Lysosomes and protein degradation

Evidence from studies on mouse peritoneal macrophages using the inhibitor pepstatin confirms lysosomal involvement in basal protein degradation, and extends its relevance to degradation of long half-life and analogue containing proteins. Studies on the ability of MRC-5 (a limited life-span fibroblast line) cells to selectively degrade analogue-containing proteins are described. These indicate that this capacity is retained even in very old cells; indeed such cells show an increased proportion of rapidly-degradable proteins. Analogue containing proteins bind preferentially to lysosomal membranes, and like liver cytosol proteins of short half-life, are selectively endocytosed and degraded by certain cells in culture. Thus membrane binding allowing selective entry to the lysosomal system may be important in controlling rate of degradation of both intracellular and extracellular protein. A method potentially allowing for determination of the rate of autophagy in cells, is described. This should enable further assessment of the quantitative involvement of lysosomes in protein degradation.     

Intracellular protein degradation: basis of a self-regulating mechanism for the proteolysis of endogenous proteins

The intracellular basal proteolysis system, as distinct from the lysosomal system, is important in sustaining a high flux of proteins required for maintenance, growth and adaptability of cells. Its activity automatically fluctuates with changes in protein synthetic activity, but with a considerably slower response time, since the two processes are only indirectly or passively linked. Since as much as one-third of intracellular proteolysis in mammalian cells is directed as nascent proteins, the consequences are more fully discussed in relation to cell growth state. During rapid growth, cells have to accumulate more than double their original protein mass in order to achieve a 100% increase between divisions. The effects of reducing protein synthesis by inducing quiescence, serum step-down or cycloheximide treatment on intracellular proteolysis are considered, and the possibility that this leads to enhanced degradation of existing proteins has been explored. No substantial evidence was found to support this latter notion. The basal proteolysis system is seen as a constitutive, pervasive and broad-spectrumed collection of hydrolytic enzymes. It destroys proteins randomly, having no means of distinguishing young from old, aberrant from normal. The rate of demise of protein substrates depends on two factors, the ease of access of the hydrolytic enzymes to their peptide bonds, and the length of time that any species of protein remains at risk to this hydrolytic potential. While the former has long been recognized, the importance of the second factor in relation to the ability of proteins to become integrated in the living fabric of the cell is only beginning to be appreciated. The discussion also suggests elaborate regulatory mechanisms akin to those for protein synthesis would be unnecessary for protein degradation, especially if it can now be substantiated that substrate availability determines the turnover rates of proteins by a pervasive and relatively unlimited proteolytic system (Grisolía, 1964).     

Abnormal proteins of shortened length are preferentially degraded in the cytosol of cultured MRC5 fibroblasts

Puromycyl peptides were degraded in MRC5 fibroblasts more rapidly than normal proteins labelled for the corresponding length of time for both long and short labelling periods. The degradation of the puromycyl peptides occurred almost exclusively in the cytosol of the cells. Even when the half-lives of normal and puromycyl peptides were manipulated to be similar, proportionally more of the normal proteins were degraded in the lysosomes. The rapid degradation of the puromycyl peptides was not due to the inhibition of protein synthesis brought about by puromycin but was due to the structure of the substrates themselves. The degree and intracellular site of degradation of puromycyl peptides closely mimic those of abnormal (missense) proteins containing amino acid analogues.     

On the lysosomal degradation of neurofibromin and its phosphorylation in cultured melanocytes.

Neurofibromatosis type 1 (NF1) is one of the most common inherited disorders in humans. Most of the NF1 gene mutations result in a reduction of the amount of neurofibromin to about 50%. Recently, we found that the level of neurofibromin can be regulated post-translationally through the alteration of its half-life. Here, we investigated whether lysosomes are involved in this post-translational regulation in cultured melanocytes of NF1 patients and controls. When the lysosomal degradation was inhibited by chloroquine, an increase of neurofibromin by a factor of 2 to 3, correlating with an increased half-life, was measured. Incubation with phosphoprotein-phosphatase inhibitors also increased the neurofibromin content in melanocytes. Investigations on phosphorylation of neurofibromin revealed a basal phosphorylation in melanocytes cultured with growth factor-deprived medium that increased upon incubation with the growth stimulators PMA or bFGF. Because both factors are also able to increase the half-life of neurofibromin, we suggest its phosphorylation to be an important step in protecting neurofibromin against specific lysosomal degradation.     

Endocytosis of liposomes containing lysosomal proteins increases intracellular protein degradation in growing L-132 cells

We have used a new approach to test the possible participation of lysosomes in the degradation of long-lived proteins. Rat liver lysosomal proteins were introduced, via multilamellar liposomes, into L-132 cells. Viability and protein synthesis were not impaired by this treatment. The liposomal content was released into the lysosomes of the cultured cells, as revealed by ferritin uptake and electron microscopy. Degradation rates of long-lived proteins increased with the uptake of lysosomal proteases. However, the increased protein degradation of chloroquine and leupeptin, in contrast to the inhibition by these reagents of the increased protein degradation of cells 'starved' of serum (step-down conditions). This approach opens a new way of investigating the degradation of intracellular proteins in cultured cells.     

Infulence of hormones and medium composition on the degradation of phosphoenolpyruvate carboxykinase (GTP) and total protein in Reuber H35 cells.

Reuber H35 cells were pulse-labeled with radioactive leucine and the influence of hormones, serum, and amino acids on protein degradation was investigated during a subsequent chase period. Radioactive, immunoprecipitable phosphoenolpyruvate carboxykinase (GTP) (EC 4.1.1.32) had a half-life of 5 to 6 hours which was not influenced by either N6, O2-dibutyryl adenosine 3':5'-monophosphate, dexamethasone, or insulin. The rate of phosphoenolpyruvate carboxykinase degradation was the same under steady state conditions as during the approach to a new steady state following hormonal induction or deinduction of the enzyme. Therefore, hormonal regulation of enzyme activity in vivo is the result of changes in the rate of enzyme synthesis. The rate of proteolysis for total cell proteins was increased under nutritional step-down conditions produced by the removal of serum or amino acids, or both, from the medium. This effect was completely prevented by insulin. Cycloheximide and puromycin, but not actinomycin D or cordycepin, inhibited protein degradation under step-down conditions but did not further decrease the basal rate of proteolysis measured in the presence of either insulin or serum plus amino acids. There was a good correlation between changes in proteolysis produced by serum and amino acids and changes in the degradation rate of phosphoenolpyruvate carboxykinase. Also, inhibition of proteolysis with cycloheximide and puromycin was accompanied by a decrease in the degradation rate for enzyme antigen. It is suggested that nutritional step-down leads either to the synthesis or activation of a proteolytic system.     

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.     

Investigation of the mechanism of protein turnover in HeLa S-3 cells by incubation at elevated temperatures

An apparent paradox relating to the degradation of endogenous proteins in HeLa S-3 cells occurs at 45 degrees C, at which their proteolysis is considerably enhanced in vitro but completely inhibited in vivo. No significant differences in rates of degradation of short-lived (nascent) and long-lived ('existing') proteins synthesised at 37 degrees C were found when chased at temperatures up to 43 degrees C, but at 45 degrees C degradation of both categories was reduced to zero in vivo. Synthesis of protein was suppressed at temperatures above 41 degrees C, being reduced by up to 60% at 43 degrees C. Proteolysis in vitro proceeded 1.6-1.7 times faster at 45 degrees C than at 37 degrees C and neutral pH. Evidence is presented for the involvement of the basal system; the findings both in vivo and in vitro do not seem to implicate the lysosomal system, no firm indication being obtained of its 'induction' at elevated temperatures. The results are discussed in terms of the arrest of intracellular circulation at elevated temperatures, thereby reducing the delivery rate of proteins as substrates of the intracellular basal proteolytic enzyme system to negligible levels (i.e., to the frequency of encounters due solely to the diffusion of protein molecules with the cytoplasm).     

When the chains do not break: the role of USP10 in physiology and pathology

Deubiquitination is now understood to be as important as its partner ubiquitination for the maintenance of protein half-life, activity, and localization under both normal and pathological conditions. The enzymes that remove ubiquitin from target proteins are called deubiquitinases (DUBs) and they regulate a plethora of cellular processes. DUBs are essential enzymes that maintain intracellular protein homeostasis by recycling ubiquitin. Ubiquitination is a post-translational modification where ubiquitin molecules are added to proteins thus influencing activation, localization, and complex formation. Ubiquitin also acts as a tag for protein degradation, especially by proteasomal or lysosomal degradation systems. With ~100 members, DUBs are a large enzyme family; the ubiquitin-specific peptidases (USPs) being the largest group. USP10, an important member of this family, has enormous significance in diverse cellular processes and many human diseases. In this review, we discuss recent studies that define the roles of USP10 in maintaining cellular function, its involvement in human pathologies, and the molecular mechanisms underlying its association with cancer and neurodegenerative diseases. We also discuss efforts to modulate USPs as therapy in these diseases.Subject terms: Cell biology, Cell signalling     

Effects of platelet-derived growth factor on degradation, nuclear translocation of nonhistone proteins, and DNA synthesis.

Stimulation of resting normal rat kidney fibroblasts, prelabeled with [3H]leucine, by platelet-derived growth factor (PDGF) caused inhibition of cellular protein degradation and a parallel increased nuclear translocation of 3H-labeled nonhistone proteins (3H-NHP) and DNA synthesis. Nuclear translocation of these proteins was independent of protein synthesis. Fractionation of the nuclear 3H-NHP in a pH gradient of 2.5-6.5 showed that the protein fractions with a high degree of proteolysis in resting cells corresponded to the protein fractions with a high extent of translocation in stimulated cells, suggesting that degradation and translocation of these proteins may be related. PDGF inhibited cellular uptake of [3H]chloroquine, suggesting that PDGF inhibits NHP degradation via the lysosomal pathway. These observations support the hypothesis that PDGF induces NHP translocation to the nucleus by inhibiting lysosomal degradation of these proteins.     

The degradation of endogenous and exogenous proteins in cultured smooth muscle cells

The pathways of degradation followed by endogenous proteins in cultured smooth muscle cells were compared with the well-characterized lysosomal pathway involved in the degradation of apolipoprotein B of endocytosed LDL. Under conditions in which lysosomal activity towards ¹²⁵I-labeled LDL was almost completely inhibited by chloroquine and/or ammonium chloride, the degradation of short-lived and abnormal proteins, assessed by the release of [³H]phenylalanine, was reduced by only 10–17%. The basal rate of degradation of long-lived proteins was reduced by about 30% by the same inhibitors while the accelerated proteolysis found under nutrient-poor conditions could be completely accounted for by the lysosomal system as defined by these lysosomotrophic agents. Temperature studies indicated differences between the mechanisms involved in the degradation of long-lived proteins (E_a = 18 kcal/mol) and short-lived proteins (E_a = 10 kcal/mol). Arrhenius plots for the degradation of endogenous proteins showed no transitions between 15 and 37°C in contrast to the breakdown of LDL which ceased below 20°C. The results indicate that the degradation of rapid-turnover proteins is largely extralysosomal and that a significant breakdown of long-lived proteins occurs also outside lysosomes.     

Disruption of the autophagy-lysosome pathway is involved in neuropathology of the nclf mouse model of neuronal ceroid lipofuscinosis

Variant late-infantile neuronal ceroid lipofuscinosis, a fatal lysosomal storage disorder accompanied by regional atrophy and pronounced neuron loss in the brain, is caused by mutations in the CLN6 gene. CLN6 is a non-glycosylated endoplasmic reticulum (ER)-resident membrane protein of unknown function. To investigate mechanisms contributing to neurodegeneration in CLN6 disease we examined the nclf mouse, a naturally occurring model of the human CLN6 disease. Prominent autofluorescent and electron-dense lysosomal storage material was found in cerebellar Purkinje cells, thalamus, hippocampus, olfactory bulb and in cortical layer II to V. Another prominent early feature of nclf pathogenesis was the localized astrocytosis that was evident in many brain regions and the more widespread microgliosis. Expression analysis of mutant Cln6 found in nclf mice demonstrated synthesis of a truncated protein with a reduced half-life. Whereas the rapid degradation of the mutant Cln6 protein can be inhibited by proteasomal inhibitors, there was no evidence for ER stress or activation of the unfolded protein response in various brain areas during postnatal development. Age-dependent increases in LC3-II, ubiquitinated proteins, and neuronal p62-positive aggregates were observed, indicating a disruption of the autophagy-lysosome degradation pathway of proteins in brains of nclf mice, most likely due to defective fusion between autophagosomes and lysosomes. These data suggest that proteasomal degradation of mutant Cln6 is sufficient to prevent the accumulation of misfolded Cln6 protein, whereas lysosomal dysfunction impairs constitutive autophagy promoting neurodegeneration.     

Accelerated degradation of mislocalized UDP-glucuronosyltransferase family 1 (UGT1) proteins in Gunn rat hepatocytes

Gunn rat is a hyperbilirubinemic rat strain that is inherently deficient in the activity of UDP-glucuronosyltransferase form 1A1 (UGT1A1). A premature termination codon is predicted to produce truncated UGT1 proteins that lack the COOH-terminal 116 amino acids in Gunn rat. Pulse-chase experiments using primary cell cultures showed that the truncated UGT1A1 protein in Gunn rat hepatocytes was synthesized similarly to wild-type UGT1A1 protein in normal Wistar rat hepatocytes. However, the truncated UGT1A1 protein was degraded rapidly with a half-life of about 50 min, whereas the wild-type UGT1A1 protein had a much longer half-life of about 10 h. The rapid degradation of truncated UGT1A1 protein was inhibited partially but not completely by treating Gunn rat hepatocytes with proteasome inhibitors such as carbobenzoxy-Leu-Leu-leucinal and lactacystin. By contrast, neither the lysosomal cysteine protease inhibitor nor the calpain inhibitor slowed the degradation. Our findings show that the absence of UGT1 protein from Gunn rat hepatocytes is due to rapid degradation of the truncated UGT1 protein by the proteasome and elucidate the molecular basis underlying the deficiency in bilirubin glucuronidation.     

Regulation of lamp2a levels in the lysosomal membrane

The selective degradation of cytosolic proteins in lysosomes by chaperone-mediated autophagy depends, at least in part, on the levels of a substrate receptor at the lysosomal membrane. We have previously identified this receptor as the lysosome-associated membrane protein type 2a (lamp2a) and showed that levels of lamp2a at the lysosomal membrane directly correlate with the activity of the proteolytic pathway. Here we show that levels of lamp2a at the lysosomal membrane are mainly controlled by changes in its half-life and its distribution between the lysosomal membrane and the matrix. The lysosomal degradation of lamp2a requires the combined action of at least two different proteolytic activities at the lysosomal membrane. Lamp2a is released from the membrane by the action of these proteases, and then the truncated lamp2a is rapidly degraded within the lysosomal matrix. Membrane degradation of lamp2a is a regulated process that is inhibited in the presence of substrates for chaperone-mediated autophagy and under conditions that activate that type of autophagy. Uptake of substrate proteins also results in transport of some intact lamp2a from the lysosomal membrane into the matrix. This fraction of lamp2a can be reinserted back into the lysosomal membrane. The traffic of lamp2a through the lysosomal matrix is not mediated by vesicles, and lamp2a reinsertion requires the lysosomal membrane potential and protein components of the lysosomal membrane. The distribution of lamp2a between the lysosomal membrane and matrix is a dynamic process that contributes to the regulation of lysosomal membrane levels of lamp2a and consequently to the activity of the chaperone-mediated autophagic pathway.     

Regeneration of cilia in starved Tetrahymena thermophila involves induced synthesis of ciliary proteins but not synthesis of membrane lipids.

The synthesis of ciliary-membrane phospholipids and ciliary proteins was studied after deciliation in starving Tetrahymena thermophila cells. Deciliated cells regenerated the new ciliary membrane without any induced phospholipid synthesis. The constant cell volume found during the regrowth of the cilia suggests that renewal of ciliary membranes takes place by insertion of intracellular membrane material into the cell surface. In contrast with the absence of induced phospholipid synthesis during ciliary regeneration, the synthesis of ciliary proteins was found to be induced. This enhanced synthetic activity was made possible by an increased rate of intracellular protein degradation in regenerating cells. It was found that the extent of the induced synthesis strongly depends upon the growth conditions of the cells before starvation. Furthermore, it was shown that the degree of induced protein synthesis is greater for higher-molecular-weight ciliary proteins than for lower-molecular-weight species.     

Inhibition of pyruvate carboxylase degradation and total protein breakdown by lysosomotropic agents in 3T3-L1 cells.

1. Exposure to [3H]biotin during the differentiation of 3T3-L1 cells to adipocytes selectively labelled pyruvate carboxylase (EC 6.4.1.1). A subsequent incubation of labelled cells permitted the measurement of the degradation rate constant of this mitochondrial enzyme. 2. In medium without serum, pyruvate carboxylase was degraded with a half-life of 64 h, considerably longer than that found for average cell protein. The long half-life is commensurate with the enzyme being catabolized when whole mitochondria are destroyed. 3. The breakdown of pyruvate carboxylase was inhibited to a greater extent than the breakdown of total cell protein by insulin, NH4Cl and inhibitors of lysosomal proteinases, suggesting that the enzyme is degraded by the autophagic lysosomal system of the cell. 4. The above evidence implies that whole mitochondria are degraded in lysosomes, a conclusion that agrees with earlier electron-microscopic evidence showing mitochondria within autophagic vacuoles. 5. A second degradative pathway must be invoked to account for the breakdown of mitochondrial proteins of short half-life.     

Inhibition of lysosomal protein degradation inhibits the basal degradation of 3-hydroxy-3-methylglutaryl coenzyme A reductase

The effect of inhibiting lysosomal protein degradation on the activity of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase was determined using a mouse mammary cell line (TS-85) which expresses a temperature-sensitive mutation in the ubiquitin degradative pathway. Incubating cells for 18 hr in medium containing 20 mM NH4Cl did not alter total protein synthesis or cell growth, but it did inhibit the rate of total protein degradation by 19%, which is consistent with the known inhibitory effect of NH4Cl on lysosomal protein degradation. NH4Cl treatment also resulted in an increase (81% +/- 20) in HMG-CoA reductase activity. The increase in reductase activity was not correlated with changes in the phosphorylation state of the enzyme or with alteration in the relative rate of reductase synthesis. However, the basal degradation rate of the reductase was significantly inhibited, and after NH4Cl treatment, the half-life of the enzyme increased from 4.0 +/- 0.4 hr to 8.3 +/- 0.8 hr. The change in the rate of reductase degradation can account completely for the increase in reductase activity observed in NH4Cl-treated cells. The accelerated degradation of HMG-CoA reductase induced by 25-hydroxycholesterol treatment was not affected by either NH4Cl or by inactivation of the ubiquitin degradative pathway. Therefore, two different mechanisms may be responsible for the accelerated degradation and basal degradation of HMG-CoA reductase. The latter can be inhibited by NH4Cl and may imply that under basal conditions the enzyme may be degraded in lysosomes.     

Degradation of oxidized extracellular proteins by microglia

In living organisms a permanent oxidation of protein oxidation occurs. The degradation of intracellular oxidized proteins is intensively studied, but knowledge about the fate of oxidatively modified extracellular proteins is still limited. We studied the fate of exogenously added oxidized proteins in microglial cells. Both primary microglial cells and RAW cells are able to remove added oxidized laminin and myelin basic protein from the extracellular environment. Moderately oxidized proteins are degraded most efficiently, whereas strongly oxidized proteins are taken up by the microglial cells without an efficient degradation. Activation of microglial cells enhances the selective recognition and degradation of moderately oxidized protein substrates by proteases. Inhibitor studies also revealed an involvement of the lysosomal and the proteasomal system in the degradation of extracellular proteins. These studies let us conclude that microglial cells are able to remove oxidized proteins from the extracellular environment in the brain.     

Degradation of structurally characterized proteins injected into HeLa cells. Effects of intracellular location and the involvement of lysosomes

Thirty-five proteins of known x-ray structure were radioiodinated and injected into HeLa cells. The cells were then cultured in the presence or absence of the lysosomotropic agents, ammonium chloride and chloroquine. These compounds did not inhibit the degradation of an injected protein unless its half-life was greater than 45 h. Among the more stable proteins the extent of inhibition was proportional to their half-lives. These results indicate that all injected proteins are transferred to lysosomes at comparable rates such that the fraction of a specific protein degraded in lysosomes depends upon its rate of degradation in the cytosol. That is, basal autophagy is nonselective in HeLa cells. The intracellular location of each injected protein was measured by homogenization of injected cells in sucrose and differential sedimentation or by extraction in buffers containing Triton X-100. Solubilities of the injected proteins ranged from 6 to 89%, and stabilities of 10 proteins, originally extracellular in function, were inversely proportional to their solubility. These results illustrate the potential importance of subcellular location on protein stability in the cytosol.