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.
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.
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...
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...
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.
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.
... 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 ...
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.
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.
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.
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...
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...
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.
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.
... 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 ...
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.
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Papers by Alfred Goldberg