Biomedical subjects

M Rechsteiner

Publications and source records attributed to M Rechsteiner.

At least 73 records · Page 4Linked to original sources

PubMed1991

The C terminus of mouse ornithine decarboxylase confers rapid degradation on dihydrofolate reductase. Support for the pest hypothesis.

Several years ago, we proposed that polypeptide regions rich in proline (P), glutamic acid (E), serine (S), and threonine (T) (PEST) target intracellular proteins for destruction (Rogers, S., Wells, R., and Rechsteiner, M. (1986) Science 234, 364-368). To test the PEST hypothesis, we have produced chimeric proteins in which the N or C terminus of mouse dihydrofolate reductase is extended by the PEST-containing C terminus of mouse ornithine decarboxylase. Oligonucleotides encoding the 37 C-terminal residues of mouse ornithine decarboxylase (mODC) or equivalent lengths of dissimilar amino acids were inserted at appropriate sites in a dihydrofolate reductase (DHFR) expression vector. The various fusion proteins were expressed in Escherichia coli and purified to homogeneity by enzyme affinity chromatography. All purified fusion proteins exhibited similar abilities to convert dihydrofolate to tetrahydrofolate, thereby demonstrating that the attachment of peptide extensions to either terminus did not prevent the proper folding of DHFR. Metabolic stabilities of the radioiodinated fusion proteins were assayed in rabbit reticulocyte lysate or Xenopus egg extract. Proteolysis was found to be energy-dependent with mODC-DHFR fusion proteins being degraded from 2 to almost 40-fold faster than the parental DHFR molecule or DHFR fusion proteins bearing non-PEST extensions. Deletion of most of the PEST region from the mODC extension resulted in a significantly more stable fusion protein. Rapid proteolysis of DHFR proteins containing intact mODC extensions provides support for the PEST hypothesis.

Amino Acid Sequence↗

PubMed1991

Multicatalytic and 26 S ubiquitin/ATP-stimulated proteases in maturing rabbit red blood cells.

Rabbit red blood cells of various ages were separated on Percoll gradients and the activities of two large cytosolic proteases were measured. Both the multicatalytic protease (MCP), assayed by hydrolysis of fluorigenic peptides, and the 26 S ubiquitin/ATP-stimulated protease, assayed by degradation of ubiquitin-lysozyme conjugates, declined 3-fold or less during maturation of rabbit reticulocytes to erythrocytes. The ability of MCP to hydrolyze three classes of peptides decreased in parallel indicating that the 20 S protease is not significantly remodeled during red blood cell maturation.

Adenosine Triphosphate↗

PubMed1990

Ubiquitin-mediated degradation of histone H3 does not require the substrate-binding ubiquitin protein ligase, E3, or attachment of polyubiquitin chains.

Radioiodinated histone H3 was incubated with ubiquitin, the ubiquitin-activating enzyme E1, and one of three ubiquitin carrier proteins, reticulocyte E2(20K) or E2(32K) or the yeast RAD6 product. Although the resulting ubiquitin-histone conjugates were synthesized in the absence of the substrate-binding protein E3, they were nevertheless degraded by purified rabbit reticulocyte 26 S protease. In contrast, unmodified histone H3 remained intact upon challenge with the 26 S ubiquitin/ATP-dependent enzyme. Conjugates produced by the RAD6 protein were better proteolytic substrates than those formed by reticulocyte E2 unless ubiquitin molecules with altered lysines were used for conjugate synthesis. Substitution of methylated ubiquitin or ubiquitin molecules in which lysine 48 was converted to arginine by site-directed mutation produced histone conjugates that were degraded at slow but measurable rates. Since methylated ubiquitin molecules are incapable of forming branched polyubiquitin chains, these results demonstrate that neither ubiquitin "trees" nor the substrate binding factor E3 is absolutely required for ubiquitin-dependent degradation of histone H3 in vitro.

Animals↗

PubMed1990

The use of ubiquitin-peptide extensions as protein kinase substrates.

Four ubiquitin-peptide extensions prepared as cloned products in E. coli were tested as casein kinase II substrates. Two extensions containing the sequence Ser-Glu-Glu-Glu-Glu-Glu were readily phosphorylated by partially purified rabbit reticulocyte casein kinase II. The other two fusion proteins, which lack a consensus phosphorylation site for casein kinase II, did not serve as substrates under identical reaction conditions. Native ubiquitin was not phosphorylated by reticulocyte casein kinase II, nor have we observed its phosphorylation in crude extracts from HeLa cells, mouse liver, or Xenopus eggs. Ubiquitin's apparent lack of phosphorylatable residues coupled with its remarkable heat stability and rapid migration on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels make the protein an attractive carrier for carboxyl-terminal peptides containing specific phosphorylation sites. Such ubiquitin extension proteins should prove valuable as protein kinase substrates.

Amino Acid Sequence↗

PubMed1990

Ubiquitin metabolism in ts85 cells, a mouse carcinoma line that contains a thermolabile ubiquitin activating enzyme.

Red blood cell-mediated microinjection was used to introduce radioiodinated ubiquitin into ts85 cells, a mouse cell line that contains a thermolabile ubiquitin-activating enzyme (E1). The proportion of ubiquitin present as histone conjugates, high molecular weight conjugates, and free molecules was then determined by gel electrophoresis and autoradiography. When ts85 cells were incubated at the nonpermissive temperature, 39.5 degrees C, high molecular weight conjugates accumulated. This unexpected result was confirmed by Western blot analyses. To determine whether ubiquitin conjugates formed under nonpermissive conditions or merely persisted after the temperature increase, ts85 cells were incubated at 39.5 degrees C to generate large amounts of conjugates and then shifted to 42 degrees C. The higher temperature resulted in a 25% reduction in conjugates, but upon return to 39.5 degrees C, the ubiquitin conjugates were restored to pre-42 degrees C amounts. Since all changes in ubiquitin conjugate levels occurred above 39.5 degrees C, ts85 cells can couple ubiquitin to cellular proteins even after prolonged culture at nonpermissive temperatures. Western blot analyses showed that less than 10% of the E1 molecules present in ts85 cells at 31 degrees C remained after 2 h at 39.5 degrees C. However, when 125I-ubiquitin was added to extracts from heated ts85 cells an apparent high molecular weight form of E1 and thiol ester adducts between ubiquitin and the E2 carrier proteins were detected by electrophoresis at 4 degrees C. Considering both in vivo and in vitro demonstrations that heated ts85 cells retain the ability to conjugate ubiquitin to endogenous proteins, considerable caution must be exercised in the design and interpretation of proteolysis experiments using this mutant cell line.

Animals↗

PubMed1990

Antibody caging of a nuclear-targeting signal.

We have developed a technique for reversibly masking a peptide-targeting signal. A fluoresceinated derivative of the simian virus 40 large tumor antigen nuclear-targeting signal was synthesized and cross-linked to bovine serum albumin. The conjugated protein was efficiently transported into rat liver nuclei unless the peptide-targeting signal was sterically hindered by binding of an anti-fluorescein antibody. Addition of free 5-aminofluorescein competed for antibody binding and rapidly restored nuclear accumulation of the derivatized bovine serum albumin. General use of hapten derivatization and anti-hapten antibodies for caging portions of macromolecular surfaces can be extended to a variety of proteins, including antibodies themselves.

Amino Acid Sequence↗

PubMed1990

PEST sequences are signals for rapid intracellular proteolysis.

Many features of a protein can enhance its degradation within cells. Unlike thermal lability or oxidizability which increase the likelihood of damage, PEST sequences are built-in signals for destruction. These stretches of polypeptide chain rich in proline (P), glutamate (E), serine (S) and threonine (T) are invariably found in rapidly degraded enzymes, transcriptional factors and components of receptor signalling pathways. They are, by contrast, rarely present among long-lived cellular proteins. In this brief essay, I review the properties of PEST sequences, recent experimental support for the idea that they are indeed proteolytic signals, and possible pathways for catabolism of PEST proteins.

Amino Acid Sequence↗

PubMed1989

Synthesis of peptides as cloned ubiquitin extensions.

Oligonucleotides encoding four peptides ranging in length from 10 to 21 amino acids were cloned between the Afl2 and KpnI sites in the ubiquitin expression vector, pNMHUb. Escherichia coli AR13, a strain that contains a temperature-sensitive lambda repressor, was transformed by the plasmids, and upon shift to 42 degrees C, the cells produced large amounts of ubiquitin extended at its carboxyl terminus by each of the four peptides. Following a simple three-step purification of the ubiquitin fusion proteins, the peptides were cleaved from ubiquitin using a ubiquitin-alpha-protein hydrolase isolated from rabbit reticulocytes. The released ubiquitin molecules were shown to be competent in conjugation assays, and amino acid analyses of the purified peptides revealed that full length products were obtained in all cases. Since peptide yields varied from 2 to 4 mg/liter of culture medium, ubiquitin extensions provide an attractive alternative to solid phase synthesis for the production of peptides.

Amino Acid Sequence↗

PubMed1989

Proteolysis in heat-stressed HeLa cells. Stabilization of ubiquitin correlates with the loss of proline endopeptidase.

When intact HeLa cells were incubated at 45 degrees C, there was progressive inactivation of proline endopeptidase. Rapid loss of the enzyme did not occur in extracts maintained at 45 degrees C. Since Western blots of sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels showed no decrease in the immunoreactive 70-kDa proline endopeptidase band, its in vivo disappearance apparently results from irreversible denaturation or modification. Loss of proline endopeptidase activity was paralleled by reduced degradation of injected ubiquitin and bovine serum albumin. In contrast, proteolysis of injected lysozyme or pancreatic trypsin inhibitor was barely affected. Electrophoretic analysis of ubiquitin or bovine serum albumin retrieved from heated HeLa cells showed that the injected proteins were intact. Thus, the presence of proline endopeptidase appears to be required for initial cleavage of these two substrates, but it has not been shown that the enzyme is directly responsible. Selective stabilization of a subset of the injected proteins does, however, demonstrate the existence of distinct proteolytic pathways in HeLa cytosol.

Animals↗

PubMed1989

Degradation of structurally characterized proteins injected into HeLa cells. Comparison with their stability in rabbit reticulocyte lysate.

We recently reported (Rogers, S. W., and Rechsteiner, M. (1988) J. Biol. Chem. 263, 19833-19842) the metabolic stabilities of 35 structurally characterized proteins following their injection into HeLa cells. In this study 22 proteins from that set were radioiodinated, and their susceptibilities to proteolysis were measured in reticulocyte lysate. Degradation rates varied from less than 1% per h to almost 25% per h. ATP stimulated the degradation of 21 of the proteins with nucleotide enhancement typically in the range 2- to 3-fold. When structural features of the 22 proteins were compared with their degradation rates in lysate, no correlation was found with respect to charge, size, thermal stability, or N-terminal acylation. Furthermore, relative rates of proteolysis in lysate correlated only marginally with the metabolic stabilities of the 22 proteins as measured 24-48 h after injection into HeLa cells. Degradation rates for the 22 proteins in lysate did, however, show a strong correlation with their rates of turnover immediately after injection into the human cells. Since the enhanced proteolysis observed for many proteins soon after injection is thought to reflect disruption of HeLa cytoskeletal assemblies, this correlation provides further evidence that diffusibility or location can affect intracellular protein stability.

Animals↗

PubMed1989

Identification of the long ubiquitin extension as ribosomal protein S27a.

Two proteins of unknown function are encoded by 3' in-frame extensions of ubiquitin genes. The polypeptides are synthesized as an additional 52 or 76-80 amino acids on the C terminus of ubiquitin, an unusual arrangement conserved in man, yeast and plants (J. Callis and R. Vierstra, personal communication). Although not homologous to each other or to ubiquitin, both extension proteins are highly basic and contain patterns of cysteine and histidine similar to those proposed to form 'zinc fingers'. The longer C-terminal extension protein (CEP80) is 30% lysine and arginine and, when denatured, behaves like a small cationic protein. Its properties after isolation in physiological conditions, however, suggested that CEP80 is part of an RNA-protein complex. Using the antibodies that confirmed the presence of CEP80 in eukaryotic cells, we show here that the protein is located on ribosomes. Immunoblotting of rat 40S subunit proteins specifically identifies CEP80 as ribosomal protein S27a.

Animals↗

PubMed1989

PEST regions, proteolysis and cell cycle progression.

How eucaryotic cells control cell division is both a major intellectual challenge and a problem of considerable medical importance. A decade of studies on oncogenes has made it abundantly clear that protein kinases play a central role in regulating mitosis. In this essay, I review a number of recent discoveries providing evidence that selective proteolysis, like phosphorylation, is an important mechanism for ordering metabolic events within the cell cycle.

Adenosine Triphosphate↗

PubMed1988

Degradation of structurally characterized proteins injected into HeLa cells. Basic measurements.

Thirty-five proteins of known x-ray structure were labeled by chloramine-T radioiodination or by reaction with 125I-Bolton-Hunter reagent and introduced into HeLa cells using red cell-mediated microinjection. Degradation rates of the injected proteins were then determined over the next 50 h by measuring the release of soluble isotope to the culture medium. Control experiments demonstrated that the measured rates were not compromised by proteolysis within RBCs, the presence of unfused RBCs, or degradation of protein released from RBCs to the medium. Degradation of some injected proteins was faster during the first 12 h after fusion than at later times, apparently a response of HeLa cells to trypsinization. However, all proteins exhibited first-order degradation rates between 24 and 48 h post injection. Except for seven proteins, stabilities measured during this interval were unaffected by the labeling procedure. Reductive methylation was used to choose among the seven discordant values, and half-lives for the 35 proteins ranged from 16 h for lysozyme to 214 h for yeast alcohol dehydrogenase. Since half-lives for six of the injected proteins closely match values obtained by in vivo measurements, we consider our estimates of the metabolic stabilities of the injected proteins to be generally accurate. Therefore, the half-lives obtained by microinjection should prove useful in the search for relationships between protein structure and intracellular stability.

Autoradiography↗

PubMed1988

Degradation of structurally characterized proteins injected into HeLa cells. Effects of intracellular location and the involvement of lysosomes.

Thirty-five proteins of known x-ray structure were radioiodinated and injected into HeLa cells. The cells were then cultured in the presence or absence of the lysosomotropic agents, ammonium chloride and chloroquine. These compounds did not inhibit the degradation of an injected protein unless its half-life was greater than 45 h. Among the more stable proteins the extent of inhibition was proportional to their half-lives. These results indicate that all injected proteins are transferred to lysosomes at comparable rates such that the fraction of a specific protein degraded in lysosomes depends upon its rate of degradation in the cytosol. That is, basal autophagy is nonselective in HeLa cells. The intracellular location of each injected protein was measured by homogenization of injected cells in sucrose and differential sedimentation or by extraction in buffers containing Triton X-100. Solubilities of the injected proteins ranged from 6 to 89%, and stabilities of 10 proteins, originally extracellular in function, were inversely proportional to their solubility. These results illustrate the potential importance of subcellular location on protein stability in the cytosol.

Half-Life↗

PubMed1988

Degradation of structurally characterized proteins injected into HeLa cells. Tests of hypotheses.

We have compared sequence and structural features of 35 proteins to their metabolic stabilities in HeLa cells. No relationship was observed between the half-life of an injected protein and its subunit molecular weight, isoelectric point, hydrophobicity, thermostability, surface charge density, or N-terminal residue. Other properties, including susceptibility to oxidation, specific combinations of amino acids, secondary structure composition, and solvent exposed residues, also failed to correlate with protein stability. Although a weak inverse correlation was obtained when stability was compared to asparagine and glutamine content, we conclude that the degradation of an injected protein is unlikely to be related to any single structural parameter. Rather, we hypothesize that it results from an interplay between subcellular location and still poorly defined surface features of the injected proteins.

Amino Acid Sequence↗

PubMed1988

Extended reading frame of a ubiquitin gene encodes a stable, conserved, basic protein.

Antibodies specific for the 80-amino acid hypothetical protein encoded by the in-frame, 3'-extension of a human ubiquitin gene were produced in rabbits by immunization with a 14-residue synthetic peptide. When used to probe HeLa cell extracts for the non-ubiquitin product of this natural fusion gene, the antipeptide sera detected a protein with an apparent molecular weight of 16,000 Da by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. An immunoreactive protein of identical mobility was detected in organisms ranging from Acanthamoeba to man, indicating that the extension protein, like ubiquitin, is highly conserved. The immunoreactive protein was isolated from calf thymus, and direct sequencing revealed the first 16 amino acids to be identical to those predicted from the extension portion of the human cDNA. Thus, ubiquitin was no longer present at the amino terminus. The purified bovine extension protein failed to react with a ubiquitin-specific antibody indicating the absence of isopeptide-linked ubiquitin as well. Moreover, by denaturing gel permeation chromatography the extension has a molecular weight of 10,000 Da, a value that corresponds more closely to the size of the extension alone (9,000 Da) than to the intact fusion protein (17,500 Da). The extension protein, which was found in both cytoplasmic and nuclear fractions of HeLa cells, persisted at high levels when protein synthesis was blocked with cycloheximide or puromycin. These results show that the 80-residue extension protein is the stable, processed product of the ubiquitin fusion gene.

Amino Acid Sequence↗

PubMed1988

Regulation of enzyme levels by proteolysis: the role of pest regions.

Enzymes can be regulated in a variety of ways. Readily reversible mechanisms, such as phosphorylation, are frequently used by cells to control metabolic pathways. Less often, enzyme levels are regulated by changing the rate at which the protein is destroyed. Although these changes, too, are reversible through protein synthesis, large variations in enzyme concentration can be produced in very short periods of time by combinations of transcriptional control, translational control and rapid degradation. We recently examined the primary sequences of proteins whose intracellular half-lives are less than two hours. With a single exception, each short-lived protein contains one or more regions rich in proline (P), glutamic acid (E), serine (S) and threonine (T). These PEST regions range in length from 12 to 60 residues, and they are often flanked by possibly charged amino acids. Similar inspection of 35 more stable, structurally characterized proteins revealed only three weak PEST regions. All PEST proteins appear to be important regulatory molecules, and their fast turnover surely reflects a metabolic requirement for rapid changes in their concentrations. Known PEST proteins include oncogene products, key enzymes and components of signal pathways. In addition, there are a number of PEST-containing proteins that are suspected of being rapidly degraded. These proteins include Drosophila homeotic proteins (e.g., notch, snake, caudal, ftz and even-skipped) and a host of yeast cdc mutants. PEST regions, which target the molecules containing them for destruction, thus appear to be widely distributed among metabolically unstable proteins.

Amino Acid Sequence↗

PubMed1987

Purification of two high molecular weight proteases from rabbit reticulocyte lysate.

We have purified two high molecular weight proteases approximately 400-fold from rabbit reticulocyte lysate. Both enzymes hydrolyze 125I-alpha-casein and 4-methylcoumaryl-7-amide peptides with tyrosine, phenylalanine, or arginine at the P1 position. Both are inhibited by hemin, thiol reagents, chymostatin, and leupeptin. They differ, however, by other criteria. Degradation of 125I-lysozyme-ubiquitin conjugates and succinyl-Leu-Leu-Val-Tyr-4-methylcoumaryl-7-amide by the larger 26 S protease is stimulated by ATP. Based on sedimentation, gel filtration, and nondenaturing polyacrylamide gel electrophoresis, the ATP-dependent protease has a molecular weight of 1,000,000 +/- 100,000 and is a multisubunit complex. The smaller 20 S protease has a molecular weight of 700,000 +/- 20,000 and is composed of 8-10 separate subunits with Mr values between 21,000 and 32,000. It does not require nucleotides for degradation of protein or peptide substrates. This smaller enzyme is similar, if not identical, to the "multicatalytic proteinase complex" first described by Wilk and Orlowski (Wilk, S., and Orlowski, M. (1983) J. Neurochem. 40, 842-849).

Adenosine Triphosphate↗