-stop receptors. Following internalization, the wild type A(2B) adenosine receptor recycled rapidly to the cell surface, whereas the Gln(325)-stop receptor did not recycle. Deletion of the COOH-terminus of the A(2B) adenosine receptor beyond Leu(330) switches internalization from an arrestin- and clathrin-dependent pathway to one that is dynamin dependent but arrestin and clathrin independent. The presence of a Type II PDZ motif appears to be essential for arrestin- and clathrin-dependent internalization, as well as recycling of the A(2B) adenosine receptor following prolonged agonist addition.

Analysis of the Potential Role of GluA4 Carboxyl-Terminus in PDZ Interactions

BackgroundSpecific delivery to synapses of α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptors with long-tailed subunits is believed to be a key event in many forms of activity-dependent changes in synaptic strength. GluA1, the best characterized long-tailed AMPA receptor subunit, contains a C-terminal class I PDZ binding motif, which mediates its interaction with scaffold and trafficking proteins, including synapse-associated protein 97 (SAP97). In GluA4, another long-tailed subunit implicated in synaptic plasticity, the PDZ motif is blocked by a single proline residue. This feature is highly conserved in vertebrates, whereas the closest invertebrate homologs of GluA4 have a canonical class I PDZ binding motif. In this work, we have examined the role of GluA4 in PDZ interactions.Methodology/Principal FindingsDeletion of the carboxy-terminal proline residue of recombinant GluA4 conferred avid binding to SAP97 in cultured cells as shown by coimmunoprecipitation, whereas wild-type GluA4 did not associate with SAP97. Native GluA4 and SAP97 coimmunoprecipitated from mouse brain independently of the GluA1 subunit, supporting the possibility of in vivo PDZ interaction. To obtain evidence for or against the exposure of the PDZ motif by carboxyterminal processing of native GluA4 receptors, we generated an antibody reagent specific for proline-deleted GluA4 C-terminus. Immunoprecipitation and mass spectrometric analyses indicated that the carboxyl-terminus of native GluA4 AMPA receptors is intact and that the postulated single-residue cleavage does not occur to any significant extent.Conclusion/SignificanceWe conclude that native GluA4 receptors are not capable of canonical PDZ interactions and that their association with SAP97 is likely to be indirect.

Abstract 5578: Cell Polarity Factor Par3 Binds SPTLC1 Modulating Monocyte/Macrophage Serine Palmitoyltransferase Activity, De Novo Ceramide Synthesis and Chemotaxis

Elevated sphingolipids associate with increased cardiovascular disease. Conversely, atherosclerosis is reduced in mice by blocking de novo synthesis of sphingolipids catalyzed by serine palmitoyltransferase (SPT). The SPT enzyme is composed of the SPTLC1 and 2 subunits, and we have identified a novel protein-protein interaction between SPTLC1 and the PDZ protein Partitioning Defective Protein 3 (Par3). Mammalian SPTLC1 orthologs have a highly conserved C-terminus that conforms to a type II PDZ protein interaction motif, and by screening PDZ domain protein arrays with a SPTLC1 C-terminal peptide we found it bound the 3 rd PDZ domain of Par3. Overlay and immuno-precipitation assays confirmed this interaction and indicate Par3 associates with the SPTLC1/2 holoenzyme by binding the C-terminal PDZ motif of SPTLC1, and this binding can re-localize SPTLC1 to the periphery of the cell at sites of cell-cell contact and in cell protrusions. The SPTLC1/2-Par3 complex was detected at endogenous expression levels in mouse liver and primary macrophages demonstrating its physiologic existence, and siRNA inhibition of Par3 in human THP-1 monocytes significantly reduced SPT activity and the de novo synthesis of ceramide and sphingomyelin by nearly 40%. Given monocyte recruitment into inflamed vessels promotes atherosclerosis, and since Par3 and sphingolipids have been associated with polarized cell migration, we tested and found that Par3 and SPTLC1 are required for the ability of THP-1 monocytes to migrate towards monocyte chemoattractant protein 1 (MCP-1). Knockdown of Par3 significantly reduced MCP-1 induced chemotaxis of THP-1 monocytes, as did knockdown of SPTLC1, and this Par3 effect depended upon SPT activity. Migration in response to other inflammatory G-protein coupled chemokines including fractalkine and fMLP was also inhibited in these cells and in primary mouse macrophages treated with the anti-atherosclerotic SPT inhibitor myriocin. In conclusion, we have identified a novel SPTLC1-Par3 interaction that promotes monocyte serine palmitoyltransferase activity and chemotaxis towards inflammatory chemokine signals.

Agonist Dose-dependent Phosphorylation by Protein Kinase A and G Protein-coupled Receptor Kinase Regulates β2 Adrenoceptor Coupling to Gi Proteins in Cardiomyocytes

Adrenoceptors receptors (ARs) play a pivotal role in regulating cardiovascular response to catecholamines during stress. beta(2)ARs, prototypical G protein-coupled receptors (GPCRs), expressed in animal hearts, display dual coupling to both G(s) and G(i) proteins to control the adenylyl cyclase-cAMP dependent protein kinase A (PKA) pathway to regulate contraction responses. Here, we showed that the beta(2)AR coupling to G(i) proteins was agonist dose-dependent and occurred only at high concentrations in mouse cardiac myocytes. Both the beta(2)AR-induced PKA activity, measured by fluorescence resonance energy transfer (FRET) imaging, and the increase in myocyte contraction rate displayed sensitivity to the G(i) inhibitor pertussis toxin (PTX). Further studies revealed that activated beta(2)ARs underwent PKA phosphorylation at a broad range of agonist concentrations. Disruption of the PKA phosphorylation sites on the beta(2)AR blocked receptor/G(i) coupling. However, a sufficient beta(2)AR/G(i) coupling was also dependent on the G protein-coupled receptor kinase (GRK)-mediated phosphorylation of the receptors, which only occurred at high concentrations of agonist (> or = 100 nm). Disruption of the GRK phosphorylation sites on the beta(2)AR blocked receptor internalization and coupling to G(i) proteins, probably by preventing the receptor's transportation to access G(i) proteins. Furthermore, neither PKA nor GRK site mutated receptors displayed sensitivity to the G(i)-specific inhibitor, G(i)CT. Together, our studies revealed distinct roles of PKA and GRK phosphorylation of the beta(2)AR for agonist dose-dependent coupling to G(i) proteins in cardiac myocytes, which may protect cells from overstimulation under high concentrations of catecholamines.

Cell Polarity Factor Par3 Binds SPTLC1 and Modulates Monocyte Serine Palmitoyltransferase Activity and Chemotaxis

Elevated sphingolipids have been associated with increased cardiovascular disease. Conversely, atherosclerosis is reduced in mice by blocking de novo synthesis of sphingolipids catalyzed by serine palmitoyltransferase (SPT). The SPT enzyme is composed of the SPTLC1 and -2 subunits, and here we describe a novel protein-protein interaction between SPTLC1 and the PDZ protein Par3 (partitioning defective protein 3). Mammalian SPTLC1 orthologs have a highly conserved C terminus that conforms to a type II PDZ protein interaction motif, and by screening PDZ domain protein arrays with an SPTLC1 C-terminal peptide, we found it bound the third PDZ domain of Par3. Overlay and immunoprecipitation assays confirmed this interaction and indicate Par3 is able to associate with the SPTLC1/2 holoenzyme by binding the C-terminal SPTLC1 PDZ motif. The physiologic existence of the SPTLC1/2-Par3 complex was detected in mouse liver and macrophages, and short interfering RNA inhibition of Par3 in human THP-1 monocytes significantly reduced SPT activity and de novo ceramide synthesis by nearly 40%. Given monocyte recruitment into inflamed vessels is thought to promote atherosclerosis, and because Par3 and sphingolipids have been associated with polarized cell migration, we tested whether the ability of THP-1 monocytes to migrate toward MCP-1 (monocyte chemoattractant protein 1) depended upon Par3 and SPTLC1 expression. Knockdown of Par3 significantly reduced MCP1-induced chemotaxis of THP-1 monocytes, as did knockdown of SPTLC1, and this Par3 effect depended upon SPT activity and was blunted by ceramide treatment. In conclusion, protein arrays were used to identify a novel SPTLC1-Par3 interaction that associates with increased monocyte serine palmitoyltransferase activity and chemotaxis toward inflammatory signals.

Sticky Fingers

See related articles, pages 758–769 The multifunctional Ca2+ and calmodulin dependent protein kinase (CaMK)II is a serine threonine kinase that plays increasingly evident and important roles in regulating ion channels in heart and other excitable tissues. CaMKII activation initially requires an increase in intracellular Ca2+ that leads to binding of calcified calmodulin (Ca2+/CaM) to the CaMKII regulatory domain. This activating Ca2+/CaM signal is amplified within the CaMKII holoenzyme, which is composed of 10 to 12 monomers arranged in a wheel and spoke pattern (Figure, A), by an autophosphorylation process. Autophosphorylation of Thr286/287 (numbering varies by isoform) markedly increases the avidity of Ca2+/CaM binding to CaMKII but also renders CaMKII constitutively active even in the absence of Ca2+/CaM binding.1 Autophosphorylation of Thr286/287 prevents the reassociation of the autoinhibitory region with the CaMKII catalytic domain, leaving the autophosphorylated kinase in the open and active configuration. Reactive oxygen species modify a pair of methionines (281/282) near the autophosphorylation site to convert CaMKII activity from Ca2+/CaM-dependent to Ca2+/CaM-independent by a process that mirrors autophosphorylation (Figure, A).2 In this issue of Circulation Research , El-Haou et al3 have provided exciting new insights into the association of CaMKII with the MAGUK protein SAP97 by showing that a PDZ motif on the C terminus of KV4.3 partners with the SAP97-CaMKII module to enable CaMKII-dependent gating effects on KV4.3 channels. CaMKII-mediated increases in the transient outward current ( I to) require binding of the SAP97-CaMKII module to the KV4.3 C terminus. These findings provide a molecular framework for understanding how excitable tissues tune repolarization to manage …

Membrane targeting and intracellular trafficking of the human sodium-dependent multivitamin transporter in polarized epithelial cells.

The human sodium-dependent multivitamin transporter (hSMVT) mediates sodium-dependent uptake of biotin in renal and intestinal epithelia. To date, however, there is nothing known about the structure-function relationship or targeting sequences in the hSMVT polypeptide that control its polarized expression within epithelia. Here, we focused on the role of the COOH-terminal tail of hSMVT in the targeting and functionality of this transporter. A full-length hSMVT-green fluorescent protein (GFP) fusion protein was functional and expressed at the apical membrane in renal and intestinal cell lines. Microtubule disrupting agents disrupted the mobility of trafficking vesicles and impaired cell surface delivery of hSMVT, which was also prevented in cells treated with dynamitin (p50), brefeldin, or monensin. Progressive truncation of the COOH-terminal tail impaired the functionality and targeting of the transporter. First, biotin transport decreased by approximately 20-30% on deletion of up to 15 COOH-terminal amino acids of hSMVT, a decrease mimicked solely by deletion of the terminal PDZ motif (TSL). Second, deletions into the COOH-terminal tail (between residues 584-612, containing a region of predicted high surface accessibility) resulted in a further drop in hSMVT transport (to approximately 40% of wild-type). Third, apical targeting was lost on deletion of a helical-prone region between amino acids 570-584. We conclude that the COOH tail of hSMVT contains several determinants important for polarized targeting and biotin transport.

Ancient, independent evolution and distinct molecular features of the novel human T-lymphotropic virus type 4

BackgroundHuman T-lymphotropic virus type 4 (HTLV-4) is a new deltaretrovirus recently identified in a primate hunter in Cameroon. Limited sequence analysis previously showed that HTLV-4 may be distinct from HTLV-1, HTLV-2, and HTLV-3, and their simian counterparts, STLV-1, STLV-2, and STLV-3, respectively. Analysis of full-length genomes can provide basic information on the evolutionary history and replication and pathogenic potential of new viruses.ResultsWe report here the first complete HTLV-4 sequence obtained by PCR-based genome walking using uncultured peripheral blood lymphocyte DNA from an HTLV-4-infected person. The HTLV-4(1863LE) genome is 8791-bp long and is equidistant from HTLV-1, HTLV-2, and HTLV-3 sharing only 62–71% nucleotide identity. HTLV-4 has a prototypic genomic structure with all enzymatic, regulatory, and structural proteins preserved. Like STLV-2, STLV-3, and HTLV-3, HTLV-4 is missing a third 21-bp transcription element found in the long terminal repeats of HTLV-1 and HTLV-2 but instead contains unique c-Myb and pre B-cell leukemic transcription factor binding sites. Like HTLV-2, the PDZ motif important for cellular signal transduction and transformation in HTLV-1 and HTLV-3 is missing in the C-terminus of the HTLV-4 Tax protein. A basic leucine zipper (b-ZIP) region located in the antisense strand of HTLV-1 and believed to play a role in viral replication and oncogenesis, was also found in the complementary strand of HTLV-4. Detailed phylogenetic analysis shows that HTLV-4 is clearly a monophyletic viral group. Dating using a relaxed molecular clock inferred that the most recent common ancestor of HTLV-4 and HTLV-2/STLV-2 occurred 49,800 to 378,000 years ago making this the oldest known PTLV lineage. Interestingly, this period coincides with the emergence of Homo sapiens sapiens during the Middle Pleistocene suggesting that early humans may have been susceptible hosts for the ancestral HTLV-4.ConclusionThe inferred ancient origin of HTLV-4 coinciding with the appearance of Homo sapiens, the propensity of STLVs to cross-species into humans, the fact that HTLV-1 and -2 spread globally following migrations of ancient populations, all suggest that HTLV-4 may be prevalent. Expanded surveillance and clinical studies are needed to better define the epidemiology and public health importance of HTLV-4 infection.

The Interaction between Human Papillomavirus Type 16 and FADD Is Mediated by a Novel E6 Binding Domain

High-risk strains of human papillomavirus, such as types 16 and 18, have been etiologically linked to cervical cancer. Most cervical cancer tissues are positive for both the E6 and E7 oncoproteins, since it is their cooperation that results in successful transformation and immortalization of infected cells. We have reported that E6 binds to tumor necrosis factor receptor 1 and to Fas-associated death domain (FADD) and, in doing so, prevents E6-expressing cells from responding to apoptotic stimuli. The binding site of E6 to FADD localizes to the first 23 amino acids of FADD and has now been further characterized by the use of deletion and site-directed mutants of FADD in pull-down and functional assays. The results from these experiments revealed that mutations of serine 16, serine 18, and leucine 20 obstruct FADD binding to E6, suggesting that these residues are part of the E6 binding domain on FADD. Because FADD does not contain the two previously identified E6 binding motifs, the LxxphiLsh motif, and the PDZ motif, a novel binding domain for E6 has been identified on FADD. Furthermore, peptides that correspond to this region can block E6/FADD binding in vitro and can resensitize E6-expressing cells to apoptotic stimuli in vivo. These results demonstrate the existence of a novel E6 binding domain.

Implicaton of tetraspanin‐enriched microdomains (TEMs) in hepatitis C virus†

Implicaton of tetraspanin‐enriched microdomains (TEMs) in hepatitis C virus†