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1.
Diabetes ; 50(1): 24-31, 2001 Jan.
Article in English | MEDLINE | ID: mdl-11147790

ABSTRACT

Insulin receptor substrate (IRS)-1 protein expression is markedly reduced in many insulin-resistant states, although the mechanism for this downregulation is unclear. In this study, we have investigated the early events in the insulin pathway that trigger the degradation of IRS-1. Incubation of the adipocytes with insulin induced a fast electrophoretic mobility shift of IRS-1 and a subsequent degradation of the protein. Wortmannin and rapamycin blocked this mobility shift of IRS-1, maintained the insulin-induced tyrosine phosphorylation of IRS-1, and blocked its degradation. In contrast, a glycogen synthase kinase 3 inhibitor, a mitogen-activated protein kinase/extracellular-regulated kinase inhibitor, and various protein kinase C inhibitors had no effect. Incubation with okadaic acid increased the serine/threonine phosphorylation of IRS-1 and its degradation, mimicking insulin, and its effect was prevented by the proteasome inhibitor lactacystin, as well as by rapamycin. Treatment of the cells with the tyrosine phosphatase inhibitor orthovanadate in the presence of insulin or okadaic acid partially inhibited the degradation of IRS-1. We propose that a rapamycin-dependent pathway participates as a negative regulator of IRS-1, increasing its serine/threonine phosphorylation, which triggers degradation. Thus, regulation of serine/threonine versus tyrosine phosphorylation may modulate IRS-1 degradation, affecting insulin sensitivity.


Subject(s)
Phosphoproteins/metabolism , Serine/metabolism , Threonine/metabolism , 3T3 Cells , Androstadienes/pharmacology , Animals , Cysteine Endopeptidases/metabolism , Enzyme Inhibitors/pharmacology , Insulin/pharmacology , Insulin Receptor Substrate Proteins , Mice , Multienzyme Complexes/metabolism , Okadaic Acid/pharmacology , Phosphorylation , Proteasome Endopeptidase Complex , Protein Kinase Inhibitors , Sirolimus/pharmacology , Time Factors , Tyrosine/metabolism , Vanadates/pharmacology , Wortmannin
2.
J Biol Chem ; 270(16): 9039-42, 1995 Apr 21.
Article in English | MEDLINE | ID: mdl-7721816

ABSTRACT

Apolipoprotein E (apoE), particularly the e4 allele, is genetically linked to the incidence of Alzheimer's disease. In vitro, apoE has been shown to bind beta-amyloid (A beta), an amyloidogenic peptide that aggregates to form the primary component of senile plaques. In previous work, we demonstrated that apoE3 from tissue culture medium binds to A beta with greater avidity than apoE4 (LaDu, M. J., Falduto, M. T., Manelli, A. M., Reardon, C. A., Getz, G. S., and Frail, D. E. (1994) J. Biol. Chem. 269, 23403-23406). This is in contrast to data using purified apoE isoforms as substrate for A beta (Strittmatter, W. J., Weisgraber, K. H., Huang, D. Y., Dong, L.-M., Salvesen, G. S., Pericak-Vance, M., Schmechel, D., Saunders, A. M., Goldgaber, D., and Roses, A. D. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 8098-8102). Here we resolve this apparent discrepancy by demonstrating that the preferential binding of A beta to apoE3 is attenuated and even abolished with purification, a process that includes delipidation and denaturation. We compared the A beta binding capacity of unpurified apoE isoforms from both tissue culture medium and intact human very low density lipoproteins with that of apoE purified from these two sources. The interaction of human A beta-(1-40)-peptide and apoE was analyzed by nonreducing SDS-polyacrylamide gel electrophoresis followed by Western immunoblotting for either A beta or apoE immunoreactivity. While the level of the apoE3.A beta complex was approximately 20-fold greater compared with the apoE4.A beta complex in unpurified conditioned medium, apoE3 and apoE4 purified from this medium bound to A beta with comparable avidity. Moreover, using endogenous apoE on very low density lipoproteins from plasma of apoE3/3 and apoE4/4 homozygotes, apoE3 was again a better substrate for A beta than apoE4. However, apoE purified from these plasma lipoproteins exhibited little isoform specificity in binding to A beta. These results suggest that native preparations of apoE may be a more physiologically relevant substrate for A beta binding than purified apoE and further underscore the importance of subtle differences in apoE conformation to its biological activity.


Subject(s)
Amyloid beta-Peptides/metabolism , Apolipoproteins E/metabolism , Alzheimer Disease/etiology , Apolipoproteins E/isolation & purification , Culture Media , Humans , Lipoproteins, VLDL/metabolism , Protein Denaturation
3.
Biochemistry ; 33(16): 4847-64, 1994 Apr 26.
Article in English | MEDLINE | ID: mdl-8161544

ABSTRACT

The amino-terminal fragment (ATF) of urokinase-type plasminogen activator is a two domain protein which consists of a growth factor and a kringle domain. The 1H, 13C, and 15N chemical shifts of this protein have been assigned using heteronuclear two- and three-dimensional NMR experiments on selective and uniformly 15N- and 15N/13C-labeled protein isolated from mammalian cells that overexpress the protein. The chemical shift assignments were used to interpret the NOE data which resulted in a total of 1299 NOE restraints. The NOE restraints were used along with 27 phi angle restraints and 21 hydrogen-bonding restraints to produce 15 low energy structures. The individual domains in the structures are highly converged, but the two domains are structurally independent. The root mean square deviations (rmsd) between residues 11-46 in the growth factor domain and the mean atomic coordinates were 0.99 +/- 0.2 for backbone heavy atoms and 1.65 +/- 0.2 for all non-hydrogen atoms. For residues 55-130 in the kringle domain, the rmsd was 0.84 +/- 0.2 for backbone heavy atoms and 1.42 +/- 0.2 for all non-hydrogen atoms. The overall structures of the individual domains are very similar to the structures of homologous proteins. However, important structural differences between the growth factor and other homologous proteins were observed in the region which has been implicated in binding the urokinase receptor which may explain, in part, why other growth factors show no appreciable affinity for the urokinase receptor.


Subject(s)
Peptide Fragments/chemistry , Urokinase-Type Plasminogen Activator/chemistry , Amino Acid Sequence , Animals , Cells, Cultured , Kringles , Magnetic Resonance Spectroscopy , Mammals , Models, Molecular , Molecular Sequence Data , Protein Structure, Secondary , Recombinant Proteins/chemistry , Solutions
4.
Biochemistry ; 31(51): 12713-8, 1992 Dec 29.
Article in English | MEDLINE | ID: mdl-1463742

ABSTRACT

A method to obtain uniformly isotopically labeled (15N and 15N/13C) protein from mammalian cells is described. The method involves preparation of isotopically labeled media consisting of amino acids isolated from bacterial and algal extracts supplemented with cysteine and enzymatically synthesized glutamine. The approach is demonstrated by producing 15N-labeled and 15N/13C-labeled urokinase from Sp2/0 cells and successfully growing Chinese hamster ovary (CHO) cells on the labeled media. Thus, using the procedures described, isotopically labeled proteins that have been expressed in mammalian cells can be prepared, allowing them to be studied by heteronuclear multidimensional NMR techniques.


Subject(s)
Isotope Labeling/methods , Recombinant Proteins , Amino Acids/analysis , Animals , CHO Cells/enzymology , Carbon Isotopes , Cells, Cultured , Cricetinae , Culture Media/analysis , Cysteine , Glutamine , Magnetic Resonance Spectroscopy , Nitrogen Isotopes , Urokinase-Type Plasminogen Activator
5.
J Protein Chem ; 9(6): 663-72, 1990 Dec.
Article in English | MEDLINE | ID: mdl-1963533

ABSTRACT

The gene for human preprorenin was obtained from total RNA prepared from primary human chorion cells. An expression vector was constructed containing an SV40 early promoter, a human preprorenin cDNA, bovine growth hormone poly-A addition signal, and a dihydrofolate reductase (dhfr) expression cassette. This vector was inserted into the DXB-11 Chinese hamster ovary (CHO) cell line. The recombinant protein was exported by CHO cells into the tissue culture media. At harvest the prorenin levels ranged from approximately 1-5 mg/L. For prorenin isolation the cell culture supernatants were processed by filtration, concentration, dialysis, and batch extraction. Preparative-scale isolation of prorenin was accomplished using blue-dye chromatography and size-exclusion chromatography. The isolated prorenin yielded a single SDS-gel band with Mr approximately 40,000. The proprotein was characterized with respect to N-terminal sequence and N-linked sugar composition. Trypsin-activated renin prepared from the proprotein was characterized with respect to N-terminal sequence and pH-activity profile. Enzyme activity was measured with a newly developed fluorogenic peptide substrate containing the P6-P'3 sequence of human angiotensinogen.


Subject(s)
Enzyme Precursors/biosynthesis , Recombinant Proteins/biosynthesis , Renin/biosynthesis , Amino Acid Sequence , Animals , Base Sequence , Cell Line , Chromatography, High Pressure Liquid , Cloning, Molecular , Cricetinae , Cricetulus , DNA Restriction Enzymes , Enzyme Precursors/chemistry , Enzyme Precursors/isolation & purification , Gene Expression , Humans , Molecular Sequence Data , Oligonucleotide Probes , Plasmids , Renin/chemistry , Renin/isolation & purification , Transfection/genetics
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