Characterization of the structure and intermolecular interactions between the connexin 32 carboxyl-terminal domain and the protein partners synapse-associated protein 97 and calmodulin.

In Schwann cells, connexin 32 (Cx32) can oligomerize to form intracellular gap junction channels facilitating a shorter pathway for metabolite diffusion across the layers of the myelin sheath. The mechanisms of Cx32 intracellular channel regulation have not been clearly defined. However, Ca(2+), pH, and the phosphorylation state can regulate Cx32 gap junction channels, in addition to the direct interaction of protein partners with the carboxyl-terminal (CT) domain. In this study, we used different biophysical methods to determine the structure and characterize the interaction of the Cx32CT domain with the protein partners synapse-associated protein 97 (SAP97) and calmodulin (CaM). Our results revealed that the Cx32CT is an intrinsically disordered protein that becomes α-helical upon binding CaM. We identified the GUK domain as the minimal SAP97 region necessary for the Cx32CT interaction. The Cx32CT residues affected by the binding of CaM and the SAP97 GUK domain were determined as well as the dissociation constants for these interactions. We characterized three Cx32CT Charcot-Marie-Tooth disease mutants (R219H, R230C, and F235C) and identified that whereas they all formed functional channels, they all showed reduced binding affinity for SAP97 and CaM. Additionally, we report that in RT4-D6P2T rat schwannoma cells, Cx32 is differentially phosphorylated and exists in a complex with SAP97 and CaM. Our studies support the importance of protein-protein interactions in the regulation of Cx32 gap junction channels and myelin homeostasis.

ALIX/AIP1 is required for NP incorporation into Mopeia virus Z-induced virus-like particles.

During virus particle assembly, the arenavirus nucleoprotein (NP) associates with the viral genome to form nucleocapsids, which ultimately become incorporated into new virions at the cell membrane. Virion release is facilitated by the viral matrix Z protein through its interaction with the cellular endosomal sorting complex required for transport (ESCRT) machinery. However, the mechanism of nucleocapsid incorporation into virions is not well understood. Here, we demonstrate that ALIX/AIP1, an ESCRT-associated host protein, is required for the incorporation of the NP of Mopeia virus, a close relative of Lassa virus, into Z-induced virus-like particles (VLPs). Furthermore, we show that the Bro1 domain of ALIX/AIP1 interacts with the NP and Z proteins simultaneously, facilitating their interaction, and we identify residues 342 to 399 of NP as being necessary for its interaction with ALIX/AIP1. Our observations suggest a potential role for ALIX/AIP1 in linking Mopeia virus NP to Z and the budding apparatus, thereby promoting NP incorporation into virions.

Disruption of Mtmr2 produces CMT4B1-like neuropathy with myelin outfolding and impaired spermatogenesis.

Mutations in MTMR2, the myotubularin-related 2 gene, cause autosomal recessive Charcot-Marie-Tooth (CMT) type 4B1, a demyelinating neuropathy with myelin outfolding and azoospermia. MTMR2 encodes a ubiquitously expressed phosphatase whose preferred substrate is phosphatidylinositol (3,5)-biphosphate, a regulator of membrane homeostasis and vesicle transport. We generated Mtmr2-null mice, which develop progressive neuropathy characterized by myelin outfolding and recurrent loops, predominantly at paranodal myelin, and depletion of spermatids and spermatocytes from the seminiferous epithelium, which leads to azoospermia. Disruption of Mtmr2 in Schwann cells reproduces the myelin abnormalities. We also identified a novel physical interaction in Schwann cells, between Mtmr2 and discs large 1 (Dlg1)/synapse-associated protein 97, a scaffolding molecule that is enriched at the node/paranode region. Dlg1 homologues have been located in several types of cellular junctions and play roles in cell polarity and membrane addition. We propose that Schwann cell-autonomous loss of Mtmr2-Dlg1 interaction dysregulates membrane homeostasis in the paranodal region, thereby producing outfolding and recurrent loops of myelin.

Craniofacial dysmorphogenesis including cleft palate in mice with an insertional mutation in the discs large gene.

The discs large (Dlg) protein, or synapse-associated protein 97 (SAP97), is a member of the membrane-associated guanylate kinase family of multidomain scaffolding proteins which recruits transmembrane and signaling molecules to localized plasma membrane sites. Murine dlg is the homologue of the Drosophila dlg tumor suppressor gene. The loss of dlg function in Drosophila disrupts cellular growth control, apicobasal polarity, and cell adhesion of imaginal disc epithelial cells, resulting in embryonic lethality. In this study, we isolated a mutational insertion in the murine dlg locus by gene trapping in totipotent embryonic stem cells. This insertion results in a truncated protein product that contains the N-terminal three PSD-95/DLG/ZO-1 domains of Dlg fused to the LacZ reporter and subsequently lacks the src homology 3 (SH3), protein 4.1 binding, and guanylate kinase (GUK)-like domains. The Dlg-LacZ fusion protein is expressed in epithelial, mesenchymal, neuronal, endothelial, and hematopoietic cells during embryogenesis. Mice homozygous for the dlg mutation exhibit growth retardation in utero, have hypoplasia of the premaxilla and mandible, have a cleft secondary palate, and die perinatally. Consistent with this phenotype, Dlg-LacZ is expressed in mesenchymal and epithelial cells throughout palatal development. Our genetic and phenotypic analysis of dlg mutant mice suggests that protein-protein interactions involving the SH3, protein 4.1 binding, and/or GUK-like domains are essential to the normal function of murine Dlg within craniofacial and palatal morphogenesis.

Effects of the dimeric PSD-95 inhibitor UCCB01-144 in mouse models of pain, cognition and motor function.

NMDAR antagonism shows analgesic action in humans and animal pain models, but disrupts cognitive and motor functions. NMDAR-dependent NO production requires tethering of the NMDAR to neuronal NO synthase (nNOS) by the postsynaptic density protein-95 (PSD-95). Perturbing the NMDAR/PSD-95/nNOS interaction has therefore been proposed as an alternative analgesic mechanism. We recently reported that UCCB01-125, a dimeric PSD-95 inhibitor with limited blood-brain-barrier permeability, reduced mechanical hypersensitivity in the complete Freund's adjuvant (CFA) inflammatory pain model, without disrupting cognitive or motor functions. Here, we investigated the analgesic efficacy in the CFA model of UCCB01-144, a PSD-95 inhibitor with improved blood-brain-barrier permeability. To extend the comparison of UCCB01-125 and UCCB01-144, we also tested both compounds in the spared nerve injury (SNI) model of neuropathic pain. Potential cognitive effects of UCCB01-144 were examined using the social transmission of food preference (STFP) test and the V-maze test, and motor coordination was assessed with the rotarod test. UCCB01-144 (10mg/kg) reversed CFA-induced mechanical hypersensitivity after 1h, and completely normalised sensitivity after 24h. In the SNI model, UCCB01-144 (30mg/kg) partially reversed hypersensitivity after 1h, but no effect was observed after 24h. UCCB01-125 did not affect SNI-induced hypersensitivity. Rotarod performance was unaffected by UCCB01-144, but 30mg/kg UCCB01-144 impaired performance in the STFP test. Collectively, UCCB01-144 reversed both CFA and SNI-induced hypersensitivity, but the efficacy in the SNI model was only transient. This suggests that enhanced BBB permeability of PSD-95 inhibitors improves the analgesic action in neuropathic pain states.

Association of the kinesin superfamily motor protein KIF1Balpha with postsynaptic density-95 (PSD-95), synapse-associated protein-97, and synaptic scaffolding molecule PSD-95/discs large/zona occludens-1 proteins.

Mutation in KIF1B, a kinesin superfamily motor protein, causes a peripheral neuropathy known as Charcot-Marie-Tooth disease type 2A (CMT2A). Little is known, however, about how a defective KIF1B gene leads to CMT2A. Here we report that KIF1Balpha, one of the two splice variants of KIF1B, directly interacts through its C-terminal postsynaptic density-95 (PSD-95)/discs large/zona occludens (PDZ) domain-binding motif with PDZ proteins including PSD-95/synapse-associated protein-90 (SAP90), SAP97, and synaptic scaffolding molecule (S-SCAM)-90 (SAP90). KIF1Balpha selectively interacts with PSD-95, SAP97, and S-SCAM in yeast two-hybrid, pull-down, and in vivo coimmunoprecipitation experiments. KIF1Balpha, SAP97, and S-SCAM are widely distributed to both dendrites and axons of cultured neurons and are enriched in the small membrane fraction of the brain. In the flotation assay, KIF1Balpha cofractionates and coimmunoprecipitates with PSD-95, SAP97, and S-SCAM. These results suggest that the PSD-95 family proteins and S-SCAM have a novel function as KIF1Balpha receptors, linking KIF1Balpha to its specific cargos, and are involved in peripheral neuropathies.

Acute amnestic encephalopathy in amyloid-ß oligomer-injected mice is due to their widespread diffusion in vivo.

Amyloid-ß (Aß) oligomers are the suspected culprit as initiators of Alzheimer's disease (AD). However, their diffusion in the brain remains unknown. Here, we studied Aß oligomers' dissemination and evaluated their in vivo toxicity. Wild-type mice were injected with 50 pmol of synthetic Aß oligomers (of different size) in the hippocampus. Oligomers diffused largely in the brain as soon as 1 hour and up to 7 days after injection. A transient encephalopathy with memory impairment was induced by this unique injection. The immunoreactivity of the postsynaptic marker PSD95 was diffusely decreased. Similar results (both on memory and PSD95 immunoreactivity) were obtained with delipidated and high molecular weight oligomers (>50 kDa) but not with smaller assemblies. Tau hyperphosphorylation was observed in the oligomer-injected brains. Finally, fos immunostaining was increased in Aß-derived diffusible ligands-injected mice, suggesting neuronal hyperactivity. Rapid and widespread diffusion of Aß oligomers was demonstrated in vivo and associated with decreased synaptic markers and memory deficits which gives new insight to the pathogenicity of Aß.

Analysis of PHA-1 reveals a limited role in pharyngeal development and novel functions in other tissues.

PHA-1 encodes a cytoplasmic protein that is required for embryonic morphogenesis and attachment of the foregut (pharynx) to the mouth (buccal capsule). Previous reports have in some cases suggested that PHA-1 is essential for the differentiation of most or all pharyngeal cell types. By performing mosaic analysis with a recently acquired pha-1 null mutation (tm3671), we found that PHA-1 is not required within most or all pharyngeal cells for their proper specification, differentiation, or function. Rather, our evidence suggests that PHA-1 acts in the arcade or anterior epithelial cells of the pharynx to promote attachment of the pharynx to the future buccal capsule. In addition, PHA-1 appears to be required in the epidermis for embryonic morphogenesis, in the excretory system for osmoregulation, and in the somatic gonad for normal ovulation and fertility. PHA-1 activity is also required within at least a subset of intestinal cells for viability. To better understand the role of PHA-1 in the epidermis, we analyzed several apical junction markers in pha-1(tm3671) homozygous embryos. PHA-1 regulates the expression of several components of two apical junction complexes including AJM-1-DLG-1/discs large complex and the classical cadherin-catenin complex, which may account for the role of PHA-1 in embryonic morphogenesis.

UCCB01-125, a dimeric inhibitor of PSD-95, reduces inflammatory pain without disrupting cognitive or motor performance: comparison with the NMDA receptor antagonist MK-801.

Excessive N-Methyl-d-aspartate receptor (NMDAR)-dependent production of nitric oxide (NO) is involved in the development and maintenance of chronic pain states, and is mediated by postsynaptic density protein-95 (PSD-95). By binding to both the NMDAR and neuronal NO synthase (nNOS), PSD-95 mediates a specific coupling between NMDAR activation and NO production. NMDAR antagonism shows anti-nociceptive action in humans and animal models of chronic pain but is associated with severe disturbances of cognitive and motor functions. An alternative approach to modulate the NMDAR-related activity is to perturb the NMDAR/PSD-95/nNOS complex by targeting PSD-95, thereby decreasing NO production without interfering with the NMDAR ion channel function. Here, we compared the effects of a dimeric PSD-95 inhibitor, UCCB01-125, and the NMDAR antagonist, MK-801, on mechanical hypersensitivity in the complete Freund's adjuvant (CFA) model of inflammatory pain. To examine side-effect profiles we also compared the effects of UCCB01-125 and MK-801 in tests of attention, long-term memory, and motor performance. When administered concurrently with CFA, both MK-801 and UCCB01-125 prevented the development of CFA-induced mechanical hypersensitivity 1 and 24 h after treatment. Moreover, UCCB01-125 was found to reverse CFA-induced hypersensitivity when administered 24 h after CFA treatment, an effect lasting for at least 3 days. At the dose reducing hypersensitivity, MK-801 disrupted attention, long-term memory, and motor performance. By contrast, even high doses of UCCB01-125 were devoid of side-effects in these tests. The data suggest that PSD-95 inhibition is a feasible strategy to prevent both development and maintenance of chronic inflammatory pain, while avoiding NMDAR antagonism-related side-effects.

Cracking phase diagram for the dynamics of an enzyme.

We measure the ensemble averaged deformation of an enzyme for an oscillating applied force. From the low frequency divergence of the mechanical susceptibility for the hinge motion of guanylate kinase we obtain a nonequilibrium phase diagram in the frequency-force plane. A phase line separates linear elasticity dynamics from softer (viscoelastic) dynamics. The hinge motion corresponds to crossing this phase line (not to a soft linear elastic mode). The phase line is dramatically shifted in the closed state compared to the open state of the enzyme.

Effect of a Surfactant on the Antimicrobial Activity of Sodium Hypochlorite Solutions

The objective of the present study was to evaluate the antimicrobial activity of sodium hypochlorite (NaOCl) associated with a surfactant. Seventy single-rooted extracted human teeth were inoculated with Enterococcus faecalis, and incubated for 21 days (37 °C). The groups were distributed according to the irrigation solution used during root canal preparation: 5%, 2.5% and 1% NaOCl; 5%, 2.5% and 1% Hypoclean(r), a solution containing a surfactant (cetrimide) associated with NaOCl. Three microbiological samples were collected from each tooth: S1 - before instrumentation; S2 - immediately after instrumentation; and S3 - after a seven-day period. Data were submitted to ANOVA and Tukey test with 5% significance level. The results showed that immediately after root canal preparation (S2), E. faecalis was eliminated in all the experimental groups. However, after 7 days (S3), only the groups in which Hypoclean was used, remained contamination-free, including Hypoclean associated with 1% NaOCl, while the root canals irrigated with 1% NaOCl only, presented the highest percentage of bacterial growth. In conclusion, the addition of surfactant increased the antimicrobial activity of 1% NaOCl to levels similar to 5% NaOCl.

O objetivo da presente pesquisa foi avaliar a atividade antimicrobiana de hipoclorito de sódio (NaOCl), associado a um tensoativo. Setenta dentes humanos monorradiculares extraídos foram inoculados com Enterococcus faecalis e incubados durante 21 dias (37 °C). Os grupos foram distribuídos de acordo com a solução irrigadora utilizada no preparo do canal: hipoclorito de sódio a 5%, 2,5% e 1%; Hypoclean(r) a 5%, 2,5% e 1% - uma solução contendo um surfactante (cetrimida) associado com NaOCl. Três amostras microbiológicas foram coletadas de cada dente: S1 - antes de instrumentação; S2 - imediatamente após a instrumentação; e S3 - após um período de sete dias. Os dados foram submetidos à análise de variância e teste de Tukey com 5% de nível de significância. Os resultados mostraram que imediatamente após o preparo do canal radicular (S2), o E. faecalis foi eliminado em todos os grupos experimentais. No entanto, após 7 dias (S3), apenas os grupos em que se utilizou Hypoclean permaneceram livres de contaminação, incluindo Hypoclean 1%, enquanto que os canais radiculares irrigados apenas com hipoclorito de sódio 1% apresentaram a mais elevada percentagem de crescimento bacteriano. Em conclusão, a adição de surfactante aumentou a atividade antimicrobiana de 1% de NaOCl a níveis semelhantes aos do NaOCl 5% .

MAGI1 recruits Dll1 to cadherin-based adherens junctions and stabilizes it on the cell surface.

Delta-Notch signaling plays an essential role in cell fate determination in many tissue types, including the central nervous system. Although the signaling mechanism of Notch has been extensively studied, the behaviors of its ligands are not well understood. In the present study, we found that, in the developing neural tube, Dll1(Delta-like 1) was mainly localized on the processes extending from nascent neurons toward both the pia and the ventricle and accumulated at apical termini, where adherens junctions (AJs) were formed. To understand the mechanism of Dll1 localization, we searched for binding proteins for Dll1 and identified a scaffolding molecule, MAGI1. In the developing spinal cord, MAGI1 mRNA was highly expressed in the ventricular zone, where Dll1 mRNA was expressed. MAGI1 protein accumulated at the AJs formed around the termini of apically extending processes and was partially colocalized with Dll1. MAGI1 bound not only to Dll1 but also to N-cadherin-beta-catenin complexes. In cultured AJ-forming fibroblasts, MAGI1 was localized at AJs, and Dll1 was recruited to these AJs through binding to MAGI1. In addition, Dll1 was stabilized on the cell surface by MAGI1. Taken together, these results suggest that Dll1 is presented on the surface of AJs formed at the apical termini of processes through interaction with MAGI1 to activate Notch on neighboring cells in the developing central nervous system.

Liposomes for microcompartmentation of enzymes and their influence on catalytic activity.

Modular systems for protein coupling have been applied for anchoring enzyme molecules on liposome surfaces. Two cytoplasmic model enzymes, alpha-amylase from Escherichia coli (EC. 3.2.1.1) and guanylate kinase from Saccharomyces cerevisiae (EC. 2.7.4.8), were directly coupled by a histidine-tag or indirectly via strep-tag and streptavidin or streptactin linker to a liposome membrane. Though the catalytic properties of the enzymes are generally maintained, stability and specific activity of the enzymes are modified after coupling and are especially influenced by the lipid used for the liposome assembly.

Requirement of N-terminal cysteines of PSD-95 for PSD-95 multimerization and ternary complex formation, but not for binding to potassium channel Kv1.4.

The PSD-95 family of PSD-95/Discs large/ZO-1 (PDZ) domain-containing proteins plays a role in the clustering and localization of specific ion channels and receptors at synapses. Previous studies have shown that PSD-95 forms multimers through an N-terminal region (termed the N-segment) and that the multimerization of PSD-95 is critical for its ability to cluster Shaker-type potassium channel Kv1.4 in heterologous cells. We show here that the PSD-95 N-segment functions as a multimerization domain only when located at the N-terminal end of a heterologous protein. A pair of N-terminal cysteines, Cys3 and Cys5, is essential for the ability of PSD-95 to self-associate and to form cell surface clusters with Kv1.4. However, PSD-95 mutants lacking these cysteine residues retain their ability to associate with membranes and to bind to Kv1.4. Unlike wild type PSD-95, the cysteine mutant of PSD-95 cannot form a ternary complex with Kv1.4 and the cell adhesion molecule Fasciclin II. These results suggest that the N-terminal cysteines are essential for PSD-95 multimerization and that multimerization is required for simultaneous binding of multiple membrane protein ligands by PSD-95.

Three isoforms of synaptic scaffolding molecule and their characterization. Multimerization between the isoforms and their interaction with N-methyl-D-aspartate receptors and SAP90/PSD-95-associated protein.

The synaptic scaffolding molecule (S-SCAM) has been identified as a protein interacting with SAP90/PSD-95-associated protein (SAPAP) (also called guanylate kinase-associated protein/hDLG-associated protein). S-SCAM has six PDZ (we have numbered them PDZ-0 to -5), two WW, and one guanylate kinase (GK) domains and interacts with N-methyl-D-aspartate (NMDA) receptor via PDZ-5 and SAPAP via the GK domain. We have identified here shorter isoforms of S-SCAM that start at the 164th or 224th methionine, and we renamed the original one, S-SCAMalpha, the middle one, S-SCAMbeta, and the shortest one, S-SCAM-gamma. S-SCAMbeta and -gamma have five PDZ (PDZ-1 to -5), two WW, and one GK domains. S-SCAMalpha interacted with S-SCAMbeta and -gamma through the region containing PDZ-4 and -5. The region containing both of PDZ-4 and -5 is sufficient for the clustering of NMDA receptors and forms a dimer in gel filtration, suggesting that S-SCAM forms multimers via the interaction between the C-terminal PDZ domains and assembles NMDA receptors into clusters. S-SCAMbeta and -gamma also interacted with SAPAP, suggesting that the N-terminal region of the GK domain is not necessary for the interaction. Finally, we have identified the interaction of the PDZ domains of S-SCAM with the GK domain of PSD-95/SAP90. S-SCAM, PSD-95/SAP90, and SAPAP are colocalized at least in some part in brain. Therefore, S-SCAM, PSD-95/SAP90, and SAPAP may form a complex in vivo.