multi-PDZ Domain Protein Research Articles

A PDZ scaffolding/CaM-mediated pathway in Cryptochrome signaling.

Cryptochromes are cardinal constituents of the circadian clock, which orchestrates daily physiological rhythms in living organisms. A growing body of evidence points to their participation in pathways that have not traditionally been associated with circadian clock regulation, implying that cryptochromes may be subject to modulation by multiple signaling mechanisms. In this study, we demonstrate that human CRY2 (hCRY2) forms a complex with the large, modular scaffolding protein known as Multi-PDZ Domain Protein 1 (MUPP1). This interaction is facilitated by the calcium-binding protein Calmodulin (CaM) in a calcium-dependent manner. Our findings suggest a novel cooperative mechanism for the regulation of mammalian cryptochromes, mediated by calcium ions (Ca2+ ) and CaM. We propose that this Ca2+ /CaM-mediated signaling pathway may be an evolutionarily conserved mechanism that has been maintained from Drosophila to mammals, most likely in relation to its potential role in the broader context of cryptochrome function and regulation. Further, the understanding of cryptochrome interactions with other proteins and signaling pathways could lead to a better definition of its role within the intricate network of molecular interactions that govern circadian rhythms.

Characterization of the adipogenic protein E4orf1 from adenovirus 36 through an in silico approach.

Adenovirus 36 (Ad-36) is related to human obesity due to its adipogenic activity mediated by the early 4 open reading frame 1 (E4orf1) protein. Mechanisms underlying the adipogenic effect of E4orf1 are not completely understood; however, the proliferation and differentiation of fat cells are increased through the activation of the phosphatidyl inositol 3 kinase pathway by binding proteins containing PDZ domain. This study characterized E4orf1 tridimensional structure and analyzed its interactions with PDZ domain-containing proteins in order to provide new information about the behavior of this viral protein and its targets, which could provide an interesting druggable target for obesity-related cardiometabolic alterations. In silico strategies such as homology modeling, docking, and molecular dynamics (MD) were used to study the interaction of E4orf1 with five PDZ domains of disk large homolog 1 (PDZ-1 and PDZ-2), membrane-associated guanylate kinase 1 (PDZ-3), and multi-PDZ domain protein 1 (PDZ-7 and PDZ-10). Mutagenesis analysis of selected residues was performed to evaluate their effects on the stabilization of E4orf1:PDZ complexes. MD simulations showed that the E4orf1:PDZ10 complex was more stable than the othersones. The highly hydrophobic residues at the C-terminal region (114-125) of the E4orf1 are essential in the initial phase stabilization of the complexes. Moreover, the residues 80-85 in the core region contribute to longer stabilization of the E4orf1:PDZ10 complex, a result that was confirmed by in silico mutagenesis. In conclusion, E4orf1 forms a stable complex with PDZ10 domain, and the residues 80-85 are of particular importance. The characterization of E4orf1 interactions with PDZ domains provides an initial approach to discover druggable targets for Ad-36-induced obesity.

MiR-363 acts as a tumor suppressor in osteosarcoma cells by inhibiting PDZD2.

PDZ domain containing 2 (PDZD2) is a multi-PDZ domain protein that promotes the proliferation of insulinoma cells, and is upregulated during prostate tumorigenesis. However, the function of PDZD2 in other cancers, including osteosarcoma (OS), remains unclear. Dysregulation of microRNAs (miRNAs) contributes to tumor initiation, proliferation and metastasis, via the regulation of their target genes. The present study investigated the functions of miR-363 and PDZD2 in MG-63 OS cells. The results revealed that MG-63 cells contained low levels of miR-363, and that overexpression of miR-363 in MG-63 cells significantly inhibited the vitality, proliferation, and colony formation ability of the cells, but promoted their apoptosis and G1/S arrest by regulating proliferating cell nuclear antigen (PCNA) and caspase-3 expression. Additionally, miR-363 impaired the migration and invasion of MG-63 cells by regulating the epithelial-mesenchymal transition (EMT) phenotype. Notably, a bioinformatics analysis and luciferase reporter assay indicated that PDZD2 was a direct target of miR-363. miR-363 overexpression reduced PDZD2 protein levels and knockdown of PDZD2 suppressed the colony formation, migration and invasion of MG-63 cells, but promoted their apoptosis by regulating expression of PCNA, caspase-3, and the EMT phenotype. In vivo studies further confirmed that miR-363 functioned as tumor suppressor, by inhibiting tumor growth, promoting cell apoptosis, and reducing PDZD2 and PCNA levels and the prevalence of the EMT phenotype in tumor tissues. The present data demonstrated that downregulation of the tumor suppressor miR-363 may be involved in the development of osteosarcoma via regulation of PDZD2.

Probing the Architecture of a Multi-PDZ Domain Protein: Structure of PDZK1 in Solution

The scaffolding protein PDZK1 has been associated with the regulation of membrane transporters. It contains four conserved PDZ domains, which typically recognize a 3-5-residue long motif at the C terminus of the binding partner. The atomic structures of the individual domains are available but their spatial arrangement in the full-length context influencing the binding properties remained elusive. Here we report a systematic study of full-length PDZK1 and deletion constructs using small-angle X-ray scattering, complemented with biochemical and functional studies on PDZK1 binding to known membrane protein partners. A hybrid modeling approach utilizing multiple scattering datasets yielded a well-defined, extended, asymmetric L-shaped domain organization of PDZK1 in contrast to a flexible "beads-on-string" model predicted by bioinformatics analysis. The linker regions of PDZK1 appear to play a central role in the arrangement of the four domains underlying the importance of studying scaffolding proteins in their full-length context.

Design and verification of halogen-bonding system at the complex interface of human fertilization-related MUP PDZ5 domain with CAMK’s C-terminal peptide

Calmodulin-dependent protein kinase (CAMK) is physiologically activated in fertilized human oocytes and is involved in the Ca2+ response pathways that link the fertilization calmodulin signal to meiosis resumption and cortical granule exocytosis. The kinase has an unstructured C-terminal tail that can be recognized and bound by the PDZ5 domain of its cognate partner, the multi-PDZ domain protein (MUP). In the current study, we reported a rational biomolecular design of halogen-bonding system at the complex interface of CAMK’s C-terminal peptide with MUP PDZ5 domain by using high-level computational approaches. Four organic halogens were employed as atom probes to explore the structural geometry and energetic property of designed halogen bonds in the PDZ5–peptide complex. It was found that the heavier halogen elements such as bromine Br and iodine I can confer stronger halogen bond but would cause bad atomic contacts and overlaps at the complex interface, while fluorine F cannot form effective halogen bond in the complex. In addition, the halogen substitution at different positions of peptide’s aromatic ring would result in distinct effects on the halogen-bonding system. The computational findings were then verified by using fluorescence analysis; it is indicated that the halogen type and substitution position play critical role in the interaction strength of halogen bonds, and thus the PDZ5–peptide binding affinity can be improved considerably by optimizing their combination.

A genome-wide significant association on chromosome 2 for footrot resistance/susceptibility in Swiss White Alpine sheep.

Footrot is one of the most important causes of lameness in global sheep populations and is characterized by a bacterial infection of the interdigital skin. As a multifactorial disease, its clinical representation depends not only on pathogen factors and environmental components but also on the individual resistance/susceptibility of the host. A genetic component has been shown in previous studies; however, so far no causative genetic variant influencing the risk of developing footrot has been identified. In this study, we genotyped 373 Swiss White Alpine sheep, using the ovine high-density 600k SNP chip, in order to run a DNA-based comparison of individuals with known clinical footrot status. We performed a case-control genome-wide association study, which revealed a genome-wide significant association for SNP rs418747104 on ovine chromosome 2 at 81.2Mb. The three best associated SNP markers were located at the MPDZ gene, which codes for the multiple PDZ domain crumbs cell polarity complex component protein, also known as multi-PDZ domain protein 1 (MUPP1). This protein is possibly involved in maintaining the barrier function and integrity of tight junctions. Therefore, we speculate that individuals carrying MPDZ variants may differ in their footrot resistance/susceptibility due to modified horn and interdigital skin integrity. In conclusion, our study reveals that MPDZ might represent a functional candidate gene, and further research is needed to explore its role in footrot affected sheep.

PDZRN3 destabilizes endothelial cell-cell junctions through a PKCζ-containing polarity complex to increase vascular permeability.

Endothelial cells serve as a barrier between blood and tissues. Maintenance of the endothelial cell barrier depends on the integrity of intercellular junctions, which is regulated by a polarity complex that includes the ζ isoform of atypical protein kinase C (PKCζ) and partitioning defective 3 (PAR3). We revealed that the E3 ubiquitin ligase PDZ domain-containing ring finger 3 (PDZRN3) regulated endothelial intercellular junction integrity. Endothelial cell-specific overexpression of Pdzrn3 led to early embryonic lethality with severe hemorrhaging and altered organization of endothelial intercellular junctions. Conversely, endothelial-specific loss of Pdzrn3 prevented vascular leakage in a mouse model of transient ischemic stroke, an effect that was mimicked by pharmacological inhibition of PKCζ. PDZRN3 regulated Wnt signaling and associated with a complex containing PAR3, PKCζ, and the multi-PDZ domain protein MUPP1 (Discs Lost-multi-PDZ domain protein 1) and targeted MUPP1 for proteasomal degradation in transfected cells. Transient ischemic stroke increased the ubiquitination of MUPP1, and deficiency of MUPP1 in endothelial cells was associated with decreased localization of PKCζ and PAR3 at intercellular junctions. In endothelial cells, Pdzrn3 overexpression increased permeability through a PKCζ-dependent pathway. In contrast, Pdzrn3 depletion enhanced PKCζ accumulation at cell-cell contacts and reinforced the cortical actin cytoskeleton under stress conditions. These findings reveal how PDZRN3 regulates vascular permeability through a PKCζ-containing complex.

Planar cell polarity 조절단백질 Wdpcp와 multi-PDZ domain protein 1 (MUPP1)의 PDZ 결합

Protein-protein interactions regulate the subcellular localization and function of receptors, enzymes, and cytoskeletal proteins. Proteins containing the postsynaptic density-95/disks large/zonula occludens-1 (PDZ) domain have potential to act as scaffolding proteins and play a pivotal role in various processes, such as synaptic plasticity, neural guidance, and development, as well as in the pathophysiology of many diseases. Multi-PDZ domain protein 1 (MUPP1), which has 13 PDZ domains, has a scaffolding function in the clustering of surface receptors, organization of signaling complexes, and coordination of cytoskeletal dynamics. However, the cellular function of MUPP1 has not been fully elucidated. In the present study, a yeast two-hybrid system was used to identify proteins that interacted with the N-terminal PDZ domain of MUPP1. The results revealed an interaction between MUPP1 and Wdpcp (formerly known as Fritz). Wdpcp was identified as a planar cell polarity (PCP) effector, which is known to have a role in collective cell migration and cilia formation. Wdpcp bound to the PDZ1 domain but not to other PDZ domains of MUPP1. The C-terminal end of Wdpcp was essential for the interaction with MUPP1 in the yeast two-hybrid assay. This interaction was further confirmed in a glutathione S-transferase (GST) pull-down assay. When coexpressed in HEK-293T cells, Wdpcp was coimmunoprecipitated with MUPP1. In addition, MUPP1 colocalized with Wdpcp at the same subcellular region in cells. Collectively, these results suggest that the MUPP1-Wdpcp interaction could modulate actin cytoskeleton dynamics and polarized cell migration.

Structure-based identification of CaMKIIα-interacting MUPP1 PDZ domains and rational design of peptide ligands to target such interaction in human fertilization.

The recognition and association between Ca(2+)/calmodulin-activated protein kinase II-α (CaMKIIα) and multi-PDZ domain protein 1 (MUPP1) plays an important role in sperm acrosome reaction and human fertilization, which is mediated by the binding of CaMKIIα's C-terminal tail to one or more PDZ domains of the scaffolding protein MUPP1. In this study, we attempt to identify the CaMKIIα-interacting MUPP1 PDZ domains and to design peptide ligands that can potently target and then competitively disrupt such interaction. Here, a synthetic biology approach was proposed to systematically characterize the structural basis, energetic property, dynamic behavior and biological implication underlying the intermolecular interactions between the C-terminal peptide of CaMKIIα and all the 13 PDZ domains of MUPP1. These domains can be grouped into four clusters in terms of their sequence, structure and physiochemical profile; different clusters appear to recognize different classes of PDZ-binding motifs. The cluster 3 includes two members, i.e. MUPP1 PDZ 5 and 11 domains, which were suggested to bind class II motif Φ-X-Φ(-COOH) of the C-terminal peptide SGAPSV(-COOH) of CaMKIIα. Subsequently, the two domains were experimentally measured as the moderate- and high-affinity binders of the peptide by using fluorescence titration (dissociation constants K d=25.2±4.6 and 0.47±0.08µM for peptide binding to PDZ 5 and 11, respectively), which was in line with theoretical prediction (binding free energies ΔG total=-7.6 and -9.2kcal/mol for peptide binding to PDZ 5 and 11, respectively). A systematic mutation of SGAPSV(-COOH) residues suggested few favorable amino acids at different residue positions of the peptide, which were then combined to generate a number of potent peptide mutants for PDZ 11 domain. Consequently, two peptides (SIAPNV(-COOH) and SIVMNV(-COOH)) were identified to have considerably improved affinity with K d increase by ~tenfold relative to wild type peptide. Thus, the two peptides are considered as promising lead entities to develop therapeutic molecular agents with high efficacy and specificity to target CaMKIIα-MUPP1 interaction. Other five designed peptides (SILPSV(-COOH), SGLPNV(-COOH), SIVMSV(-COOH), SIVPNV(-COOH) and SIAMNV(-COOH)) possessed comparable affinity with the wild type, and they may be further optimized to obtain higher potency.

Muskelin과 multi-PDZ domain protein 1 (MUPP1) 단백질의 PDZ 도메인을 통한 결합

Protein-protein interactions have a critical role in the regulation of many cellular functions. Postsynaptic density-95/disks large/zonula occludens-1 (PDZ) domain is one of domains that mediate protein-protein interactions. PDZ domains typically bind to the specific motif at the carboxyl (C)-terminal end of partner proteins. Multi-PDZ domain protein 1 (MUPP1), which has 13 PDZ domains, serves a scaffolding function for structure proteins and signaling proteins, but the cellular function of MUPP1 has not been fully elucidated. We used the yeast two-hybrid system to identify proteins that interact with PDZ domains of MUPP1. We found an interaction between MUPP1 and muskelin. Muskelin was recently identified as a GABA A receptor (GABA A R) α1 subunit binding protein and known to have a role in receptor endocytosis and degradation. Muskelin bound to the 3 rd PDZ domain, but not to other PDZ domains of MUPP1. The C-terminal end of muskelin was essential for the interaction with MUPP1 in the yeast two-hybrid assay. When co-expressed in HEK-293T cells, muskelin but not the C-terminal deleted muskelin was co-immunoprecipitated with MUPP1. In addition, MUPP1 co-localized with muskelin at the same subcellular region in cells. These findings collectively suggest that MUPP1 or its interacting proteins could modulate GABAAR trafficking and turnover through the interaction with muskelin.

Biochemical and structural characterization of MUPP1-PDZ4 domain from Mus musculus.

Specific protein-protein interactions are important for biological signal transduction. The postsynaptic density-95, disc-large, and zonulin-1 (PDZ) domain is one of the most abundant protein interaction modules. Multi-PDZ-domain protein 1 (MUPP1), as a scaffold protein, contains 13 PDZ domains and plays an important role in cytoskeletal organization, cell polarity, and cell proliferation. The study on PDZ domain of MUPP1 helps to understand the mechanisms and functions of MUPP1. In the present study, the fourth PDZ domain of MUPP1 (MUPP1-PDZ4) from Mus musculus was cloned, expressed, purified, and characterized. The MUPP1-PDZ4 domain was subcloned into a pET-vector and expressed in Escherichia coli. Affinity chromatography and size-exclusion chromatography were used to purify the protein. MUPP1-PDZ4 protein was a monomer with a molar mass of 16.4 kDa in solution and had a melting point of 60.3°C. Using the sitting-drop vapor-diffusion method, MUPP1-PDZ4 protein crystals were obtained in a solution (pH 7.0) containing 2% (v/v) polyethylene glycol 400, 0.1 M imidazole, and 24% (w/v) polyethylene glycol monoethyl ether 5000. Finally, the crystal was diffracted with 1.6 Å resolution. The crystal structure showed that MUPP1-PDZ4 domain contained three α-helices and six β-strands in the core. The GLGI motif, L562/A564 on the β-strand B, and H605/V608/L612 on the α-helix B formed a PDZ binding pocket which could bind to the C-terminal of the binding partners. This biochemical and structural information will provide insights into how PDZ binds to its target peptide and the theoretical foundation for the function of MUPP1.

Cadms/SynCAMs/Necls/TSLCs와 multi-PDZ domain protein MUPP1 단백질의 결합

Cell adhesion molecules determine the cell-cell binding and the interactions between cells and extracellular signals. Cell-cell junctional complexes, which maintain the structural integrity of tissues, consist of more than 50 proteins including multi-PDZ domain protein 1 (MUPP1). MUPP1 contains 13 postsynaptic density-95/disks large/zonula occludens-1 (PDZ) domains and serves a scaffolding function for transmembrane proteins and cytoskeletal proteins or signaling proteins, but the mechanism how MUPP1 links and stabilizes the juxtamembrane proteins has not yet been elucidated. We used the yeast two-hybrid system to identify proteins that interact with PDZ domains of MUPP1. We found an interaction between MUPP1 and cell adhesion molecule 1 (Cadm1, also known as SynCAM1, Necl-2, or TSLC1). Cadm1 bound to the second PDZ domain of MUPP1. The carboxyl (C)-terminal end of Cadm1 has a type II PDZ-association motif (-Y-F-I) which was essential for the interaction with MUPP1 in the yeast two-hybrid assay. MUPP1 also bound to the C-terminal cytoplasmic tail region of other Cadm family members (Cadm2, Cadm3, and Cadm4). In addition, these protein-protein interactions were observed in the glutathione S-transferase (GST) pull-down assay and by coimmunoprecipitation. Anti-MUPP1 antibody co-immunoprecipitated Cadm1 and Cadm4 with MUPP1 from mouse brain extracts. These results suggest that MUPP1 could mediate interaction between Cadms and cytoskeletal proteins.

Parkin과 Multi-PDZ Domain Protein (MUPP1) 단백질 간의 PDZ 결합

세포표면 수용체와 통로가 적절히 기능하려면 특정 세포 내 위치로 배치되고 조절되어야 한다. PSD95/Dlg/Zo-1 (PDZ) 도메인은 이러한 배치와 조절을 매개하는 다양한 단백질들을 인식하고 이 단백질들이 서로 결합하는데 관여한다. MUPP1은 13개의 PDZ domain을 가지는 단백질로서 여러 구조 단백질 및 신호전달 단백질과 상호 결합하지만, MUPP1이 어떻게 안정화되며, 어떻게 신호전달과정에 관여하는지에 대해 아직 명확히 밝혀지지 않았다. 본 연구에서 MUPP1의 PDZ 도메인과 상호 작용하는 단백질을 규명하기 위하여 효모 two-hybrid 방법을 이용하였고, Parkin이 MUPP1과 결합하는 것을 확인하였다. Parkin은 E3 ubiquitin ligase로서, Parkin 유전자의 기능상실 돌연변이는 autosomal recessive juvenile parkinsonism을 일으키는 것으로 알려져 있다. Parkin은 MUPP1의 12번째 PDZ domain과 결합하지만, 다른 PDZ 도메인과는 결합하지 않았다. Parkin의 C-말단부위는 II 형 PDZ-결합모티프를 가지고 있는데, 이 모티프가 MUPP1과의 결합에 필수적임을 확인하였다. HEK-293T 세포에 MUPP1과 Parkin을 동시에 발현하여 발현위치를 확인한 결과 세포내의 같은 위치에서 발현하였다. 또한 Parkin은 MUPP1을 강하게 유비퀴틴화 하였다. 이러한 결과들은 MUPP1이 Parkin의 기질이며, Parkin에 의한 유비퀴틴화에 의해 MUPP1의 기능 혹은 안정성이 조절될 수 있음을 시사한다. The localization to specific subcellular sites and the regulation of cell surface receptors and channels are crucial for proper functioning. Postsynaptic density-95/Disks large/Zonula occludens-1 (PDZ)-domain is involved in recognition of and interaction between various proteins, by which the localization and the regulation are mediated. Multi-PDZ domain protein 1 (MUPP1) contains 13 PDZ domains. MUPP1 serves a scaffolding function for structure proteins and signaling proteins, but the mechanism how MUPP1 is stabilized and signalized has not yet been elucidated. We used the yeast two-hybrid system to identify proteins that interact with PDZ domains of MUPP1. We found an interaction between MUPP1 and Parkin. Parkin is an E3 ubiquitin ligase. Loss-of-function mutations of Parkin gene are known to cause an autosomal recessive juvenile parkinsonism. Parkin bound to the <TEX>$12^{th}$</TEX> PDZ domain, but not to other PDZ domains of MUPP1. The C-terminal end of Parkin has a type II PDZ-association motif, which was essential for the interaction with MUPP1 in the yeast two-hybrid assay. When co-expressed in HEK-293T cells, Parkin co-localized with MUPP1. When co-expressed with ubiquitin in HEK-293T cells, MUPP1 has been strongly ubiquitinated by Parkin. These findings collectively suggest that MUPP1 is a novel substrate of Parkin and its function or stability could be modulated by Parkin-mediated ubiquitination.

Neuronal cell-surface protein neurexin 1 interaction with multi-PDZ domain protein MUPP1

Location of membrane proteins is often stabilized by PDZ domain-containing scaffolding proteins. Using the yeast two-hybrid screening, we found that neurexin 1 interacted with multi-PDZ domain protein 1 (MUPP1) through PDZ domain. Neurexin 2 and 3 also interacted with MUPP1. MUPP1 and neurexin 1 were co-localized in cultured cells. These results suggest a novel mechanism for localizing neurexin 1 to synaptic sites.

Identification of a Receptor for Neuropeptide VGF and Its Role in Neuropathic Pain

VGF (nonacronymic) is a neuropeptide precursor that plays multiple roles in regulation of energy balance, reproduction, hippocampal synaptic plasticity, and pain. Data from a number of pain models showed significant up-regulation of VGF in sensory neurons. TLQP-21, one of the VGF-derived neuropeptides, has been shown to induce a hyperalgesic response when injected subcutaneously into the hind paw of mice. However, the precise role of VGF-derived neuropeptides in neuropathic pain and the molecular identity of the receptor for VGF-derived peptides are yet to be investigated. Here we identified gC1qR, the globular heads of the C1q receptor, as the receptor for TLQP-21 using chemical cross-linking combined with mass spectrometry analysis. TLQP-21 caused an increase in intracellular Ca(2+) levels in rat macrophages and microglia. Inoculation of TLQP-21-stimulated macrophages into rat hind paw caused mechanical hypersensitivity. The increase in intracellular Ca(2+) levels in macrophages was attenuated by either siRNA or neutralizing antibodies against gC1qR. Furthermore, application of the gC1qR-neutralizing antibody to rats with partial sciatic nerve ligation resulted in a delayed onset of nerve injury-associated mechanical hypersensitivity. These results indicate that gC1qR is the receptor for TLQP-21 and plays an important role in chronic pain through activation of macrophages. Because direct association between TLQP-21 and gC1qR is required for activation of macrophages and causes hypersensitivity, disrupting this interaction may be a useful new approach to develop novel analgesics.

Changes in the expression levels of tight junction components during reconstruction of tight junction from mucosal lesion by intestinal ischemia/reperfusion

Tight junction (TJ) is composed of the most apical components of the intercellular junctional complex in epithelial cells; TJ has cell polarity and functions as a major determinant of epithelial barrier function. In this study, to clarify the components of TJ required for its reconstruction and functional acquisition, we examined the changes in intestinal mucosal structure that depended on mucosal lesion by intestinal I/R, that is, the changes in mRNA and protein expression of the claudin family and scaffold proteins. We used an in vivo intestinal I/R model made using the spring scale and surgical sutures, and examined the mRNA and protein expression levels of TJ components by real-time RT-PCR and Western blotting, respectively. Changes in mRNA and protein expression levels of TJ components by intestinal I/R were observed. Among them, characteristic changes were observed in claudin-2 and claudin-4. In addition, the expression behavior of multi-PDZ domain protein (MPDP) mRNA was similar to that of claudin-4. In conclusion, in the recovery process of TJ from mucosal lesion by intestinal I/R, it was suggested that claudin-2 and claudin-4 strongly participate in the reconstruction and functional acquisition of TJ, respectively. Furthermore, it was suggested that MPDZ, which is scaffold protein, also has an important role in these processes.

The multi-PDZ domain protein-1 (MUPP-1) expression regulates cellular levels of the PALS-1/PATJ polarity complex

MUPP-1 (multi-PDZ domain protein-1) and PATJ (PALS-1-associated tight junction protein) proteins are closely related scaffold proteins and bind to many common interactors including PALS-1 (protein associated with Lin seven) a member of the Crumbs complex. Our goal is to understand how MUPP-1 and PATJ and their interaction with PALS-1 are regulated in the same cells. We have shown that in MCF10A cells there are at least two different and co-existing complexes, PALS-1/MUPP-1 and PALS-1/PATJ. Surprisingly, MUPP-1 levels inversely correlated with PATJ protein levels by acting on the stabilization of the PATJ/PALS-1 complex. Upon MUPP-1 depletion, the increased amounts of PATJ are in part localized at the migrating front of MCF10A cells and are able to recruit more PAR3 (partition defective 3). All together these data indicate that a precise balance between MUPP-1 and PATJ is achieved in epithelial cells by regulating their association with PALS-1.

Changes in protein and mRNA expression levels of claudin family after mucosal lesion by intestinal ischemia/reperfusion

Ischemia/reperfusion (I/R) injury of the intestine is the leading cause of organ dysfunction after restoration of blood flow after diverse events, including shock and intestinal transplantation. I/R injury must be overcome for successful small intestinal transplantation. Tight junctions (TJ) are the most apical component of the intercellular junctional complex in epithelial cells; they establish cell polarity and functioning as major determinants of epithelial barrier function. Among the proteins that comprise TJ, the claudin family is thought to play a crucial role in homeostasis in multicellular organisms. Therefore, the aim of this study was to examine the changes in function of TJ and behavior of the claudin family during intestinal I/R. Wistar/ST rats underwent intestinal ischemia by using the spring scale and surgical suture for 1h, followed by 24h of reperfusion. We examined the changes in area under the blood concentration curve (AUC) after oral administration of FD-4, which is a paracellular marker, and claudin-1, -2, -4, and -7 mRNA and protein expression levels in ileum. The structure of ileal mucosa was partly damaged and its function was diminished by intestinal I/R until 3h after reperfusion, but were almost recovered 24h after reperfusion. However, a time difference was shown between the recoveries of mucosal structure and function. Furthermore, a difference in the expression among various kinds of claudin was found. It was suggested that claudin-4 and multi-PDZ domain protein, which is a scaffolding protein, regulate intestinal paracellular permeability during intestinal I/R. Moreover, the changes in the expression level of claudin-2 were unique.

Hypertonic Stress Promotes the Upregulation and Phosphorylation of Zonula Occludens 1

Tight junction molecules form a barrier between adjacent cells and mediate the cells’ ability to develop membranes that constitute boundaries of different compartments within the body. Membranes with selective ion and water passage are important for the electrolyte and water homeostasis in the kidney. Due to their role in the urinary concentration process, renal medullary cells are exposed to hyperosmotic stress. Therefore, we were interested in the question of how mouse inner medullary collecting duct cells (mIMCD3) manage to maintain their cell-cell contacts, despite hypertonicity-induced cell shrinkage. Employing mRNA expression analysis, we found that the zonula occludens type 1 (Zo-1), multi-PDZ domain protein 1 (MUPP1) and cortactin mRNA levels were upregulated in a tonicity-dependent manner. Using Western blot analysis, immunoprecipitation and immunofluorescence, we show that the Zo-1 protein is upregulated, phosphorylated and linked to the actin cytoskeleton in response to hypertonic stress. After cell exposure to hypertonicity, rearrangement of the actin cytoskeleton resulted in a stronger colocalization of actin fibres with Zo-1. Urea, which generates hyperosmolality, but no transcellular gradient, did not induce changes in Zo-1 protein expression or actin rearrangement. This data indicates that Zo-1 is a response protein to inner medullary tonicity and that extracellular stressors can promote Zo-1 protein expression, tyrosine phosphorylation and cytoskeleton association.

Striatal Involvement in Human Alcoholism and Alcohol Consumption, and Withdrawal in Animal Models

Different regions of the striatum may have distinct roles in acute intoxication, alcohol seeking, dependence, and withdrawal. The recent advances are reviewed and discussed in our understanding of the role of the dorsolateral striatum (DLS), dorsomedial striatum (DMS), and ventral striatum in behavioral responses to alcohol, including alcohol craving in abstinent alcoholics, and alcohol consumption and withdrawal in rat, mouse, and nonhuman primate models. Reduced neuronal activity as well as dysfunctional connectivity between the ventral striatum and the dorsolateral prefrontal cortex is associated with alcohol craving and impairment of new learning processes in abstinent alcoholics. Within the DLS of mice and nonhuman primates withdrawn from alcohol after chronic exposure, glutamatergic transmission in striatal projection neurons is increased, while GABAergic transmission is decreased. Glutamatergic transmission in DMS projection neurons is also increased in ethanol withdrawn rats. Ex vivo or in vivo ethanol exposure and withdrawal causes a long-lasting increase in NR2B subunit-containing NMDA receptor activity in the DMS, contributing to ethanol drinking. Analyses of neuronal activation associated with alcohol withdrawal and site-directed lesions in mice implicate the rostroventral caudate putamen, a ventrolateral segment of the DMS, in genetically determined differences in risk for alcohol withdrawal involved in physical association of the multi-PDZ domain protein, MPDZ, with 5-HT(2C) receptors and/or NR2B. Alterations of dopaminergic, glutamatergic, and GABAergic signaling within different regions of the striatum by alcohol is critical for alcohol craving, consumption, dependence, and withdrawal in humans and animal models.