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PubMed2024

Abundance of the Membrane Proteome in Yeast Cells Lacking Spc1, a Non-catalytic Subunit of the Signal Peptidase Complex.

The signal peptidase complex (SPC) mediates processing of signal peptides of secretory precursors. But, recent studies show that the eukaryotic SPC also cleaves internal transmembrane segments of some membrane proteins, and its non-catalytic subunit, Spc1/SPCS1 plays a critical role in this process. To assess the impact of Spc1 on membrane proteostasis, we carried out quantitative proteomics of yeast cells with and without Spc1. Our data show that the abundance of the membrane proteome in yeast cells lacking Spc1 is in general reduced compared to that in wild-type cells, implicating its role in controlling the cellular levels of membrane proteins.

Saccharomyces cerevisiae↗
PubMed1979

Characterization of byssochlamyopeptidase A.

The enzyme properties of byssochlamyopeptidase A, a chymosin-like enzyme produced by Byssochlamys fulva were studied. The enzyme was shown to be electrophoretically and immunochemically pure. Most metallic cations had negligible effect, whereas Hg2+ greatly suppressed the enzyme activity. N-Bromosuccinimide and I2 completely inactivated the enzyme. For milk clotting at pH 4.6-6.6, the enzyme was less sensitive to pH than pepsin. The substrate specificity of the enzyme was studied by incubation of the enzyme at 37 degrees C with whole and individual casein fractions at pH 6.6 and pH 3.0, respectively. The proteolysis by the enzyme was found to be most extensive for alphas-, less for kappa-, and least for beta-casein. Studies with different synthetic dipeptides and tripeptides revealed that byssochlamyopeptidase A exhibits specificity for Phe-Tyr and Gly-Phe-Phe, but the enzyme did not hydrolyze Ac-Phe-Tyr(I2).

Ascomycota↗
PubMed2006

Nonsteroidal anti-inflammatory drugs repress beta-secretase gene promoter activity by the activation of PPARgamma.

Epidemiological evidence suggests that nonsteroidal anti-inflammatory drugs (NSAIDs) decrease the risk for Alzheimer's disease (AD). Certain NSAIDs can activate the peroxisome proliferator-activated receptor-gamma (PPARgamma), which is a nuclear transcriptional regulator. Here we show that PPARgamma depletion potentiates beta-secretase [beta-site amyloid precursor protein cleaving enzyme (BACE1)] mRNA levels by increasing BACE1 gene promoter activity. Conversely, overexpression of PPARgamma, as well as NSAIDs and PPARgamma activators, reduced BACE1 gene promoter activity. These results suggested that PPARgamma could be a repressor of BACE1. We then identified a PPARgamma responsive element (PPRE) in the BACE1 gene promoter. Mutagenesis of the PPRE abolished the binding of PPARgamma to the PPRE and increased BACE1 gene promoter activity. Furthermore, proinflammatory cytokines decreased PPARgamma gene transcription, and this effect was supressed by NSAIDs. We also demonstrate that in vivo treatment with PPARgamma agonists increased PPARgamma and reduced BACE1 mRNA and intracellular beta-amyloid levels. Interestingly, brain extracts from AD patients showed decreased PPARgamma expression and binding to PPRE in the BACE1 gene promoter. Our data strongly support a major role of PPARgamma in the modulation of amyloid-beta generation by inflammation and suggest that the protective mechanism of NSAIDs in AD involves activation of PPARgamma and decreased BACE1 gene transcription.

Aged↗
PubMed1975

Purification of S1 muclease from Takadiastase by affinity chromatography on single-stranded DNA-acrylamide columns.

When S1 nuclease from Takadiastase was partially purified according to previously reported methods, it showed a 10 to 15 fold increase in specificactivity. Although such preparations were highly active on single-stranded DNA, they had traces of activity on native DNA and were contaminated by T1-RNase. The S1 enzyme was further purified by a single step of affinity chromatography on single-stranded DNA-acrylamide column to a final purification of 275-fold. This preparation was free of T1-RNase and had an absolute specificity for single-stranded DNA.

Acrylamides↗
PubMed2026

Dual plasmepsin IX and X inhibitors are refractory to development of resistance.

Artemisinin-based combination therapies (ACTs) remain the cornerstone of malaria treatment, but emerging resistance threatens their efficacy. The potential for the development of drug resistance against plasmepsin X (PMX)-selective inhibitors and dual plasmepsin IX/X (PMIX/X) inhibitors was investigated in Plasmodium falciparum. A series of PMX-selective (WM4, WM76, WM92) and PMIX/X dual inhibitors (WM382, WM09, WM42) were characterised for potency against parasite growth and enzyme inhibition. In vitro selection experiments showed that all compounds had a high barrier to resistance, although parasites with reduced sensitivity to PMX‑selective inhibitors could still be selected. Resistance mechanisms involved pmx gene amplification and point mutations (D245N, S315P, S359P, I363L) that alter inhibitor binding. Recombinant expression and Michaelis-Menten kinetics demonstrated that these mutations impair drug binding whilst preserving PMX catalytic function. Reverse genetics confirmed that introducing these mutations into the pmx gene resulted in decreased potency of the inhibitors. In this study, resistance to the PMIX/X dual inhibitors evaluated here could not be selected, despite prolonged selection pressure. Antimalarial Resistome Barcoding (AReBar) assays confirmed the absence of pre-existing resistance to either inhibitor class. Critically, PMIX/X dual inhibitors maintained efficacy against parasites with decreased sensitivity to PMX-selective compounds. These findings demonstrate that dual PMIX/X inhibitors present a substantially higher barrier to resistance than PMX-selective inhibitors, informing antimalarial drug development strategies and highlighting dual-target inhibition as a promising approach to mitigate resistance risks.

Aspartic Acid Endopeptidases↗
PubMed1979

Protein degradation in health and disease. Introduction: the classification of proteinases.

Twenty years after B.S. Hartley's 1960 review, on which the present scheme for classification of the proteinases is based, most of the new information that has been obtained appears fully consistent with Hartley's views. A slight amendment is proposed of the name of the four groups of these enzymes to 'serine', 'cysteine', 'aspartic' and 'metallo'-proteinases.

Animals↗
PubMed1977

Mechanism of acid protease catalysis based on the crystal structure of penicillopepsin.

A proposed mechanism for the catalytic hydrolysis of peptide bonds by acid proteases is similar in many respects to the Zn-carbonyl mechanism previously derived for carboxypeptidase A. In the acid proteases the electrophilic component is the proton shared by Asp-32 and Asp-215; Tyr-75 donates its proton to the amide nitrogen of the scissile bond and an OH- ion from a water molecule bound between the carboxyl group of Asp-32 and the substrate attacks the carbonyl carbon atom.

Affinity Labels↗
PubMed1976

4-Chloro-7-nitrobenzo-2-oxa-1,3-diazole as a reactivity probe for the investigation of the thiol proteinases. evidence that ficin and bromelain may lack carboxyl groups conformationally equivalent to that of aspartic acid-158 of papain.

1. 4-Chloro-7-nitrobenzo-2-oxa-1,3-diazole (Nbd chloride) was used as a reactivity probe to characterize the active centres of papin (EC 3.4.22.2), ficin (EC 3.4.22.3) and bromelain (EC 3.4.22.4). 2. In the pH range 0-8 Nbd chloride probably exists mainly as a monocation, possibly with the proton located on N-1 of the oxadiazole ring. 3. Spectroscopic evidence is presented for the intermediacy of Meisenheimer-type adducts in the reaction of Nbd chloride with nucleophiles. 4. The pH-dependence of the second-order rate constants (k) of the reactions of the three enzymes with Nbd chloride was determined at 25 degrees C, I = 0.1 mol/litre in 6.7% (v/v) ethanol in the pH range 2.5-5, where, at least for papain and ficin, the reactions occur specifically with their active-centre thiol groups. The pH-k profile for the papain reaction is bell-shaped (pKaI = 3.24, pKaII = 3.44 and k = 86M(-1)-s(-1), whereas that for ficin is sigmoidal (pKa = 3.6, k = 0.36M(-1)-s(-1), the rate increasing with increasing pH. The profile for the bromelain reaction appears to resemble that for the ficin reaction, but is complicated by amino-group labelling. 5. The bell-shaped profile of the papain reaction is considered to arise from the reaction of the thiolate ion of cysteine-25, maintained in acidic media by interaction with the side chain of histidine-159, with the Nbd chloride monocation hydrogen-bonded at its nitro group to the un-ionized form of the carboxyl group of aspartic acid-158. The lack of acid catalysis in the corresponding reactions of ficin and probably of bromelain suggests that these enzymes may lack carboxyl groups conformationally equivalent to that of aspartic acid-158 of papain. The possible consequences of this for the catalytic sites of these enzymes is discussed.

4-Chloro-7-nitrobenzofurazan↗
PubMedDate not supplied

The primary structure of mitochondrial aspartate aminotransferase from pig heart: peptides obtained by cleavage at basic residues.

Results obtained as part of a study of the primary structure of mitochondrial aspartate aminotransferase from pig heart are described. In particular, the S-aminoethylated protein was digested with trypsin and with the lysine specific protease from A. mellea. In the first case peptides contained 221 out of the total of 401 amino acid residues in the protein were obtained. By contrast the digest with A. mellea protease was not examined exhaustively and six peptides containing 49 amino acid residues were isolated. Digestion of the trifluoroacetylated and S-aminoethylated protein with A. mellea protease yielded a mixture of large fragments three of which, containing 89 amino acid residues, are described here. The combined results of these three digests yielded 66.6% of the total structure, concentrated mainly in the N-terminal half of the protein.

Amino Acid Sequence↗
PubMed1977

N-terminal amino acid sequences of acid proteases: acid proteases from Penicillium roqueforti and Rhizopus chinensis and alignment with penicillopepsin and mammalian proteases.

The amino-terminal sequence (33 residues) of the acid protease from Penicillium roqueforti has been determined with an automated sequencer. The amino-terminal sequence of Rhizopus pepsin (published by Sepulveda, P., Jackson, K. W. & Tang, J. (1975) Biochem. Biophys. Res. Commun. 63, 1106-1112) has been extended from 27 residues to 39 residues. Also, it was found that two forms of Rhizopus pepsin differ in position 15, where Rhizopus pepsin I has an isoleucine and Rhizopus pepsin II a valine residue. The new sequences have been aligned with the amino-terminal sequences of penicillopepsin (EC 3.4.23.7), pig pepsin (EC 3.4.23.1), calf chymosin (EC 3.4.23.4), human pepsin (EC 3.4.23.2), human gastricsin (EC 3.4.23.3), and cow pepsin (EC 3.4.23.1). Residues 31-35 (numbering based on pig pepsin, Tang, J., Sepulveda, P., Marciniszyn, Jr., J., Chen, K.S.C., Huang, W.-Y. , Tao, N., Liu, D. & Lanier, P. (1973) Proc. Natl. Acad. Sci. U.S.A. 70, 3437-3739) are identical in all enzymes. This section contains one of the two aspartic acids (Asp-32) implicated in the active site. The similarity of the sequences provides strong evidence for the homology of these acid proteases.

Amines↗
PubMed1976

Preparation of fully active ficin from Ficus glabrata by covalent chromatography and characterization of its active centre by using 2,2'-depyridyl disulphide as a reactivity probe.

1. Fully active ficin (EC 3.4.22.3) containing 1 mol of thiol with high reactivity towards 2,2'-dipyridyl disulphide (2-Py-S-S-2-Py) at pH4.5 per mol of protein was prepared from the dried latex of Ficus glabrata by covalent chromatography on a Sepharose-glutathione-2-pyridyl disulphide gel. 2. Ficin thus prepared is a mixture of ficins I-IV and ficin G, in which ficins II and III predominate. The various ficins exhibit similar reactivity characteristics towards 2-Py-S-S-2-Py. 3. Use of 2-Py-S-S-2-Py as a reactivity probe demonstrates (a) that in ficin, as in papain (EC 3.4.22.2), the active-centre thiol and imidazole groups interact to provide a nucleophilic state at pH values of approx. 6 additional to the uncomplicated thiolate ion that predominates at pH values over 9, and (b) a structural difference between ficin and papain that leads to a much higher rate of reaction of 2-Py-S-S-2-Py with ficin than with papain at pH values 3-4. This difference is suggested to include a lack in ficin of a carboxyl group conformationally equivalent to that of aspartic acid-158 in papain. 4. The high electrophilicity of the 2-Py-S-S-2PyH+ monocation allows directly the detection of the exposure of the buried thiol group of ficin at pH values below 4.

2,2'-Dipyridyl↗
PubMed1977

Penicillopepsin: 2.8 A structure, active site conformation and mechanistic implications.

The crystal structure of penicillopepsin, an extracellular acid protease isolated from the mold Penicillium janthinellum, has been determined at 2.8 A resolution by the method of multiple isomorphous replacement. The resulting electron density map computed from the native structure factor amplitudes and MIR phases has an overall mean figure of merit of 0.90. The molecule is decidedly nonspherical, with the majority of residues in beta-structure. There is an 18-stranded mixed beta-sheet which forms the structural core in the region of the active site. This site, identified by the covalent binding of two EPNP molecules to Asp-32 and Asp-215, is located in a deep groove which divides the molecule into two approximately equal lobes. Both aspartic acid residues in the active site are in intimate contact with one another and the carboxyl group of Asp-32 makes two other important hydrogen-bonded contacts: one with Ser-35 and the other with the main chain peptide bond between Thr-216 and Gly-217. A proposed mechanism for acid protease catalysis is similar in many aspects to that proposed for carboxypeptidase A. The electrophilic component which polarizes the substrate carbonyl bond in the acid proteases is the proton shared between the beta-carboxyl groups of Asp-32 and Asp-215. The beta-carboxyl group of Asp-32 removes a proton from a water molecule bound between this side chain and the substrate; the resultant OH- attacks the carbonyl carbon atom of the substrate molecule. The phenolic -OH group of Tyr-75 donates its proton to the amide nitrogen of the scissile bond of the substrate.

Amino Acid Sequence↗