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M Rechsteiner

Publications and source records attributed to M Rechsteiner.

At least 55 records · Page 3Linked to original sources
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↗
PubMed1987

Do myc, fos and E1A function as protein phosphatase inhibitors?

The oncogenic proteins myc, fos and E1A bear striking resemblance to protein phosphatase inhibitors 1 and 2. Both sets of proteins possess several regions rich in proline (P), glutamic acid (E), serine (S) and threonine (T). In addition to PEST sequences four of the five proteins contain clusters of arginine-arginine pairs. On the basis of these similarities, I suggest that myc, fos and E1A are protein phosphatase inhibitors.

Adenovirus Early Proteins↗
PubMed1987

Microinjection of ubiquitin: intracellular distribution and metabolism in HeLa cells maintained under normal physiological conditions.

Radioiodinated ubiquitin was introduced into HeLa cells by erythrocyte-mediated microinjection. Subsequent electrophoretic analyses revealed that the injected ubiquitin molecules were rapidly conjugated to HeLa proteins. At equilibrium, 10% of the injected ubiquitin was conjugated to histones and 40% was distributed among conjugates of higher molecular weight. Although the remaining ubiquitin molecules appeared to be unconjugated, the free pool of ubiquitin decreased by one-third and additional conjugates were present when electrophoresis was performed at low temperature under nonreducing conditions. Molecular weights of these labile conjugates suggest that they are ubiquitin adducts in thiolester linkage to activating enzymes. Despite the fairly rapid degradation of injected ubiquitin (t1/2 approximately 10-20 h), the size distribution of ubiquitin conjugates within interphase HeLa cells remained constant for at least 24 h after injection. The intracellular locations of ubiquitin and ubiquitin conjugates were determined by autoradiography, by differential sedimentation of subcellular fractions in sucrose, and by extraction of injected cells with buffer containing Triton X-100. Free ubiquitin was found mostly in the cytosolic or Triton X-100-soluble fractions. As expected, histone conjugates were located predominately in the nuclear fraction and exclusively in the Triton X-100-insoluble fraction. Although high molecular weight conjugates were enriched in the Triton X-100-insoluble fraction, their size distribution was similar to that of soluble conjugates. When injected HeLa cells were exposed to cycloheximide to inhibit protein synthesis, the size distribution of ubiquitin conjugates was similar to that found in untreated cells. Moreover, high molecular weight conjugates decreased less than 20% after inhibition of protein synthesis. These results indicate that most ubiquitin conjugates are not newly synthesized proteins which have been marked for destruction.

Erythrocytes↗
PubMed1987

Microinjection of ubiquitin: changes in protein degradation in HeLa cells subjected to heat-shock.

Ubiquitin was radiolabeled by reaction with 125I-Bolton-Hunter reagent and introduced into HeLa cells using erythrocyte-mediated microinjection. The injected cells were then incubated at 45 degrees C for 5 min (reversible heat-shock) or for 30 min (lethal heat-shock). After either treatment, there were dramatic changes in the levels of ubiquitin conjugates. Under normal culture conditions, approximately 10% of the injected ubiquitin is linked to histones, 40% is found in conjugates with molecular weights greater than 25,000, and the rest is unconjugated. After heat-shock, the free ubiquitin pool and the level of histone-ubiquitin conjugates decreased rapidly, and high molecular weight conjugates predominated. Formation of large conjugates did not require protein synthesis; when analyzed by two-dimensional electrophoresis, the major conjugates did not co-migrate with heat-shock proteins before or after thermal stress. Concomitant with the loss of free ubiquitin, the degradation of endogenous proteins, injected hemoglobin, BSA, and ubiquitin was reduced in heat-shocked HeLa cells. After reversible heat-shock, the decrease in proteolysis was small, and both the rate of proteolysis and the size of the free ubiquitin pool returned to control levels upon incubation at 37 degrees C. In contrast, neither proteolysis nor free ubiquitin pools returned to control levels after lethal heat-shock. However, lethally heat-shocked cells degraded denatured hemoglobin more rapidly than native hemoglobin and ubiquitin-globin conjugates formed within them. Therefore, stabilization of proteins after heat-shock cannot be due to the loss of ubiquitin conjugation or inability to degrade proteins that form conjugates with ubiquitin.

Autoradiography↗
PubMed1987

Ubiquitin-mediated pathways for intracellular proteolysis.

Ubiquitination is one of several ways in which cells modify their proteins. As for phosphorylation or acetylation, there are distinct enzymes for adding and removing Ub from the surfaces of protein substrates. The dynamic equilibration of Ub with cellular proteins is also typical of most posttranslational modifications. Ubiquitination differs, however, in that the added group is large compared to acetate or phosphate. Its size must provide great potential for recognition by other cellular proteins. Ub may be the cell's reversible cross-linking reagent, covalently bound to protein substrates at one end and noncovalently associated with various Ub binding proteins at the other. It is likely that one ubiquitin binding protein is a component of the 26S ATP-dependent protease. The presence of Ub on histones and on the lymphocyte homing receptor suggests that ubiquitination does not serve exclusively to mark proteins for degradation. There are probably various ubiquitin binding proteins since Ub appears to be a multifunctional protein that affects chromatin structure, intracellular proteolysis, cellular interactions, and the stress response. This abundant protein may serve as an intracellular barometer whose distribution among several pools regulates a variety of processes.

Animals↗
PubMed1986

Degradation of proteins microinjected into HeLa cells. The role of substrate flexibility.

Increasing the flexibility of a protein enhances its susceptibility to defined proteases in vitro. To ascertain whether flexibility also affects protein stability in vivo, radioiodinated proteins with similar structures, but dissimilar flexibilities, were introduced into HeLa cells using red cell-mediated microinjection. Intracellular proteolysis was then measured as the rate of release of 125I-tyrosine into the medium. Ribonuclease A was considerably more resistant to degradation by purified proteases or in reticulocyte lysate than its flexible derivatives ribonuclease S and S-protein. In contrast, all three proteins were equally stable within HeLa cells. Like the results obtained for RNases, the rates of degradation of trypsin inhibitors, trypsin analogs, and their complexes correlated with flexibility in reticulocyte lysate. However, the intracellular half-lives of anhydrotrypsin and various proteinaceous trypsin inhibitors were not affected upon formation of enzyme-inhibitor complexes. Furthermore, trypsinogen was degraded more slowly than the structurally similar anhydrotrypsin in HeLa cells, although trypsinogen has additional segmental flexibility in its activation domain. Electrophoretic analyses revealed that trypsin-inhibitor complexes remained intact following injection into HeLa cells, and that neither free inhibitors nor anhydrotrypsin formed Triton-stable complexes with soluble cytoplasmic proteins. The observation that the components of the trypsin-inhibitor complexes were degraded simultaneously indicates that neither constituent unfolded prior to the onset of proteolysis. These studies provide evidence that RNases, trypsin, and trypsin inhibitors are degraded by an intracellular proteolytic pathway(s) which recognizes surface features of the folded proteins.

Glycoproteins↗