Nonselective autophagy of cytosolic enzymes by isolated rat hepatocytes.

Seven cytosolic enzymes with varying half-lives (ornithine decarboxylase, 0.9 h; tyrosine aminotransferase, 3.1 h; tryptophan oxygenase, 3.3 h; serine dehydratase, 10.3 h; glucokinase, 12.7 h; lactate dehydrogenase, 17.0 h; aldolase, 17.4 h) were found to be autophagically sequestered at the same rate (3.5%/h) in isolated rat hepatocytes. Autophagy was measured as the accumulation of enzyme activity in the sedimentable organelles (mostly lysosomes) of electrodisrupted cells in the presence of the proteinase inhibitor leupeptin. Inhibitors of lysosomal fusion processes (vinblastine and asparagine) allowed accumulation of catalytically active enzyme (in prelysosomal vacuoles) even in the absence of proteolytic inhibition, showing that no inactivation step took place before lysosomal proteolysis. The completeness of protection by leupeptin indicates, furthermore, that a lysosomal cysteine proteinase is obligatorily required for the initial proteolytic attack upon autophagocytosed proteins. The experiments suggest that sequestration and degradation of normal cytosolic proteins by the autophagic-lysosomal pathway is a nonselective bulk process, and that nonautophagic mechanisms must be invoked to account for differential enzyme turnover.

The age dependence of intracellular proteolysis: changes of the substrate proteins.

Liver cytosol proteins of young (4--6 months) and old (18--27 months) rats were degraded in vitro by papain, pronase, trypsin, pepsin, cathepsin D from rat liver and a soluble lysosomal enzyme mixture from rat liver. We could demonstrate the capability of the latter enzyme mixture to degrade proteolytically the cytosol proteins of young animals about 20% faster than those of the older animal group. Digesting radioactive labelled "young" cytosol in the presence of unlabelled "old" cytosol the possibility could be excluded, that this effect was due to an inhibitor of macromolecular size present in the "old" cytosol.

Preparation and characterization of monoclonal antibodies against ornithine decarboxylase.

In order to develop a method for the immunocytochemical detection of ornithine decarboxylase (ODC), EC 4.1.1.17, we have prepared and characterized monoclonal antibodies (MAbs) against ODC. The primary structure of rat ODC (Rattus Norvegicus) was used for the selection of an epitope by computer calculations. The epitope (P16), a hexadecapeptide representing ODC-(345-360), was synthesized by means of solid phase peptide synthesis and coupled to a carrier protein. A bovine serum albumin conjugate of the P16 peptide was used as the immunogen for the production of MAbs in mice. Hybridoma clones were screened and the specificity of the monoclonal antibodies was tested in an ELISA utilizing a thyroglobulin conjugate of the hexadecapeptide. Two hybridoma cell lines were developed, i.e., MP16-2 and MP16-3. The epitope specificity of the MAbs produced by these cell lines was characterized in an ELISA using a set of small peptides representing parts of the P16 hexadecapeptide chain. MP16-2 recognized the ODC-(355-360) portion whereas MP16-3 reacted with the ODC-(345-350) part of the hexadecapeptide. Further studies showed that both MAbs also recognized native ODC but not the inhibited (i.e., ODC labelled with 3H-DFMO) enzyme indicating that the selected epitope was associated with the active site of ODC or a locus in its direct vicinity.

Localization of cathepsin H and its inhibitor in the skin and other stratified epithelia.

The rat-skin-derived cysteine proteinase, so-called BANA-hydrolase, which is capable of hydrolysing benzoylarginine naphthylamide and leucine naphthylamide was shown to be immunologically identical to cathepsin H purified from rat liver. The enzyme was immunocytochemically localized in the basal cell layer of rat epidermis. A natural inhibitor of cathepsin H with a molecular weight of about 13,000 was mainly localized in the keratinizing cell layers and showed only a weak reaction in the basal cells. Thus, cathepsin H appears to be a characteristic feature of the proliferating cell layer, whereas the cysteine-proteinase inhibitor is a characteristic feature of keratinizing cells.

Surface hydrophobicity and intracellular degradation of proteins.

Cellular proteins turn over with rates varying more than 1000 fold and all organelles are involved in this process of renewal. Although the surface of protein molecules bears many peptide bonds, which could be potential cleavage sites for proteases, only a small fraction of all cellular proteins is subject to very rapid turnover, with half-lives of less than one hour in mammalian cells. Many of these proteins play key roles in basic regulatory mechanisms. One of the features that make proteins short-lived is surface hydrophobicity which is increased in nascent polypeptide chains before association with chaperones, in oligomeric proteins before the association of the monomers, and in many enzymes in absence of their substrates. Cellular proteases tend to act most rapidly on peptide bonds involving (or near to) apolar amino acids. A striking correlation has been found between the half-lives of cellular proteins and their surface hydrophobicity. Clusters of hydrophobic residues appear to be necessary for ubiquitination. Apolar amino acid residues that are hidden can be exposed after oxidation of proteins or after binding of ubiquitin molecules. Additionally, tetraubiquitin exposes many hydrophobic residues which are essential for targeting of the ubiquitinated substrate proteins to a 50 kDa-subunit of the 26S-protease.

The fates of proteins in cells.

Nascent polypeptide chains are in a dangerous situation as soon as they leave their place of birth, the channel of the large ribosomal subunit: more than 20 different pathways for the degradation of proteins exist in cells. Chaperones protect and guide the young protein molecules and support their correct foldings. Targeting signals direct the proteins to the organelles of their destination. The lysosome is the site of random degradation, while the proteasome is highly selective. Although these two organelles provide the most important pathways for the degradation of long- and short-lived proteins, other pathways with roles in deciding the fate of cellular proteins must also be considered.

Intracellular protein catabolism. IX. Hydrophobicity of substrate proteins is a molecular basis of selectivity.

Double-labeled cytosol proteins from rat liver (3H in short-lived, 14C in long-lived proteins) were fractionated by using siliconized glass-beads, phenylsepharose and octylsepharose. Always the short-lived proteins are more tightly bound to the hydrophobic matrix. The same distribution was found with monkey liver substrate proteins. Therefore it is concluded that the different degrees of exposure of superficial hydrophobic areas on substrate protein molecules are a molecular basis of selectivity of the intracellular protein catabolism.

Cooperation of various subcellular fractions in protein degradation in vitro.

In order to test the possibilities in protein degradation between cell organelles comparatively, [3H]- and [14C]-leucine short-time labelled subcellular fractions from rat liver were incubated with each other at pH 6.9. All fractions tested were able to degrade short-lived proteins from foreign fractions, whereby the lysosomal supernatant fraction showed the highest proteolytic activity, which declines in the sequence: lysosomes--nuclei--mitochondria--cytosol--microsomes. Short-lived cytosolic proteins were especially suited as substrate for neutral proteases from all other fractions, but also microsomal, mitochondrial and nuclear proteins were well degraded by foreign fractions in comparison with the substrate autoproteolysis. Therefore in vivo manyfold cooperations between several organelles in protein catabolism seem to be possible.

Proteome analysis by three-dimensional protein separation: turnover of cytosolic proteins in hepatocytes.

We performed a three-dimensional separation of pulse-chase dual-labelled rat liver cytosolic proteins using hydrophobic interaction chromatography, isoelectric focusing, and SDS gel electrophoresis. Due to very different expression rates but similar size and pI of rat liver cytosolic proteins, we demonstrate the impossibility of successful two-dimensional separations of such complex protein mixtures. A pre-fractionation of proteins by hydrophobic interaction chromatography is therefore recommended prior to two-dimensional gel electrophoresis. Our studies confirmed the correlation between protein turnover rates and surface hydrophobicity.

Very fast purification of ornithine decarboxylase with high yield from mouse kidney and generation of a monoclonal antibody.

Based on methods for ornithine-decarboxylase purification published previously we developed an improved procedure for purification of the enzyme from the kidneys of testosterone-treated NMRI mice. Advantages of the new procedure are, that inactivation of the enzyme during purification is largely reduced by fast methods for purification and by the use of proteinase inhibitors. That way we got pure ornithine decarboxylase within 60 h with a yield of about 70%. A part of the highly purified ornithine decarboxylase was used for the generation of monoclonal antibodies.

Post-translational arginylation of ornithine decarboxylase from rat hepatocytes.

Ornithine decarboxylase (ODC) was purified 6500-fold from NMRI mouse kidneys under conditions designed to inhibit degradation by proteinases. The enzyme was homogeneous by SDS/polyacrylamide-gel electrophoresis, and the specific activity was among the highest reported. The yield was 70%. A monoclonal antibody against this preparation was generated and used in studies to investigate the half-life of ODC in cultured rat hepatocytes labelled with [35S]methionine. This value was 39 +/- 4 min and was unchanged when either NH4Cl (as a lysosomotropic agent) or leupeptin (as a lysosomal proteinase inhibitor) was added to the culture medium. Thus the intracellular turnover of ODC in cultured hepatocytes occurs mainly in extra-lysosomal compartments. Arginylation of rat ODC was investigated in vitro by incubation with L-[3H]arginyl-tRNA, and the incorporation of the label was compared with that of total cytosolic proteins. Arginylated ODC had a specific radioactivity 8600 times that of the bulk of cytosolic protein. Edman degradation of this ODC showed that the post-translational arginylation occurred only at the alpha-amino end of the enzyme. The inhibitor of arginyl-tRNA:protein arginyltransferase (EC 2.3.2.8), L-glutamyl-L-valyl-L-phenylalanine, increased the half-life of ODC in cultured hepatocytes from 39 min to more than 90 min. The possible significance of the preferential post-translational arginylation of ornithine decarboxylase to its rapid turnover is discussed.

Surface hydrophobicity, arginylation and degradation of cytosol proteins from rat hepatocytes.

In vitro 14C-prelabelled cytosol proteins from rat hepatocytes were incubated with [3H]arginyl-tRNA in ATP-Tris-Mg-KCl-dithioerithrol medium and arginyltransferase, subsequently treated with RNase A, and the double-labelled proteins were isolated by gel filtration. The affinity of these [3H]arginylated-14C-labelled cytosol proteins to hydrophobic surfaces was investigated with octyl-Sepharose, phenyl-Sepharose and with FPLC on phenyl-Superose (HR 5/5). All 3H/14C-ratios of the proteins in the column fractions show that arginylated proteins bind preferentially to the hydrophobic matrices: the fractions eluted first show low 3H/14C-ratios, and after addition of ethylene glycol and especially of Tween 80 the 3H/14C-ratios markedly increase. Furthermore, these arginylated proteins aggregate preferentially after incubation of the cytosol proteins for 2 h at 37 degrees C and are more rapidly degraded by endopeptidases.

Proteases and proteolysis in the lysosome.

Proteins sequestered by a non-selective bulk process within the lysosomes turn over with an apparent half-life of about 8 minutes and this rapid lysosomal proteolysis is initiated by endopeptidases, in particular by the cathepsins D and L. We describe also the cathepsins B and H which show mainly exopeptidase and only low endopeptidase activity. Especially cathepsin H is most probably the only lysosomal aminopeptidase in many cell types. Additionally, the properties of other mammalian lysosomal endo- and exopeptidases are compared. Finally, we discuss some of the conditions for the action of lysosomal proteases as the low intralysosomal pH, the high part of lysosomal thiol groups and the absence of intralysosomal proteinase inhibitors.

Autophagy and other vacuolar protein degradation mechanisms.

Autophagic degradation of cytoplasm (including protein, RNA etc.) is a non-selective bulk process, as indicated by ultrastructural evidence and by the similarity in autophagic sequestration rates of various cytosolic enzymes with different half-lives. The initial autophagic sequestration step, performed by a poorly-characterized organelle called a phagophore, is subject to feedback inhibition by purines and amino acids, the effect of the latter being potentiated by insulin and antagonized by glucagon. Epinephrine and other adrenergic agonists inhibit autophagic sequestration through a prazosin-sensitive alpha 1-adrenergic mechanism. The sequestration is also inhibited by cAMP and by protein phosphorylation as indicated by the effects of cyclic nucleotide analogues, phosphodiesterase inhibitors and okadaic acid. Asparagine specifically inhibits autophagic-lysosomal fusion without having any significant effects on autophagic sequestration, on intralysosomal degradation or on the endocytic pathway. Autophaged material that accumulates in prelysosomal vacuoles in the presence of asparagine is accessible to endocytosed enzymes, revealing the existence of an amphifunctional organelle, the amphisome. Evidence from several cell types suggests that endocytosis may be coupled to autophagy to a variable extent, and that the amphisome may play a central role as a collecting station for material destined for lysosomal degradation. Protein degradation can also take place in a 'salvage compartment' closely associated with the endoplasmic reticulum (ER). In this compartment unassembled protein chains are degraded by uncharacterized proteinases, while resident proteins return to the ER and assembled secretory and membrane proteins proceed through the Golgi apparatus. In the trans-Golgi network some proteins are proteolytically processed by Ca(2+)-dependent proteinases; furthermore, this compartment sorts proteins to lysosomes, various membrane domains, endosomes or secretory vesicles/granules. Processing of both endogenous and exogenous proteins can occur in endosomes, which may play a particularly important role in antigen processing and presentation. Proteins in endosomes or secretory compartments can either be exocytosed, or channeled to lysosomes for degradation. The switch mechanisms which decide between these options are subject to bioregulation by external agents (hormones and growth factors), and may play an important role in the control of protein uptake and secretion.

[Action of proteinase inhibitors. 4. Effect of short-term application of chymostatin on nitrogen metabolism in the digestive tract and protein metabolism in tissue]. / Untersuchungen zur Wirkung von Proteinaseinhibitoren. 4. Mitteilung Einfluss einer kurzzeitigen Applikation von Chymostatin auf den N-Umsatz im Verdauungstrakt und den Gewebeproteinumsatz.

Chymostatin is an effective inhibitor of intracellular proteinases in vitro. In the present experiment male rats were injected intraperitonealy during a 3 days period twice daily with a solution containing 0,9 mg Chymostatin per 100 g live weight. Reference animals received a control injection containing the same solvents but no chymostatin. During this period a daily nitrogen balance was made and metabolic faecal nitrogen and true digestibility of nitrogen were estimated using 15N-labelled animals. Furthermore, apparent biological half lives of proteins in liver and intestinal tissues were determined following the decay curves for radioactivity in proteins 48 hours after injection of L-[5-3H]-arginine und L-[guanido-14C]-arginine. The fractional rate of protein synthesis in tissues was measured by a 6 hours continuous infusion technique with L-[U-14C]-tyrosine and L-[U-14C]-leucine. Among the parameters estimated only the apparent biological half lives of proteins in liver and intestinal tissues were influenced by chymostatin. However, the prolonged half lives seemed to be rather an effect of an increased reutilisation of amino acids resulting from the intracellular protein breakdown than a decreased rate of protein degradation. The in vivo effect of the proteinase inhibitor was by far inferior compared with the action in vitro. Factors like distribution, degradation and excretion of the inhibitor could be responsible for the moderate in vivo action of chymostatin.

Membrane sialoglycoprotein from human erythrocytes activates lysosomal proteinases.

The capacity of membrane glycoproteins to interact with proteinases was investigated in the model system: Membrane sialoglycoprotein from human erythrocytes (glycophorin) and lysosomal proteinases from rat liver. Glycophorin was found to stimulate the activity of a lysosomal proteinase mixture up to about 150% at pH 6.9. Cathepsin L was found to be the primarily stimulated proteinase. The stoichiometry in the saturation range of the dose-response curve waas about 10 to 20 molecules glycophorin per molecule cathepsin L. The mechanism of the activation is unknown. Interactions of this type may be of importance for the regulation of cell proliferation on the level of cell membranes.