Degradation of a monoclonal anti-mu chain antibody in a human surface IgM-positive B cell line starts in prelysosomal vesicle

The surface IgM-mediated endocytosis and intracellular transport of an anti-F(c mu)5 mAb was studied by using subcellular fractionation in sucrose gradients. The results of such experiments showed that antibody was initially endocytosed in vesicles of low density, and later transferred to a presumably lysosomal compartment of higher density. SDS-PAGE analysis of gradient fractions showed that high Mr degradation fragments of the endocytosed antibody were formed in the low density vesicles before terminal degradation could be recorded. The partial degradation of the antibody was not blocked by low temperature or enzyme inhibitors, such as leupeptin and benzyloxycarbonyl-phenylalanylalanine-diazomyethyl-ketone, all of which severely retarded terminal degradation. The data also suggested that the recycling of partially degraded antibody to the cell surface employed a pool of such low density prelysosomal vesicles.     

Autophagy is not a main contributor to the degradation of phospholipids in tobacco cells cultured under sucrose starvation conditions

Net degradation of cellular components occurs in plant cells cultured under starvation conditions, and autophagy contributes to the degradation of intracellular proteins. In this study, we investigated the degradation of membrane phospholipids by autophagy in cultured tobacco (Nicotiana tabacum) cells. The amounts of total phospholipids and a major phospholipid, phosphatidylcholine (PC), decreased, whereas phosphorylcholine, a degradation product of PC, increased in response to deprivation of sucrose. The addition of glycerol to the culture medium inhibited both the degradation of phospholipids and the concomitant increase of phosphorylcholine. Glycerol, however, did not block autophagy, which was assessed by the accumulation of autolysosomes in the presence of a cysteine protease inhibitor. On the other hand, 3-methyladenine, an inhibitor of autophagy, did not affect the net degradation of PC. We labeled intracellular phospholipids by loading cells with a fluorochrome-labeled fatty acid and chased it under sucrose-free conditions. Glycerol slowed down the decrease in the amount of fluorochrome-labeled PC, suggesting that it inhibits the degradation process of PC. These results show that phospholipids are degraded by mechanisms different from autophagy in tobacco cells cultured under sucrose-free conditions.     

Autophagy in tobacco BY-2 cells cultured under sucrose starvation conditions: isolation of the autolysosome and its characterization

Tobacco culture cells carry out a large-scale degradation of intracellular proteins in order to survive under sucrose starvation conditions. We have previously suggested that this bulk degradation of cellular proteins is performed by autophagy, where autolysosomes formed de novo act as the major lytic compartments. The digestion process in autolysosomes can be retarded by addition of the cysteine protease inhibitor E-64c to the culture medium, resulting in the accumulation of autolysosomes. In the present study, we have investigated several properties of autolysosomes in tobacco cells. Electron microscopy showed that the autolysosomes contain osmiophilic particles, some of which resemble partially degraded mitochondria. It also revealed the presence of two kinds of autolysosome precursor structures; one resembled the isolation membrane and the other the autophagosome of mammalian cells. Immunofluorescence microscopy showed that autolysosomes contain acid phosphatase, in accordance with cytochemical enzyme analyses by light and electron microscopy in a previous study. Autolysosomes isolated by cell fractionation on Percoll gradients showed the localization of acid phosphatase, vacuolar H(+)-ATPase and cysteine protease. These results show that starvation-induced autophagy in tobacco cells follows a macroautophagic-type response similar to that described for other eukaryotes. However, our results indicate that, although the plant vacuole is often described as being equivalent to the lysosome of the animal cell, a new low pH lytic compartment-the autolysosome-also contributes to proteolytic degradation when tobacco cells are subjected to sucrose deprivation.     

HDL3 degradation by proteases in isolated rat intestinal mucosal cells

Human 125I-labelled HDL3 is degraded by isolated rat intestinal mucosal cells. In our experimental conditions, lipoprotein degradation occurred by two different mechanisms. In one, lipoprotein was degraded within the cell, following binding and internalisation. In the other, degradation occurred in the medium, which seemed to contain protease activity released from cells during incubation. Though lipoprotein-deficient serum apparently interfered with degradation in the medium, bovine serum albumin had no such effect. The lysosomal inhibitor, chloroquine, reduced degradation by 60% without inhibiting HDL binding. Intestinal cell extracts contained at least two different proteases, with pH optima of 4.5 and 8.0, respectively. Comparing HDL and LDL degradation on a molar basis, more HDL particles were degraded by the cell-free extracts at pH 4.5. This degradation was activated by dithiothreitol and was inhibited by iodoacetic acid. From these observations we conclude that HDL3 is taken up by the rat intestinal mucosal cell through a specific binding site and subsequently degraded by a thiol-dependent protease in the lysosome.     

Ubiquitin-proteasome-dependent degradation of apolipoprotein B100 in vitro

Apolipoprotein B100 (apoB) is a large secretory protein that forms very low density lipoprotein in liver. An in vitro degradation assay was developed using rabbit reticulocyte (RR) lysate in order to investigate the mechanism of intracellular degradation of newly synthesized apoB by the ubiquitin-proteasome pathway. [³H]apoB, isolated from [³H]leucine pulsed/chased Hep G2 cells, was degraded 51% when incubated for 2 h at 37°C in an assay mixture that included RR lysate (source of the ubiquitin conjugation system and proteasome) and an exogenous ATP regenerating system. ApoB degradation was ATP-dependent and degradation fragments were not observed suggesting that the very large apoB molecule was extensively degraded. ApoB degradation was decreased to 50% when potent proteasome inhibitors, clasto-lactacystin β-lactone (10 μM) or MG-132 (50 μM), were added to the reaction mixture, but was not affected by the cysteine protease inhibitor, E-64, or the serine protease inhibitor, phenylmethylsulfonyl fluoride. ApoB degradation was inhibited by the mutant ubiquitin protein K48R and by ubiquitin aldehyde, an inhibitor of ubiquitin-protein isopeptidases. During incubation ubiquitination of apoB increased even as apoB was being degraded. These results suggest that in vitro degradation of apoB, a large secretory protein that is normally found in the endoplasmic reticulum (ER) lumen or associated with the ER membrane, was proteasome-dependent and involved both ubiquitination and deubiquitination steps.     

Examination of the contribution of vacuolar proteases to intracellular protein degradation in Chara corallina.

The contribution of proteases in the central vacuole of Chara corallina internodal cells to overall cellular protein degradation was examined. I measured the decrease in the trichloroacetic acid (TCA)-precipitable radioactivity in the cell for a 6-d chase period after labeling cellular proteins with [3H]leucine. The kinetics of [3H]leucine-labeled protein disappearance showed that the half-life of the cellular soluble proteins was 4 to 5 d. This value did not change when cells were treated with (2S,3S)-trans-epoxysuccinyl-L-leucylamido- 3-methyl-butane ethyl ester, a permeant inhibitor of cysteine proteases. This inhibitor mostly inhibited bovine serum albumin-degrading activity in the vacuole. I also measured the release of TCA-soluble radioactivity from the TCA-insoluble fraction in the cell. This experiment showed that 13% of [3H]leucine-labeled cellular proteins were degraded in 1 d. This value agreed well with the half-life obtained for soluble proteins in the above experiment. This value did not change even when both trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane, a cysteine protease inhibitor, and pepstatin A, an aspartic protease inhibitor, were introduced into the vacuole. With this operation, bovine serum albumin-degrading activity in the vacuole was almost completely inhibited. These data suggest that the cytoplasmic but not the vacuolar proteases contribute to cellular protein turnover in Chara internodal cells.     

Internalization and stepwise degradation of heparan sulfate proteoglycans in rat hepatocytes.

Intracellular transport and degradation of membrane anchored heparan sulfate proteoglycans (HSPGs) were studied in cultured rat hepatocytes labeled with [35S]sulfate and [3H]glucosamine. Pulse chase experiments showed that membrane anchored HSPGs were constitutively transported to the cell surface after completion of polymerization and modification of the glycosaminoglycan chains in the Golgi apparatus. The intact HSPGs had a relatively short residence time at the cell surface and in non-degrading compartments (T(1/2) approximately 2-3 h), while [35S]sulfate labeled degradation products were found in lysosomes, and to a lesser extent in late endosomes. These degradation products which were free heparan sulfate chains with little or no protein covalently attached, were approximately half the size of the original glycosaminoglycan chains and were the only degradation intermediate found in the course of HSPG catabolism in these cells. In cells incubated in the presence of the microtubule perturbant vinblastine, or in the presence of the vacuolar ATPase inhibitor bafilomycin A1, and in cells incubated at 19 degrees C, the endocytosed HSPGs were retained in endosomes and no degradation products were detected. Disruption of lysosomes with glycyl-phenylalanine 2-naphthylamide (GPN) revealed a GPN resistant degradative compartment with both intact and partially degraded HSPGs. This compartment probably corresponds to late endosomes. Treatment of hepatocytes with the thiol protease inhibitor leupeptin inhibited the final degradation of the protein moiety of the HSPGs. The protein portion seems to be degraded completely before the glycosaminoglycan chains are cleaved. The degradation of the glycosaminoglycan chains is rapid and complete with one observable intermediate.     

Degradation of transplanted rat liver mitochondrial-outer-membrane proteins in hepatoma cells.

Reductively [3H]methylated 3H mitochondrial-outer-membrane vesicles from rat liver and vesicles where monoamine oxidase has been derivatized irreversibly by [3H]-pargyline have been deliberately miscompartmentalized by heterologous transplantation into hepatoma (HTC) cells by poly(ethylene glycol)-mediated vesicle-cell fusion. Fluorescein-conjugated mitochondrial-outer-membrane vesicles have also been used to show that transplanted material is patched, capped and internalized. Reductively methylated outer-membrane proteins and monoamine oxidase are destroyed at the same rate (t1/2 24 h). Mitochondrial-outer-membrane proteins are not degraded at the same rate as HTC plasma-membrane proteins, endogenous cell protein, or endocytosed protein. Transplanted radiolabelled mitochondrial-outer-membrane proteins accumulate intracellularly in structures that are distinct from plasma membrane and lysosomes. However, when mitochondrial-outer-membrane vesicles derivatized with [14C]sucrose are transplanted, the acid-soluble degradation products accumulate in the lysosomal fraction. [14C]Sucrose-conjugated HTC cell plasma membrane accumulates in intracellular structures that are again distinct from plasma membrane and lysosomes. In contrast with the above observations, homologously transplanted mitochondrial-outer-membrane proteins from rat liver are destroyed in hepatocytes at rates that are remarkably similar (t1/2 60-70 h) to the rates in rat liver in vivo [Evans & Mayer (1982) Biochem. Biophys. Res. Commun. 107, 51-58].     

Modulation of red cell vesiculation by protease inhibitors

Release of vesicles from human red cell membranes was induced either by ATP-depletion or by incubation of the cells in presence of sonicated dimyristoylphosphatidylcholine (DMPC) vesicles. Vesicles released from ATP-depleted red cells but not the DMPC-induced vesicles contained degradation products of band 3 protein. Furthermore, in ATP-depleted erythrocytes proteolytic breakdown products could be demonstrated that were not detected in cells incubated with DMPC. Proteolysis was neither significantly affected by the protease inhibitor N-alpha-tosyl-L-lysine chloromethyl ketone (TLCK) nor by other protease inhibitors tested in this study (diisopropylfluorophosphate, N-ethylmaleimide and phenylmethylsulfonyl fluoride). Both vesiculation processes were inhibited in a concentration dependent way by TLCK while other protease inhibitors did not significantly influence membrane vesiculation. Phase contrast microscopy showed that TLCK diminished the DMPC-induced formation of echinocytes which is known to precede vesicle release. These results suggest that the influence of TLCK on membrane vesiculation is not primarily due to inhibition of proteolysis but to a direct interaction of the inhibitor with the intrinsic domain of the erythrocyte membrane.     

Further Evidence for Stachyose and Sucrose/H+ Antiporters on the Tonoplast of Japanese Artichoke (Stachys sieboldii) Tubers

Vacuoles of Japanese artichoke (Stachys sieboldii) tubers accumulate up to 180 mM stachyose ([alpha]-galactose-[1->6]-[alpha]-galactose-[1->6]-[alpha]-glucose-[1 <->2]-[beta]-fructose) against a concentration gradient, probably by means of an active stachyose/H+ antiporter situated on the tonoplast. The goal of this study was to use isolated tonoplast vesicles to provide further evidence for the existence of such a transport mechanism. Therefore, vesicles were prepared from purified vacuoles of dormant tubers. ATP- and pyrophosphate (PPi)-dependent fluorescence quenching of the [delta]pH probe 9-amino-6-chloro-2-methoxyacridine (ACMA) indicated that these vesicles were capable of building up a pH gradient ([delta]pH, inside acid). The potent V-type H+-ATPase inhibitor bafilomycin prevented the formation of a [delta]pH in the vesicles. Bafilomycin (as well as nitrate, but not vanadate) also inhibited ATP hydrolysis, confirming the tonoplast origin of the isolated vesicles. Addition of stachyose (or sucrose, but not of mannitol) to energized vesicles caused a recovery of ACMA fluorescence, indicating a sugar-dependent dissipation of [delta]pH. The rate of fluorescence recovery was dependent on the external sugar concentration used. It displayed a single saturable response to increasing sugar concentrations. Apparent Km values of 52 and 25 mM were computed for stachyose and sucrose antiporter activities, respectively. It was also demonstrated that energized vesicles showed a much higher rate of [14C]stachyose (3 mM) and [14C]sucrose (1 mM) uptake than deenergized vesicles. The results obtained with isolated tonoplast vesicles were very similar to those obtained earlier with intact vacuoles and, therefore, confirm the existence of active stachyose and sucrose/H+ antiporters on the tonoplast of Stachys tuber vacuoles.     

The Involvement of Tissue-Type Plasminogen Activator in Parietal Endoderm Outgrowth

When F9 stem cells are treated in suspension with retinoic acid, they differentiate into embryoid bodies (EBs) consisting of an inner core of undifferentiated stem cells surrounded by an outer layer of visceral endoderm (VE). When these EBs are plated onto a fibronectin (FN)-coated substrate, VE-derived parietal endoderm (PE) cells migrate onto the substrate. It has been suggested that increased levels of tPA associated with the emerging PE cells may help mediate PE outgrowth. We now show that goat anti-human tPA, an anticatalytic antibody that crossreacts with mouse tPA, and a panel of serine protease inhibitors partially inhibit PE outgrowth. Extracellular matrix (ECM) degradation analysis demonstrates that PE cell-mediated degradation of [³H]proline-labeled ECM is time- and cell concentration-dependent. A serine protease inhibitor reduced the extent of degradation, suggesting that tPA might play a role in PE outgrowth by cleaving the ECM. In support of this contention, we demonstrate that incubation of purified FN with conditioned medium plus plasminogen results in FN proteolysis. The degradation of FN is blocked by either serine protease inhibitors or goat anti-human tPA. Our data suggest that enhanced production of tPA during PE outgrowth may facilitate the migratory behavior of PE cells by mediating the degradation of ECM components such as FN.     

Degradation of zinc-metallothionein in monolayer cultures of rat hepatocytes

The degradation of zinc-metallothionein (MT) was studied in monolayer cultures of adult rat hepatocytes. Hepatocytes were incubated overnight in serum-free medium containing either [35S]cysteine or [3H]leucine and 100 microM zinc to induce MT synthesis. Total cellular 35S-MT was measured in the heat-stable extract of cell homogenate and quantified by fast protein liquid chromatography. When zinc was removed from the medium, 35S-MT turnover was almost 3-fold faster than that of [3H]Leu protein (t1/2 = 11 and 29 hr, respectively). The decrease in the cellular level of 35S-MT reflected degradation since less than 1% of total cellular 35S-MT was secreted into the medium. The rate of MT degradation was inversely proportional to cellular zinc content. In contrast, the degradation of [3H]Leu protein was not affected by changes in cellular zinc concentration. Chloroquine, a lysosomotrophic amine, and tosyl lysine chloromethyl ketone, an inhibitor of trypsin-like neutral protease activity, inhibited 35S-MT degradation by 65% and 50%, respectively, when cells were incubated in medium with 1 microM zinc. Turnover of [3H]Leu protein, but not 35S-MT, was enhanced by insulin deprivation. These data suggest that the degradation of hepatic MT (i) is primarily regulated by cellular zinc content and (ii) occurs in both lysosomal and nonlysosomal compartments.     

Effect of protease inhibitors on androgen receptor analysis in rat prostate cytosol

Prostate androgen receptors are liable to proteolytic digestion during in vitro analysis; thus, various proteolytic enzyme inhibitors were tested for their ability to improve the androgen receptor assay. The serine (phenylmethylsulfonylflouride, aprotinin, p-aminobenzamidine) and thiol-senine (leupeptin, bacitracin) protease inhibitors individually present in the homogenization buffer significantly increased the measurable androgen binding sites by 30-35% in rat prostate cytosol as determined by saturation analysis with [3H]-17 beta-hydroxy-17-methyl- 4,9 11-estratrien-3-one (R-1881) for 20 hr at 4 degrees C. The apparent binding affinity was also increased by these compounds. Various combinations were tried and aprotinin/bacitracin was found to be additive in effect. This combination was also shown to prevent receptor degradation as determined by sucrose density gradient centrifugation. The carboxyl protease inhibitor, pepstatin A, was ineffective in improving the receptor assay. Rabbit bile, an inhibitor of seminin, interfered with receptor binding thus rendering it ineffective for use in saturation analysis. The results show that the use of serine-thiol protease inhibitors significantly improves the cytosol androgen receptor yield and assay sensitivity; therefore, we recommend routine inclusion of these compounds(s) in the homogenization buffer for androgen receptor assays.     

Ketone-body metabolism in tumour-bearing rats.

During starvation for 72 h, tumour-bearing rats showed accelerated ketonaemia and marked ketonuria. Total blood [ketone bodies] were 8.53 mM and 3.34 mM in tumour-bearing and control (non-tumour-bearing) rats respectively (P less than 0.001). The [3-hydroxybutyrate]/[acetoacetate] ratio was 1.3 in the tumour-bearing rats, compared with 3.2 in the controls at 72 h (P less than 0.001). Blood [glucose] and hepatic [glycogen] were lower at the start of starvation in tumour-bearing rats, whereas plasma [non-esterified fatty acids] were not increased above those in the control rats during starvation. After functional hepatectomy, blood [acetoacetate], but not [3-hydroxybutyrate], decreased rapidly in tumour-bearing rats, whereas both ketone bodies decreased, and at a slower rate, in the control rats. Blood [glucose] decreased more rapidly in the hepatectomized control rats. Hepatocytes prepared from 72 h-starved tumour-bearing and control rats showed similar rates of ketogenesis from palmitate, and the distribution of [1-14C] palmitate between oxidation (ketone bodies and CO2) and esterification was also unaffected by tumour-bearing, as was the rate of gluconeogenesis from lactate. The carcinoma itself showed rapid rates of glycolysis and a poor ability to metabolize ketone bodies in vitro. The results are consistent with the peripheral, normal, tissues in tumour-bearing rats having increased ketone-body and decreased glucose metabolic turnover rates.     

Macrophage endosomes contain proteases which degrade endocytosed protein ligands

Rabbit alveolar macrophages rapidly internalize and degrade mannosylated bovine serum albumin (125I-mannose-BSA). Trichloroacetic acid-soluble degradation products appear in the cells as early as 6 min after uptake at 37 degrees C, and in the extracellular medium after 10 min. Incubation of endocytic vesicles containing this ligand in isotonic buffers at pH 7.4 + ATP resulted in intravesicular proteolysis, which was inhibited by monensin, nigericin, or ammonium chloride. At pH 5.0, degradation proceeded rapidly and was abolished by lysis of the vesicles with 0.1% Triton X-100. Readdition of lysosomes to the incubation mixture did not increase the rate of prelysosomal degradation. Proteolysis of 125I-mannose-BSA was optimal at pH 4.5, and inhibited by low concentrations of the cathepsin D inhibitor pepstatin A. After subcellular fractionation of the macrophages on Percoll gradients, 125I-mannose-BSA sedimented with prelysosomal vesicles and was not transported to secondary lysosomes. Addition of pepstatin A to extracellular medium during internalization of prebound 125I-mannose-BSA partially inhibited degradation of ligand, and resulted in transfer of undegraded 125I-mannose-BSA to lysosomes after 20 min. Using 125I-bovine serum albumin as a substrate for the protease in the presence of 0.1% Triton X-100, we have shown that as much as 36% of the total pepstatin A-sensitive activity sediments with nonlysosomal membranes. After intraendosomal iodination using lactoperoxidase, a labeled protease was isolated by affinity chromatography on pepstatin-agarose. The labeled protease, which had a subunit size of 46 kDa, was detected in endocytic vesicles after 5 min of internalization. These results suggest that a cathepsin D-like protease is responsible for the degradation of 125I-mannose-BSA in macrophages, and that this ligand is degraded in a prelysosomal vesicle.     

Mitochondrial matrix granules in soft tissues. II. Isolation and initial characterization of a calcium-precipitable, soluble lipoprotein subfraction from brown fat and liver mitochondria

Degradation of ¹²⁵I-labelled HDL ([¹²⁵I]HDL) was measured in isolated rat hepatocytes that had been preincubated with [¹²⁵I]HDL and then reincubated in fresh medium without [¹²⁵I]HDL. About 5 % of the [¹²⁵I]HDL associated with the cells in advance were degraded per hour at 37 °C. This in vitro degradation was inhibited about 50% by lysosomal inhibitors such as chloroquine, ammonia and leupeptin. Depolymerization of microtubuli by colchicine inhibited the degradation of [¹²⁵I]HDL to about 65–75 % of the control cells. Cytochalasin B (CB), a destabilizer of microfilaments, had a less marked effect on the degradation in vitro. Degradation of [¹²⁵I]HDL associated with cells in vivo after intravenous injection was also studied in isolated cells. About 8.5% of the [¹²⁵I]HDL associated with the cells in vivo were degraded per hour in the isolated cells. The effects of ammonia, chloroquine, leupeptin and colchicine on HDL degradation were similar for [¹²⁵I]HDL taken up in vivo and in vitro. Subcellular fractionation by centrifugation in sucrose gradients indicated that [¹²⁵I]HDL associated with hepatocytes in vivo are primarily accumulated in lysosomes. [¹²⁵I]HDL associated with the cells in vitro are located in organelles whose distribution coincides with that of 5′-nucleotidase. These organelles may be endocytic vesicles. It is concluded that the internalization of [¹²⁵I]HDL in rat hepatocytes is relatively slow. The intracellular degradation of the apoproteins of HDL is at least partly lysosomal.     

Intracellular localization and degradation of diphtheria toxin

The internalization of surface-bound diphtheria toxin (DT) in BS-C-1 cells correlated with its appearance in intracellular endosomal vesicles; essentially no toxin appeared within secondary lysosomal vesicles. In contrast, internalized epidermal growth factor (EGF) was localized within both endosomal and lysosomal vesicles. Upon preincubation of cells with leupeptin, a lysosomal protease inhibitor, a threefold increase in the accumulation of EGF into lysosomes was observed. Under identical conditions, essentially all of the diphtheria toxin remained within endosomes (less than 2% of the intracellular diphtheria toxin accumulated in the lysosomal fraction), indicating that the inability to detect diphtheria toxin in lysosomes was not due to its rapid turnover within this vesicle. Following internalization of EGF or DT, up to 40% of the ligand appeared in the medium as TCA-soluble radioactivity. EGF degradation was partially leupeptin-sensitive and markedly NH4Cl-sensitive, indicating lysosomal degradation. In contrast, DT A-fragment degradation was resistant to these inhibitors, while B-fragment showed only partial sensitivity. These data suggest that the bulk of endocytosed diphtheria toxin is localized within endosomes and degraded by a pathway essentially independent of lysosomes.     

Regulation of intracellular casein degradation by secreted milk proteins

Intracellular degradation of newly synthesised casein was measured by a pulse-chase method in freshly prepared goat mammary explants. After incubation in medium containing L-[5-3H]proline, explants were washed and cultured again in unlabelled medium containing 5 mM proline; at intervals up to 24 h the amount of radiolabel incorporated in casein was measured. Tissue was obtained in week 33 of lactation after goats had been milked incompletely in one gland (the test gland) for 24 weeks; the contra-lateral (control) gland was milked normally. In explants from the control gland, casein was not degraded during or after secretion: L-[5-3H]proline incorporated in casein increased to a maximum value which was maintained through the chase period. For four out of five goats, explants from the test gland showed a decrease in total [3H]casein radiolabel at 0-4 h of the chase, indicating that a proportion of casein was degraded during secretion. Intracellular casein degradation was also observed when control gland explants were cultured in chase medium containing a goat whey fraction known to inhibit casein production and milk secretion (Wilde, C.J. et al., (1987) Biochem. J. 242, 285-288). This suggests that the greater volume of residual milk left by incomplete milking reduced secretory efficiency, rendering casein susceptible to intracellular degradation, and that this occurred through the action of a secreted milk constituent, which acts as a chemical feedback inhibitor of milk secretion.     

4-Hydroxy-2-nonenal-modified glyceraldehyde-3-phosphate dehydrogenase is degraded by cathepsin G

Degradation of oxidized or oxidatively modified proteins is an essential part of the antioxidant defenses of cells. 4-Hydroxy-2-nonenal (HNE), a major reactive aldehyde formed by lipid peroxidation, causes many types of cellular damage. It has been reported that HNE-modified proteins are degraded by the ubiquitin–proteasome pathway or, in some cases, by the lysosomal pathway. However, our previous studies using U937 cells showed that HNE-modified glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is degraded by an enzyme that is sensitive to a serine protease inhibitor, diisopropyl fluorophosphate (DFP), but not a proteasome inhibitor, MG-132, and that its degradation is not catalyzed in the acidic pH range where lysosomal enzymes are active. In the present study, we purified an HNE-modified GAPDH-degrading enzyme from a U937 cell extract to a final active fraction containing two proteins of 28 kDa (P28) and 27 kDa (P27) that became labeled with [³H]DFP. Using peptide mass fingerprinting and a specific antibody, P28 and P27 were both identified as cathepsin G. The degradation activity was inhibited by cathepsin G inhibitors. Furthermore, a cell extract from U937 cells transfected with a cathepsin G-specific siRNA hardly degraded HNE-modified GAPDH. These results suggest that cathepsin G plays a role in the degradation of HNE-modified GAPDH.     

Redox pacing of proteome turnover: influences of glutathione and ketonemia

In early starvation tissue protein degradation increases, however in later starvation proteolysis declines so as to pace gradual atrophy during synthetic failure. Secondary decline of proteolytic pathways under progressive nutritional desperation is unexplained. After several days of starvation tissue GSH is partly depleted and GSSG/GSH is increased, followed by onset of ketonemia from fat breakdown. Ketone bodies inexplicably delay net muscle protein loss. Recent studies identify a proteome subset of more than 200 proteins with reactive sulfhydryl sites as candidates for coordinate redox control of diverse cell functions. Ketones cause protein sulfhydryl oxidation and protein S-glutathionylation. Here, redox-responsive proteolytic pathways were bio-assayed by release of [3H]leucine from rat myocardium under non-recirculating perfusion. More than 75% of myocardial protein degradation was inhibited and defined by infusion of diamide (100 microM) under constant physiologic concentrations of complete amino acids. Diamide-inhibitable proteolysis includes all lysosomal and some extra-lysosomal proteolysis. Following diamide washout, the reversal of proteolytic inhibitory action was greatly enhanced by artificial repletion of GSH by supra-physiologic extra-cellular GSH (1mM) exposure. Therefore, GSH maintains much of constitutive protein degradation in a primary tissue bioassay. Physiologic acetoacetate infusion (5mM) inhibited redox-responsive protein degradation. Uniformly [3H]leucine labeled 3T3 cells exhibited similar redox-dependent and redox-independent subcomponents of protein degradation. Independent of ketones, steady state cathepsin B reaction rate ex vivo was graded in proportion to the GSH concentration without GSSG, and inversely proportional to the GSSG/GSH redox ratio with inhibitory threshold at 0.5% oxidized. Linkage of some cysteine protease reaction rates to the interplay between GSH-GSSG/GSH status and ketonemia is suggested among transcendent mechanisms coordinating and pacing proteome turnover under prolonged starvation. The possibility of pre-emptive, redox coordination of distinct proteolytic pathways is speculatively discussed.