Surface plasmon resonance analysis of the binding of high‐risk mucosal HPV E6 oncoproteins to the PDZ1 domain of the tight junction protein MAGI‐1

The E6 oncoproteins from high‐risk mucosal human papillomavirus (HPV) induce cervical cancer via two major activities, the binding and the degradation of the p53 protein and PDZ domain‐containing proteins. Human MAGI‐1 is a multi‐PDZ domain protein implicated into protein complex assembly at cell–cell contacts. High‐risk mucosal HPV E6 proteins interact with the PDZ1 domain of MAGI‐1 via a C‐terminal consensus binding motif. Here, we developed a medium throughput protocol to accurately measure by surface plasmon resonance affinity constants of protein domains binding to peptidic sequences produced as recombinant fusions to the glutathione‐S‐transferase (GST). This approach was applied to measure the binding of MAGI‐1 PDZ1 to the C‐termini of viral or cellular proteins. Both high‐risk mucosal HPV E6 C‐terminal peptides and cellular partners of MAGI‐1 PDZ1 bind to MAGI‐1 PDZ1 with comparable dissociation constants in the micromolar range. MAGI‐1 PDZ1 shows a preference for C‐termini with a valine at position 0 and a negative charge at position ?3, confirming previous studies performed with HPV18 E6. A detailed combined analysis via site‐directed mutagenesis of the HPV16 C‐terminal peptide and PDZ1 indicated that interactions mediated by charged residues upstream the PDZ‐binding motif strongly contribute to binding selectivity of this interaction. In addition, our work highlighted the K₄₉₉ residue of MAGI‐1 as a novel determinant of binding specificity. Finally, we showed that MAGI‐1 PDZ1 also binds to the C‐termini of LPP and Tax proteins, which were already known to bind to PDZ proteins but not to MAGI‐1. Copyright © 2010 John Wiley & Sons, Ltd.     

Formation of a physiological complex between TRPV2 and RGA protein promotes cell surface expression of TRPV2

The transient receptor potential, sub-family Vanilloid (TRPV)(2) cation channel is activated in response to extreme temperature elevations in sensory neurons. However, TRPV2 is widely expressed in tissues with no sensory function, including cells of the immune system. Regulation of GRC, the murine homolog of TRPV2 has been studied in insulinoma cells and myocytes. GRC is activated in response to certain growth factors and neuropeptides, via a mechanism that involves regulated access of the channel to the plasma membrane. This is likely to be an important primary control mechanism for TRPV2 outside the CNS. Here, we report that a regulated trafficking step controls the access of TRPV2 to the cell surface in mast cells. In mast cells, elevations in cytosolic cAMP are sufficient to drive plasma membrane localization of TRPV2. We have previously proposed that the recombinase gene activator protein (RGA), a four-transmembrane domain, intracellular protein, associates with TRPV2 during the biosynthesis and early trafficking of the channel. We use a polyclonal antibody to RGA to confirm the formation of a physiological complex between RGA and TRPV2. Finally, we show that over-expression of the RGA protein potentiates the basal surface localization of TRPV2. We propose that trafficking and activation mechanisms intersect for TRPV2, and that cAMP mobilizing stimuli may regulate TRPV2 localization in non-sensory cells. RGA participates in the control of TRPV2 surface levels, and co-expression of RGA may be a key component of experimental systems that seek to study TRPV2 physiology.     

Different pharmacological profile of two closely related endocannabinoid ester analogs

The pharmacological and neuroprotective properties of two ester analogs of the endocannabinoids, arachidonoylethyleneglycol (AA-EG) and alpha,alpha,-dimethyl arachidonoylethyleneglycol (DMA-EG), were investigated. We examined the interaction of both compounds with cannabinoid receptors (CB1 and CB2) and their efficacy in functional assays. In competition binding assays, AA-EG and DMA-EG had low potency to displace the CB1/CB2 agonist [3H]CP-55,940 in membrane preparations expressing rodent or human receptors. Binding data correlate with low efficacy of both compounds as regards to inhibition of adenylyl cyclase activity. It was also shown that DMA-EG resists hydrolysis by rat brain membranes while AA-EG undergo complete splitting under these conditions. In the cannabinoid tetrad, AA-EG induced hypomotility, analgesia, catalepsy and decreased rectal temperature indicating cannabimimetic activity. By contrast, DMA-EG was completely inactive in the same models. DMA-EG and AA-EG potently protected rat cortical neurons in culture against oxygen deprivation at nanomolar concentrations. In glutamate-induced damage, the compounds were less active protecting neurons at micromolar concentrations. The data obtained indicate that the ester endocannabinoid template can be used for the development of new compounds with potent biological activity lacking some of the undesirable behavioral side effects.     

Conflict over multiple-partner mating between males and females of the polygynandrous common lizards

The optimal number of mate partners for females rarely coincides with that for males, leading to a potential sexual conflict over multiple-partner mating. This suggests that the population sex ratio may affect multiple-partner mating and thus multiple paternity. We investigate the relationship between multiple paternity and the population sex ratio in the polygynandrous common lizard (Lacerta vivipara). In six populations the adult sex ratio was biased toward males, and in another six populations the adult sex ratio was biased toward females, the latter corresponding to the average adult sex ratio encountered in natural populations. In males the frequency and the degree of polygyny were lower in male-biased populations, as expected if competition among males determines polygyny. In females the frequency of polyandry was not different between treatments, and polyandrous females produced larger clutches, suggesting that polyandry might be adaptive. However, in male-biased populations females suffered from reduced reproductive success compared to female-biased populations, and the number of mate partners increased with female body size in polyandrous females. Polyandrous females of male-biased populations showed disproportionately more mating scars, indicating that polyandrous females of male-biased populations had more interactions with males and suggesting that the degree of multiple paternity is controlled by male sexual harassment. Our results thus imply that polyandry may be hierarchically controlled, with females controlling when to mate with multiple partners and male sexual harassment being a proximate determinant of the degree of multiple paternity. The results are also consistent with a sexual conflict in which male behaviors are harmful to females.     

RGA protein associates with a TRPV ion channel during biosynthesis and trafficking

TRPV ion channels transduce a range of temperature stimuli. We proposed that analysis of the protein-protein interactions made by TRPV2 might give insight into the key issues surrounding this channel. These issues include the potential functional significance of TRPV2 in non-sensory tissues, the molecules involved in transducing its activation signal(s) and the mechanism by which its trafficking to the cell surface is regulated. Here we describe the interaction of TRPV2 channel with the RGA gene product. RGA is a four-transmembrane domain, intracellularly localized protein. RGA associates with TRPV2 in a rat mast cell line that is a native context for both proteins. The interaction between TRPV2 and RGA is transient and occurs intracellularly. RGA does not accompany TRPV2 to the cell surface. Formation of the TRPV2/RGA complex is dependent upon a cellular glycosylation event, suggesting that RGA may play a chaperone or targeting role for TRPV2 during the maturation of the ion channel protein. These data record a novel protein-protein interaction for TRPV2 and provide a foundation for future study of the potential regulatory contribution of RGA to TRPV2 function.     

Cutting edge: chemoattractant receptor-homologous molecule expressed on Th2 cells plays a restricting role on IL-5 production and eosinophil recruitment

PGs play key regulatory roles in inflammation and immunity. PGD2, released from mast cells and Th2 cells during allergic responses, has recently been shown to target a novel receptor, chemoattractant receptor-homologous molecule expressed TH2 cells (CRTH2), in addition to the classic PGD (DP) receptor. CRTH2 is expressed on Th2 cells and eosinophils and mediates chemotaxis of these cells to PGD2. Thus, CRTH2 is thought to be a key receptor mediating eosinophil and Th2 cell recruitment during allergic responses. To examine the role of CRTH2 in this context in vivo, we generated CRTH2 knockout mice. Surprisingly, in an allergic inflammatory model of asthma, CRTH2 knockout mice showed enhanced eosinophil recruitment into the lung compared with wild-type littermate mice. This is consistent with our observation that CRTH2 knockout cells produce significantly higher amounts of IL-5 and IL-3 in vitro. These results suggest a nonredundant role of CRTH2 in restricting eosinophilia and allergic response in vivo.     

Biochemical complementation of the betalain biosynthetic pathway in Portulaca grandiflora by a fungal 3,4-dihydroxyphenylalanine dioxygenase

3,4-Dihydroxyphenylalanine (DOPA) dioxygenase from Amanitamuscaria catalyses the key reaction of betalain biosynthesis, namely the conversion of DOPA to betalamic acid by a 4,5-ring-opening reaction. In addition, it catalyses a 2,3 opening which yields the fungal pigment muscaflavin, a compound that has never been found in plants. In this work, a cDNA clone (DodA) encoding A. muscaria DOPA-dioxygenase was expressed in white Portulacagrandiflora petals, using the particle bombardment technique. Transformation resulted in the formation of yellow and violet spots that contained betalain pigments and muscaflavin, indicating that the fungal enzyme was expressed and active in plants, and could complement the plant betalain biosynthetic pathway. The presence of muscaflavin in transformed plants indicates a difference in the specificity of the plant and A.muscaria enzymes. Received: 24 February 1997 / Accepted: 20 May 1997     

LNCaP prostatic adenocarcinoma cells derived from low and high passage numbers display divergent responses not only to androgens but also to retinoids

In the present paper, two strains of LNCaP cells derived from the same source (American Type Culture Collection), but studied either at a low passage number (LP) or at a high passage number (HP), were compared in their response to R1881 (a synthetic androgen), all-trans-retinoic acid (atRA), and 1,25-dihydroxycholecalciferol (VD₃). [³H]Thymidine incorporation and epidermal growth factor receptor (EGF-R) binding were measured as parameters related to the proliferative response of the cells. The secretion of prostate-specific antigen (PSA) and the mRNA expression of PSA, prostatic acid phosphatase (PAP), and diazepam-binding inhibitor (DBI) were used as parameters reflecting differentiated function. Marked differences were noted in the response of LP and HP cells to androgens. [³H]Thymidine incorporation displayed a bell-shaped dose-response curve in both strains. The amplitude of the response, however, was much higher in HP cells and growth inhibition at high levels of R1881 was only observed in LP cells. On the contrary, androgen induction of PSA secretion and PSA mRNA expression, as well as the expression of PAP was much more pronounced in LP cells, whereas DBI expression was not altered according to passage number. LP cells and HP cells also displayed striking differences in their response to atRA. An up to 6-fold stimulation of [³H]thymidine incorporation was observed in LP cells, whereas in HP cells the only significant effect was growth inhibition. VD₃, on the contrary, inhibited [³H]thymidine incorporation to a comparable degree in LP and HP cells. Only marginal effects of atRA and VD₃ were observed on PSA secretion. In both LP and HP cells EGF-R levels were increased by androgens and to a slight extent also by atRA and VD₃. It is concluded that LP and HP LNCaP cells display markedly divergent responses not only to androgens but also to atRA. The proliferative rather than antiproliferative effects of atRA in some strains of LNCaP should caution against the uncontrolled use of these agents, or of drugs affecting their metabolism, in patients with prostate cancer.