Papers by Alfred Goldberg
Proceedings of the National Academy of Sciences, 1971
The rate of degradation of cell proteins in Escherichia coli was studied under various conditions... more The rate of degradation of cell proteins in Escherichia coli was studied under various conditions that affect levels of charged tRNA. Protein breakdown increased markedly when the synthesis of valyl-tRNA was prevented in strains containing temperature-sensitive valyl-tRNA synthetase or when the formation of N -formylmethionyl-tRNA was inhibited with trimethoprim. Conversely, protein breakdown decreased in a valine auxotroph-administered valine or an analog capable of attachment to the valyl-tRNA. It appears that changes in the levels of aminoacyl-tRNA regulate protein breakdown by mechanisms similar to those controlling the synthesis of ribosomal RNA. These experiments also demonstrate that protein synthesis is not essential for protein degradation and suggest that the inhibition of degradation in starving cells by chloramphenicol is a secondary effect of the accumulation of charged tRNA.
Journal of Biological Chemistry, 1978
A simple relationship has been derived that may be useful in determining the energy production in... more A simple relationship has been derived that may be useful in determining the energy production in the tricarboxylic acid cycle in most tissues or whole organisms during fasting or exercise. The ratio of the rate of acetyl group oxidation to the rate of O2 consumption is nearly constant (0.34), irrespective of the proportion of glucose and fatty acids oxidized in a tissue or an organism. This relationship is derived by multiplying the respiratory quotient by the ratio of acetyl group oxidation to total CO2 production.
Brain, 2021
Agents that raise cyclic guanosine monophosphate (cGMP) by activating protein kinase G increase 2... more Agents that raise cyclic guanosine monophosphate (cGMP) by activating protein kinase G increase 26S proteasome activities, protein ubiquitination and degradation of misfolded proteins. Therefore, they may be useful in treating neurodegenerative and other diseases caused by an accumulation of misfolded proteins. Mutations in myelin protein zero (MPZ) cause the peripheral neuropathy Charcot-Marie-Tooth type 1B (CMT1B). In peripheral nerves of a mouse model of CMT1B, where the mutant MPZS63del is expressed, proteasome activities are reduced, mutant MPZS63del and polyubiquitinated proteins accumulate and the unfolded protein response (p-eif2α) is induced. In HEK293 cells, raising cGMP stimulated ubiquitination and degradation of MPZS63del, but not of wild-type MPZ. Treating S63del mice with the phosphodiesterase 5 inhibitor, sildenafil—to raise cGMP—increased proteasome activity in sciatic nerves and reduced the levels of polyubiquitinated proteins, the proteasome reporter ubG76V-GFP an...
It is now clear that the marked loss of muscle mass that occurs with disuse, denervation or in ma... more It is now clear that the marked loss of muscle mass that occurs with disuse, denervation or in many systemic diseases (cancer cachexia, sepsis, acidosis, various endocrine disorders) is due primarily to accelerated degradation of muscle proteins, especially myofibrillar components. Recent work primarily in Dr. Goldberg's laboratory had suggested that in these diverse conditions, the enhancement of muscle proteolysis results mainly from activation of the Ub-proteasome degradative pathway. In various experimental models of atrophy, rat muscles show a common series of changes indicative of activation of this pathway, including increases in MRNA for Ub and proteasome subunits, content of ubiquitinated proteins, and sensitivity to inhibitors of the proteasome. In order to understand the muscle atrophy seen in weightlessness, Dr. Goldberg's laboratory is collaborating with Dr. Baldwin in studies to define the changes in these parameters upon hind-limb suspension. Related experimen...
Journal of Biological Chemistry, 1980
Guanosine-5'-diphosphate-3'-diphosphate (ppGpp) and the Regulation of Protein Breakdown in Escher... more Guanosine-5'-diphosphate-3'-diphosphate (ppGpp) and the Regulation of Protein Breakdown in Escherichia coZi*
Journal of Bacteriology, 1980
Glycerol:oxidized nicotinamide adenine dinucleotide (NAD+) 2-oxidoreductase (EC 1.1.1.6), an indu... more Glycerol:oxidized nicotinamide adenine dinucleotide (NAD+) 2-oxidoreductase (EC 1.1.1.6), an inducible enzyme for anaerobic glycerol catabolism in Klebsiella aerogenes, was purified and found to have a molecular weight of 79,000 by gel electrophoresis. The protein seemed to be enzymatically active either as a dimer of a 40,000-dalton peptide at pH 8.6 or as a tetramer of 160,000 molecular weight at pH 7.0. The enzyme activity was present at high levels in cells growing anaerobically on glycerol, but disappeared with a half-life of about 45 min if molecular oxygen was introduced to the culture. In contrast, no such phenomenon occurred with dihydroxyacetone kinase activity, the second enzyme in the pathway. Immunochemical analysis showed that the inactivation of the oxidoreductase did not involve degradation of the protein. Furthermore, subunits of the active and inactive forms of the enzyme were indistinguishable in size on polyacrylamide gel electrophoresis in the presence of sodium...
Methods in Molecular Biology, 2018
Rapid, gentle, isolation of 26S proteasomes from cells or tissues is an essential step for studie... more Rapid, gentle, isolation of 26S proteasomes from cells or tissues is an essential step for studies of the changes in proteasome activity and composition that can occur under different physiological or pathological conditions and in response to pharmacological agents. We present here three different approaches to affinity purify or to prepare proteasome-rich cell fractions. 1) The first method uses affinity tags fused to proteasome subunits and has been useful in several cell lines (see Table 1) for studies of proteasome structure by cryo-electron microscopy and composition by mass spectrometry. 2) A second method uses the proteasome's affinity for a ubiquitin-like (UBL) domain and can be used to purify these particles from any cell or tissue. This method does not require expression of a tagged subunit and has proven very useful to investigate how proteasomes activity changes in different physiological states (e.g. fasting or aging), with neurodegenerative diseases, and with drugs or hormones that cause subunit phosphorylation. 3) A third, simple method that is based on the 26S proteasome's high molecular weight (about 2.5 MDa), concentrates these particles greatly by differential centrifugation. This method maintains the association of proteasomes with ubiquitin (Ub) conjugates and many other loosely-associated regulatory proteins and is useful to study changes in proteasome composition under different conditions (see Table 2). 7. To study how proteasome composition changes during in vitro incubations and the effects on Ub conjugate binding, protein-bound affinity gels were washed 3 times with 150 μL wash buffer. Spin at 300 × g for 1 min. The bound FLAG-tagged proteasomes were incubated at either 4° or 37 °C for 20 min [Note 8] before the final wash and elution.
Stress-Inducible Cellular Responses, 1996
In all cells and organelles, there exist multiple molecular chaperones, which not only can facili... more In all cells and organelles, there exist multiple molecular chaperones, which not only can facilitate the proper folding, transport and assembly of multimeric structures, but also appear to function in intracellular protein degradation. Recent findings in E. coli indicate that the major chaperones of the Hsp70 (DnaK) and Hsp60 (GroEL) families and their cofactors (DnaJ, GrpE or GroEL and Trigger Factor) associate with certain short-lived proteins (e.g. mutant polypeptides or regulatory proteins) and promote their degradation by the ATP-dependent proteases, La (lon or ClpP). Moreover, ATPases of ClpA/B family not only function in ATP-dependent proteolysis in association with the Clp protease, but by themselves can facilitate or act as chaperones in protein assembly. In eukaryotes, Hsp70 and their cofactors, the DnaJ homologs, are essential for the ubiquitination of certain abnormal and regulatory proteins and in the breakdown of certain polyubiquitinated polypeptides by 26S proteasome. It is likely that the chaperones function in proteolysis either as elements that faciliate the recognition of unfolded proteins or that the chaperones partially unfold substrates to make them more susceptible to proteases or ubiquitinating enzymes.
Methods in Molecular Biology, 2018
Rates of degradation by the ubiquitin proteasome system depend not only on rates of ubiquitinatio... more Rates of degradation by the ubiquitin proteasome system depend not only on rates of ubiquitination but also on the level of proteasome activity, which can be regulated through phosphorylation of proteasome subunits. Many protein kinases have been proposed to influence proteasomal activity. However, for only two is there strong evidence that phosphorylation of a specific 26S subunit enhances the proteasome's capacity to degrade ubiquitinated proteins and promotes protein breakdown in cells: 1) Protein Kinase A (PKA), which after a rise in cAMP phosphorylates the 19S subunit Rpn6, and 2) Dual Tyrosine Receptor Kinase 2 (DYRK2), which during S through M phases of the cell cycle phosphorylates the 19S ATPase subunit Rpt3. These findings were made possible by the development of methods to rapidly purify 26S proteasomes and to assay their multiple activities. The use of pulse-chase isotopic methods showed that PKA activation in cells enhances the degradation of short-lived cell proteins and misfolded proteins that cause neurodegenerative diseases, while DYRK2 enhances the breakdown of long-lived proteins and promotes progression through the cell cycle. The methods discussed here should be useful in clarifying the roles of other kinases and other post-translational modifications of proteasome subunits.
Ciba Foundation Symposium 75 - Protein Degradation in Health and Disease, 2008
Experiments with metabolic inhibitors in vivo indicate that intracellular protein degradation req... more Experiments with metabolic inhibitors in vivo indicate that intracellular protein degradation requires the continuous production of ATP. We have established soluble cell-free preparations from rabbit reticulocytes, rat liver, and Escherichia coli that degrade abnormal protein in an ATP-dependent fashion. These enzymes appear to be responsible for the selective breakdown of abnormal protein that may result from mutations, biosynthetic errors or intracellular denaturation. Experiments with inhibitors indicate that this process and the degradation of many short-lived normal proteins does not occur in the lysosome. The cell-free extracts prepared from these crude extracts hydrolyse [14C] globin by a process stimulated 2--3-fold by ATP and to a lesser extent by GTP, CTP or UTP. These activities degrade globin to large peptides which are then cleaved by soluble peptidases. The ATP-stimulated protease that partially purified from rat liver cytoplasm is also stimulated by pyrophosphate. This protease has an apparent molecular weight of 480,000. In contrast, the E. coli enzyme has an apparent molecular weight of 115,000 and is completely dependent on ATP, after partial purification by ion exchange and gel chromatography. This enzyme can be distinguished from six other proteolytic enzymes from E. coli active at pH 7.8. E. coli contains, in addition, four proteases that are not stimulated by ATP and degrade globin to acid-soluble material. We have also demonstrated in E. coli and reticulocytes other proteases that appear specific for small protein substrates and may play a role in the later steps in protein breakdown. The ATP-stimulated endoproteases appear to catalyse the rate-limiting steps in intracellular protein breakdown. However, the actual role of ATP in the degradative process is not known.
Journal of Biological Chemistry, 1987
A crucial enzyme in the pathway for protein degradation in Eecherichia coli is protease La, an AT... more A crucial enzyme in the pathway for protein degradation in Eecherichia coli is protease La, an ATPhydrolyzing protease encoded by the lon gene. This enzyme degrades various proteins to small polypeptides containing 10-20 amino acid residues. To learn more about its energy requirement, we determined the number of ATP molecules hydrolyzed by the purified protease for each peptide bond cleaved. The enzyme hydrolyzed about 2 molecules of ATP for each new amino group generated with casein, bovine serum albumin, glucagon, or guanidinated casein as substrates, even though these proteins differ up to 20-fold in size and 3-4-fold in rates of hydrolysis of peptide bonds. Similar values for the stoichiometry (from 1.9 to 2.4) were obtained using fluorescamine or 2,4,6-trinitrobenzene sulfonic acid to estimate the appearance of new amino groups. These values appeared lower at 1 m M than at 10 m M M e +. The coupling between ATP and peptide bond hydrolysis appeared very tight. However, when the protease was assayed under suboptimal conditions (e& at lower pH or with ADP present), many more ATP molecules (from 3.5 to 12) were consumed per peptide bond cleaved. Our data would indicate that the early steps in protein degradation consume almost as much energy (2 ATPs for each cleavage) as does the formation of peptide bonds during protein synthesis. * These studies have been supported by research grants from the National Institute of Neurological and Communicative Disorders and Stroke and from Biogen Research Corporation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Journal of Biological Chemistry, 1987
The interaction of protein substrates with protease La from Escherichia coli enhances its ability... more The interaction of protein substrates with protease La from Escherichia coli enhances its ability to hydrolyze ATP and peptide bonds. These studies were undertaken to clarify how unfolded proteins allosterically stimulate this ATPase activity. The tetrameric protease can bind four molecules of ATP, which activates proteolysis, or four molecules of ADP, which inhibits enzymatic activity. Protein substrates stimulate binding of the nonhydrolyzable ATP analog [3H] adenyl-5'yl imidodiphosphate, although they do not increase the net binding of [3H]ATP or [3H]ADP. Once bound, ATP is quickly hydrolyzed to ADP, which remains noncovalently associated with protease La even through repeated gel filtrations. Exposure to protein substrates (e.g. denatured bovine serum albumin at 37 "C) induces the release of all the bound ADP from the enzyme. Nonhydrolyzable ATP analogs bound to the enzyme were not released by these substrates. Proteins that are not degraded (e.g. native bovine serum albumin) and oligopeptides that only bind to the catalytic site do not induce ADP release. Thus, polypeptide substrates have to interact with an allosteric site to induce this effect. The protein-induced ADP release is inhibited by high concentrations of Mg2+ and is highly temperature-dependent. Protein substrates promoted [3H]ATP binding in the presence of ADP and Mg2+ (i.e. ATP-ADP exchange) and reduced the ability of ADP to inhibit the enzyme's peptidase and ATPase activities. These results indicate that: 1) ADP release is a rate-limiting step in protease La function; 2) bound ADP molecules inhibit protein and ATP hydrolysis in vivo; 3) denatured proteins interact with the enzyme's regulatory site and promote ADP release, ATP binding, and their own hydrolysis.
Journal of Biological Chemistry, 1979
... coli* (Received for publication, November 14, 1977, and in revised form, February 20, 1979)Ko... more ... coli* (Received for publication, November 14, 1977, and in revised form, February 20, 1979)Koko Murakami,$ Richard Voellmy,§ and Alfred L. Goldberg1 From the Department of Physiology, Harvard Medical School, Boston, Massachusetts 02115 ...
Journal of Biological Chemistry, 1992
The clpB gene in Escherichia coli encodes a heatshock protein that is a close homolog of the clpA... more The clpB gene in Escherichia coli encodes a heatshock protein that is a close homolog of the clpA gene product. The latter is the ATPase subunit of the multimeric ATP-dependent protease Ti (Clp) in E. coli, which also contains the 21-kDa proteolytic subunit (ClPP). The clpB gene product has been purified to near homogeneity by DEAE-Sepharose and heparin-agarose column chromatographies. The purified ClpB consists of a major 93-kDa protein and a minor 79-kDa polypeptide as analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Upon gel filtration on a Superose-6 column, it behaves as a 350-kDa protein. Thus, ClpB appears to be a tetrameric complex of the 93-kDa subunit. The purified ClpB has ATPase activity which is stimulated S-10-fold by casein. It is also activated by insulin, but not by other proteins, including globin and denatured bovine serum albumin. ClpB cleaves adenosine 5'-(cr,&methylene)-triphosphate as rapidly as ATP, but not adenosine 5'-@,y-methylene)-triphosphate. GTP, CTP, and UTP are hydrolyzed 15-25% as well as ATP. ADP strongly inhibits ATP hydrolysis with a Ki of 34 PM. ClpB has a K , for ATP of 1.1 mM, and casein increases its V,,, for ATP without affecting its K,. A Mg2+ concentration of 3 mM is necessary for half-maximal ATP hydrolysis. Mn2+ supports ATPase activity as well as Mg2+, and Ca2+ has about 20% their activity. Anti-ClpB antiserum does not cross-react with ClpA nor does anti-ClpA antiserum react with ClpB. In addition, ClpB cannot replace ClpA in supporting the casein-degrading activity of ClpP. Thus, ClpB is distinct from ClpA in its structural and biochemical properties despite the similarities in their sequences. Escherichia coli contain at least two distinct endoproteases, proteases La and Ti, that require ATP and Mg2+ for activity. The first ATP-requiring protease discovered was protease La, the lon gene product (1, 2). This enzyme is a heat-shock protein (3, 4) and plays an essential role in the degradation of most abnormal proteins (1-6) and certain short-lived regulatory proteins (6). It is composed of four identical subunits, each (87 kDa) of which contains a site for ATP hydrolysis (7-9). One important feature of this enzyme is that protein
Journal of Biological Chemistry, 1977
Journal of Biological Chemistry, 1978
Journal of Biological Chemistry, 1985
To characterize the system(s) responsible for degradation of short-lived and long-lived proteins ... more To characterize the system(s) responsible for degradation of short-lived and long-lived proteins in mammalian cells, we compared the concentrations of ATP required for the degradation of these classes of proteins in growing hamster fibroblasts. By treating CHEF-18 cells with increasing concentrations of dinitrophenol and 2-deoxyglucose, it was possible to reduce their steady-state ATP content by different amounts (up to 98%). These treatments caused a rapid decrease in the degradation of both short- and long-lived proteins. Removal of the inhibitors led to a prompt restoration of ATP and proteolysis. As ATP content fell below normal levels (about 3.1 mM), rates of proteolysis decreased in a graded biphasic fashion. Reduction in ATP by up to 90% (as may occur in anoxia or injury) decreased proteolysis up to 50%; and with further loss of ATP, protein breakdown fell more sharply. Degradation of both classes of proteins was inhibited by 80% when ATP levels were reduced by 98%. The levels of ATP required for the breakdown of short- and long-lived proteins were indistinguishable. Protein synthesis was much more sensitive to a decrease in ATP content than protein breakdown and fell by 50% when ATP was reduced by only 15%. Chloroquine, an inhibitor of lysosome function, did not reduce the degradation of either class of proteins in growing cells, but it did inhibit the enhanced degradation of long-lived proteins upon removal of serum (in accord with previous studies). Thus, in growing fibroblasts, an ATP-dependent nonlysosomal process appears responsible for the hydrolysis of both short- and long-lived proteins.
Journal of Biological Chemistry, 1982
Treatment of isolated rat skeletal muscles with the Ca2+ ionophores, A23181 or ionomycin, increas... more Treatment of isolated rat skeletal muscles with the Ca2+ ionophores, A23181 or ionomycin, increased overall protein degradation 45-140%. Removal of extracellular Ca2+ reduced overall proteolysis and most of the stimulation by A23187. Treatment of the muscles with the sulfhydryl inhibitor, mersalyl, completely inactivated the Ca2'-activated protease without altering overall protein breakdown or the stimulation by A23187. This agent did not inhibit the lysosomal protease, cathepsin B, in the muscle; however, leupeptin and Ep-475, which inhibit this enzyme in intact cells, decreased the stimulation of proteolysis by Ca2+. Thus, this effect does not require the Ca2+-activated enzyme, but seems to involve lysosomal proteases, Prostaglandin E, (PGE2) and its precursor, arachidonic acid, were previously shown to stimulate protein degradation in rat muscle through an effect on lysosomal function. We tested whether the enhancement of muscle proteolysis by CaZ+ ionophores may result from increased synthesis o f PGE2. A23187 increased release of PGEz and PGF2, by the muscles 3-4-fold. High extracellular potassium also markedly promotes muscle proteolysis, apparently by increasing intracellular Ca2+, and this treatment also stimulates prostaglandin production. Indomethacin and aspirin, which inhibit the cyclooxygenase, and mepacrine, which inhibits the Ca"-activated phospholipase A,, markedly reduced the increase in prostaglandin production. These agents also reduced the enhancement of protein degradation by Ca2+ or high K+. Thus, Ca2+ appears to promote protein breakdown by stimulating synthesis of PGE2, which in turn activates the lysosomal apparatus.
Journal of Biological Chemistry, 1994
The major proteins in the lumen of the endoplasmic reticulum (ER) are thought to function in Ca2+... more The major proteins in the lumen of the endoplasmic reticulum (ER) are thought to function in Ca2+ sequestration or as "molecular chaperones" in the folding and assembly of membrane or secreted proteins. Based on the ability of many chaperones to bind selectively to unfolded proteins and to dissociate from them upon ATP hydrolysis, we developed an affinity chromatography method to isolate proteins with these characteristics from pancreatic or liver ER. Seven ER proteins bound selectively to denatured protein columns and were specifically eluted by ATP (lo-' M) but not by a nonhydrolyzable ATP analog. These proteins were identified with antibodies and microsequencing as the ER chaperone BiP (grp78), grp94, calreticulin, a novel 46-kDa protein that binds azido-ATP, as well as three members of the thioredoxin superfamily: protein-disulfide isomerase, ERp72, and a previously reported SO-kDa protein (pS0). This set of seven proteins bound to and was eluted with ATP from a variety of denatured proteins, including histone, gelatin, a fetoprotein, thyroglobulin, lysozyme, casein, and IgG. The release of grp94, protein-disulfide isomerase, ERp72, calreticulin, and pS0 was stimulated by Ca2+ in the presence of ATP. These proteins thus appear to function as Ca'*-dependent chaperones, which may account for the Caz+ and ATP requirement for protein folding in the ER. The endoplasmic reticulum (ER)' is a major site of Ca2+ storage within the cell, and several ER lumenal Ca2+-binding proteins, including calreticulin, ERp72, and grp94, are believed to play important roles in this process (1-3). The ER is also the initial site in the secretory pathway where membrane and secretory proteins are folded and assembled. This
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Papers by Alfred Goldberg