Defective jejunal and colonic salt absorption and alteredNa+/H+ exchanger 3 (NHE3) activity in NHE regulatory factor 1 (NHERF1) adaptor protein-deficient mice

We investigated the role of the Na⁺/H⁺ exchanger regulatory factor 1 (NHERF1) on intestinal salt and water absorption, brush border membrane (BBM) morphology, and on the NHE3 mRNA expression, protein abundance, and transport activity in the murine intestine. NHERF1-deficient mice displayed reduced jejunal fluid absorption in vivo, as well as an attenuated in vitro Na⁺ absorption in isolated jejunal and colonic, but not of ileal, mucosa. However, cAMP-mediated inhibition of both parameters remained intact. Acid-activated NHE3 transport rate was reduced in surface colonocytes, while its inhibition by cAMP and cGMP was normal. Immunodetection of NHE3 revealed normal NHE3 localization in the BBM of NHERF1 null mice, but NHE3 abundance, as measured by Western blot, was significantly reduced in isolated BBM from the small and large intestines. Furthermore, the microvilli in the proximal colon, but not in the small intestine, were significantly shorter in NHERF1 null mice. Additional knockout of PDZK1 (NHERF3), another member of the NHERF family of adaptor proteins, which binds to both NHE3 and NHERF1, further reduced basal NHE3 activity and caused complete loss of cAMP-mediated NHE3 inhibition. An activator of the exchange protein activated by cAMP (EPAC) had no effect on jejunal fluid absorption in vivo, but slightly inhibited NHE3 activity in surface colonocytes in vitro. In conclusion, NHERF1 has segment-specific effects on intestinal salt absorption, NHE3 transport rates, and NHE3 membrane abundance without affecting mRNA levels. However, unlike PDZK1, NHERF1 is not required for NHE3 regulation by cyclic nucleotides. N. Broere and M. Chen share first authorship and are listed in alphabetical order.     

Prevention of Glutamate-Evoked Cytotoxicity

Introduction: Melanoma differentiation-associated gene – 9 (MDA-9)/Syntenin has become an increasingly popular focus for investigation in numerous cancertypes. Originally implicated in melanoma metastasis, it has diverse cellular roles and is consistently identified as a regulator of tumor invasion and angiogenesis. As a potential target for inhibiting some of the most lethal aspects of cancer progression, further insight into the function of MDA-9/Syntenin is mandatory.

Areas covered: Recent literature and seminal articles were reviewed to summarize the latest collective understanding of MDA-9/Syntenin’s role in normal and cancerous settings. Insights into its participation in developmental processes are included, as is the functional significance of the N- and C-terminals and PDZ domains of MDA-9/Syntenin. Current reports highlight the clinical significance of MDA-9/Syntenin expression level in a variety of cancers, often correlating directly with reduced patient survival. Also presented are assessments of roles of MDA-9/Syntenin in cancer progression as well as its functions as an intracellular adapter molecule.

Expert opinion: Multiple studies demonstrate the importance of MDA-9/Syntenin in tumor invasion and progression. Through the use of novel drug design approaches, this protein may provide a worthwhile therapeutic target. As many conventional therapies do not address, or even enhance, tumor invasion, an anti-invasive approach would be a worthwhile addition in cancer therapy.     

Roles of the PDZ‐binding motif of HPV 16 E6 protein in oncogenic transformation of human cervical keratinocytes

The high‐risk human papillomavirus E6 proteins have been shown to interact with and lead to degradation of PDZ‐domain‐containing proteins through its carboxy‐terminal motif. This PDZ‐binding motif plays important roles in transformation of cultured cells and carcinogenesis of E6‐transgenic mice. However, its biological effects on the natural host cells have not been elucidated. We have examined its roles in an in vitro carcinogenesis model for cervical cancer, in which E6 and E7 together with activated HRAS (HRAS^G12V) can induce tumorigenic transformation of normal human cervical keratinocytes. In this model, E6Δ151 mutant, which is defective in binding to PDZ domains, almost lost tumorigenic ability, whereas E6SAT mutant, which is defective in p53 degradation showed activity close to wild‐type E6. Interestingly, we found decreased expression of PAR3 in E6‐expressing cells independently of E6AP, which has not been previously recognized. Therefore, we knocked down several PDZ‐domain containing proteins including PAR3 in human cervical keratinocytes expressing E7, HRAS^G12V and E6Δ151 to examine whether depletion of these proteins can restore the tumorigenic ability. Single knockdown of SCRIB, MAGI1 or PAR3 significantly but partially restored the tumorigenic ability. The combinatorial knockdown of SCRIB and MAGI1 cooperatively restored the tumorigenic ability, and additional depletion of PAR3 further enhanced the tumorigenic ability surpassing that induced by wild‐type E6. These data highlight the importance of the carboxy‐terminal motif of the E6 protein and downregulation of PAR3 in tumorigenic transformation of human cervical keratinocytes.     

The epithelial brush border Na+/H+ exchanger NHE3 associates with the actin cytoskeleton by binding to ezrin directly and via PDZ domain-containing Na+/H+ exchanger regulatory factor (NHERF) proteins

1. The Na(+)/H(+) exchanger NHE3 associates with the actin cytoskeleton by binding ezrin both directly and indirectly. Both types of interaction are necessary for acute regulation of NHE3. Most acute regulation of NHE3 occurs by changes in trafficking via effects on exocytosis and/or endocytosis. However, NHE3 activity can also be regulated without changing the surface expression of NHE3 (change in turnover number). 2. A positive amino acid cluster in the a-helical juxtamembrane region in the COOH-terminus of NHE3 (amino acids K516, R520 and R527) is necessary for binding to the protein 4.1, ezrin, radixin, moesin (FERM) domain III of ezrin. Direct binding of NHE3 to ezrin is necessary for many aspects of basal trafficking, including basal exocytosis, delivery from the synthetic pathway and movement of NHE3 in the brush border (BB), which probably contributes to endocytosis over a prolonged period of time. 3. In addition, NHE3 binds indirectly to ezrin. The PDZ domain-containing proteins Na(+)/H(+) exchanger regulatory factor (NHERF) 1 and NHERF2, as intermediates in linking NHE3 to ezrin, are necessary for many aspects of NHE3 regulation. The binding of NHERF-ezrin/radixin/moesin to NHE3 occurs in the cytosolic domain of NHE3 between amino acids 475 and 689. This NHERF binding is involved in the formation of the NHE3 complex and restricts NHE3 mobility in the BB. However, it is dynamic; for example, changing in some cases of signalling. Furthermore, NHERF binding is necessary for lysophosphatidic acid stimulation of NHE3 and inhibition of NHE3 by Ca(2+), cAMP and cGMP.     

Regulation of drug transporters by PDZ adaptor proteins and nuclear receptors

Drug transporters have been suggested to be involved in various aspects of pharmacokinetics. Identification and characterization of drug transporters have given us a scientific basis for understanding drug disposition, as well as the molecular mechanisms of drug interaction and inter-individual/inter-species differences. On the other hand, regulatory mechanisms of drug transporters are still poorly understood, and information is limited to induction and down-regulation of drug transporters by various microsomal enzyme inducers. Little is known about the molecular machinery that directly interacts with the drug transporters. As a first step to clarify such molecular mechanisms, recent studies have identified PDZ (PSD-95/Discs-large/ZO-1) domain-containing proteins that directly interact with the so-called PDZ binding motif located at the C-terminus of drug transporters. Some of the PDZ proteins have been suggested to regulate transporters via at least two pathways, i.e. stabilization at the cell-surface and direct modulation of transporter function. Therefore, it is possible that membrane transport of therapeutic agents is not only governed by the drug transporters themselves, but also indirectly by PDZ proteins. The PDZ proteins are classified as a family, the members of which are thought to have distinct, but also redundant physiological roles. The purpose of this review article is to summarize the available knowledge on protein interactions and functional modulation of drug transporters.     

The adaptor Lnk (SH2B3): an emerging regulator in vascular cells and a link between immune and inflammatory signaling

A better knowledge of the process by which inflammatory extracellular signals are relayed from the plasma membrane to specific intracellular sites is a key step to understand how inflammation develops and how it is regulated. This review focuses on Lnk (SH2B3) a member, with SH2B1 and SH2B2, of the SH2B family of adaptor proteins that influences a variety of signaling pathways mediated by Janus kinase and receptor tyrosine kinases. SH2B adaptor proteins contain conserved dimerization, pleckstrin homology, and SH2 domains. Initially described as a regulator of hematopoiesis and lymphocyte differentiation, Lnk now emerges as a key regulator in hematopoeitic and non hematopoeitic cells such as endothelial cells (EC) moderating growth factor and cytokine receptor-mediated signaling. In EC, Lnk is a negative regulator of TNF signaling that reduce proinflammatory phenotype and prevent EC from apoptosis. Lnk is a modulator in integrin signaling and actin cytoskeleton organization in both platelets and EC with an impact on cell adhesion, migration and thrombosis. In this review, we discuss some recent insights proposing Lnk as a key regulator of bone marrow-endothelial progenitor cell kinetics, including the ability to cell growth, endothelial commitment, mobilization, and recruitment for vascular regeneration. Finally, novel findings also provided evidences that mutations in Lnk gene are strongly linked to myeloproliferative disorders but also autoimmune and inflammatory syndromes where both immune and vascular cells display a role. Overall, these studies emphasize the importance of the Lnk adaptor molecule not only as prognostic marker but also as potential therapeutic target.     

Part I: parkin-associated proteins and Parkinson's disease

Parkin is an E3 ligase that plays an important role in the ubiquitin/proteosome pathway responsible for protein degradation events. Mutations in parkin result in a loss-of-function and lead to Parkinson's disease, a progressive neurological disorder of movement. Presumably, this occurs due to the toxic build-up of proteins that are no longer effectively cleared/degraded by the parkin-dependent ubiqutin/proteosome pathway. To date, three types of proteins have been shown to interact with parkin. Firstly, the E2 ubiquitin conjugating proteins called UbcH7 and UbcH8 interact with parkin. Secondly, putative substrates interacting with parkin include a synaptic vesicle associated GTPase named CDCrel-1; a G protein-coupled receptor named Pael; a novel from of alpha-synuclein; and an alpha-synuclein interacting protein synphilin-1. Thirdly and more recently, a PDZ domain containing scaffolding protein CASK/Lin2 has been shown to interact with the PDZ binding motif of parkin. A network of PDZ-interacting proteins has potential to form a complex web of molecules that surround parkin and regulate its subcellular localisation and function.