Amino acid control of intracellular protein degradation.
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Amino Acids↗
Biomedical subjects
G E Mortimore
Publications and source records attributed to G E Mortimore.
Uptake and intracellular fate of [14C]sucrose-insulin in perfused rat livers.
Insulin was covalently linked to [14C]sucrose by means of cyanuric chloride to provide a label that would remain entrapped within the vacuolar system. The uptake of the conjugate by the perfused rat liver was rapid (half-life = 2.9 min), competitively inhibited by native insulin, and abolished by alkali denaturation. As assessed by its distribution on self-generating gradients of colloidal silica-povidone, label in lysosome-enriched samples of liver taken at different times after the addition of the conjugate moved progressively during 15 min from the plasma membrane into an intermediate peak and then to dense lysosomal fractions. After 30-60 min, the label had equilibrated throughout the lysosomal-vacuolar system. The initial movement from the plasma membrane to the intermediate peak occurred between 2 and 5 min. Because label in the peak could be physically separated from the lysosomal marker, beta-acetylglucosaminidase, by dispersing the sample through the gradient mixture before centrifugation rather than layering it, we concluded that the intermediate particles in question were not lysosomal in nature. On gel-filtration chromatography, label extracted from the intermediate peak did not move with insulin but rather as a broad band of lower molecular weight products, suggesting that insulin is subject to early proteolytic attack within a nonlysosomal compartment.
Acetylglucosaminidase↗
Multiphasic control of hepatic protein degradation by regulatory amino acids. General features and hormonal modulation.
Previous studies with livers from fed rats perfused in the single-pass mode have shown that regulatory amino acids (Leu, Tyr, Gln, Pro, Met, His, and Trp) as a group as well as leucine alone inhibit deprivation-induced protein degradation optimally at 0.5 and 4 times (X) normal plasma amino acid concentrations. However, they lose inhibitory effectiveness almost completely within a narrow zone centered at normal (1 X) levels (Pösö, A. R., Wert, J. J., Jr., and Mortimore, G.E. (1982) J. Biol. Chem. 257, 12114-12120; Pösö, A. R., and Mortimore, G. E. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 4270-4274). We now report similar effects for tyrosine and glutamine and suggest that this multiphasic dose response is a general feature of the regulatory group. Insulin (2.4 micrograms h-1) selectively modulated the response by abolishing the zonal loss, whereas glucagon (10 micrograms h-1) blocked the initial inhibition (0.5 X); proteolytic suppression was restored at 4 X normal plasma levels. Although the zonal loss of inhibition at 1 X was associated with a near maximal increase in the volume density of macroautophagy, the vacuoles differed from those induced by stringent amino acid deprivation in containing 4.5-fold more smooth than rough endoplasmic reticulum and thus represented a separate population. Surprisingly, the leucine analog, L-alpha-hydroxyisocaproate, elicited multiphasic responses identical to those of L-leucine, including inhibition at 0.1 mM (equivalent to 0.5 X Leu). Inasmuch as alpha-ketoisocaproate is not effective at this concentration, the initial suppression of protein degradation could be mediated from a site that recognizes structural features common to leucine and its hydroxyl analog.
Amino Acids↗
RNA degradation in perfused rat liver as determined from the release of [14C]cytidine.
The degradation of RNA in the cyclically perfused rat liver was determined from the release of labeled cytidine from RNA that had been previously labeled with [6-14C]orotic acid in vivo. Because cytidine is not appreciably degraded in rat liver (its deamination to uridine is virtually nil) or produced in significant amounts from free 5'-nucleotides, its release will directly reflect net RNA breakdown. This conclusion was substantiated by the fact that the specific radioactivity of released cytidine equaled that of CMP in RNA and remained unchanged for 180 min of perfusion. The initial rate of [14C]cytidine accumulation was slow, but after 10-20 min it increased abruptly by more than 4-fold and remained virtually constant. The addition of 0.5 mM unlabeled cytidine effectively prevented the reutilization of label and increased the rate of labeled cytidine release by an amount representing 13% of the maximal rate of cytidine accumulation. Rates of RNA degradation, measured between 20 and 60 min in the presence of 0.5 mM unlabeled cytidine, averaged 1.00 +/- 0.05 mg h-1 liver-1 (100-g rat), the equivalent of 65% of total RNA per day. This accelerated value, which was about 4-fold larger than the initial rate, is believed to be the direct consequence of amino acid deprivation since, in separate experiments, the increase was completely suppressed by the addition of plasma amino acids (Lardeux, B. R., and Mortimore, G. E. (1987) J. Biol. Chem. 262, 14514-14519). These findings demonstrate the potential value of cytidine as a marker for following moment-to-moment regulatory alterations in RNA degradation in the isolated liver or hepatocyte preparation.
Animals↗
Amino acid and hormonal control of macromolecular turnover in perfused rat liver. Evidence for selective autophagy.
The control of RNA degradation by amino acids, insulin, and glucagon was investigated in perfused livers of fed rats previously labeled in vivo with [6-14C] orotic acid; rates were determined from the release of [14C]cytidine in the presence of 0.5 mM cytidine to suppress reutilization. Studies with cyclically perfused livers showed that plasma amino acids at 10 times (10X) normal concentrations inhibited RNA breakdown by 85%. Similar inhibition was obtained with a known regulatory amino acid mixture (Leu, Met, Pro, Trp, and His), whereas leucine alone (0.8 mM) decreased degradation by 47%. Perfusions carried out in the single-pass mode with graded levels of plasma amino acids revealed that the acceleration of RNA degradation over the full range of amino acid deprivation (0 to 10X normal levels) was the same as that for protein breakdown (3.19 and 3.15% h-1, respectively), and both were equally suppressed by insulin (2.4 micrograms h-1). Glucagon (10 micrograms h-1), though, was far less effective in stimulating RNA than protein turnover. A direct comparison of the two dose responses revealed a strong dissociation at 1 and 2 times normal amino acid levels. These findings support the notion that RNA and protein are degraded within a single macroautophagic compartment during amino acid and insulin deprivation. Glucagon, however, appeared to induce a second pathway in which the proportion of sequestered RNA to protein was selectively reduced. Electron micrographs showed that the ratio of vacuoles containing rough as compared with smooth endoplasmic reticulum was decreased by nearly 80% under these conditions.
Amino Acids↗
Regulation of lysosomal fusion during deprivation-induced autophagy in perfused rat liver.
Previous studies have demonstrated that autophagic vacuoles induced in hepatocytes of the perfused rat liver can be effectively separated from secondary lysosomes in self-generating gradients of colloidal silica/polyvinylpyrrolidone. Here we show that, as autophagy is increased by graded amino acid deprivation, the lysosomal marker N-acetyl-beta-D-glucosaminidase shifts progressively from dense to buoyant gradient fractions, an effect attributable to enhanced lysosome-autophagosome fusion and the formation of additional autolysosomes. Both the amount of the shift and the aggregate volume of autolysosomes were directly proportional to the increase in protein degradation above basal. Because the total quantity of protein sequestered in the vacuoles is also a direct function of deprivation-induced proteolysis, the relative specific activity of the marker enzyme in autolysosomes (enrichment) would be expected to remain constant over the full range of deprivation; numerical estimates of enrichment, in fact, did not differ from the mean (43.9) by more than 5%. The close correspondence between cytoplasmic sequestration and fusion was maintained, despite a greater than 3-fold increase in the average volume of individual vacuoles. This suggests the existence of an intrinsic regulatory mechanism which limits the number of fusions per vacuole, on the basis of information derived from vacuolar volume.
Amino Acids↗
Inhibition of intracellular protein degradation by ethanol in perfused rat liver.
Ethanol (50 mM) inhibited proteolysis in the perfused rat liver during stringent amino acid deprivation and also in the presence of normal and 10 times normal concentrations of plasma amino acids. The concentration-response curve of ethanol reached a plateau after 5 mM in both the presence and the absence of normal plasma amino acids, suggesting inhibition by oxidation products of ethanol. Intracellular glutamine, tyrosine and proline increased in concentration with ethanol, but the increases were too small to explain the observed inhibition of proteolysis. The uptake of 125I-asialofetuin was slightly decreased and the output of ammonia increased in the presence of ethanol. These, together with a significant suppression of basal proteolysis in the presence of amino acids, suggest that lysosomal function was directly affected. Electron-microscopic examination of lysosomal components showed that the aggregate volume of autophagosomes (initial vacuoles) were significantly smaller in livers perfused with ethanol than in controls. However, the equivalent volume of autolysosomes (degradative vacuoles) was the same in both groups. According to these results, ethanol inhibits protein degradation in the liver by two discrete mechanisms: one decreasing the formation of autophagic vacuoles and the other involving lysosomotropic inhibition, possibly via ammonia.
Amino Acids↗
Intracellular protein catabolism and its control during nutrient deprivation and supply.
The continuous turnover of intracellular protein and other macromolecules is a basic cellular process that serves, among other functions, to regulate cytoplasmic content and provide amino acids for ongoing oxidative and biosynthetic reactions during nutrient deprivation. The intensity of breakdown and pattern of regulation, though, vary widely among cells. Rat hepatocytes, for example, exhibit high absolute rates of proteolysis and regulatory effects that diminish during starvation, while corresponding responses in skeletal and cardiac muscle move in the opposite direction. It is also becoming apparent that effects of insulin and other acute regulatory agents on muscle breakdown are limited to nonmyofibrillar components. The latter may be sequestered and degraded within autophagic vacuoles, whereas myofibrillar proteins require an initial attack by calcium-dependent proteases in the cytosol. By contrast, most if not all of the breakdown of resident (long-lived) proteins as well as RNA in the hepatocyte can be explained by lysosomal mechanisms. The uptake of cytoplasmic components by lysosomes can be divided into two major categories, macroautophagy and micro- or basal autophagy. The first is induced by amino acid or insulin/serum deprivation. In the hepatocyte, amino acids alone can regulate this process almost instantaneously over two thirds of the full range of proteolysis, 4.5% to 1.5% per hour. Glucagon, cyclic AMP, and beta-agonists also stimulate macroautophagy in hepatocytes but have opposite effects in skeletal and cardiac myocytes. Basal autophagy differs from the macro type in that the cytoplasmic "bite" is smaller and sequestration is not acutely regulated. It is, however, adaptively decreased during starvation in parallel with absolute rates of basal turnover. Since endoplasmic reticulum comprises an appreciable fraction of the vacuolar content, volume sequestration would be compatible with the known heterogeneity of individual protein turnover if some proteins (or altered proteins) selectively bind to membranes. The amino acid control of macroautophagy in the hepatocyte is accomplished by a small group of direct inhibitors (Leu, Tyr/Phe, Gln, Pro, Met, Trp, and His) and the permissive effect of alanine whereas only leucine is involved in myocytes and adipocytes. Of unusual interest is the fact that the inhibitory amino acid group alone evokes responses in perfused livers that are identical to those of a complete plasma mixture at 0.5 and 4 times normal plasma levels but loses effectiveness almost completely at normal concentrations.(ABSTRACT TRUNCATED AT 400 WORDS)
Amino Acids↗
The lysosomal pathway of intracellular proteolysis in liver: regulation by amino acids.
The bulk of cellular protein in hepatocytes is sequestered and degraded by two classes of autophagy, (a) an overt or macro form, and (b) microautophagy. Macroautophagy is rapidly induced by amino acid deprivation and the administration of glucagon and suppressed by amino acids and insulin. Amino acids appear to be its primary regulator since liver perfusion studies have shown that it can be inhibited almost completely and proteolysis decreased from maximal (4.5% hr) to basal rates (1.7%/hr) by 4 times normal plasma amino acid concentrations. The resulting alterations in the aggregate volume of autophagic vacuoles are associated with proportional changes in the amount of cytoplasmic protein sequestered and in rates of protein degradation. Since the apparent turnover of autophagic vacuoles is 0.087 min-1, the pools of sequestered protein at all levels of macroautophagic stimulation are sufficient to account fully for the observed rates of accelerated rate of proteolysis. Microautophagy differs from the former in that the cytoplasmic 'bite' is smaller and it is not subject to acute physiological regulation. It is, however, dramatically decreased to near zero during refeeding after prior starvation. These and other findings indicate that it is adaptively regulated, possibly as a consequence of alterations in the amount of smooth endoplasmic reticulum. The amino acid control of accelerated protein degradation appears to involve direct inhibition by a small group of amino acids (Leu, Tyr/Phe, Gln, Pro, Met, Trp, and His) and the permissive action of alanine. Of unusual interest is the fact that, whereas the inhibitory amino acid group evokes responses identical to a complete amino acid mixture at 0.5x and 4x normal amino acid concentrations, it loses its effectiveness at normal levels; similar responses have been shown for leucine alone. The loss of effectiveness at normal concentrations is abolished by the addition of 0.5 mM alanine which by itself is not directly inhibitory. No other amino acid can replace alanine. These findings suggest a novel role for alanine that could be of importance in linking energy demands to proteolysis. A hypothetical model for proteolytic regulation by leucine and the other inhibitory amino acids is presented.
Amino Acids↗
Amino acid control of hepatic protein degradation: novel requirement for alanine.
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Alanine↗
Requirement for alanine in the amino acid control of deprivation-induced protein degradation in liver.
Protein degradation in liver is actively controlled by a small group of inhibitory amino acids--leucine, tyrosine (or phenylalanine), glutamine, proline, histidine, tryptophan, and methionine. Other evidence, however, suggests that one or more of the remaining 12 noninhibitory amino acids is also required for suppression of proteolysis at normal concentrations. This question was investigated in livers of fed rats perfused in the single-pass mode. The deletion of alanine at normal (1x), but not at 4x or 10x normal, plasma amino acid concentrations evoked a near-maximal acceleration of protein degradation. No other noninhibitory amino acid was effective. Because alanine alone was not directly inhibitory and its omission was not associated with a decrease in inhibitory amino acid pools, alanine was presumed to act as a coregulator in the expression of inhibitory activity. When tested alone, the inhibitory group was as effective as the complete mixture at 0.5x and 4x levels, but it lost its suppressive ability within a narrow zone of concentration centered slightly above 1x. The addition of 1x (0.48 mM) alanine completely restored the inhibition. Pyruvate and lactate could be effectively substituted, but only at concentrations 10-20 times greater than that of alanine. These, together with earlier findings, indicate the existence of a regulatory complex that recognizes specific amino acids and transmits positive and negative signals to proteolytic sites. The results also suggest that alanine can provide an important regulatory link between energy demands and protein degradation.
Alanine↗
Liver perfusion: an in vitro technique for the study of intracellular protein turnover and its regulation in vivo.
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Amino Acids↗
Lysosomal pathways in hepatic protein degradation: regulatory role of amino acids.
Cytoplasmic protein in hepatocytes is continuously internalized and degraded by two lysosomal processes, 1) overt or macroautophagy, and 2) microautophagy, the latter involving dense bodies. The first is acutely regulated by amino acids, insulin, and glucagon; the second is also alterable, but responses are slower and probably adaptive in nature, as suggested in starvation-refeeding. Internalized protein in lysosomes was independently assessed from 1) lysosomal volumes and hepatocyte protein concentration and 2) its degradation products in liver homogenates; quantitative agreement was obtained between the two determinations. Agreement was equally close when the results were used to predict rates of hepatic proteolysis over the full range, assuming that k for all sequestered protein is equal to the turnover of macroautophagic vacuoles (0.087 min-1). These findings strongly indicate that internalization is an obligatory step in both phases of protein degradation. Acute proteolytic responses to amino acids were studied in rat livers perfused in the single-pass mode. Twelve amino acids exhibited no suppressive activity at the upper physiological limit of plasma amino acid concentrations (4 X normal), whereas seven were fully effective as a group. Of these, leucine was the most inhibitory at 4 X, but was inactive as 1 X. Deletion of the other regulatory amino acids from normal plasma mixtures, however, accelerated proteolysis strikingly as did omission of glutamine, glutamate, alanine, and glycine. Of the latter, only glutamine was directly inhibitory although its effectiveness was low. Restraint to protein degradation at normal plasma concentrations appears to be complex and to involve specific amino acid regulators as well as glucogenic amino acids in ways not yet understood.
Amino Acids↗
Quantitative correlation between proteolysis and macro- and microautophagy in mouse hepatocytes during starvation and refeeding.
Cytoplasmic protein in hepatocytes is sequestered and degraded by two general classes of lysosomes, overt autophagic vacuoles (macroautophagy) and dense bodies (microautophagy). Volumes of the apparent space in each class that contain the internalized protein, together with estimates of cytoplasmic protein concentration, were used as a basis for predicting rates of protein degradation by the lysosomal system in livers of fed, 48-hr starved, and starved-refed mice. Assuming that the turnover of all sequestered protein is equal to that previously determined in overt autophagic vacuoles (0.087 min-1), we obtained close agreement between predicted and observed rates in the three conditions studied. The two autophagic components, though, exhibited different patterns of regulation. Microautophagy followed a downward course through starvation and into refeeding, a trend that explained fully the fall in absolute rates of protein degradation during starvation. By contrast, macroautophagy remained constant throughout starvation but was virtually abolished with refeeding. Whereas regulation of the latter can be explained largely by immediate responses to the supply of amino acids, present evidence together with results of others indicate that microsequestration could be linked to functional and quantitative alterations in the smooth endoplasmic reticulum. Both types of regulation contributed equally to the marked suppression of proteolysis during cytoplasmic regrowth.
Animals↗
Role of particle interaction on distribution of liver lysosomes in colloidal silica.
Rat liver mitochondrial-lysosomal fractions were separated on gradients of colloidal silica. Lysosomal enzymes were distributed bimodally. The dense peak (1.117 g/ml) was nearly free from contaminants; beta-N-acetyl-D-glucosaminidase was enriched nearly 60-fold. By contrast, the buoyant peak (1.085 g/ml) co-sedimented with mitochondria, microsomes, peroxisomes, and Golgi particles. Decreasing the amount of protein layered on the gradient medium or dispersing a full sample through it shifted lysosomal marker from the buoyant to the dense peak. Thus the majority of lysosomes in the two peaks appeared to have equivalent densities. Electron microscopic examination of particles separated from gradients with layered samples showed that the dense peak contained most of the dense bodies, whereas the buoyant peak was relatively enriched in autophagic vacuoles. Dispersion, however, shifted autophagic vacuoles from the buoyant to the dense peak without affecting the distribution of dense bodies. We conclude that the bulk of buoyant particles act as a sieve to retard the density equilibration of autophagic vacuoles without specifically affecting other lysosomal enzyme-containing components.
Acetylglucosaminidase↗
Metabolic alterations and distribution of rat liver lysosomes in colloidal silica.
Lysosome-enriched samples from normal fed rat livers have been shown to separate into dense and buoyant bands when layered on self-generating gradients of colloidal silica. The hypothesis that the buoyant peak is comprised largely of autophagic vacuoles that are selectively restrained by sedimenting mitochondria was tested in livers subjected to conditions known to accelerate autophagy or suppress it. In all cases, a sharp peak of beta-N-acetyl-D-glucosaminidase activity was evident when autophagy was present but was undetectable when it was eliminated or virtually eliminated. Moreover, the sensitivity to osmotic shock of buoyant peak particles, but not those of the dense band, was increased after autophagic stimulation. The total amount of enzyme that moved to buoyant fractions with the induction of autophagy generally correlated with the degree of autophagic acceleration. However, with maximal autophagy, the buoyant peak was broad, and only 70% was capable of being dislodged and returned to the dense peak by dispersing samples in the gradient medium and/or reducing the sample size. On the other hand, at roughly half-maximal stimulation and lower, the buoyant distribution was sharply defined and completely reversible. These findings support the above hypothesis and suggest that the buoyant peak could be used as a measure of autophagy in the physiological range of regulation.
Acetylglucosaminidase↗
Multifunctional control of amino acids of deprivation-induced proteolysis in liver. Role of leucine.
The control of intracellular proteolysis by amino acids was investigated in rat livers perfused in the single pass mode with various mixtures at multiples of normal plasma concentrations. Twelve amino acids together exhibited no suppressive activity while the remainder (Leu, Phe, Tyr, Gln, Pro, His, Trp, and Met) were inhibitory. Because (a) the effects of phenylalanine and tyrosine were equal and nonadditive, and (b) the former is rapidly converted to the latter, tyrosine rather than phenylalanine was considered to be an active regulator. The inhibitory group, minus phenylalanine, duplicated responses of the complete mixture between 0 and 4 times (4x) normal plasma levels. When tested individually, leucine was the strongest inhibitor, suppressing deprivation-accelerated proteolysis by 63% at 4x and 10x. However, at lower concentrations its effects differed dramatically from those of the regulatory mixture in showing a sharp modal loss of responsiveness at approximately 1x, a finding which suggests that proteolytic regulation at normal levels is achieved largely by the remaining 6 amino acids. Leucine responses from 1x to 4x (0.2 to 0.8 mM) were mimicked by equimolar additions of alpha-ketoisocaproate. The fact that 0.8 mM alpha-ketoisocaproate increased intracellular leucine by only 7.8% of the value obtained with equimolar (4x) leucine supports observations of others that transamination limits interconversion between leucine and its alpha-ketoacid to 4-10% of total alpha-ketoisocaproate utilization at this level. We conclude that the effects of leucine and alpha-ketoisocaproate on hepatic proteolysis are mediated by separate pathways.
Amino Acids↗
Regulation of phenylalanine hydroxylase activity by phenylalanine in vivo, in vitro, and in perfused rat liver.
We show that phenylalanine is able to control the extent of activation and, as a result, the catalytic activity of rat liver phenylalanine hydroxylase in vivo, in perfused liver, and in vitro. Both phosphorylated and unphosphorylated enzyme activities are controlled by phenylalanine activation and, overall, this mechanism appears to be a major means of regulating the enzyme's activity in rat liver. At normal phenylalanine levels in vivo, phenylalanine hydroxylase is at most 1-4% activated, and phosphorylated enzyme (glucagon-induced) appears at most 5-7% activated under similar conditions. In both cases, a phenylalanine load increased the percentage of activated enzyme found in vivo to about 40% of maximal. In perfused rat livers, a plasma phenylalanine concentration of only 4 times normal induced a 4-fold increase in the amount of activated enzyme present and a corresponding functional increase in the rate of phenylalanine hydroxylation by the tissue. Under the latter conditions, more than 25% of the amino acid could be hydroxylated in a single pass through the organ. Purified phosphorylated phenylalanine hydroxylase must be activated to be catalytically active. The activation with phenylalanine, at equilibrium, is a cooperative process, and the phosphorylated enzyme is activated more rapidly at pH 6.8 and 8.0 and at lower phenylalanine concentration than the unphosphorylated species. Overall, phosphorylation appears to allow phenylalanine hydroxylase to be more easily activated at relatively low phenylalanine concentrations.
Animals↗