Control of Neuronal Size Homeostasis by Trophic Factor–mediated Coupling of Protein Degradation to Protein Synthesis

We demonstrate that NGF couples the rate of degradation of long-lived proteins in sympathetic neurons to the rate of protein synthesis. Inhibiting protein synthesis rate by a specific percentage caused an almost equivalent percentage reduction in the degradation rate of long-lived proteins, indicating nearly 1:1 coupling between the two processes. The rate of degradation of short-lived proteins was unaffected by suppressing protein synthesis. Included in the pool of proteins that had increased half-lives when protein synthesis was inhibited were actin and tubulin. Both of these proteins, which had half-lives of several days, exhibited no degradation over a 3-d period when protein synthesis was completely suppressed. The half-lives of seven other long-lived proteins were quantified and found to increase by 84–225% when protein synthesis was completely blocked.Degradation–synthesis coupling protected cells from protein loss during periods of decreased synthesis. The rate of protein synthesis greatly decreased and coupling between degradation and synthesis was lost after removal of NGF. Uncoupling resulted in net loss of cellular protein and somatic atrophy. We propose that coupling the rate of protein degradation to that of protein synthesis is a fundamental mechanism by which neurotrophic factors maintain homeostatic control of neuronal size and perhaps growth.     

Ubiquitin as a degradation signal.

For many short-lived eukaryotic proteins, conjugation to ubiquitin, yielding a multiubiquitin chain, is an obligatory pre-degradation step. The conjugated ubiquitin moieties function as a 'secondary' signal for degradation, in that their posttranslational coupling to a substrate protein is mediated by amino acid sequences of the substrate that act as a primary degradation signal. We report that the fusion protein ubiquitin--proline--beta-galactosidase (Ub-P-beta gal) is short-lived in the yeast Saccharomyces cerevisiae because its N-terminal ubiquitin moiety functions as an autonomous, primary degradation signal. This signal mediates the formation of a multiubiquitin chain linked to Lys48 of the N-terminal ubiquitin in Ub-P-beta gal. The degradation of Ub-P-beta gal is shown to require Ubc4, one of at least seven ubiquitin-conjugating enzymes in S.cerevisiae. Our findings provide the first direct evidence that a monoubiquitin moiety can function as an autonomous degradation signal. This generally applicable, cis-acting signal can be used to manipulate the in vivo half-lives of specific intracellular proteins.     

Kinetics of protein degradation in diploid and trisomic human fibroblasts

The degradation rate of long-lived and short-lived proteins was determined in diploid fibroblasts and fibroblasts with trisomy 7 derived from human embryos. Two fractions of proteins were detected in the exponentially growing diploid fibroblasts with half-lives (T 1/2) 37 and 19 hours. The rate of protein degradation increases in diploid fibroblasts as they approach confluence and protein fractions with T 1/2 30, 18 and 12 hours appear. The rate of protein degradation in trisomic fibroblasts does not change for the long-lived and short-lived proteins and is the same in both exponential (T 1/2 31 and 14 hours) and stationary phase (T 1/2 33 and 17 hours). The relative amount of the short-lived proteins in trisomic fibroblasts in the stationary phase decreased as compared with the one in diploid fibroblasts. It is apparent that a mechanism of regulation of protein catabolism in trisomic fibroblasts is impaired.     

The degradation signal in a short-lived protein

Our previous work has shown that the amino-terminal residue of a short-lived protein is a distinct component of the protein's degradation signal. To define the complete signal, otherwise identical dihydrofolate reductase test proteins bearing different extensions and either a "stabilizing" or a "destabilizing" amino-terminal residue were expressed in the yeast S. cerevisiae and their in vivo half-lives compared. The amino-terminal degradation signal is shown to comprise two distinct determinants. One, discovered previously, is the protein's amino-terminal residue. The second determinant, identified in the present work, is a specific lysine residue whose function in the degradation signal is not dependent on the unique amino acid sequences in the vicinity of the residue. The mechanistic significance of the second determinant is illuminated by the finding that in a targeted, short-lived protein, a chain of branched ubiquitin-ubiquitin conjugates is confined to a lysine residue that has been identified in the present work as the second determinant of the degradation signal.     

E3 ubiquitin ligases as regulators of membrane protein trafficking and degradation

Ubiquitination is a regulated post-translational modification that conjugates ubiquitin (Ub) to lysine residues of target proteins and determines their intracellular fate. The canonical role of ubiquitination is to mediate degradation by the proteasome of short-lived cytoplasmic proteins that carry a single, polymeric chain of Ub on a specific lysine residue. However, protein modification by Ub has much broader and diverse functions involved in a myriad of cellular processes. Monoubiquitination, at one or multiple lysine residues of transmembrane proteins, influences their stability, protein-protein recognition, activity and intracellular localization. In these processes, Ub functions as an internalization signal that sends the modified substrate to the endocytic/sorting compartments, followed by recycling to the plasma membrane or degradation in the lysosome. E3 ligases play a pivotal role in ubiquitination, because they recognize the acceptor protein and hence dictate the high specificity of the reaction. The multitude of E3s present in nature suggests their nonredundant mode of action and the need for their controlled regulation. Here we give a short account of E3 ligases that specifically modify and regulate membrane proteins. We emphasize the intricate network of interacting proteins that contribute to the substrate-E3 recognition and determine the substrate's cellular fate.     

Regulation of intracellular protein degradation in IMR-90 human diploid fibroblasts

Human diploid fibroblasts (IMR-90) regulate their overall rates of proteolysis in response to the composition of the culture medium and the ambient temperature. The magnitude and, in some cases, the direction of the response depend on the half-lives of the cellular proteins that are radioactively labeled and the time chosen for measurements of protein degradation. Fetal calf serum, insulin, fibroblast growth factor, epidermal growth factor, and amino acids selectively regulate catabolism of long-lived proteins without affecting degradation of short-lived proteins. Fetal calf serum reduces degradative rates of long-lived proteins and is maximally effective at a concentration of 20%, but the effect of serum on proteolysis is evident only for the first 24 hr. Insulin inhibits degradation of long-lived proteins in the presence or absence of glucose and amino acids in the medium, but is maximally effective only at high concentrations (10(-5) M). Amino acid deprivation increases degradative rates of long-lived proteins for the first 6 hr, but then decreases their catabolism for the subsequent 20 hr. Lowered temperature is the only condition tested that significantly alters degradative rates of short-lived proteins. Although cells incubated at 27 degrees C have reduced rates of degradation for both short-lived and long-lived proteins compared to cells at 37 degrees C, lowered temperature reduces catabolism of long-lived proteins to a greater extent.     

Differential degradation of intracellular proteins in human skin fibroblasts of mitotic and mitomycin-C (MMC)-induced postmitotic differentiation states in vitro

Rates of degradation of short- and long-lived proteins were analysed in homogeneous fibroblast cultures of mitotic or mitomycin C (MMC)-induced postmitotic states. When the highly mitotic MFII type cells--the major cell type of so called "early passage" or "young" fibroblasts--differentiate into MFIII type cells, the last mitotic fibroblast type, and further into MMC-induced postmitotic fibroblasts, the degradation of short-lived proteins increases by a factor of 1.4, resulting in significantly reduced half-lives of these proteins in the postmitotic fibroblasts. From the highly mitotic MFII to the final postmitotic PMFVI-type cells via the intermediates MFIII, PMFIV and PMFV, the half lives (t1/2) of short-lived proteins decrease by a total of 122 min in average, from 362 to 240 min. Degradation of long-lived proteins did not change significantly from cell type MFII to PMFVI. As analysed by two-dimensional (2D)-gel electrophoresis the half-lives of the mitotic and postmitotic cell-type-specific proteins except one, protein PIVa (33 kDa; Pi 5.0), range between 33.2 h and 62.9 h. Protein PIVa, the first protein specific for postmitotic cells, is initially expressed 18 h after the induction of the postmitotic state by mitomycin C (MMC) and has a half-life of approximately 66 min. This may indicate that protein PIVa could function as one possible regulatory factor controlling the postmitotic differentiation state.     

Intracellular protein degradation in cultured bovine lens epithelial cells

Summary Although several proteases have been identified in homogenates of cultured epithelial cells of the eye lens and in lens tissues, there is little information regarding intracellular protein degradation in intact lens cells in vitro. Cultured lens cells may be useful in the study of intracellular protein degradation in the lens, a tissue with a wide range of protein half-lives. This is of interest because alterations in protein turnover in the lens have been implicated in cataract formation. This study examines intracellular protein degradation in cultured bovine lens epithelial cells (BLEC). Cell cultures were incubated with radiolabeled leucine to label intracellular proteins. Protein degradation was measured by monitoring the release of trichloroacetic-acid-soluble radioactivity into the culture medium. The average half-life of long-lived proteins (half-life >50 h) was typically about 57 h in serum-supplemented medium. Average rates of degradation of long-lived proteins increased by up to 73% when fetal bovine serum was withdrawn from the culture medium. Serum had no effect on the degradation of short-lived proteins (half-life <10 h). Degradation of long-lived proteins in the presence and absence of serum was further studied in cultured BLEC from population doubling level (PDL) 2 to 43. Average half-life of proteins in serum-supplemented medium was 52 to 58 h and did not vary significantly as a function of PDL. Degradation rates in serum-free medium increased approximately twofold up to PDL 7, but returned by PDL 25 to original levels, which were maintained through PDL 43. This work was supported in part by grants from U. S. Department of Agriculture contract 53-3K06-5-10, Massachusetts Lions Eye Research Fund, Inc., and the Daniel and Florence Guggenheim Foundation. D. A. E. is a recipient of a National Eye Institute postdoctoral fellowship.     

Recognition of influenza virus proteins by cytotoxic T lymphocytes

Recombinant DNA techniques have been used to express the proteins of influenza virus individually. Target cells expressing single viral proteins were then used to identify the molecules recognized by cytotoxic T lymphocytes (CTLS). Results have shown that, contrary to expectation, the majority of the proteins recognized by class I major histocompatibility complex-restricted CTLS are not transmembrane glycoproteins. Experiments with deletion mutants of the nucleoprotein (NP) gene showed that transport of epitopes to the membrane for recognition by CTLS was independent of a definable signal sequence. In addition, the epitopes recognized were contained within short linear sequences of amino acids, and rapid degradation of large NP fragments within the target cell did not prevent recognition by CTLS. These results led to the suggestion that the epitopes recognized by class-I-restricted CTLS resulted from degradation of viral proteins. If so, the epitopes should, like those for class-II-restricted T cells, be replaceable in vitro with short synthetic peptides. Five different epitopes of NP have now been demonstrated that can be defined with short peptides in vitro. Each peptide is recognized with a specific class I molecule (Db, Kk, Kd and HLA B37). This has been extended to the influenza matrix protein, and a peptide epitope defined that is recognized by human CTLS in association with HLA-A2. The question arose as to whether a similar phenomenon would be found with viral proteins which are naturally inserted in the target cell membrane. A mutant haemagglutinin has been produced that lacks a hydrophobic signal sequence. This protein is expressed as a short-lived, unglycosylated, intracellular protein. However, target cells expressing this molecule were recognized efficiently by CTLS raised to the wild-type haemagglutinin and vice versa. These and more recent results with non-viral glycoproteins are consistent with the existence of a mechanism for degrading viral (and perhaps host) proteins and exposing them at the cell surface for recognition by cytotoxic T cells in association with class I molecules of the major histocompatibility complex.     

Thermolability of ubiquitin-activating enzyme from the mammalian cell cycle mutant ts85

Ubiquitin, a 76 residue protein, occurs in eucaryotic cells either free or covalently joined to a variety of protein species. Previous work suggested that ubiquitin may function as a signal for attack by proteinases specific for ubiquitin-protein conjugates. We show that the mouse cell line ts85 , a previously isolated cell cycle mutant, is temperature-sensitive in ubiquitin-protein conjugation, and that this effect is due to the specific thermolability of the ts85 ubiquitin-activating enzyme (E1). From E1 thermoinactivation kinetics in mixed (wild-type plus ts85 ) extracts, and from copurification of the determinant of E1 thermolability with E1 in ubiquitin-affinity chromatography, we conclude that the determinant of E1 thermolability is contained within the E1 polypeptide. ts85 cells fail to degrade otherwise short-lived intracellular proteins at the nonpermissive temperature (accompanying paper), demonstrating that degradation of the bulk of short-lived proteins in this higher eucaryotic cell proceeds through a ubiquitin-dependent pathway. We discuss possible roles of ubiquitin-dependent pathways in DNA transactions, the cell cycle, and the heat shock response.     

The mitochondrial probe rhodamine 123 inhibits in isolated hepatocytes the degradation of short-lived proteins

The fluorescent dye rhodamine 123 (R123) decreases the intracellular ATP levels and also inhibits the degradation of short-lived proteins in isolated hepatocytes. This inhibition affects lysosomal and, to some extent, non-lysosomal mechanisms. The degradation of short-lived proteins decreases more when ATP levels are less than 40% of those in control cells, in contrast to the reported linear correlation between ATP levels and degradation of long-lived proteins. R123 provides a powerful probe for clarifying the proteolytic mechanisms involved in degradation of short-lived proteins and the ATP requirements in protein degradation. Indeed, as illustrated, the results suggest different mechanisms for the degradation of short- and long-lived proteins. Moreover, they provide a warning for the clinical use of this reagent.     

Structural disorder serves as a weak signal for intracellular protein degradation

Targeted turnover of proteins is a key element in the regulation of practically all basic cellular processes. The underlying physicochemical and/or sequential signals, however, are not fully understood. This issue is particularly pertinent in light of the recent recognition that intrinsically unstructured/disordered proteins, common in eukaryotic cells, are extremely susceptible to proteolytic degradation in vitro. The in vivo half-lives of proteins were determined recently in a high-throughput study encompassing the entire yeast proteome; here we examine whether these half-lives correlate with the presence of classical degradation motifs (PEST region, destruction-box, KEN-box, or the N-terminal residue) or with various physicochemical characteristics, such as the size of the protein, the degree of structural disorder, or the presence of low-complexity regions. Our principal finding is that, in general, the half-life of a protein does not depend on the presence of degradation signals within its sequence, even of ubiquitination sites, but correlates mainly with the length of its polypeptide chain and with various measures of structural disorder. Two distinct modes of involvement of disorder in degradation are proposed. Susceptibility to degradation of longer proteins, containing larger numbers of residues in conformational disorder, suggests an extensive function, whereby the effect of disorder can be ascribed to its mere physical presence. However, after normalization for protein length, the only signal that correlates with half-life is disorder, which indicates that it also acts in an intensive manner, that is, as a specific signal, perhaps in conjunction with the recognition of classical degradation motifs. The significance of correlation is rather low; thus protein degradation is not determined by a single characteristic, but is a multi-factorial process that shows large protein-to-protein variations. Protein disorder, nevertheless, plays a key signalling role in many cases.     

Role of proteasomes in the degradation of short-lived proteins in human fibroblasts under various growth conditions

Degradation of proteins in the cells occurs by proteasomes, lysosomes and other cytosolic and organellar proteases. It is believed that proteasomes constitute the major proteolytic pathway under most conditions, especially when degrading abnormal and other short-lived proteins. However, no systematic analysis of their role in the overall degradation of truly short-lived cell proteins has been carried out. Here, the degradation of short-labelled proteins was examined in human fibroblasts by release of trichloroacetic acid-soluble radioactivity. The kinetics of degradation was decomposed into two, corresponding to short- and long-lived proteins, and the effect of proteasomal and lysosomal inhibitors on their degradation, under various growth conditions, was separately investigated. From the degradation kinetics of proteins labelled for various pulse times it can be estimated that about 30% of newly synthesised proteins are degraded with a half-life of approximately 1h. These rapidly degraded proteins should mostly include defective ribosomal products. Deprivation of serum and confluent conditions increased the degradation of the pool of long-lived proteins in fibroblasts without affecting, or affecting to a lesser extent, the degradation of the pool of short-lived proteins. Inhibitors of proteasomes and of lysosomes prevented more than 80% of the degradation of short-lived proteins. It is concluded that, although proteasomes are responsible of about 40-60% of the degradation of short-lived proteins in normal human fibroblasts, lysosomes have also an important participation in the degradation of these proteins. Moreover, in confluent fibroblasts under serum deprivation, lysosomal pathways become even more important than proteasomes in the degradation of short-lived proteins.     

Multiple growth-associated nuclear proteins immunoprecipitated by antisera raised against human c-myc peptide antigens.

Different antisera raised against various regions of the human c-myc protein were used to identify four human c-myc proteins with apparent molecular masses in sodium dodecyl sulfate-polyacrylamide gels ranging from 64 to 68 kilodaltons (phosphoproteins pp64 and pp67 and nonphosphorylated proteins p65 and p68). pp64 and p65 were the major detectable c-myc proteins, and pp67 and p68 were minor but specific components of the immunoprecipitates. The c-myc proteins were all localized in the cell nucleus. Accumulation of [35S]methionine-labeled p65 was observed after pulse-labeling and chase, suggesting that the stable p65 c-myc protein is generated posttranslationally from short-lived precursors. pp64, pp67, and p68 possessed short half-lives and may therefore be precursors of the stable p65. Confirmation of the nuclear localization of the human c-myc proteins was obtained by immunofluorescent staining. The human c-myc proteins were revealed as a pattern of punctate nuclear staining with, particularly for p65, nucleolar enhancement that left an unstained annulus surrounding the nucleolus.     

Effects of centrifugation on the degradation of short-lived proteins in exponentially growing cultured cells

The degradation mechanisms of short-lived proteins in cultured cells are unknown, probably due to the lack of procedures which specifically affect the degradation of these proteins. We found that centrifugation of cultured cells, growing either in monolayer or in suspension, between 5000 and 25,000g for 30 min, inhibits (more than 50%) the degradation of short-lived proteins but not of long-lived proteins. Protein synthesis or cell viability is not affected. Centrifugation also disorganizes the Golgi apparatus, as checked by routine electron microscopy, and inhibits the degradation of endocytosed proteins (a lysosomal process which is controlled by the Golgi apparatus). Using different centrifugation speeds, a good correlation was found between alteration of the Golgi apparatus and inhibition of protein degradation.     

A conserved ubiquitin- and ESCRT-dependent pathway internalizes human lysosomal membrane proteins for degradation

The lysosome is an essential organelle to recycle cellular materials and maintain nutrient homeostasis, but the mechanism to down-regulate its membrane proteins is poorly understood. In this study, we performed a cycloheximide (CHX) chase assay to measure the half-lives of approximately 30 human lysosomal membrane proteins (LMPs) and identified RNF152 and LAPTM4A as short-lived membrane proteins. The degradation of both proteins is ubiquitin dependent. RNF152 is a transmembrane E3 ligase that ubiquitinates itself, whereas LAPTM4A uses its carboxyl-terminal PY motifs to recruit NEDD4-1 for ubiquitination. After ubiquitination, they are internalized into the lysosome lumen by the endosomal sorting complexes required for transport (ESCRT) machinery for degradation. Strikingly, when ectopically expressed in budding yeast, human RNF152 is still degraded by the vacuole (yeast lysosome) in an ESCRT-dependent manner. Thus, our study uncovered a conserved mechanism to down-regulate lysosome membrane proteins.

A study of how lysosomal membrane proteins are down-regulated reveals a conserved pathway involving ubiquitination of the membrane protein and subsequent internalization into the lysosome lumen by the ESCRT machinery for degradation.     

Hyperthermia stimulates energy-proteasome-dependent protein degradation in cultured myotubes

Previous studies suggest that elevated temperature stimulates protein degradation in skeletal muscle, but the intracellular mechanisms are not fully understood. We tested the role of different proteolytic pathways in temperature-dependent degradation of long- and short-lived proteins in cultured L6 myotubes. When cells were cultured at different temperatures from 37 to 43 degrees C, the degradation of both classes of proteins increased, with a maximal effect noted at 41 degrees C. The effect of high temperature was more pronounced on long-lived than on short-lived protein degradation. By using blockers of individual proteolytic pathways, we found evidence that the increased degradation of both long-lived and short-lived proteins at high temperature was independent of lysosomal and calcium-mediated mechanisms but reflected energy-proteasome-dependent degradation. mRNA levels for enzymes and other components of different proteolytic pathways were not influenced by high temperature. The results suggest that hyperthermia stimulates the degradation of muscle proteins and that this effect of temperature is regulated by similar mechanisms for short- and long-lived proteins. Elevated temperature may contribute to the catabolic response in skeletal muscle typically seen in sepsis and severe infection.     

Towards the control of intracellular protein turnover: mitochondrial Lon protease inhibitors versus proteasome inhibitors

Cellular protein homeostasis results from the combination of protein biogenesis processes and protein quality control mechanisms, which contribute to the functional state of cells under normal and stress conditions. Proteolysis constitutes the final step by which short-lived, misfolded and damaged intracellular proteins are eliminated. Protein turnover and oxidatively modified protein degradation are mainly achieved by the proteasome in the cytosol and nucleus of eukaryotic cells while several ATP-dependent proteases including the matrix protease Lon take part in the mitochondrial protein degradation. Moreover, Lon protease seems to play a major role in the elimination of oxidatively modified proteins in the mitochondrial matrix. Specific inhibitors are commonly used to assess cellular functions of proteolytic systems as well as to identify their protein substrates. Here, we present and discuss known proteasome and Lon protease inhibitors. To date, very few inhibitors of Lon have been described and no specific inhibitors of this protease are available. The current knowledge on both catalytic mechanisms and inhibitors of these two proteases is first described and attempts to define specific non-peptidic inhibitors of the human Lon protease are presented.