Residues forming a hydrophobic pocket in ARF3 are determinants of GDP dissociation and effector interactions

Three residues of human ADP-ribosylation factor 3 (ARF3) (F51, W66 and Y81) cluster into a hydrophobic pocket in the inactive, GDP-bound protein. Disruption of the hydrophobic pocket with mutations at these residues increased the rate of GDP dissociation and association, but not always that of GTPgammaS. Several of the same mutants were found to be defective, often selectively, in binding different ARF effectors in two-hybrid assays. These results highlight three features of these hydrophobic residues in regulating (1) the rate of GDP dissociation, (2) the conformational changes that promote GTP binding and (3) their role in binding target proteins.     

Confocal multilaser focusing and single-laser characterization of ultraviolet excitable stains of cellular preparations

BACKGROUND: The aims of this study were (1) to realign cellular preparations when spots and structures are excited by different lasers of a confocal laser scanning microscope (multilaser studies); (2) to avoid the use of realigment methods by selecting fluorochromes that can be excited by only one laser (single-laser experiments). METHODS: In multilaser studies, we used propidium iodide fluorescent beads, as well as tetramethyl rhodamine isothiocyanate (TRITC), fluorescein isothiocyanate (FITC), and 4'-6 diamidino-2-phenylindole (DAPI)-stained human cancer lines. They were excited using HeNe, argon, and ultraviolet (UV) argon laser lines of a confocal laser scanning microscope. Single-laser experiments using UV excitation only were performed using europium as a model for magnetic resonance paramagnetic contrast agents. Nuclei of human cancer lines and tissue were counterstained by DAPI and cytoplasms were labeled with ELF-97 substrates. Factor analysis of medical images (FAMIS) and correlation methods were used to realign shifted images, focus images, and characterize each fluorochrome when necessary. RESULTS: In multilaser studies, superimposition of factor images corrected Z shifts and correlation methods provided X, Y correction values. In single-laser experiments, each fluorochrome was clearly distinguished in the group of fluorochromes. Estimated images in both studies showed colocalizations of structures. CONCLUSIONS: It is possible to characterize differences in the focus and alignment of fluorescent probes and to correct them. It is also possible to study colocalization of UV excitable fluorochromes (DAPI, ELF-97, europium) in cellular and tissular preparations via multilaser or single-laser experiments.     

Mechanism of formation of the complex between transferrin and bismuth, and interaction with transferrin receptor 1

The kinetics and thermodynamics of Bi(III) exchange between bismuth mononitrilotriacetate (BiL) and human serum transferrin as well as those of the interaction between bismuth-loaded transferrin and transferrin receptor 1 (TFR) were investigated at pH 7.4-8.9. Bismuth is rapidly exchanged between BiL and the C-site of human serum apotransferrin in interaction with bicarbonate to yield an intermediate complex with an effective equilibrium constant K(1) of 6 +/- 4, a direct second-order rate constant k(1) of (2.45 +/- 0.20) x 10(5) M(-1) s(-1), and a reverse second-order rate constant k(-1) of (1.5 +/- 0.5) x 10(6) M(-1) s(-1). The intermediate complex loses a single proton with a proton dissociation constant K(1a) of 2.4 +/- 1 nM to yield a first kinetic product. This product then undergoes a modification in its conformation followed by two proton losses with a first-order rate constant k(2) = 25 +/- 1.5 s(-1) to produce a second kinetic intermediate, which in turn undergoes a last modification in the conformation to yield the bismuth-saturated transferrin in its final state. This last process rate-controls Bi(III) uptake by the N-site of the protein and is independent of the experimental parameters with a constant reciprocal relaxation time tau(3)(-1) of (3 +/- 1) x 10(-2) s(-1). The mechanism of bismuth uptake differs from that of iron and probably does not involve the same transition in conformation from open to closed upon iron uptake. The interaction of bismuth-loaded transferrin with TFR occurs in a single very fast kinetic step with a dissociation constant K(d) of 4 +/- 0.4 microM, a second-order rate constant k(d) of (2.2 +/- 1.5) x 10(8) M(-1) s(-1), and a first-order rate constant k(-d) of 900 +/- 400 s(-1). This mechanism is different from that observed with the ferric holotransferrin and implies that the interaction between TFR and bismuth-loaded transferrin probably takes place on the helical domain of the receptor which is specific for the C-site of transferrin and HFE. The relevance of bismuth incorporation by the transferrin receptor-mediated iron acquisition pathway is discussed.     

Cyclic enterobacterial common antigen: Potential contaminant of bacterially expressed protein preparations

We have previously reported the identification of the cyclic enterobacterial common antigen (ECA(CYC)) polysaccharide in E. coli strains commonly used for heterologous protein expression (PJA Erbel et al., J. Bacteriol. 185 (2003): 1995). Following this initial report, interactions among several NMR groups established that characteristic N -acetyl signals of ECA(CYC) have been observed in (15)N-(1)H HSQC spectra of samples of various bacterially-expressed proteins suggesting that this water-soluble carbohydrate is a common contaminant. We provide NMR spectroscopic tools to recognize ECA(CYC) in protein samples, as well as several methods to remove this contaminant. Early recognition of ECA-based NMR signals will prevent time-consuming analyses of this copurifying carbohydrate.     

Characterization of a novel human breast cancer associated gene (BCA3) encoding an alternatively spliced proline-rich protein

As part of an integrated study of breast cancer gene expression, partial cDNAs were cloned from normal and tumor breast cells by subtractive-hybridization and differential display cloning. The DNA sequence for one of these breast cancer associated genes was used to construct the larger 1319 bp BCA3 cDNA sequence using ESTs without assigned names or functions. High-level BCA3 mRNA expression was found in breast and prostate tumor cell lines whereas normal breast and prostate tissues have low-level expression. Further analysis revealed possible functional domains and alternative splicing of BCA3 that we confirmed by RT-PCR analysis. Immunohistochemistry revealed that the protein is expressed in breast tumor cells in vivo, and not in surrounding stromal tissue.     

Assessment of putative protein targets derived from the SARS genome

The ability to rapidly and reliably develop hypotheses on the function of newly discovered protein sequences requires systematic and comprehensive analysis. Such an analysis, embodied within the DS GeneAtlas pipeline, has been used to critically evaluate the severe acute respiratory syndrome (SARS) genome with the goal of identifying new potential targets for viral therapeutic intervention. This paper discusses several new functional hypotheses on the roles played by the constituent gene products of SARS, and will serve as an example of how such assignments can be developed or extended on other systems of interest.     

Insulin-induced up-regulated uncoupling protein-1 expression is mediated by insulin receptor substrate 1 through the phosphatidylinositol 3-kinase/Akt signaling pathway in fetal brown adipocytes

To investigate the role of insulin receptor substrate-1 (IRS-1) and its downstream signaling in insulin-induced thermogenic differentiation of brown adipocytes, we have reconstituted IRS-1-deficient fetal brown adipocytes (IRS-1(-/-)) with wild-type IRS-1 (IRS-1(wt)). The lack of IRS-1 resulted in the inability of insulin to induce IRS-1-associated phosphatidylinositol 3-kinase (PI 3-kinase) activity and Akt phosphorylation in IRS-1(-/-) brown adipocytes. In addition, these cells showed an impairment in activating alpha-Akt, beta-Akt, and gamma-Akt isoforms upon insulin stimulation. Reconstitution of IRS-1(-/-) brown adipocytes with IRS-1(wt) restored the IRS-1/PI 3-kinase/Akt signaling pathway. Treatment of wild-type brown adipocytes with insulin for 24 h up-regulated uncoupling protein-1 (UCP-1) expression and transactivated the UCP-1 promoter; this effect was abolished in the absence of IRS-1 or in the presence of an Akt inhibitor and further recovered after IRS-1(wt) reconstitution. Neither UCP-2 nor UCP-3 was up-regulated by insulin in wild-type and IRS-1-deficient brown adipocytes. Insulin stimulated the expression of CCAAT/enhancer-binding protein alpha (C/EBPalpha) and its DNA binding activity in wild-type brown adipocytes but not in IRS-1(-/-) cells. However, insulin stimulation of both C/EBPalpha expression and binding activity was restored after IRS-1(wt) reconstitution of deficient cells. Retrovirus-mediated expression of C/EBPalpha and peroxisome proliferator-activated receptor gamma in IRS-1(-/-) brown adipocytes up-regulated UCP-1 protein content and transactivated UCP-1 promoter regardless of insulin stimulation. Both C/EBPalpha and peroxisome proliferator-activated receptor gamma reconstituted FAS mRNA expression, but only C/EBPalpha restored insulin sensitivity in the absence of IRS-1. Finally, reconstitution of IRS-1(-/-) brown adipocytes with the IRS-1 mutants IRS-1(Phe-895), which lacks IRS-1/growth factor receptor binding protein 2 binding but not IRS-1/p85-PI 3-kinase binding, or with IRS-1(Tyr-608/Tyr-628/Tyr-658), which only binds p85-PI 3-kinase, induced UCP-1 expression and transactivated the UCP-1 promoter. These data provide strong evidence for an essential role of IRS-1 through the PI 3-kinase/Akt signaling pathway inducing UCP-1 gene expression by insulin.