Effect of Glycation of Hemoglobin on its Interaction with Trifluoperazine

Trifluoperazine (TFZ), a phenothiazine drug, penetrates into human erythrocytes and releases oxygen by interaction with hemoglobin. TFZ-induced oxygen release from hyperglycemic erythrocytes isolated from diabetic patients is considerably less compared to that from the cells of normoglycemic individuals. In diabetes mellitus, hemoglobin is significantly glycated by glucose. Non-glycated hemoglobin, HbA₀ and its major glycated analog, HbA_1c have been separated from the blood samples of diabetic patients. TFZ releases considerable amount of oxygen from HbA₀, but very little from HbA_1c. Spectrofluorimetric studies reveal that TFZ forms excited state complexes with both HbA₀ and HbA_1c. Titration of HbA₀ with TFZ in a spectrophotometric study exhibits two isosbestic points. Similar experiment with HbA_1c causes gradual loss of the Soret peak without appearance of any isosbestic point indicating a possibility of heme loss during interaction, which is also supported by gel filtration experiment and SDS-PAGE experiment followed by heme staining. The results suggest that drug action on hemoglobin is influenced by glycation-induced structural modification of the protein.     

HCPTPA, a protein tyrosine phosphatase that regulates vascular endothelial growth factor receptor-mediated signal transduction and biological activity

Angiogenesis is a tightly controlled process in which signaling by the receptors for vascular endothelial growth factor (VEGF) plays a key role. In order to define signaling pathways downstream of VEGF receptors (VEGFR), the kinase domain of VEGFR2 (Flk-1) was used as a bait to screen a human fetal heart library in the yeast two-hybrid system. One of the signaling molecules identified in this effort was HCPTPA, a low molecular weight, cytoplasmic protein tyrosine phosphatase. Although HCPTPA possesses no identifiable phosphotyrosine binding domains (i.e. SH2 or phosphotyrosine binding domains), it bound specifically to active, autophosphorylated VEGFR2 but not to a mutated, kinase-inactive VEGFR2. Recombinant VEGFR2 and endogenous VEGFR2 were substrates for recombinant HCPTPA, and HCPTPA was co-expressed with VEGFR2 in endothelial cell lines, suggesting that HCPTPA may be a negative regulator of VEGFR2 signal transduction. To pursue this possibility, an adenovirus directing the expression of HCPTPA was constructed. When used to infect cultured endothelial cells, this adenovirus directed high level expression of HCPTPA that resulted in impairment of VEGF-mediated VEGFR2 autophosphorylation and mitogen-activated protein kinase activation. Adenovirus-mediated overexpression of HCPTPA also inhibited VEGF-induced cellular responses (endothelial cell migration and proliferation) and inhibited angiogenesis in the rat aortic ring assay. Taken together, these findings indicate that HCPTPA may be an important regulator of VEGF-mediated signaling and biological activity. Potential interactions with other signaling pathways and possible therapeutic implications are discussed.     

Stimulation of DNA glycosylase activity of OGG1 by NEIL1: functional collaboration between two human DNA glycosylases

The eukaryotic 8-oxoguanine-DNA glycosylase 1 (OGG1) provides the major activity for repairing mutagenic 7,8-dihydro-8-oxoguanine (8-oxoG) induced in the genome due to oxidative stress. Earlier in vitro studies showed that, after excising the base lesion, the human OGG1 remains bound to the resulting abasic (AP) site in DNA and does not turn over efficiently. The human AP-endonuclease (APE1), which cleaves the phosphodiester bond 5' to the AP site, in the next step of repair, displaces the bound OGG1 and thus increases its turnover. Here we show that NEIL1, a DNA glycosylase/AP lyase specific for many oxidized bases but with weak 8-oxoG excision activity, stimulates turnover of OGG1 in a fashion similar to that of APE1 and carries out betadelta-elimination at the AP site. This novel collaboration of two DNA glycosylases, which do not stably interact with each other, in stimulating 8-oxoguanine repair is possible because of higher AP site affinity and stronger AP lyase activity of NEIL1 relative to OGG1. Comparable levels of NEIL1 and OGG1 in some human cells raise the possibility that NEIL1 serves as a backup enzyme to APE1 in stimulating 8-oxoG repair in vivo.     

Trafficking defects of a novel autosomal recessive distal renal tubular acidosis mutant (S773P) of the human kidney anion exchanger (kAE1)

Autosomal dominant and recessive distal renal tubular acidosis (dRTA) can be caused by mutations in the anion exchanger 1 (AE1 or SLC4A1) gene, which encodes the erythroid chloride/bicarbonate anion exchanger membrane glycoprotein (eAE1) and a truncated kidney isoform (kAE1). The biosynthesis and trafficking of kAE1 containing a novel recessive missense dRTA mutation (kAE1 S773P) was studied in transiently transfected HEK-293 cells, expressing the mutant alone or in combination with wild-type kAE1 or another recessive mutant, kAE1 G701D. The kAE1 S773P mutant was expressed at a three times lower level than wild-type, had a 2-fold decrease in its half-life, and was targeted for degradation by the proteasome. It could not be detected at the plasma membrane in human embryonic kidney cells and showed predominant endoplasmic reticulum immunolocalization in both human embryonic kidney and LLC-PK1 cells. The oligosaccharide on a kAE1 S773P N-glycosylation mutant (N555) was not processed to the complex form indicating impaired exit from the endoplasmic reticulum. The kAE1 S773P mutant showed decreased binding to an inhibitor affinity resin and increased sensitivity to proteases, suggesting that it was not properly folded. The other recessive dRTA mutant, kAE1 G701D, also exhibited defective trafficking to the plasma membrane. The recessive kAE1 mutants formed dimers like wild-type AE1 and could hetero-oligomerize with wild-type kAE1 or with each other. Hetero-oligomers of wild-type kAE1 with recessive kAE1 S773P or G701D, in contrast to the dominant kAE1 R589H mutant, were delivered to the plasma membrane.     

High Levels of Orexin A in the Brain of the Mouse Model for Phenylketonuria: Possible Role of Orexin A in Hyperactivity Seen in Children with PKU

Phenylketonuria (PKU) is a metabolic disorder caused by phenylalanine hydroxylase deficiency leading to increased levels of phenylalanine in the brain. Hyperactivity is reportedly induced by a high level of orexin A, and therefore orexin A content was studied in the PKU mice. Hypothalamus and brain stem had higher levels of orexin A compared to cerebrum and cerebellum both in wild type and PKU mice brains as observed by radioimmunoassay method. Interestingly, all these regions of the brain in PKU mouse showed a higher level of orexin A compared to the wild type. Heart and plasma also had higher levels of orexin A in PKU compared to the wild type. Immunohistochemical analysis revealed an increased number of orexin A–stained cells in the brain and heart of PKU mouse compared to the wild type. This is the first report of increased level of orexin in the PKU mouse brain. Hyperactivity is commonly observed in children with PKU; thus these findings suggest that orexin A is a contributing factor for the hyperactivity.     

High-density functional display of proteins on bacteriophage lambda

We designed a bacteriophage lambda system to display peptides and proteins fused at the C terminus of the head protein gpD of phage lambda. DNA encoding the foreign peptide/protein was first inserted at the 3' end of a DNA segment encoding gpD under the control of the lac promoter in a plasmid vector (donor plasmid), which also carried lox P(wt) and lox P(511) recombination sequences. Cre-expressing cells were transformed with this plasmid and subsequently infected with a recipient lambda phage that carried a stuffer DNA segment flanked by lox P(wt) and lox P(511) sites. Recombination occurred in vivo at the lox sites and Amp(r) cointegrates were formed. The cointegrates produced recombinant phage that displayed foreign protein fused at the C terminus of gpD. The system was optimised by cloning DNA encoding different length fragments of HIV-1 p24 (amino acid residues 1-72, 1-156 and 1-231) and the display was compared with that obtained with M13 phage. The display on lambda phage was at least 100-fold higher than on M13 phage for all the fragments with no degradation of displayed products. The high-density display on lambda phage was superior to that on M13 phage and resulted in selective enrichment of epitope-bearing clones from gene-fragment libraries. Single-chain antibodies were displayed in functional form on phage lambda, strongly suggesting that correct disulphide bond formation takes place during display.This lambda phage display system, which avoids direct cloning into lambda DNA and in vitro packaging, achieved cloning efficiencies comparable to those obtained with any plasmid system. The high-density display of foreign proteins on bacteriophage lambda should be extremely useful in studying low-affinity protein-protein interactions more efficiently compared to the M13 phage-based system.     

F3/contactin acts as a functional ligand for Notch during oligodendrocyte maturation

Axon-derived molecules are temporally and spatially required as positive or negative signals to coordinate oligodendrocyte differentiation. Increasing evidence suggests that, in addition to the inhibitory Jagged1/Notch1 signaling cascade, other pathways act via Notch to mediate oligodendrocyte differentiation. The GPI-linked neural cell recognition molecule F3/contactin is clustered during development at the paranodal region, a vital site for axoglial interaction. Here, we show that F3/contactin acts as a functional ligand of Notch. This trans-extracellular interaction triggers gamma-secretase-dependent nuclear translocation of the Notch intracellular domain. F3/Notch signaling promotes oligodendrocyte precursor cell differentiation and upregulates the myelin-related protein MAG in OLN-93 cells. This can be blocked by dominant negative Notch1, Notch2, and two Deltex1 mutants lacking the RING-H2 finger motif, but not by dominant-negative RBP-J or Hes1 antisense oligonucleotides. Expression of constitutively active Notch1 or Notch2 does not upregulate MAG. Thus, F3/contactin specifically initiates a Notch/Deltex1 signaling pathway that promotes oligodendrocyte maturation and myelination.     

Postendocytotic trafficking of the follicle-stimulating hormone (FSH)-FSH receptor complex

Although the fates of the internalized hormone-receptor complexes formed by the lutropin/choriogonadotropin and the TSH receptors have been examined in some detail, much less is known about the fate of the internalized FSH-FSH receptor (FSHR) complex.Using biochemical and imaging approaches we show here that the majority of the internalized FSH-FSHR complex accumulates in endosomes and subsequently recycles back to the cell surface where the bound, intact hormone dissociates back into the medium. Only small amounts of FSH and the FSHR are routed to a lysosomal degradation pathway, and the extent of FSH-induced down-regulation of the cell surface and total FSHR is minimal. This pathway was detected in heterologous (human kidney 293T) cells transfected with the rat (r) or human (h) FSHR as well as in a mouse Sertoli cell line (MSC-1) or a mouse granulosa cell line (KK-1) transfected with the rFSHR.Additional experiments using a series of C-terminal deletions of the rFSHR and the hFSHR showed that the recycling of the internalized FSH-FSHR complex and the extent of hFSH-induced down-regulation is dictated by a short stretch of amino acids present at the extreme C-terminal end of the receptor.We conclude that most of the internalized FSH-FSHR complex is recycled back to the cell surface, that this recycling pathway is highly dependent on amino acid residues present near the C terminus of the FSHR, and that it is an important determinant of the extent of down-regulation of the FSHR.