Identification of the structural and functional boundaries of the multidrug resistance protein 1 cytoplasmic loop 3

Multidrug resistance protein (MRP) 1 is a member of the ABCC branch of the ATP binding cassette (ABC) transporter superfamily that can confer resistance to natural product chemotherapeutic drugs and transport a variety of conjugated organic anions, as well as some unconjugated compounds in a glutathione- (GSH-) dependent manner. In addition to the two tandemly repeated polytopic membrane-spanning domains (MSDs) typical of ABC transporters, MRP1 and its homologues MRP2, -3, -6, and -7 contain a third NH(2)-terminal MSD. The cytoplasmic loop (CL3) connecting this MSD, but apparently not the MSD itself, is required for MRP1 leukotriene C(4) (LTC(4)) transport activity, substrate binding and appropriate trafficking of the protein to the basolateral membrane. We have used a baculovirus dual-expression system to produce various functionally complementing fragments of MRP1 in insect Sf21 cells to precisely define the region in CL3 that is required for activity and substrate binding. Using a parallel approach in polarized MDCK-I cells, we have also defined the region of CL3 that is required for basolateral trafficking. The CL3 NH(2)- and COOH-proximal functional boundaries have been identified as Cys(208) and Asn(260), respectively. Cys(208) also corresponds to the NH(2)-proximal boundary of the region required for basolateral trafficking in MDCK-I cells. However, additional residues downstream of the CL3 COOH-proximal functional boundary extending to Lys(270) were found to be important for basolateral localization. Finally, we show that regions in CL3 necessary for LTC(4) binding and transport are also required for binding of the photoactivatable GSH derivative azidophenacyl-GSH.     

The role of C-terminal tyrosine phosphorylation in the regulation of SHP-1 explored via expressed protein ligation

The protein-tyrosine phosphatase SHP-1 plays a variety of roles in the "negative" regulation of cell signaling. The molecular basis for the regulation of SHP-1 is incompletely understood. Whereas SHP-1 has previously been shown to be phosphorylated on two tail tyrosine residues (Tyr(536) and Tyr(564)) by several protein-tyrosine kinases, the effects of these phosphorylation events have been difficult to address because of the intrinsic instability of the linkages within a protein-tyrosine phosphatase. Using expressed protein ligation, we have generated semisynthetic SHP-1 proteins containing phosphotyrosine mimetics at the Tyr(536) and Tyr(564) sites. Two phosphonate analogues were installed, phosphonomethylenephenylalanine (Pmp) and difluorophosphonomethylenephenylalanine (F(2)Pmp). Incorporation of Pmp at the 536 site led to 4-fold stimulation of the SHP-1 tyrosine phosphatase activity whereas incorporation at the 564 site led to no effect. Incorporation of F(2)Pmp at the 536 site led to 8-fold stimulation of the SHP-1 tyrosine phosphatase activity and 1.6-fold at the 564 site. A combination of size exclusion chromatography, phosphotyrosine peptide stimulation studies, and site-directed mutagenesis led to the structural model in which tyrosine phosphorylation at the 536 site engages the N-Src homology 2 domain in an intramolecular fashion relieving basal inhibition. In contrast, tyrosine phosphorylation at the 564 site has the potential to engage the C-Src homology 2 domain intramolecularly, which can modestly and indirectly influence catalytic activity. The finding that phosphonate modification at each of the 536 and 564 sites can promote interaction with the Grb2 adaptor protein indicates that the intramolecular interactions fostered by post-translational modifications of tyrosine are not energetically strong and susceptible to intermolecular competition.     

Differential regulation of Cl- transport proteins by PKC in Calu-3 cells

Cl- transport proteins expressed in a Calu-3 airway epithelial cell line were differentiated by function and regulation by protein kinase C (PKC) isotypes. mRNA expression of Cl- transporters was semiquantitated by RT-PCR after transfection with a sense or antisense oligonucleotide to the PKC isotypes that modulate the activity of the cystic fibrosis transmembrane conductance regulator [CFTR (PKC-epsilon)] or of the Na/K/2Cl (NKCC1) cotransporter (PKC-delta). Expression of NKCC1 and CFTR mRNAs and proteins was independent of antisense oligonucleotide treatment. Transport function was measured in cell monolayers grown on a plastic surface or on filter inserts. With both culture methods, the antisense oligonucleotide to PKC-epsilon decreased the amount of PKC-epsilon and reduced cAMP-dependent activation of CFTR but not alpha(1)-adrenergic activation of NKCC1. The antisense oligonucleotide to PKC-delta did not affect CFTR function but did block alpha(1)-adrenergic activation of NKCC1 and reduce PKC-delta mass. These results provide the first evidence for mRNA and protein expression of NKCC1 in Calu-3 cells and establish the differential regulation of CFTR and NKCC1 function by specific PKC isotypes at a site distal to mRNA expression and translation in airway epithelial cells.     

Ploidy race distributions since the Last Glacial Maximum in the North American desert shrub, Larrea tridentata

• 1 A classic biogeographic pattern is the alignment of diploid, tetraploid and hexaploid races of creosote bush (Larrea tridentata) across the Chihuahuan, Sonoran and Mohave Deserts of western North America. We used statistically robust differences in guard cell size of modern plants and fossil leaves from packrat middens to map current and past distributions of these ploidy races since the Last Glacial Maximum (LGM).
• 2 Glacial/early Holocene (26–10 ¹⁴C kyr bp or thousands of radiocarbon years before present) populations included diploids along the lower Rio Grande of west Texas, 650 km removed from sympatric diploids and tetraploids in the lower Colorado River Basin of south‐eastern California/south‐western Arizona. Diploids migrated slowly from lower Rio Grande refugia with expansion into the northern Chihuahuan Desert sites forestalled until after ~4.0 ¹⁴C kyr bp . Tetraploids expanded from the lower Colorado River Basin into the northern limits of the Sonoran Desert in central Arizona by 6.4 ¹⁴C kyr bp . Hexaploids appeared by 8.5 ¹⁴C kyr bp in the lower Colorado River Basin, reaching their northernmost limits (~37°N) in the Mohave Desert between 5.6 and 3.9 ¹⁴C kyr bp .
• 3 Modern diploid isolates may have resulted from both vicariant and dispersal events. In central Baja California and the lower Colorado River Basin, modern diploids probably originated from relict populations near glacial refugia. Founder events in the middle and late Holocene established diploid outposts on isolated limestone outcrops in areas of central and southern Arizona dominated by tetraploid populations.
• 4 Geographic alignment of the three ploidy races along the modern gradient of increasingly drier and hotter summers is clearly a postglacial phenomenon, but evolution of both higher ploidy races must have happened before the Holocene. The exact timing and mechanism of polyploidy evolution in creosote bush remains a matter of conjecture.

     

Juvenile hormone catabolism and oviposition in the codling moth, Cydia pomonella, as functions of age, mating status, and hormone treatment.

In vitro catabolism of juvenile hormone (JH) in haemolymph of adult female Cydia pomonella was ascribed mainly to juvenile hormone esterase (JHE) activity. No significant differences were noted between virgin and mated females 0-96 h post-emergence. Changes in JHE activity did not appear dependent upon fluctuations in JH titre; conversely, changes in JHE activity could not explain the changes in JH titres. Maximal JHE activity was recorded at 24 h (331.47 +/- 47.25 pmol/h/microl; 355.93 +/- 36.68 pmol/h/microl, virgin; mated insects, respectively) and preceded the peak in JH titres at 48 h. Topical application of JH II (10 ng-10 microg) or fenoxycarb (50 ng) enhanced JHE activity up to 640 and 56%, respectively. Treatment upon emergence with 10 microg JH II induced enzymic activity for less than 24 h, and when 10 microg JH II or 50 ng fenoxycarb were applied, circulating JH titres returned to control levels within 24 h. Oviposition was highly sensitive to exogenous JH and declined significantly with dosages >100 pg. To allow a degree of oocyte maturation before JH treatment, the hormone was administered at 6, 12, 24, or 48 h post-emergence and/or females were mated. Neither measure "protected" the system; oviposition declined immediately after JH application.     

Determinants of the substrate specificity of multidrug resistance protein 1: role of amino acid residues with hydrogen bonding potential in predicted transmembrane helix 17

Human multidrug resistance protein 1 (MRP1) confers resistance to many natural product chemotherapeutic agents and actively transports structurally diverse organic anion conjugates. We previously demonstrated that two hydrogen-bonding amino acid residues in the predicted transmembrane 17 (TM17) of MRP1, Thr(1242) and Trp(1246), were important for drug resistance and 17beta-estradiol 17-(beta-d-glucuronide) (E(2)17betaG) transport. To determine whether other residues with hydrogen bonding potential within TM17 influence substrate specificity, we replaced Ser(1233), Ser(1235), Ser(1237), Gln(1239), Thr(1241), and Asn(1245) with Ala and Tyr(1236) and Tyr(1243) with Phe. Mutations S1233A, S1235A, S1237A, and Q1239A had no effect on any substrate tested. In contrast, mutations Y1236F and T1241A decreased resistance to vincristine but not to VP-16, doxorubicin, and epirubicin. Mutation Y1243F reduced resistance to all drugs tested by 2-3-fold. Replacement of Asn(1245) with Ala also decreased resistance to VP-16, doxorubicin, and epirubicin but increased resistance to vincristine. This mutation also decreased E(2)17betaG transport approximately 5-fold. Only mutation Y1243F altered the ability of MRP1 to transport both leukotriene 4 and E(2)17betaG. Together with our previous results, these findings suggest that residues with side chain hydrogen bonding potential, clustered in the cytoplasmic half of TM17, participate in the formation of a substrate binding site.     

Cationic polypeptides are required for antibacterial activity of human airway fluid

In a search for direct evidence leading to the biological relevance of airway secretions in innate host defense, we characterized the antibacterial function of cationic polypeptides within minimally manipulated nasal fluid. In this study, we show that cationic antimicrobial polypeptides are responsible for most of the bactericidal activity of whole nasal fluid. The removal of cationic polypeptides using a cation-exchange resin ablated the activity of nasal fluid against Escherichia coli, Listeria monocytogenes, and Pseudomonas aeruginosa. By using a novel proteomic approach, we identified a dozen cationic peptides and proteins within nasal fluid, all of which either are known antimicrobial polypeptides or have other proposed roles in host defense. Of the three most abundant cationic polypeptides in nasal fluid, lysozyme was more effective than either lactoferrin or secretory leukoprotease inhibitor in restoring the antibacterial activity of the cationic polypeptide-depleted fluid against a mucoid cystic fibrosis isolate of P. aeruginosa.     

Comparison of parasitism by Cotesia glomerata with bacterial infection and wounding in Pieris brassicae: induction of new haemolymph polypeptides and changes in humoral immune response

In Pieris brassicae, parasitism by Cotesia glomerata and bacterial infection are differentiated with respect to haemolymph protein arrays, and production or suppression of antibacterial agents. Bacteriolytic activity in haemolymph from parasitized larvae was slightly, but significantly, higher 24h post-treatment than that of untreated and wounded controls. Micrococcus lysodeikticus- or lipopolysaccharide-(LPS) injected insects exhibited an 11-fold greater response than those parasitized. At 24h post-treatment, antibacterial activity against Escherichia coli was observed in haemolymph from all but untreated larvae. Injection of Grace's medium, M. lysodeikticus or LPS, caused a greater than threefold response than parasitization or wounding. The protein banding patterns of parasitized hosts did not correspond to those of the other treatments. Two parasitoid-induced proteins (38 and 128 kDa) were examined. Both were found in parasitized insects, not in those wounded, injected with Grace's medium, M. lysodeikticus or LPS. Neither protein was bacteriolytic or bacteriostatic in inhibition zone assays.     

Protein film voltammetry reveals distinctive fingerprints of nitrite and hydroxylamine reduction by a cytochrome C nitrite reductase

The cytochrome c nitrite reductases perform a key step in the biological nitrogen cycle by catalyzing the six-electron reduction of nitrite to ammonium. Graphite electrodes painted with Escherichia coli cytochrome c nitrite reductase and placed in solutions containing nitrite (pH 7) exhibit large catalytic reduction currents during cyclic voltammetry at potentials below 0 V. These catalytic currents were not observed in the absence of cytochrome c nitrite reductase and were shown to originate from an enzyme film engaged in direct electron exchange with the electrode. The catalytic current-potential profiles observed on progression from substrate-limited to enzyme-limited nitrite reduction revealed a fingerprint of catalytic behavior distinct from that observed during hydroxylamine reduction, the latter being an alternative substrate for the enzyme that is reduced to ammonium in a two electron process. Cytochrome c nitrite reductase clearly interacts differently with these two substrates. However, similar features underlie the development of the voltammetric response with increasing nitrite or hydroxylamine concentration. These features are consistent with coordinated two-electron reduction of the active site and suggest that the mechanisms for reduction of both substrates are underpinned by common rate-defining processes.