We have purified a protease with characteristics of TNFa convertase from bovine spleen membranes. Peptide sequencing of the purified protein identified it as ADAM 10 (Genbank accession no. Z21961). This metalloprotease cleaves a recombinant proTNFa substrate to mature TNFa, and can cleave a synthetic peptide substrate to yield the mature TNFa amino terminus in vitro. The enzyme is sensitive to a hydroxamate inhibitor of MMPs, but insensitive to phosphoramidon. In addition, cloned ADAM 10 mediates proTNFa processing in a processing-incompetent cell line.
A 71-kDa protein (P71) with properties similar to those ofthe Escherichia coli heat shock protein DnaK has been found in extracts of HeLa cells. P71 was copurified by ATP-agarose affinity chromatography with three additional proteins of the Hsp7O family. Of these proteins, only P71 crossreacted strongly with antiserum raised against purified DnaK, and both DnaK and P71 could be phosphorylated in vitro with [y-32]ATP in a reaction that was markedly stimulated by Ca2+. In HeLa cells, P71 was found to be concentrated in mitochondria. A protein similar to P71 was also found in calf liver and yeast mitochondria.A universal response of living organisms to a variety of stress agents including elevated temperatures is the synthesis of a specific subset of proteins (reviewed in ref. 1). The heat shock protein referred to as Hsp7O has been intensively studied because of its early induction and high rate of synthesis during stress. In eukaryotic cells, Hsp7O belongs to a large gene family with complex regulatory mechanisms (2-4). These proteins share a high degree of homology, and in addition, all organisms contain constitutively expressed members, suggesting that the proteins of this group share a similar role in normally growing and stressed cells (5). A current view is that one of the major functions of the Hsp7O family of proteins is to facilitate the proper folding of proteins, as may be required for their translocation across the endoplasmic reticulum and mitochondrial membranes (6-9). Proteins that are members of the Hsp7O family are found in diverse species including bacteria. In Escherichia coli, the product of the dnaK gene is a 72-kDa heat shock protein homologous with the Hsp7O family of eukaryotes (1). Previous studies on DnaK showed that it is a phosphoprotein and that purified preparations of DnaK could be autophosphorylated in vitro (10, 11). The present report shows that one of the Hsp7O-related proteins, a 71-kDa protein (P71), is found in the mitochondria of eukaryotic cells and shares some of the characteristics of DnaK. During the preparation of this manuscript, Craig et al. (12) reported the sequence of the yeast SSCI gene. This gene codes for a Hsp7O protein that appears to be similar to P71, since it is localized in mitochondria and has a high degree of homology to DnaK. MATERIALS AND METHODSHeLa (S3) cells were grown in suspension at 37°C to a density of 6-8 x 105 cells per ml in Joklik's modified minimal essential medium (13) supplemented with 10% fetal bovine serum and 2 mM glutamine. Cells were harvested by centrifugation at 200 x g for 5 min and were washed in 10 mM Hepes-KOH (pH 7.5) containing 0.15 M NaCl. Cell pellets were either used immediately for cell fractionation or frozen in liquid nitrogen for protein purification.For cell fractionation, cells (1 x 109) were suspended in 15 ml of sucrose buffer (0.25 M sucrose/10 mM Hepes-KOH, pH 7.5), the suspension was forced through a 26-gauge needle twice, and the lysate was centrifuged at 1000 x g for 10 min at 4TC. The pellet was resusp...
The heat-shock response of Euglena gracilis was studied by pulse-labeling cells with [35S~sulfate at both the normal growth temperature (210C) and an elevated temperature (36TC). Analysis of the labeled proteins by polyacrylamide gel electrophoresis indicated that the rate of synthesis of at least 3 major and 15 minor polypeptides increased in cells grown at the higher temperature. Three of the proteins appear to be immunologically related to the ubiquitous -70-kDa heat-shock protein (Hsp7O) family. One protein of 68 kDa was found in the cytoplasm (P68Cyt) and was the major heat-shock protein in Euglena gracilis. Two other proteins, 68 and 70 kDa, were localized in mitochondria (P68,,) and chloroplasts (P7ch), respectively, and they crossreacted with a polyclonal antibody raised against the Escherichia coli heat-shock protein DnaK. Like DnaK, P68mit and P70ChI could be phosphorylated in vitro with [v-32PIATP in a reaction that was stimulated by Ca2. A protein with characteristics similar to those of P70Chi was also found in chloroplasts isolated from maize and spinach.
The primary product of the adenovirus E1A gene is a protein that is sufficient for controlling host-cell proliferation and immortalizing primary rodent cells. The mechanism by which the protein induces these cellular effects is poorly understood, but might be linked to its ability to regulate RNA transcription from a number of viral and cellular genes. The mechanism of E1A's transcriptional-activation (trans-activation) was studied here by monitoring the protein's effect on specific adenovirus promoters in two types of transcriptional systems in vitro. One of these systems consisted of extracts from transformed cells constitutively expressing E1A, and the other consisted of extracts of HeLa cells supplemented with a plasmid-encoded E1A protein purified from Escherichia coli. The results show that the E1A protein specifically stimulates transcription from adenovirus promoters; thus, the induction of cellular transcription factors is not necessary to explain the stimulation of transcription by E1A.
The interaction of adenovirus‐2 (Ad2) early region IA (EIA) protein (encoded by the 13S mRNA) with DNA was examined using EIA protein synthesized in Escherichia coli extracts directed by a plasmid containing the cloned EIA gene. Without any purification, this protein when chromatographed over calf thymus DNA immobilized on cellulose, showed at least two types of salt‐sensitive activities after associating with equal efficiency to both single‐ and double‐stranded DNA; however, a putative C‐terminal proteolytic fragment of the EIA protein (identified by immunoprecipitation with anti‐serum specific to the EIA carboxy‐terminus) showed 10‐fold greater affinity to double‐ versus single‐stranded DNA. When examined with Ad2 DNA, the EIA protein had a retention that was at least 2‐fold higher compared to calf thymus DNA, suggesting some substrate specificity. It was also found that a 1.0 M salt concentration was required for the elution of the EIA protein from pBR322 DNA containing cloned regulatory sequences of adenovirus early regions II and III. This suggests that the strength of the protein interaction depends on the target DNA sequence. Finally, addition of uninfected HeLa cell extract to bacterial extracts containing EIA‐like protein potentiated the association of the protein to double‐stranded calf thymus DNA up to 7‐fold. These data support the hypothesis that the EIA protein interacts with target DNA, presumably mediated by co‐factor(s) in an indirect fashion.
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