Degradation of abnormal proteins in intact mouse reticulocytes: accumulation of intermediates in the presence of bestatin.

Botbol, Violeta; Scornik, Oscar A.

doi:10.1073/pnas.76.2.710

Cited by 30 publications

(11 citation statements)

References 20 publications

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“…Formulating a testable hypothesis about a specific role for Pf-LAP is complicated by the fact that its inhibition did not yield any overt morphological change in the parasite other than death. However, we suspect Pf-LAP may have an essential housekeeping function in the cytosolic turnover of dipeptides (49) and perhaps acts in concert with the parasite proteasome, as has been shown for other neutral cytosolic leucine aminopeptidases pathways (50). Like PNAP, lethal amounts of proteasome inhibitors exert their effect in the ring-trophozoite transition and parasites do not progress into the later trophozoite stage (51).…”

Section: Discussionmentioning

confidence: 99%

Bestatin-based chemical biology strategy reveals distinct roles for malaria M1- and M17-family aminopeptidases

Harbut

Velmourougane

Dalal

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

134

176

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Malaria causes worldwide morbidity and mortality, and while chemotherapy remains an excellent means of malaria control, drug-resistant parasites necessitate the discovery of new antimalarials. Peptidases are a promising class of drug targets and perform several important roles during the Plasmodium falciparum erythrocytic life cycle. Herein, we report a multidisciplinary effort combining activity-based protein profiling, biochemical, and peptidomic approaches to functionally analyze two genetically essential P. falciparum metallo-aminopeptidases (MAPs), PfA-M1 and Pf-LAP. Through the synthesis of a suite of activity-based probes (ABPs) based on the general MAP inhibitor scaffold, bestatin, we generated specific ABPs for these two enzymes. Specific inhibition of PfA-M1 caused swelling of the parasite digestive vacuole and prevented proteolysis of hemoglobin (Hb)-derived oligopeptides, likely starving the parasite resulting in death. In contrast, inhibition of Pf-LAP was lethal to parasites early in the life cycle, prior to the onset of Hb degradation suggesting that Pf-LAP has an essential role outside of Hb digestion.protease | chemical-genetics | proteomics | small molecule | drug design

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Section: Discussionmentioning

confidence: 99%

Bestatin-based chemical biology strategy reveals distinct roles for malaria M1- and M17-family aminopeptidases

Harbut

Velmourougane

Dalal

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

134

176

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“…The amidase would provide correction to adducts with few APF-1 ligands at a favored rate compared with proteolysis, which would depend on specific proteases that could recognize the APF-1 component and were especially directed to molecules with several APF-1 molecules attached. Protein breakdown occurring on a multi-enzyme complex has been an attractive hypothesis because of the general failure to detect small peptides as intermediates (11). Fraction II has recently been found (unpublished data) to contain at least four separable proteins that together with APF-1 are necessary for the ATP-dependent formation of trichloroacetic acid-soluble products of protein breakdown.…”

Section: Formation Ofmentioning

confidence: 99%

Proposed role of ATP in protein breakdown: conjugation of protein with multiple chains of the polypeptide of ATP-dependent proteolysis.

Hershko¹,

Ciechanover²,

Heller³

et al. 1980

Proc. Natl. Acad. Sci. U.S.A.

633

398

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The heat-stable polypeptide ATP-dependent proteolysis factor 1 (APF-1) of the reticulocyte proteolytic system forms covalent compounds with proteins in an ATP-requiring reaction. APF-1 and lysozyme, a good substrate for ATP-dependent proteolysis, form multiple conjugates, as was shown by comigration of label from each upon gel electrophoresis.Multiple bands were also seen with other substrates of the ATP-dependent proteolytic system, such as globin or a-lactalbumin. Analysis of the ratio of APF-1 to lysozyme radioactivities and of the molecular weights of the bands indicated that they consist of increasing numbers of the APF-1 polypeptide bound to one molecule of lysozyme. The covalent linkage is probably of an isopeptide nature, because it is stable to hydroxylamine and alkali, and polylysine is able to give conjugates of APF-1. Removal of ATP after formation of the 125I-labeled APF-1 conjugates with endogenous proteins caused the regeneration of APF-1, indicating presence of an amidase. This reaction is thought to compete with proteases that may act on APF-1-protein conjugates, especially those containing several APF-1 ligands. A sequence of reactions in which the linkage of APF-1 to the substrate is followed by the proteolytic breakdown of the substrate is proposed to explain the role of ATP.Since the discovery of the energy requirement of protein breakdown by Simpson in 1953 (1), this feature has been found to be universal in a variety of biological systems (for review, see ref.2). Only recently have cell-free systems in which protein breakdown is stimulated by ATP been delineated (3-6).We have resolved the ATP-dependent proteolytic system from reticulocytes into several components (7,8). In the preceding paper (9) it was found that the heat-stable polypeptide of this system, ATP-dependent proteolysis factor 1 (APF-1), is bound to reticulocyte proteins in an ATP-specific reaction. We now show that this is due to the formation of a covalent, probably amide, linkage between the protein substrate and one or more APF-1 molecules. The role of this reaction in protein breakdown is considered. METHODS Preparation of Radiolabeled Proteins and EnzymeFractions. The purification and radiolabeling of APF-1 from rabbit reticulocytes were described in the previous article (9). Crystalline hen egg white lysozyme (Worthington) and purified bovine a-lactalbumin (a generous gift of W. Klee) were labeled with Na'25I by a method similar to that described for the radioiodination of APF-1 (9). When used in unlabeled form these proteins were treated in the same way without isotope. Globin labeled with [3H]leucine was prepared as described (7). Fraction 11 (0.5 M KCI eluate from DEAE-cellulose) was prepared from ATP-depleted rabbit reticulocytes (7). Poly(L-lysine) was obtained from Miles-Yeda (Mr 1500-8000). (vol/vol) glycerol, 0.5% NaDodSO4, and 0.5% 2-mercaptoethanol. The samples were electrophoresed on NaDodSO4/polyacrylamide slab gels (13 X 13 X 0.12 cm) with the system of Laemmli (10).Unless otherwise stated, 10-14...

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“…All three were inhibited by Zn2+ and by PHMB, a strong sulfhydryl reagent, but not by the weaker reagent NEM. AP2 was unique in being inhibited by bestatin, an inhibitor for aminopeptidases of mammalian cells (1).…”

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confidence: 99%

Neutral Peptidases in the Stroma of Pea Chloroplasts

Liu¹,

Jagendorf²

1986

Plant Physiol.

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ABSTRACIOne endopeptidase (EPI) and at least three aminopeptidases (API, AP2, and AP3) were discovered in the stroma of chloroplasts isolated from pea seedlings (Pisum sativum L.), and purified over 100-fold. EPI requires added Mg2" or Ca2" for activity, may have an additional tightly bound metal atom, and is inhibited by sulfhydryl reagents but not by serine residue-directed inhibitors. It is reversibly inhibited by dithiothreitol. Its specificity is for the bond between two adjacent Ala or Gly residues. Its molecular mass is 93 kilodaltons, estimated on a gel filtration column. Aminopeptidase activities were detected with the aid of different amino acyl-f-naphthylamides as substrates. They were resolved into at least three individual proteins by gel filtration and DEAE-cellulose chromatography, having apparent molecular masses of 269,000 (API), 84,000 (AP2), and 42,000 (AP3) daltons, respectively. Each has a unique specificity for substrates, with API hydrolyzing only the Prolyl-B-naphthylamide. None of the APs require added divalent cations for activity, but the possibility of a tightly bound metal function was suggested in AP2 and AP3 (not API) from effects of inhibitors. A probable sulfhydryl residue function was indicated for all three, from inhibition by p-hydroxymercuribenzoate and Zn2". All these peptidases had pH optima at 7.7.Specific protein turnover or selective protein degradation has been found to be an essential part of chloroplast development. In many cases there are reasons to think that the responsible proteases are located inside the chloroplasts. Proteolytic activity was postulated to be responsible for prolamellar body degradation when etiolated leaves are put in the light (5); for the removal of damaged QB apoprotein, and therefore for its high turnover rate (10, 15); for accelerated turnover of chloroplast-encoded 48 and 34.5 kD polypeptides in thylakoids lacking PSII (1 1); and for rapid degradation of NADPH:Pchlide reductase in barley etioplasts once the plants are put into the light (9). During leaf senescence, extensive protein degradations occurring in apparently intact chloroplasts are explained by an internal set of proteases (21). Chloroplast localization of peptidases was noted in wheat by Waters et al. (22). The best defined internal protease is the one in pea chloroplasts which processes ribulose 1,5-bis P carboxylase small subunit precursors imported from the cytosol; this was purified to a considerable degree ( 19).A thylakoid-located, ATP-dependent proteolytic activity was found responsible for removal of a significant fraction of newly translated proteins in pea chloroplasts, perhaps those which are mature but unassembled into larger complexes (12,14). On the other hand, an ATP-independent but Mg2+-dependent proteolytic activity seemed to be responsible for removal of newly translated proteins containing abnormal amino acids, and of prematurely terminated proteins (13). In an effort to locate the specific enzyme responsible for one or another of these activities, we have fo...

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Degradation of abnormal proteins in intact mouse reticulocytes: accumulation of intermediates in the presence of bestatin.

Abstract: Incubation of intact mouse reticulocytes with bestatin (a competitive inhibitor of aminopeptidases) produced the accumulation of low molecular weight intermediates in the degradation of puromycinyl-peptides or analog-containing proteins that had been pulse labeled with L[1-_4C]leucine. A

Cited by 30 publications

References 20 publications

Bestatin-based chemical biology strategy reveals distinct roles for malaria M1- and M17-family aminopeptidases

Bestatin-based chemical biology strategy reveals distinct roles for malaria M1- and M17-family aminopeptidases

Proposed role of ATP in protein breakdown: conjugation of protein with multiple chains of the polypeptide of ATP-dependent proteolysis.

Neutral Peptidases in the Stroma of Pea Chloroplasts

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