A.L. GOLDBERG
Department of Cellular and Molecular Physiology
Harvard Medical School
Boston, Massachusetts 02115, USA
Protease La
The Heat-Shock Response and Protein Breakdown
Other ATP-Hydrolyzing Proteases
The Proteolytic Complexes in Mammalian Cells
Prospects
Selected Reading
Intracellular proteins are continually being synthesized and degraded back to amino acids. One important function of intracellular proteolysis is to eliminate from the cell polypeptides with highly abnormal conformations as may result from mutations, biosynthetic errors and postsynthetic damage. In recent years much progress has been made in our understanding of how such abnormal proteins are recognized and selectively eliminated. Such studies have led to the discovery of new proteolytic systems with unanticipated properties, which are reviewed in this article.
One important feature of protein degradation in all cells and even within organelles, such as mitochondria or chloroplasts, is that this process requires metabolic energy. On thermodynamic grounds, peptide-bond hydrolysis should be a spontaneous process and not require ATP. Our laboratory has therefore focused on understanding this ATP requirement because it represented an important clue to discovering the responsible degradative system, and because it suggested the existence of novel biochemical mechanisms. This research led to the discovery of a new soluble proteolytic pathway in mammalian and bacterial cells, as well as in mitochondria, which requires ATP hydrolysis for function. Our knowledge of this pathway is most advanced in Escherichia coli, which were first shown to contain a new type of enzyme, an ATP-dependent protease. The best studied such enzyme is protease La from E. coli; however, as discussed below, similar enzymes appear to be widespread. For example, a similar ATP-dependent protease has been demonstrated in the matrix of mammalian mitochondria, and an ATP-requiring protease complex that acts independently of ubiquitin has recently been demonstrated in the cytosol of mammalian cells. Also, in the cytosol of eukaryotic cells, there exists a very large protease complex (1.5 kDa) that specifically degrades proteins conjugated to ubiquitin; it also requires ATP hydrolysis to function.
In E. coli the energy requirement for protein breakdown results in large part from the involvement of the ATP-dependent protease La. This enzyme is encoded by the lon gene and has been shown to catalyze initial steps in the selective breakdown of proteins with highly abnormal conformations and of certain normal proteins that are also rapidly degraded. Protease La is an ATPase as well as a protease, and these two functions are tightly coupled. In fact, for every peptide bond hydrolyzed in proteins, the enzyme consumes two ATP molecules. In other words, protease La consumes almost as much energy in cleaving peptide bonds as the cell uses to form them.
One important property of protease La is its precise regulation and its activation by protein substrates. The binding of a protein substrate to the protease leads to a marked activation of the enzyme's capacity to degrade peptide bonds, as shown with exogenous model peptides. This effect is seen only with protein substrates, such as denatured proteins, which interact with a regulatory region outside the active site and thereby cause allosteric activation. Native proteins are not hydrolyzed and do not activate the protease. This mechanism probably helps ensure that the protease does not exist in an active form in the cytosol, causing cell damage, but it becomes active only when it binds to a potential substrate (e.g., an unfolded polypeptide).
Substrates also influence the activity of this enzyme through a novel ATP-ADP exchange mechanism. The tetrameric enzyme binds up to four molecules of either ATP or ADP, but it has an even higher affinity for ADP, which is a potent inhibitor of proteolysis. Protein substrates, such as denatured albumin, both stimulate the binding of ATP analogs to the protease and also induce the release of the ADP molecules bound to the enzyme. By contrast, native proteins that are not degraded do not have these effects. Thus, in vivo, ADP molecules are normally bound to the protease and inhibit its function, until a potential substrate interacts with protease La. This step causes release of the bound ADP, promotes ATP binding and enhances proteolytic activity. These unusual properties presumably have evolved to prevent inappropriate or excessive degradation of normal cell constituents.
In the past year, the complete sequence of the lon gene, which encodes protease La, has been determined by the dideoxy method. The enzyme contains consensus sequences found in several other ATP-binding proteins. However, no two sequences homologous to the catalytic sites of any other proteases were found in this enzyme. Thus, protease La seems to represent the first of a new class of proteolytic enzymes.
The cellular content of protease La is also carefully regulated at the transcriptional level. Our studies, and those of Neidhardt, indicated that the lon gene is a heat-shock gene, i.e., one of the cellular genes that is induced at high temperatures and a variety of other stressful environments. We have shown induction of protease La in other conditions where cells generate large amounts of abnormal proteins, e.g., after incorporation of amino acid analogs or after expression of cloned foreign proteins in the bacteria. Under such conditions, the increased production of protease La seems to help prevent the accumulation of the abnormal proteins by enhancing the cell's capacity to degrade such polypeptides. Accordingly, the rates of protein breakdown and the level of protease La are reduced in E. coli mutants (htpR strains), which have a defect in the expression of heat-shock genes. Also, when wild-type cells are incubated at high temperatures (42 °C) or when they synthesize large amounts of incomplete or missense proteins at low temperatures, the cellular content of protease La increases 2 to 4 fold. Concomitantly, there is an induction of the other heat-shock genes in the bacteria. The promoter region of the lon gene contains a sequence homologous to that in promoter regions of other heat-shock proteins. In eukaryotic cells the generation of large amounts of abnormal proteins also appears to initiate the heat-shock response.
For example, microinjection of denatured proteins into frog oocytes elicits expression of heat-shock genes. Furthermore, ubiquitin, a critical component of the ATP-dependent degradative system in eukaryotic cells, is a major heat-shock protein.
In addition to protease La, E. coli has been found to contain another ATP-Mg-dependent endoprotease named protease Ti. This enzyme (Mr = 340,000) is composed of two components, both of which are required for proteolysis, but have distinct functions. One component, A (subunit Mr = 80,000), is a labile ATPase that is stabilized by ATP. The other component, P (subunit Mr = 20,000), is a heat-shock polypeptide containing a latent proteolytic site that can be labelled with diisopropyl-3 fluorophosphate. These subunits show no proteolytic activity unless they are reconstituted. The ATPase activity of the reconstituted enzyme is activated 2 to 4 fold by protein substrates. Thus, protease Ti shares many unusual properties with protease La, such as coupled ATP and protein hydrolysis and protein-activated ATPase. However, these functions in Ti are associated with distinct subunits that modify each other's activities. Since these findings are very recent ones, it is still unknown whether similar enzymes exist in other cell types.
A very similar enzyme to protease La has been found in the mitochondria from rat liver. The mitochondrial matrix contains an ATP-dependent pathway capable of completely hydrolyzing abnormal organelle proteins to amino acids. Furthermore, the mitochondrial ATP-hydrolyzing endoprotease functions independently of ubiquitin, like protease La. This enzyme is very large (550 kDa) and degrades proteins in an ATP-dependent fashion, rather than resembling the main proteolytic system in the eukaryotic cytosol. The mitochondrial enzyme even has a similar specificity for peptides as does protease La, and its ATPase activity is activated by protein substrates.
Eukaryotic cells contain a 650 kDa (19S) multifunctional complex with multiple endoproteolytic activities. This structure is composed of 9-12 subunits of 23-34 kDa and hydrolyzes basic, hydrophobic and acidic peptide substrates as well as proteins. It has been called by many names (e.g., the multicatalytic protease, macropain, LAMP), but we recently suggested the name "proteasome" to reflect its proteolytic activity and its identity with the cylindrical particle known as the "prosome" and present in the eukaryotic nucleus and cytosol. Prior studies had noted a small activation of this enzyme by ATP. Recently, using rapid chromatographic procedures and glycerol as a stabilizing agent, we isolated from skeletal muscle and liver a form of this enzyme that displays a large ATP stimulation. Hydrolysis of peptide substrates was stimulated up to 9 fold by ATP and of casein 4 to 6 fold. Neither ADP nor AMP had any effect, nor do nonhydrolyzable ATP analogs. This effect of ATP was very labile, and was lost with storage. The ATP-stimulated and independent "proteasomes" closely resemble each other (e.g., in apparent molecular weight, subunit composition and immunological reactivity). This enzyme does not require ubiquitin for activity, and thus it may be responsible for the ATP-stimulated hydrolysis of proteins that cannot be conjugated to ubiquitin (e.g., ones that lack free amino groups). Although its precise role in intracellular protein breakdown is still uncertain, this enzyme is clearly the major proteolytic activity in the cytosol of mammalian cells at neutral pH.
The elegant studies of A. Hershko, A. Ciechanover and co-workers have shown that the ATP-dependent pathway in mammalian cells requires the heat-stable polypeptide, ubiquitin. In this process, protein substrates undergo conjugation to ubiquitin by a multistep process requiring ATP. This modification marks them for rapid degradation. Our laboratory and that of Rechsteiner have identified a very large (1.5 kDa, 26S) enzyme complex within the cytosol that specifically hydrolyzes these conjugated proteins. The ubiquitin-conjugate-degrading enzyme (UCDEN) and the proteasome differ in many respects, including size, subunit composition and requirement for ATP. However, these enzymes seem to co-purify and to function in concert during ubiquitin-dependent proteolysis, and we have found that monoclonal antibodies against the proteasome block the ubiquitin-dependent pathway. Although the structural relationships between these two large enzyme complexes remain to be elucidated, it seems most likely that the proteasome functions as a subunit of the larger (26S) conjugate-degrading complex.
Appreciable progress has been made in our understanding of protein breakdown in bacteria and mammalian cells, but many important questions are still unresolved. Greater knowledge about protein turnover and the heat-shock response in parasitic organisms is highly desirable. Many similarities, and important differences, may be discovered through such studies of protozoa. For example, it has been shown that the gene for ubiquitin in trypanosomes is induced by heat shock, but it is organized in a very different fashion from that in mammals, since it contains up to 50 transcripts of ubiquitin in linear array. The significance of this interesting difference is unclear, probably because virtually nothing is known about protein breakdown, the role of ubiquitin and the heat-shock response in these microorganisms.
ATP-Dependent Proteases in Bacteria and Mitochondria
CHIN, D.T., S.A. GOFF, T. WEBSTER, T. SMITH and A.L. GOLDBERG. 1988. Sequence of the lon gene in Escherichia coli: a heat-shock gene which encodes the ATP-dependent protease La. J. Biol. Chem. 263: 1718-1728.
DESAUTELS, M. and A.L. GOLDBERG. 1982. Demonstration of an ATP-dependent vanadate sensitive endoprotease from rat liver mitochondria. J. Biol. Chem. 257: 11673-11679.
GOLDBERG, A.L., A.S. MENON, S. GOFF and D.T. CHIN. 1987. The mechanism and regulation of the ATP-dependent protease La from Escherichia coli. Biochem. Soc. Trans. 15: 809-811.
HWANG, B.J., W.J. PARK, C.H. CHUNG and A.L. GOLDBERG. 1987. Escherichia coli contains a soluble ATP-dependent protease (Ti) distinct from protease La. Proc. Natl. Acad. Sci. USA 84: 5550-5554.
KATAYAMA-FUJIMURA, Y., S. GOTTESMAN and M.R. MAURIZI. 1987. A multiple-component, ATP-dependent protease from Escherichia coli. J. Biol. Chem. 262: 4477-4485.
MENON, A.S. and A.L. GOLDBERG. 1987. Protein substrates activate the ATP-dependent protease La by promoting nucleotide binding and release of bound ADP. J. Biol. Chem. 262: 1492914934.
MENON, A.S., L. WAXMAN and A.L. GOLDBERG. 1987. The energy utilized in protein breakdown by the ATP-dependent protease (La) from Escherichia coli. J. Biol. Chem. 262: 722726.
Proteasome
APRIGO, A-P.; K. TANAKA, A.L. GOLDBERG and W.J. WELCH. 1988. Identity of the 19S 'prosome' particle with the large multifunctional protease complex of mammalian cells (the proteasome). Nature 331: 192-194.
DRISCOLL, J. and A.L. GOLDBERG. In press. The mammalian proteasome can degrade proteins in an ATP-dependent process that is independent of ubiquitin. Proc. Natl. Acad. Sci. USA.
MATTHEWS, M., J. DRISCOLL and A.L. GOLDBERG. In press. Involvement of the proteasome in different degradative processes in mammalian cells. Proc. Natl. Acad. Sci. USA.
Ubiquitin and the Ubiquitin-Conjugate-Degrading Enzyme
FAGAN, J.M., L. WAXMAN and A.L. GOLDBERG. 1987. Skeletal muscle and liver contain a soluble ATP + ubiquitin-dependent proteolytic system. Biochem. J. 243: 335-343.
HERSKHO, A. and A. CIECHANOVER. 1982. A mechanism of intracellular protein breakdown. Ann. Rev. Biochem. 51: 335-364.
HOUGH, R., G. PRATT and M. RECHSTEINER. 1986. Ubiquitin lysozyme conjugates: identification and characterisation of an ATP-dependent protease from rabbit reticulocytes. J. Biol. Chem. 261: 2400-2408.
WAXMAN, L., J.M. FAGAN and A.L. GOLDBERG. 1987. Demonstration of two distinct high molecular weight proteases in rabbit reticulocytes: one of which degrades ubiquitin conjugates. J. Biol. Chem. 262: 2451-2457.
Heat-Shock and Proteolysis
ANANTHAN, J., A.L. GOLDBERG and R. VOELLMY. 1986. Abnormal proteins serve as eukaryotic stress signals and trigger the activation of heat shock genes. Science 232: 522-524.
GOFF, S.A. and A.L. GOLDBERG. 1985. Production of abnormal proteins in E. coli stimulates transcription of lon and other heat-shock genes. Cell 41: 587-595.
GOFF, S.A., R. VOELLMY and A.L. GOLDBERG. 1988. Protein breakdown and the heat-shock response. In Ubiquitin. M. Rechsteiner, Ed.: 207-238. Plenum Press, New York.
