Regulation of the Drosophila ubiquitin ligase DIAP1 is mediated via several distinct ubiquitin system pathways.

Inhibitors of apoptosis proteins (IAPs) suppress cell death by inactivating proapoptotic regulators, and therefore play important roles in controlling apoptosis in normal and malignant cells. Many IAPs are ubiquitin ligases, and their activity is mediated via ubiquitination and subsequent degradation of their targets. Here we corroborate a previous observation that DIAP1 (Drosophila IAP1) can be degraded via a two-step mechanism: (i) limited caspase-mediated cleavage and (ii) degradation of the released fragment via the ubiquitin N-end rule pathway. Yet, we demonstrate that this pathway is not the only one involved in DIAP1 degradation, and the intact protein can be degraded independent of prior caspase cleavage. Importantly, this mode of degradation does not require the RING-finger-mediated autoubiquitinating activity of DIAP1, believed to target many RING-finger E3s for self-destruction. Our preliminary data suggest that DIAP2 mediates DIAP1 degradation, suggesting a novel regulatory loop within the apoptotic pathway. Studying the role of the autoubiquitinating activity of DIAP1, we demonstrate that it does not involve formation of Lys48-based polyubiquitin chains, but probably chains linked via Lys63. Our preliminary data suggest that the autoubiquitination serves to attenuate the ligase activity of DIAP1 towards its exogenous substrates.

Functional interaction between SEL-10, an F-box protein, and the nuclear form of activated Notch1 receptor.

The Notch signaling pathway is essential in many cell fate decisions in invertebrates as well as in vertebrates. After ligand binding, a two-step proteolytic cleavage releases the intracellular part of the receptor which translocates to the nucleus and acts as a transcriptional activator. Although Notch-induced transcription of genes has been reported extensively, its endogenous nuclear form has been seldom visualized. We report that the nuclear intracellular domain of Notch1 is stabilized by proteasome inhibitors and is a substrate for polyubiquitination in vitro. SEL-10, an F-box protein of the Cdc4 family, was isolated in a genetic screen for Lin12/Notch-negative regulators in Caenorhabditis elegans. We isolated human and murine counterparts of SEL-10 and investigated the role of a dominant-negative form of this protein, deleted of the F-box, on Notch1 stability and activity. This molecule could stabilize intracellular Notch1 and enhance its transcriptional activity but had no effect on inactive membrane-anchored forms of the receptor. We then demonstrated that SEL-10 specifically interacts with nuclear forms of Notch1 and that this interaction requires a phosphorylation event. Taken together, these data suggest that SEL-10 is involved in shutting off Notch signaling by ubiquitin-proteasome-mediated degradation of the active transcriptional factor after a nuclear phosphorylation event.

Mechanisms of ubiquitin-mediated, limited processing of the NF-kappaB1 precursor protein p105.

In most cases, target proteins of the ubiquitin system are completely degraded. In several exceptions, such as the first step in the activation of the transcriptional regulator NF-kappaB, the substrate, the precursor protein p105, is processed in a limited manner to yield the active subunit p50. p50 is derived from the N-terminal domain of p105, whereas the C-terminal domain is degraded. The mechanisms involved in this unique process have remained elusive. We have shown that a Gly-rich region (GRR) at the C-terminal domain of p50 is one important processing signal and that it interferes with processing of the ubiquitinated precursor by the 26S proteasome. Also, amino acid residues 441-454 are important for processing under non-stimulated conditions. Lys 441 and 442 serve as ubiquitination targets, whereas residues 446-454 may serve as a ligase recognition motif. Following IkappaB kinase (IKK)-mediated phosphorylation, the C-terminal domain of p105, residues 918-934, recruits the SCF(beta-TrCP) ubiquitin ligase, and ubiquitination by this complex leads to accelerated processing. The two sites appear to be recognized under different physiological conditions by two different ligases, targeting two distinct recognition motifs. We have shown that ubiquitin conjugation and processing of a series of precursors of p105 that lack the C-terminal IKK phosphorylation/TrCP binding domain, is progressively inhibited with increasing number of ankyrin repeats. Inhibition is due to docking of active NF-kappaB subunits to the ankyrin repeat domain in the C-terminal half of p105 (IkappaBgamma). Inhibition is alleviated by phosphorylation of the C-terminal domain that leads to ubiquitin-mediated degradation of the ankyrin repeat domain and release of the anchored subunits. We propose a model that may explain the requirement for two sites: a) a basal site that may be involved in co-translational processing prior to the synthesis of the ankyrin repeat domain; and b) a signal-induced site that is involved in processing/degradation of the complete molecule following cell activation, with rapid release of stored, transcriptionally active subunits.

SCF(beta)(-TrCP) ubiquitin ligase-mediated processing of NF-kappaB p105 requires phosphorylation of its C-terminus by IkappaB kinase.

Processing of the p105 precursor to form the active subunit p50 of the NF-kappaB transcription factor is a unique case in which the ubiquitin system is involved in limited processing rather than in complete destruction of the target substrate. A glycine-rich region along with a downstream acidic domain have been demonstrated to be essential for processing. Here we demonstrate that following IkappaB kinase (IkappaK)-mediated phosphorylation, the C-terminal domain of p105 (residues 918-934) serves as a recognition motif for the SCF(beta)(-TrCP) ubiquitin ligase. Expression of IkappaKbeta dramatically increases processing of wild-type p105, but not of p105-Delta918-934. Dominant-negative beta-TrCP inhibits IkappaK-dependent processing. Furthermore, the ligase and wild-type p105 but not p105-Delta918-934 associate physically following phosphorylation. In vitro, SCF(beta)(-TrCP) specifically conjugates and promotes processing of phosphorylated p105. Importantly, the TrCP recognition motif in p105 is different from that described for IkappaBs, beta-catenin and human immunodeficiency virus type 1 Vpu. Since p105-Delta918-934 is also conjugated and processed, it appears that p105 can be recognized under different physiological conditions by two different ligases, targeting two distinct recognition motifs.

Identification of the ubiquitin carrier proteins, E2s, involved in signal-induced conjugation and subsequent degradation of IkappaBalpha.

The last step in the activation of the transcription factor NF-kappaB is signal-induced, ubiquitin- and proteasome-mediated degradation of the inhibitor IkappaBalpha. Although most of the components involved in the activation and degradation pathways have been identified, the ubiquitin carrier proteins (E2) have remained elusive. Here we show that the two highly homologous members of the UBCH5 family, UBCH5b and UBCH5c, and CDC34/UBC3, the mammalian homolog of yeast Cdc34/Ubc3, are the E2 enzymes involved in the process. The conjugation reaction they catalyze in vitro is specific, as they do not recognize the S32A,S36A mutant species of IkappaBalpha that cannot be phosphorylated and conjugated following an extracellular signal. Furthermore, the reaction is specifically inhibited by a doubly phosphorylated peptide that spans the ubiquitin ligase recognition domain of the inhibitor. Cys-to-Ala mutant species of the enzymes that cannot bind ubiquitin inhibit tumor necrosis factor alpha-induced degradation of the inhibitor in vivo. Not surprisingly, they have a similar effect in a cell-free system as well. Although it is clear that the E2 enzymes are not entirely specific to IkappaBalpha, they are also not involved in the conjugation and degradation of the bulk of cellular proteins, thus exhibiting some degree of specificity that is mediated probably via their association with a defined subset of ubiquitin-protein ligases. The mechanisms that underlie the involvement of two different E2 species in IkappaBalpha conjugation are not clear at present. It is possible that different conjugating machineries operate under different physiological conditions or in different cells.

Degradation of myogenic transcription factor MyoD by the ubiquitin pathway in vivo and in vitro: regulation by specific DNA binding.

MyoD is a tissue-specific transcriptional activator that acts as a master switch for skeletal muscle differentiation. Its activity is induced during the transition from proliferating, nondifferentiated myoblasts to resting, well-differentiated myotubes. Like many other transcriptional regulators, it is a short-lived protein; however, the targeting proteolytic pathway and the underlying regulatory mechanisms involved in the process have remained obscure. It has recently been shown that many short-lived regulatory proteins are degraded by the ubiquitin system. Degradation of a protein by the ubiquitin system proceeds via two distinct and successive steps, conjugation of multiple molecules of ubiquitin to the target protein and degradation of the tagged substrate by the 26S proteasome. Here we show that MyoD is degraded by the ubiquitin system both in vivo and in vitro. In intact cells, the degradation is inhibited by lactacystin, a specific inhibitor of the 26S proteasome. Inhibition is accompanied by accumulation of high-molecular-mass MyoD-ubiquitin conjugates. In a cell-free system, the proteolytic process requires both ATP and ubiquitin and, like the in vivo process, is preceded by formation of ubiquitin conjugates of the transcription factor. Interestingly, the process is inhibited by the specific DNA sequence to which MyoD binds: conjugation and degradation of a MyoD mutant protein which lacks the DNA-binding domain are not inhibited. The inhibitory effect of the DNA requires the formation of a complex between the DNA and the MyoD protein. Id1, which inhibits the binding of MyoD complexes to DNA, abrogates the effect of DNA on stabilization of the protein.

Inhibition of NF-kappa-B cellular function via specific targeting of the I-kappa-B-ubiquitin ligase.

Activation of the transcription factor NF-kappa B is a paradigm for signal transduction through the ubiquitin-proteasome pathway: ubiquitin-dependent degradation of the transcriptional inhibitor I kappa B in response to cell stimulation. A major issue in this context is the nature of the recognition signal and the targeting enzyme involved in the proteolytic process. Here we show that following a stimulus-dependent phosphorylation, and while associated with NF-kappa B, I kappa B is targeted by a specific ubiquitin-ligase via direct recognition of the signal-dependent phosphorylation site; phosphopeptides corresponding to this site specifically inhibit ubiquitin conjugation of I kappa B and its subsequent degradation. The ligase recognition signal is functionally conserved between I kappa B alpha and I kappa B beta, and does not involve the nearby ubiquitination site. Microinjection of the inhibitory peptides into stimulated cells abolished NF-kappa B activation in response to TNF alpha and the consequent expression of E-selectin, an NF-kappa B-dependent cell-adhesion molecule. Inhibition of NF-kappa B function by specific blocking of ubiquitin ligase activity provides a novel approach for intervening in cellular processes via regulation of unique proteolytic events.

On the involvement of calpains in the degradation of the tumor suppressor protein p53.

A crude fraction that contains ubiquitin-protein ligases contains also a proteolytic activity of approximately 100 kDa that cleaves p53 to several fragments. The protease does not require ATP and is inhibited in the crude extract by an endogenous approximately 250 kDa inhibitor. The proteinase can be inhibited by chelating the Ca2+ ions, by specific cysteine proteinase inhibitors and by peptide aldehyde derivatives that inhibit calpains. Purified calpain demonstrates an identical activity that can be inhibited by calpastatin, the specific protein inhibitor of the enzyme. Thus, it appears that the activity we have identified in the extract is catalyzed by calpain. The calpain in the extract degrades also N-myc, c-Fos and c-Jun, but not lysozyme. In crude extract, the calpain activity can be demonstrated only when the molar ratio of the calpain exceeds that of its native inhibitor. Recent experimental evidence implicates both the ubiquitin proteasome pathway and calpain in the degradation of the tumor suppressor, and it was proposed that the two pathways may play a role in targeting the protein under various conditions. The potential role of the two systems in this important metabolic process is discussed.

Protein synthesis elongation factor EF-1 alpha is an isopeptidase essential for ubiquitin-dependent degradation of certain proteolytic substrates.

Targeting of different cellular proteins for conjugation and subsequent degradation via the ubiquitin pathway involves diverse recognition signals and distinct enzymatic factors. A few proteins are recognized via their N-terminal amino acid residue and conjugated by a ubiquitin-protein ligase that recognizes this residue. However, most substrates, including N-alpha-acetylated proteins that constitute the vast majority of cellular proteins, are targeted by different signals and are recognized by yet unknown ligases. In addition to the ligases, other factors may also be specific for the recognition of this subset of proteins. We have previously shown that degradation of N-terminally blocked proteins require a specific factor, designated FH, and that the factor acts along with the 26S protease complex to degrade ubiquitin-conjugated proteins (Gonen et al., 1991). Further studies have shown that FH is identical to the protein synthesis elongation factor EF-1 alpha, and that it can be substituted by the bacterial elongation factor EF-Tu (Gonen et al., 1994). This, rather surprising, finding raises two important and interesting problems. The first involves the mechanism of action of the factor and the second the possibility that protein synthesis and degradation may be regulated by a commonly shared factor. Here, we demonstrate that EF-1 alpha is a ubiquitin C-terminal hydrolase (isopeptidase) that is probably involved in trimming the conjugates to lower molecular weight forms recognized by the 26S proteasome complex. Additional findings demonstrate that its activity is inhibited specifically by tRNA. This finding raises the possibility that under anabolic conditions, when the factor is associated with AA.tRNA and GTP, it is active in protein synthesis but inactive in proteolysis. Under catabolic conditions, when the factor is predominantly found in its apo form, it is active in proteolysis.

Isolation, characterization, and partial purification of a novel ubiquitin-protein ligase, E3. Targeting of protein substrates via multiple and distinct recognition signals and conjugating enzymes.

Degradation of a protein via the ubiquitin system involves two discrete steps, conjugation of ubiquitin to the substrate and degradation of the adduct. Conjugation follows a three-step mechanism. First, ubiquitin is activated by the ubiquitin-activating enzyme, E1. Following activation, one of several E2 enzymes (ubiquitin-carrier proteins or ubiquitin-conjugating enzymes, UBCs) transfers ubiquitin from E1 to the protein substrate that is bound to one of several ubiquitin-protein ligases, E3s. These enzymes catalyze the last step in the process, covalent attachment of ubiquitin to the protein substrate. The binding of the substrate to E3 is specific and implies that E3s play a major role in recognition and selection of proteins for conjugation and subsequent degradation. So far, only a few ligases have been identified, and it is clear that many more have not been discovered yet. Here, we describe a novel ligase that is involved in the conjugation and degradation of non "N-end rule" protein substrates such as actin, troponin T, and MyoD. This substrate specificity suggests that the enzyme may be involved in degradation of muscle proteins. The ligase acts in concert with E2-F1, a previously described non N-end rule UBC. Interestingly, it is also involved in targeting lysozyme, a bona fide N-end substrate that is recognized by E3 alpha and E2-14 kDa. The novel ligase recognizes lysozyme via a signal(s) that is distinct from the N-terminal residue of the protein. Thus, it appears that certain proteins can be targeted via multiple recognition motifs and distinct pairs of conjugating enzymes. We have purified the ligase approximately 200-fold and demonstrated that it is different from other known E3s, including E3 alpha/UBR1, E3 beta, and E6-AP. The native enzyme has an apparent molecular mass of approximately 550 kDa and appears to be a homodimer. Because of its unusual size, we designated this novel ligase E3L (large). E3L contains an -SH group that is essential for its activity. Like several recently described E3 enzymes, including E6-AP and the ligase involved in the processing of p105, the NF-kappa B precursor, the novel ligase is found in mammalian tissues but not in wheat germ.

Degradation of the proto-oncogene product c-Fos by the ubiquitin proteolytic system in vivo and in vitro: identification and characterization of the conjugating enzymes.

The transcription factor c-Fos is a short-lived cellular protein. The levels of the protein fluctuate significantly and abruptly during changing pathophysiological conditions. Thus, it is clear that degradation of the protein plays an important role in its tightly regulated activity. We examined the involvement of the ubiquitin pathway in c-Fos breakdown. Using a mutant cell line, ts20, that harbors a thermolabile ubiquitin-activating enzyme, E1, we demonstrate that impaired function of the ubiquitin system stabilizes c-Fos in vivo. In vitro, we reconstituted a cell-free system and demonstrated that the protein is multiply ubiquitinated. The adducts serve as essential intermediates for degradation by the 26S proteasome. We show that both conjugation and degradation are significantly stimulated by c-Jun, with which c-Fos forms the active heterodimeric transcriptional activator AP-1. Analysis of the enzymatic cascade involved in the conjugation process reveals that the ubiquitin-carrier protein E2-F1 and its human homolog UbcH5, which target the tumor suppressor p53 for degradation, are also involved in c-Fos recognition. The E2 enzyme acts along with a novel species of ubiquitin-protein ligase, E3. This enzyme is distinct from other known E3s, including E3 alpha/UBR1, E3 beta, and E6-AP. We have purified the novel enzyme approximately 350-fold and demonstrated that it is a homodimer with an apparent molecular mass of approximately 280 kDa. It contains a sulfhydryl group that is essential for its activity, presumably for anchoring activated ubiquitin as an intermediate thioester prior to its transfer to the substrate. Taken together, our in vivo and in vitro studies strongly suggest that c-Fos is degraded in the cell by the ubiquitin-proteasome proteolytic pathway in a process that requires a novel recognition enzyme.

The 26S proteasome of the yeast Saccharomyces cerevisiae.

Proteasomes are large multicatalytic proteinase complexes found in all eukaryotic organisms investigated so far. They have been shown to play a central role in cytosolic and nuclear proteolysis. According to their sedimentation coefficients two types of these particles can be distinguished: 20S proteasomes and 26S proteasomes. In contrast to 20S proteasomes, which were mainly characterized on the basis of their ability to cleave small chromogenic peptide substrates and certain proteins in an ATP-independent manner, 26S proteasomes degrade ubiquitinylated proteins in an ATP-dependent reaction. 20S proteasomes have been found in all eukaryotes from yeast to man. So far 26S proteasomes have only been discovered in higher eukaryotes. We now report the existence of the 26S proteasome in a lower eukaryote, the yeast Saccharomyces cerevisiae. Formation of the 26S proteasome could most effectively be induced in crude extracts of heat stressed yeast cells by incubation with ATP and Mg2+ ions. This treatment yielded a protein complex, which eluted from gel filtration columns at molecular masses higher than 1500 kDa. Besides chromogenic peptide substrates, this complex cleaves ubiquitinylated proteins in an ATP-dependent fashion. In non-denaturing-PAGE, the purified 26S proteasome disintegrated and migrated as four protein bands. One of these bands could be identified as the 20S proteasome. On SDS-PAGE, the 26S proteasome showed a complex pattern of subunit bands with molecular masses between 15 and 100 kDa. Further evidence for the 20S proteasome being the proteolytically active core of the 26S proteasome was obtained by following peptide cleaving activities in extracts of yeast strains carrying mutations in various subunits of the 20S proteasome.

Protein synthesis elongation factor EF-1 alpha is essential for ubiquitin-dependent degradation of certain N alpha-acetylated proteins and may be substituted for by the bacterial elongation factor EF-Tu.

Targeting of different cellular proteins for conjugation and subsequent degradation via the ubiquitin pathway involves diverse recognition signals and distinct enzymatic factors. A few proteins are recognized via their N-terminal amino acid residue and conjugated by a ubiquitin-protein ligase that recognizes this residue. Most substrates, including the N alpha-acetylated proteins that constitute the vast majority of cellular proteins, are targeted by different signals and are recognized by yet unknown ligases. We have previously shown that degradation of N-terminally blocked proteins requires a specific factor, designated FH, and that the factor acts along with the 26S protease complex to degrade ubiquitin-conjugated proteins. Here, we demonstrate that FH is the protein synthesis elongation factor EF-1 alpha. (a) Partial sequence analysis reveals 100% identity to EF-1 alpha. (b) Like EF-1 alpha, FH binds to immobilized GTP (or GDP) and can be purified in one step using the corresponding nucleotide for elution. (c) Guanine nucleotides that bind to EF-1 alpha protect the ubiquitin system-related activity of FH from heat inactivation, and nucleotides that do not bind do not exert this effect. (d) EF-Tu, the homologous bacterial elongation factor, can substitute for FH/EF-1 alpha in the proteolytic system. This last finding is of particular interest since the ubiquitin system has not been identified in prokaryotes. The activities of both EF-1 alpha and EF-Tu are strongly and specifically inhibited by ubiquitin-aldehyde, a specific inhibitor of ubiquitin isopeptidases. It appears, therefore, that EF-1 alpha may be involved in releasing ubiquitin from multiubiquitin chains, thus rendering the conjugates susceptible to the action of the 26S protease complex.

Complete reconstitution of conjugation and subsequent degradation of the tumor suppressor protein p53 by purified components of the ubiquitin proteolytic system.

The wild-type tumor suppressor protein p53 is a short-lived protein that plays important roles in regulation of cell cycle, differentiation, and survival. Mutations that inactivate or alter the tumor suppressor activity of the protein seem to be the most common genetic change in human cancer and are frequently associated with changes in its stability. The ubiquitin system has been implicated in the degradation of p53 both in vivo and in vitro. A mutant cell line that harbors a thermolabile ubiquitin-activating enzyme, E1, fails to degrade p53 at the nonpermissive temperature. Studies in cell-free extracts have shown that covalent attachment of ubiquitin to the protein requires the three conjugating enzymes: E1, a novel species of ubiquitin-carrier protein (ubiquitin-conjugating enzyme; UBC),E2-F1, and an ubiquitin-protein ligase, E3. Recognition of p53 by the ligase is facilitated by formation of a complex between the protein and the human papillomavirus (HPV) oncoprotein E6. Therefore, the ligase has been designated E6-associated protein (E6-AP). However, these in vitro studies have not demonstrated that the conjugates serve as essential intermediates in the proteolytic process. In fact, in many cases, conjugation of ubiquitin to the target protein does not signal its degradation. Thus, it is essential to demonstrate that p53-ubiquitin adducts serve as essential proteolytic intermediates and are recognized and degraded by the 26S protease complex, the proteolytic arm of the ubiquitin pathway. In this study, we demonstrate that conjugates of p53 generated in the presence of purified, E1, E2, E6-AP, E6, ubiquitin and ATP, are specifically recognized by the 26S protease complex and degraded. In contrast, unconjugated p53 remains stable. The ability to reconstitute the system from purified components will enable detailed analysis of the recognition process and the structural motifs involved in targeting the protein for degradation.

Purification and characterization of a novel species of ubiquitin-carrier protein, E2, that is involved in degradation of non-"N-end rule" protein substrates.

Ubiquitin-carrier proteins (E2s, ubiquitin-conjugating enzymes, UBCs) participate in proteolysis by catalyzing transfer of activated ubiquitin to the protein substrates, which are bound to specific ubiquitin-protein ligases (E3s). Yeast UBC2 (RAD6) and the mammalian E2(14kDa) bind to the ligase that recognizes and is involved in the degradation of certain free amino-terminal substrates ("N-end rule" substrates). As such proteins are rather scarce, the role of these E2s in general proteolysis is probably limited. Here, we report the purification and characterization of a novel 18-kDa species of E2 from rabbit reticulocytes. Unlike most members of the E2 family, this enzyme does not adsorb to anion exchange resin in neutral pH, and it is purified from the unadsorbed material (Fraction 1). Thus, it is designated E2-F1. Like all members of the E2 family, it generates a thiol ester with ubiquitin that serves as an intermediate in the conjugation reaction. Sequence analysis revealed a significant homology to many known species of E2s. The enzyme generates multiply ubiquitinated proteins in the presence of an E3 that has not been characterized yet. Most importantly, the ubiquitination via this E2 leads to the degradation of certain non-"N-end rule" substrates such as glyceraldehyde-3-phosphate dehydrogenase (Val at the NH2 terminus) and to the ubiquitination and degradation of certain N-alpha-acetylated proteins such as histone H2A, actin, and alpha-crystallin. The enzyme is also involved in the conjugation and degradation of the tumor suppressor protein p53.

Purification and characterization of a novel protein that is required for degradation of N-alpha-acetylated proteins by the ubiquitin system.

Anion exchange chromatography of reticulocyte lysates revealed that the ubiquitin cell-free system can be resolved into two essential fractions: unadsorbed material (Fraction I) that contains ubiquitin and a high salt eluate (Fraction II) that contains the conjugating enzymes and the conjugate-degrading protease. Many proteins with exposed NH2 termini are degraded in a ubiquitin-supplemented Fraction II. However, this partially purified and reconstituted system does not degrade N-alpha-acetylated proteins. These proteins are degraded in whole lysates in a ubiquitin-dependent manner (Mayer, A. Siegel, N. R., Schwartz, A. L., and Ciechanover, A. (1989) Science 244, 1480-1483). It appears that a protein factor which is specifically required for the degradation of N-alpha-acetylated proteins is removed or inactivated during the fractionation of the lysate. Here we report the purification and characterization of a novel protein that is required along with the protease for the degradation of ubiquitin conjugates of histone H2A, an N-alpha-acetylated protein. The protein is not required for the degradation of ubiquitin conjugates of proteins with free NH2 termini. The protein, which is found in crude Fraction I, was purified approximately 200-fold by (NH4)2SO4 precipitation, Sephadex G-100 gel-filtration chromatography, Mono Q anion exchange chromatography, and an additional Sephadex G-100 gel filtration chromatography step. The protein is removed from Fraction I during the purification of ubiquitin and has not been previously recognized since the majority of the protein substrates evaluated in the cell-free system have free NH2 termini. The protein has an apparent molecular mass of approximately 92 kDa. It is a homodimer that is composed of two identical 46-kDa subunits. Initial analysis of the mechanism of action of this protein revealed that it must interact with the conjugates in order to allow proteolysis to occur. We designated the protein Factor H (Factor Hedva).

The ubiquitin-mediated proteolytic pathway: enzymology and mechanisms of recognition of the proteolytic substrates.

Degradation of proteins by the ubiquitin system involves two discrete steps. Initially, ubiquitin is covalently linked in an ATP-dependent mode to the protein substrate. The protein moiety of the conjugate is subsequently degraded by a specific protease into peptides and free amino acids with the release of free and reutilizable ubiquitin. The degradation process also requires energy. In this review we shall discuss the mechanisms involved in ubiquitin activation, selection of substrates for conjugation, and subsequent degradation of ubiquitin-conjugated proteins. In addition, we shall briefly summarize what is currently known of the role of the ubiquitin system in protein degradation in vitro and in vivo.

Degradation of proteins by the ubiquitin-mediated proteolytic pathway.

Degradation of a protein by the ubiquitin system involves two distinct processes. In the first step, ubiquitin is covalently linked in an ATP-dependent mode to the protein substrate. The protein moiety of the conjugate is then degraded by a specific protease into free amino acids, resulting in the release of free and reutilizable ubiquitin. This process also requires energy. In this review we will briefly summarize our current knowledge of the role of the ubiquitin system in protein turnover and discuss in detail the mechanism involved in selection of substrates for conjugation and in degradation of ubiquitin-conjugated proteins.

In vitro transformation of chondroprogenitor cells into osteoblasts and the formation of new membrane bone.

Mandibular condyles of fetal mice 19 to 20 days in utero were kept in an organ culture system for up to 10 days. After 2 days in culture the cartilage of the mandibular condyle appeared to have maintained all its inherent structural characteristics, including its various cell layers: chondroprogenitor, chondroblastic, and hypertrophic. After 5 days in culture no chondroblasts could be seen and, instead, the entire cartilage was occupied by hypertrophic chondrocytes. At the same time, the mesenchymal cells at the chondroprogenitor zone differentiated with osteoblasts which produced osteoid. Light microscopic examinations showed that the newly formed osteoid did not stain with acidic toluidine blue or with alcian blue, but stained intensively with the van Gieson stain and with Periodic acid-Schiff (PAS). The osteoid reacted with antibodies against type I collagen but not with antibodies against type II collagen. Electron microscopic examinations showed that the mineralization appeared to be associated with collagen fibers in bone rather than with matrix vesicles in the cartilage. The process of bone formation progressed with time and by the 10th day new bone replaced almost the entire cartilage, thus forming an expanded layer of membrane bone. This in vitro system represents an experimental model whereby undifferentiated precursor cells transform into osteoblasts with the subsequent formation of a typical membrane bone.

A serum protein inhibitor of acid lipase and its possible role in lipid accumulation in cultured fibroblasts.

Histochemical examination of L929 fibroblasts indicates massive accumulation of intracellular lipids in cells grown in medium supplemented with 10% calf serum. The present study suggests that the accumulation of triacylglycerols in these cells may be due to the inhibition of acid lipase activity by a serum component present in the culture medium. This is based on the following observations. (a) Acid lipase appears to be the major intracellular enzyme responsible for triacylglycerol catabolism in L929 cells. (b) The acid lipase is strongly inhibited by either human of calf serum. Several lines of evidence show that the inhibitor is a serum protein: it is heat-labile, non-dialysable and is destroyed by trypsin. It is present mainly in Cohn's fraction IV and has mol.wt. approx. 50000. (c) Lipid accumulation in intact cells is reduced when cells are grown on a limited supply of serum (2%) and is elevated by the addition of Cohn's fraction IV, freed of lipoproteins, to the growth medium.