Major histocompatibility complex class I-presented antigenic peptides are degraded in cytosolic extracts primarily by thimet oligopeptidase.

Nearly all peptides generated by proteasomes during protein degradation are digested rapidly to amino acids, but a few proteasomal products escape this fate and are presented to the immune system on cell surface major histocompatibility complex class I molecules. To test whether these antigenic peptides may be inherently resistant to cytosolic peptidases, six different antigenic peptides were incubated with HeLa cell extracts. All six were degraded rapidly by a process involving o-phenanthroline-sensitive metallopeptidases. One antigenic peptide, FAPGNYPAL, was rapidly destroyed in the extracts by a bestatin-sensitive exopeptidase, apparently by the puromycin-sensitive aminopeptidase. The disappearance of the other five was reduced 30-90% by a specific inhibitor of the cytosolic endopeptidase, thimet oligopeptidase (TOP) (EC ), whose physiological function(s) have been unclear and controversial. All these peptides were sensitive to pure recombinant TOP. Furthermore, upon fractionation of the extracts, the major peptidase peak that degraded the ovalbumin-derived epitope, SIINFEKL, co-purified with TOP. In the extracts, TOP also catalyzed rapid degradation of N-extended variants of SIINFEKL and of other antigenic peptides, which in vivo can serve as precursors of these major histocompatibility complex-presented epitopes. This enzyme (unlike cell proteins that promote production of antigenic peptides) is not regulated by interferon-gamma. TOP seems to be primarily responsible for the rapid breakdown of antigenic peptides in cytosolic extracts, and our related studies (A. X. Y. Mo, K. Lemerise, W. Zeng, Y. Shen, C. R. Abraham, A. L. Goldberg, and K. L. Rock, submitted for publication) indicate that TOP by destroying such peptides limits antigen presentation in vivo.

Proteasome active sites allosterically regulate each other, suggesting a cyclical bite-chew mechanism for protein breakdown.

In eukaryotes, the 20S proteasome contains two chymotrypsin-like, two trypsin-like, and two active sites shown here to have caspase-like specificity. We report that certain sites allosterically regulate each other's activities. Substrates of a chymotrypsin-like site stimulate dramatically the caspase-like activity and also activate the other chymotrypsin-like site. Moreover, substrates of the caspase-like sites inhibit allosterically the chymotrypsin-like activity (the rate-limiting one in protein breakdown) and thus can reduce the degradation of proteins by 26S proteasomes. These allosteric effects suggest an ordered, cyclical mechanism for protein degradation. We propose that the chymotrypsin-like site initially cleaves ("bites") the polypeptide, thereby stimulating the caspase-like sites. Their activation accelerates further cleavage ("chewing") of the fragments, while the chymotrypsin-like activity is temporarily inhibited. When further caspase-like cleavages are impossible, the chymotryptic site is reactivated and the cycle repeated.

The sizes of peptides generated from protein by mammalian 26 and 20 S proteasomes. Implications for understanding the degradative mechanism and antigen presentation.

Knowledge about the sizes of peptides generated by proteasomes during protein degradation is essential to fully understand their degradative mechanisms and the subsequent steps in protein turnover and generation of major histocompatibility complex class I antigenic peptides. We demonstrate here that 26 S and activated 20 S proteasomes from rabbit muscle degrade denatured, nonubiquitinated proteins in a highly processive fashion but generate different patterns of peptides (despite their containing identical proteolytic sites). With both enzymes, products range in length from 3 to 22 residues, and their abundance decreases with increasing length according to a log-normal distribution. Less than 15% of the products are the length of class I presented peptides (8 or 9 residues), and two-thirds are too short to function in antigen presentation. Surprisingly, these mammalian proteasomes, which contain two "chymotryptic," two "tryptic," and two "post-acidic" active sites, generate peptides with a similar size distribution as do archaeal 20 S proteasomes, which have 14 identical sites. Furthermore, inactivation of the "tryptic" sites altered the peptides produced without significantly affecting their size distribution. Therefore, this distribution is not determined by the number, specificity, or arrangement of the active sites (as proposed by the "molecular ruler" model); instead, we propose that proteolysis continues until products are small enough to diffuse out of the proteasomes.

Range of sizes of peptide products generated during degradation of different proteins by archaeal proteasomes.

The 20 S proteasome processively degrades cell proteins to peptides. Information on the sizes and nature of these products is essential for understanding the proteasome's degradative mechanism, the subsequent steps in protein turnover, and major histocompatibility complex class I antigen presentation. Using proteasomes from Thermoplasma acidophilum and four unfolded polypeptides as substrates (insulin-like growth factor, lactalbumin, casein, and alkaline phosphatase, whose lengths range from 71 to 471 residues), we demonstrate that the number of cuts made in a polypeptide and the time needed to degrade it increase with length. The average size of peptides generated from these four polypeptides was 8 +/- 1 residues, but ranged from 6 to 10 residues, depending on the protein, as determined by two new independent methods. However, the individual peptide products ranged in length from approximately 3 to 30 residues, as demonstrated by mass spectrometry and size-exclusion chromatography. The sizes of individual peptides fit a log-normal distribution. No length was predominant, and more than half were shorter than 10 residues. Peptide abundance decreased with increasing length, and less than 10% exceeded 20 residues. These findings indicate that: 1) the proteasome does not generate peptides according to the "molecular ruler" hypothesis, and 2) other peptidases must function after the proteasome to complete the turnover of cell proteins to amino acids.

New insights into the mechanisms and importance of the proteasome in intracellular protein degradation.

Recent studies of the 20S proteasome from Thermoplasma acidophilum have uncovered some fundamental new properties of its catalytic mechanism. Unlike conventional proteases, 20S and 26S proteasomes degrade protein substrates in a highly processive fashion. They cleave a protein substrate to small peptides before attacking another substrate molecule. This processive behavior is an inherent feature of the 20S particle not requiring cofactors or ATP hydrolysis. Recently, we have described a proteasome-like particle, HslVU, in Escherichia coli. HslVU is a two-component ATP-dependent protease composed of the proteasome-related peptidase HslV (beta-subunit) and the ATPase HslU. In active HslVU complex, cleavage of small peptides and proteins requires the presence of ATP. EM analysis revealed that HslV and HslU are both ring-shaped particles and that the active HslVU complex is a cylindrical four-ring structure, composed of HslV, a two-ring dodecamer, sandwiched between HslU rings. Elucidation of its mode of action may help us understand the role of ATP in function of the 26S proteasome. Several proteasome-specific inhibitors have been recently identified which block the function of proteasome in vivo. These agents have proven very useful to clarify the intracellular function of the proteasome. In mammalian cells, both the rapid degradation of short-lived regulatory proteins and of abnormal polypeptides and the slower degradation of long-lived proteins are blocked by these agents. Thus, in mammalian cells, the proteasome is the site for the degradation of most cell proteins. In contrast, in budding yeast, proteasome inhibitors block the degradation of short-lived proteins but not the breakdown of long-lived proteins, which can be blocked by inhibitors of vacuolar proteases. The inhibition of proteasome function in yeast and mammalian cells, presumably by causing an accumulation of unfolded proteins, triggers the expression of heat shock proteins and concomitantly increases cell resistance to high temperature and various toxic insults.

Processive degradation of proteins and other catalytic properties of the proteasome from Thermoplasma acidophilum.

Although the structure of the 20 S proteasome from Thermoplasma acidophilum has been elucidated, its enzymatic properties have not been explored in depth. Thermoplasma proteasomes, which contain one type of active site, exhibit not only "chymotrypsin-like" activity (as reported), but also some "post-glutamyl" and "trypsin-like" activities. Like eukaryotic proteasomes, its activity can be stimulated by SDS, Mg2+, and also guanidine HCl, but not urea. The enzyme was strongly inhibited by novel peptide aldehydes with hydrophobic P4 residues, and was rapidly inactivated by 3, 4-dichloroisocoumarin (DCI). DCI modified the N-terminal threonine of the catalytic beta-subunit, the presumed active site nucleophile. To define how proteins are degraded, casein was derivatized with fluorescein isothiocyanate to facilitate detection of released products by the proteasome. Many fluorescent peptides were generated, but the relative amounts of different peptides were independent of the duration of the reaction. The rate of disappearance of protein substrates paralleled the rate of appearance of small products. Unlike conventional proteases, proteasome degrades proteins processively without release of polypeptide intermediates. Upon activation by SDS, guanidine, heat (55 degrees C), or partial inhibition with DCI, proteasomes still functioned processively, but generated a different pattern of peptides under each condition. Thus, processivity is an inherent feature of the 20 S proteasome, not requiring all active sites or ATP hydrolysis.

[Level of peptides in blood serum in experimental traumatic illness]. / Uroven' peptidov v syvorotke krovi pri éksperimental'noi travmaticheskoi bolezni.

A procedure, developed for quantitative estimation of peptides in protein free blood serum, involved registration of products formed after reaction of peptide amino groups with o-phthalic aldehyde. The procedure developed was 50-100-fold more sensitive than spectrophotometry. Content of peptides in blood was altered depending on the steps of traumatic disease development. The concentration of peptides correlated with the composition of proteins in the serum.

[Activity of proteolytic enzymes and their inhibitors in experimental myocardial ischemia]. / Aktivnost' proteoliticheskikh fermentov i ikh ingibitorov pri ékperimental'noi ishemii miokarda.

Proteolytic activity and activity of endogenous inhibitors of endopeptidases (using chymotrypsin and papain) were studied in the myocardium of rats with experimental ischemia during an acute phase (60 min) and within 5 days after ligation of the left descending coronary artery; effects of the beta-adrenoblocking agent propranolol and the calcium antagonist verapamil on these activities was also studied. During the acute phase of ischemia, the activity of acid proteases was increased by 30%, that of Ca(2+)-activated neutral proteases by 15-20%. At the same time, the activity of serine proteases inhibitors was decreased while the activity of thiol protease inhibitors was increased. Within 5 days of coronary artery occlusion, Lysosomal thiol-dependent endopeptidases were activated in the myocardium; a considerably higher activity of the inhibitors of serine- and cysteine-containing endopeptidases was detected. The cardioactive drugs propranolol and verapamil affected selectively both endopeptidase activity and their inhibitors.

[Synaptosomal degradation of neuropeptides]. / Sinaptosomnoe rasshcheplenie neiropeptidov.

Proteolytic conversions of some neuropeptides possessing neurotransmitter and neuromodulator properties were studied on the synaptosomal plasma membrane level. The main goal was to describe the peptide bonds being primarily hydrolysed by membrane peptidases. The analysis of the accumulated data allowed one to make some summarizing conclusions about properties of the enzyme system responsible for the biological inactivation of the peptides in synapsis.

[The effect of cardioactive drugs on the proteolytic activity of the rat heart muscle]. / Vliianie kardioaktivnykh preparatov na proteoliticheskuiu aktivnost' serdechnoi myshtsy krys.

Cardioactive drugs--beta-adrenoblocking agents (propranolol, trasicor and cordanum) and Ca2(+)-antagonists (nifedipine and phenoptine)--exhibited both activating and inhibitory effects on proteolytic activity in rat heart tissue extract. The beta-adrenoblocking agents did not affect distinctly the activity of partially purified cathepsin D and Ca2(+)-dependent neutral protease from myocardium. Nifedipine inhibited cathepsin D by 50% and Ca2(+-dependent neutral protease--by 75%; phenoptine inhibited also the latter enzyme by 30%.

[Glutathione disulfide inhibits degradation of substance P induced by synaptosomal plasma membranes]. / Disul'fid glutationa podavliaet rasshcheplenie veshchestva P pod deistviem sinaptosomal'nykh plazmaticheskikh membran.

The effects of phosphorylation, ribosylation of proteins and formation of protein-mixed disulfides on substance P degradation under the action of synaptosomal plasma membranes were studied. It was found that only the formation of mixed disulfides between membrane proteins and oxidized glutathione affected (inhibited) the peptide degradation process. Using an oxidized glutathione fluorescent derivative, it was shown that a 50% inhibition occurs as a result of binding of 2 nmol of the glutathione residue to 1 mg of the membrane protein.

[Protein substrates of proteinases with fluorescent labels]. / Belkovye substraty proteinaz s fluorestsentnymi metkami.

Protein substrates for proteinases with double fluorescent fluorophors are synthesized. Pyridoxal-5'-phosphate, dansyl chloride and fluorescein isothiocyanate were used as fluorescent sounds for modification of globin. Phosphoyridoxyl fluorophor was present in the both substrates. The second label was either fluoresceinthiocarbamyl or dansyl fluorophor. Spectral characteristics and ability to hydrolysis of obtained substrates have been studied. The influence of some salts on fluorescent characteristics of those substrates have been analyzed. Differentiation of the hydrolyzed substrate from the initial one by ammonium sulphate is shown to be possible.

[An increase in the rate of proteolysis in vitro after treatment with oxidized glutathione]. / Uvelichenie skorosti rasshchepleniia belkov in vitro pod vliianiem okislennogo glutationa.

The effect of the formation of mixed disulphides--protein-glutathione--on the proteolysis rate was studied using soluble fractions of proteins from different rat tissues as substrates. It was shown that the binding of oxidized glutathione to proteins increases the proteolysis rate under the effect of trypsin and chymotrypsin. When the concentration of oxidized glutathione is 5.10(-4) M a 1.2-1.4-fold increase in the proteolysis rate is registered and when the concentration is 5.10(-3) M a 1.4-1.8 fold increase is observed.

[Multiple forms of cathepsin D from the human brain]. / Mnozhestvennye formy katepsina D iz mozga cheloveka.

Six peaks of the endopeptidase activity at pH 3.2 were obtained after isoelectric focusing of soluble fractions of cortex and hypothalamus of the human brain. The molecular weight of these endopeptidases are approximately 50000. All obtained endopeptidases possess almost the same Km and I50 relative to the substrate--pyridoxal globin and specific inhibitor--pepstatin. The studies of the revealed properties show that the endopeptidases are multiple forms of cathepsin D.

[Isoenzyme composition of cathepsin D from bovine hypothalamus]. / Izofermentnyi sostav katepsina D iz gipotalamusa byka.

The isoenzyme composition of cathepsin D from bovine hypothalamus was studied by isoelectric focusing. It was found that the soluble fraction of hypothalamic proteins contains five peaks of endopeptidase activity at pH 3.2. The properties studied allowed to identify these peaks of endopeptidase activity as isoenzyme forms of cathepsin D.

[Breakdown of luliberin, somatostatin and substance P as an effect of hypothalamic endopeptidases]. / Raspad liuboliberina, somatostatina i veshchestva P pod deistviem gipotalamicheskikh éndopeptidaz.

Acid and neutral proteinases were isolated with the purpose of investigating their participation in the breakdown of hypothalamic peptides and proteins. The acid proteinase was purified about 1000-fold from hypothalamus by precipitation with acetone, chromatography on SP-Sephadex G-50, gel filtration through column of G-100 and chromatography on DEAE-Sephadex A-50. The molecular weight of the enzyme was approximately 50.000. Maximal activity against hemoglobin was obtained at pH 3,2--3,5: serum albumin was split much more slowly. Hypothalamus acid proteinase was partially inhibited by beta-phenyl pyruvate, benzothonium cloride, and was completely inhibited by low concentrations of pepstatin. This proteinase splits somatostatin, Substance P and some C-fragments of Substance P. The probable sites of enzyme action on these peptides were determined by the end group dansyl technique. Neutral proteinase was isolated from the supernatant fraction(100.000 g) of a 0,3 M sucrose homogenate of bovine hypothalamus by chromatography on DEAE Sephadex A-50, gel filtration through Sephadex G-100 and rechromatography on DEAE sephadex A-50 using luliberin as substrate. The rates of breakdown of luliberin and denaturated hemoglobin were measured by fluorometric estimation of acid-soluble peptides wieht o-phthaldialdehyde. The purifed enzyme preparations have a pH optimum of activity at 7--7,5. The enzymes molecular weight was approximatelyy 30--40.000. Enzyme activity was inhibited by L-1-tosylamide-2-phenylethylchloromethyl ketone, p-chloromercuribenzoate and divalent ions Co2+, Zn2+ and was significantly enhanced by dithiothreitol. The Km values for the reaction of hydrolysis of luliberin and hemoglobin were 1,33.10(-5) and 5,2.10(-5) M respectively. The neutral proteinase from the hypothalamus cleaves luliberin, somatostatin and Substance P. Sites of action of the enzyme upon those peptides were determined by means of the dansyl technique. The acid proteinase, most likely cathepsin D, and neutral proteinase from hypothalamus, may play an important role in the formation and breakdown of peptide hormones in the hypothalamus.

[Beta-cyanoalanine synthase: Its purification and basic physico-chemical properties]. / Beta-tsianoalaninsintaza: Ochistka i osnovnye fiziko-khimicheskie svoistva

A method has been developed for the purification of beta-cyano-L-alanine synthase from etiolated 10-day-old seedlings of blue lupine. High purity preparations of the enzyme were obtained with specific activity exceeding 4000-fold that of the seedling homogenate. Preparations were homogeneous on electrophoresis in polyacrylamide gel. The yield of total activity after purification was approximately 20%. Glutamic acid is the enzyme's only N-terminal amino acid; the molecular weight of the enzyme (both native and treated with 6 M urea) is 52000. The synthase containes one mole of pyridoxal-P per mole of protein; its isoelectric point is situated at pH 4,8. The enzyme's absorption spectrum has a maximum at 410 nm i.e., in the characteristic range of many pyridoxal-U-containing enzymes. Data on the amino acid composition of the enzyme are presented.