Glutathione reductase (NADPH+GSSG+H+-->NADP(+) + 2GSH) is a homodimeric flavoenzyme of known geometry. Each subunit contains four well-defined domains and contributes essential residues to the active sites; consequently, the monomer is expected to be inactive. As part of our program to develop dimerization inhibitors of human glutathione reductase (hGR) as antimalarial agents, we mutagenized the residues 446 and 447 which, together with their counterparts on the other subunit, represent the tightest contact between the subunits [Karplus, P. A., & Schulz, G. E. (1987) J. Mol. Biol. 195, 701-729]. Wild-type human glutathione reductase and mutants of this protein were produced in plasmid-transformed Escherichia coli SG5 cells. Active enzyme species, namely, wild-type hGR, N-terminally truncated delta(1-15)hGR, and the point mutant F447P-hGR, were purified by 2',5'-ADP-Sepharose chromatography and crystallization. Inactive mutants such as G446E-hGR or the double mutants G446E/F447P-hGR and G446P/F447P-hGR were isolated by immunoadsorption chromatography. G446E/F447P-hGR was studied in detail. This mutant behaved like a poorly folded monomeric protein, as indicated by the following properties: absence of the intersubunit disulfide bridge, Cys90-Cys90'; failure to bind FAD; failure to bind NADPH and analogues thereof; a short half-life (< 4 min) in E. coli cells; and high susceptibility to trypsin in vitro. The results suggest that the sequence around G446 can control dimerization as well as domain folding. This is unexpected since the FAD-binding domain and the NADPH-binding domain occur in many different enzymes and have been regarded as autonomous folding units.(ABSTRACT TRUNCATED AT 250 WORDS)

Human RNPS1 was originally purified and characterized as a pre-mRNA splicing activator, and its role in the postsplicing process has also been proposed recently. To search for factors that functionally interact with RNPS1, we performed a yeast two-hybrid screen with a human cDNA library. Four factors were identified: p54 (also called SRp54; a member of the SR protein family), human transformer 2␤ (hTra2␤; an exonic splicing enhancer-binding protein), hLucA (a potential component of U1 snRNP), and pinin (also called DRS and MemA; a protein localized in nuclear speckles). The N-terminal region containing the serine-rich (S) domain, the central RNA recognition motif (RRM), and the C-terminal arginine/serine/proline-rich (RS/P) domain of RNPS1 interact with p54, pinin, and hTra2␤, respectively. Protein-protein binding between RNPS1 and these factors was verified in vitro and in vivo. Overexpression of RNPS1 in HeLa cells induced exon skipping in a model ␤-globin pre-mRNA and a human tra-2␤ pre-mRNA. Coexpression of RNPS1 with p54 cooperatively stimulated exon inclusion in an ATP synthase ␥-subunit pre-mRNA. The RS/P domain and RRM are necessary for the exon-skipping activity, whereas the S domain is important for the cooperative effect with p54. RNPS1 appears to be a versatile factor that regulates alternative splicing of a variety of pre-mRNAs.Most pre-mRNAs in higher eukaryotes complete accurate splicing in the nucleus, as a prerequisite for carrying the correct genetic information to the cytoplasm for translation. Constitutive splicing is highly precise and is sensitive to mutations in critical signal sequences of the pre-mRNA. Indeed, human genetic diseases are often caused by point mutations that lie in 5Ј or 3Ј splice site elements or by creation of new ones at inappropriate locations, which result in splicing defects (reviewed in references 33 and 50). On the other hand, a subset of pre-mRNAs shows sufficient flexibility for alternative potential splice sites to be used, often in a regulated way in response to tissue-specific or developmentally regulated states (reviewed in references 24, 55, and 82). This process, called alternative splicing, is a basic strategy for the regulation of eukaryotic gene expression. An unexpectedly small set of protein-coding genes, at most 30,000 has recently been estimated in the human genome (51, 72, 79). Indeed, a higher prevalence of alternative splicing than earlier estimates is likely responsible for a larger number of, and more complex, protein products (51, 64).Pre-mRNA splicing takes place within a large complex, or spliceosome, which includes the small nuclear ribonucleoprotein particles (snRNPs) U1, U2, U4/U6, and U5, together with a large number of non-snRNP protein factors. Biochemical characterization of the spliceosome, together with genetic studies in fission and budding yeast, predicted that more than 50 proteins are essential for constitutive splicing (reviewed in references 15 and 73). The members of the serine/arginine-rich protein (SR protein) family are wel...

DNA-dependent protein kinase (DNA-PK), which is involved in DNA double-strand break repair and V(D)J recombination, is comprised of a DNA-targeting component termed Ku and an ∼465-kD catalytic subunit, DNA-PK cs . Although DNA-PK phosphorylates proteins in the presence of DSBs or other discontinuities in the DNA double helix in vitro, the possibility exists that it is also activated in other circumstances via its association with additional proteins. Here, through use of the yeast two-hybrid screen, we discover that the recently identified high affinity DNA binding protein C1D interacts with the putative leucine zipper region of DNA-PK cs . Furthermore, we show that C1D can interact with DNA-PK in mammalian cells and that C1D is a very effective DNA-PK substrate in vitro. Finally, we establish that C1D directs the activation of DNA-PK in a manner that does not require DNA termini. Therefore, these studies provide a function for C1D and suggest novel mechanisms for DNA-PK activation in vivo.

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Polypeptides remaining tightly associated with isolated genomic DNA are of interest with respect to their potential involvement in the topological organization and/or function of genomic DNA. Such residual DNA-polypeptide complexes were used for raising monoclonal antibodies by in vitro immunization. Screening of a murine lambdagt11 cDNA library with these antibodies released a positive cDNA (MC1D) encoding a 16 kDa polypeptide. The cloned homologous human cDNA (HC1D) was identified in the dbest data base by partial sequence comparison, and it was sequenced full length. The cDNA-derived amino acid sequences comprise nuclear location signals but none of the known DNA-binding motifs. However, the recombinantly expressed proteins show in vitro DNA binding affinities. A polyclonal antiserum to the recombinant MC1D protein immunostains sub-nuclear structures, and it detects a residual 16 kDa polypeptide on western blots of DNA digests. These results support the conclusion that the cloned cDNAs reflect mRNAs encoding one of the chemically-resistant polypeptides which can be detected in isolated genomic DNA by sensitive techniques, e.g. by125Iodine labeling and SDS-PAGE.

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The molecular cloning of a partial cDNA to mouse glutathione reductase mRNA and of a full-length cDNA to the mRNA of the human enzyme is described. An initial cDNA clone designated AGRM-B11 was isolated by plaque-screening of an induced mouse cDNA expression library in the l g t l l vector with a rabbit antibody probe to human glutathione reductase. 2sIodine-labelled whole anti-rabbit immunoglobulin was used as second antibody. EcoRI digestion of the AGRM-B11 clone released a 720-bp fragment which was identified as a partial mouse glutathione reductase cDNA by the following techniques. (a) Escherichia coli Y1089 lysogenized with 1.GRM-B11 could be induced to synthesize a recombinant polypeptide whose antigenicity to anti-(glutathione reductase) serum was established by SDS/polyacrylamide gel electrophoresis and subsequent immunoblotting. (b) The GRM-B11 sequence, recloned in the Bluescript vector to give the plasmid pGRM-B11, was found to code for a polypeptide consisting of 242 amino acid residues exhibiting 82% identities with the known amino acid sequence of the human glutathione reductase from position 77 to 318.The insert of the pGRM-B11 plasmid was used as a bonajide nucleic acid probe to screen mouse and human cDNA libraries prepared in the i g t l l or in the AgtlO vector. The first full-length cDNA clone (AGRH-Mev10) was identified in a human cDNA library based on RNA of human placental cells. Its insert was composed of three EcoRI fragments of 720, 613 and 336 bp. The three fragments were recloned in the Bluescript vector and sequenced. The largest fragment (pGRH-B) is colinear with the mouse sequence cloned in the pGRM-B1 1 plasmid. The fragment of intermediate size (pGRH-CT) comprises the 3' end of the mRNA and the poly(A) tail while the short fragment (pGRH-NT) corresponds to the 5' region of the mRNA. The amino acid sequence deduced from the nucleotide sequences of the three subclones is identical with the known sequence of the mature glutathione reductase from human erythrocytes in all 478 positions.Glutathione reductase, a homodimeric flavoprotein of 2 x 52 kDa, catalyzes the production of glutathione (GSH) from glutathione disulfide (GSSG) at the expense of NADPH :As part of the glutathione redox cycle, the enzyme plays a role in the detoxification of reactive oxygen species [l]. In addition, by adjusting the [GSH]/[GSSG] ratio, it is involved in a number of cellular functions including the activation of dormant cells [2] and the regulation of the cell cycle [3].Most structural information is available for the enzyme from human erythrocytes. This includes the amino acid sequence [4], the three-dimensional geometry at 0.15-nm resolution [5], the binding mode of the substrates [6-81 and the

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