alpha-chymotrypsin and lactacystin

Proteolytic enzymes are often strongly affected by redox reactions, free radicals, oxidation, or oxidative stress. The 20S Proteasome and the Immuno-Proteasome are examples of major intracellular proteases whose concentration, transcription, translation, and proteolytic activity are all subject to redox regulation. Proteasomes are essential in maintaining overall protein homeostasis (or proteostasis), and their dysregulation results in detrimental phenotypes associated with various pathologies, including several common age-related diseases. Many studies have used Western blots to assess redox changes in Proteasome protein levels or RT-PCR to study RNA transcript levels, but actual measurements of proteolytic activity are far less common. Since each intact protein substrate exhibits a different proteolytic profile when incubated with proteasome or Immuno-Proteasome [± activators such as 19S or 11S (also called PA28)] and these proteolytic profiles are drastically altered if the protein substrate is denatured, for example by oxidation, heat, acetylation, or methylation. In an attempt to standardize proteasomal activity measurements small fluorogenic protein/peptide substrates were developed to test the three proteolytically active sites of the Proteasome and Immuno-Proteasome: trypsin-like, chymotrypsin-like, and caspase-like activities. Despite extensive use of fluorogenic peptide substrates to measure proteasome activity, there is an absence of a standardized set of best practices. In this study we analyze different parameters, such as sample concentration, AMC conjugated substrate concentration, duration of assay, and frequency of measurements, and examine how they impact the determination of Proteasome and Immuno-Proteasome activities using fluorogenic peptide substrates.

Topics: Acetylcysteine; Animals; Anti-Inflammatory Agents; Chymotrypsin; Cytoplasm; Humans; Hydrogen Peroxide; Mice; Oxidation-Reduction; Oxidative Stress; Oxygen; Peptide Hydrolases; Peptides; Phenotype; Proteasome Endopeptidase Complex; Trypsin

Screening of the 50000 ChemBridge compound library led to the identification of the oxadiazole-isopropylamide 1 (PI-1833) which inhibited chymotrypsin-like (CT-L) activity (IC50 = 0.60 μM) with little effects on the other two major proteasome proteolytic activities, trypsin-like (T-L) and postglutamyl-peptide-hydrolysis-like (PGPH-L). LC-MS/MS and dialysis show that 1 is a noncovalent and rapidly reversible CT-L inhibitor. Focused library synthesis provided 11ad (PI-1840) with CT-L activity (IC50 = 27 nM). Detailed SAR studies indicate that the amide moiety and the two phenyl rings are sensitive toward modifications. Hydrophobic residues, such as propyl or butyl in the para position (not ortho or meta) of the A-ring and a m-pyridyl group as B-ring, significantly improve activity. Compound 11ad (IC50 = 0.37 μM) is more potent than 1 (IC50 = 3.5 μM) at inhibiting CT-L activity in intact MDA-MB-468 human breast cancer cells and inhibiting their survival. The activity of 11ad warrants further preclinical investigation of this class as noncovalent proteasome inhibitors.

Topics: Acetylcysteine; Cell Line, Tumor; Cell Survival; Chromatography, High Pressure Liquid; Chymotrypsin; Cysteine Proteinase Inhibitors; Drug Screening Assays, Antitumor; High-Throughput Screening Assays; Humans; Indicators and Reagents; Mass Spectrometry; Oxadiazoles; Proteasome Inhibitors; Structure-Activity Relationship; Trypsin

The ubiquitin proteasome-proteolytic pathway has emerged as one of the most significant pathways in modulating protein homeostasis under both normal and disease states. The use of proteasome inhibitors (PI) has played a pivotal role in understanding protein turn over. The main objective of this work was to develop a comprehensive, fast, and reliable, yet simple in vitro assay that would allow for the identification and characterization of a wide range of PIs. The assays consist of a 96-well plate high throughput (HTP) method to assess proteasome activity in Hs578T breast cancer cell extracts, purified 20S proteasome, using a fluorogenic substrate, Suc-leu-leu-val-tyr-7-AMC, specific to the chymotrypsin-like enzymatic activity of the proteasome. We showed that the chymotrypsin-like activity of the proteasome was inhibited in the two in vitro systems, albeit to different degrees. The assay system also includes two cell-based assays consisting of a vector expressing a fusion protein of green fluorescent protein (gfp) and Mouse Ornithine Decarboxylase (MODC) in Zs578T (parental Hs578T carrying the vector that expresses the fusion protein). In the cell-based assay analyses (qualitatively by microscopy and quantitatively by flow cytometry), treatment of Zs578T with PIs prevented the degradation of MODC, accumulated gfp, indicative of increased proteasome inhibition. Because no single assay represents a definitive proof of proteasome inhibitory activity, combined, these assays should serve as a comprehensive benchmark for the identification and partial characterization of novel inhibitors. In summary, the four-step assay protocol can easily be adapted into a high throughput format to rapidly screen unknown inhibitors.

Topics: Acetylcysteine; Animals; Biological Assay; Boronic Acids; Bortezomib; Cell Extracts; Cells, Cultured; Chymotrypsin; High-Throughput Screening Assays; Inhibitory Concentration 50; Leupeptins; Mice; Models, Biological; Oligopeptides; Ornithine Decarboxylase; Protease Inhibitors; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Pyrazines; Recombinant Fusion Proteins; Time Factors

We investigated the effect of beta-amyloid (Abeta) (25-35), a cytotoxic fragment of Abeta peptide, on lipid metabolism and protein ubiquitination in cultured rat hippocampal neurons. After treatment with Abeta under conditions leading to apoptotis, as assessed by caspase activity assay, the total cell mass of lipids changed following a biphasic behavior, with an increase that reached a maximum after 16 hr of treatment, followed by a decrease. The increase at 16 hr was 15.3% in the case of phospholipids and 103.0% in the case of gangliosides and was due to enhanced biosynthesis as confirmed by increase of radioactivity incorporation (phospholipids +52.0%, gangliosides +193.1%) in cells fed with tritiated palmitic acid. No change with respect to cholesterol was observed. Strikingly, under these conditions, the ubiquitination state of cell proteins strongly increased. These effects were not observed with the (35-25) reverse sequence peptide. Similarly to Abeta, lactacystin treatment increased lipid synthesis and protein ubiquitination; only lactacystin, and not Abeta, induced a strong decrease of proteasome chimotrypsin activity. These results suggest that Abeta enhances protein ubiquitination, without inhibiting proteasomal activity, and lipid synthesis. These results may shed new light on the mechanisms of Abeta toxicity.

Topics: Acetylcysteine; Amyloid beta-Peptides; Animals; Caspases; Cells, Cultured; Chymotrypsin; Cysteine Proteinase Inhibitors; Hippocampus; Lipid Metabolism; Microscopy, Electron; Nerve Tissue Proteins; Neurons; Palmitic Acid; Peptide Fragments; Rats; Tritium; Ubiquitin

Lipopolysaccharide (LPS) is a major structural component of all Gram-negative organisms and has been implicated in Gram-negative sepsis and septic shock. In the present study, Affymetrix microarray analysis of RNA derived from murine macrophages treated with LPS in the absence or presence of the proteasome inhibitor lactacystin revealed that the vast majority of genes regulated by LPS is under control of the proteasome. Analysis of the data has revealed that the products of these genes participate in 14 distinct signaling pathways. This represents a novel approach to the identification of signaling pathways that are both toll-like receptor 4- and proteasome-dependent and may lead to the development of new drug targets in Gram-negative sepsis and septic shock.

Topics: Acetylcysteine; Animals; Cell Survival; Chymotrypsin; Gene Expression Regulation, Enzymologic; Inflammation; Lipopolysaccharides; Macrophages; Mice; Oligonucleotide Array Sequence Analysis; Proteasome Endopeptidase Complex; Shock, Septic; Signal Transduction; Toll-Like Receptor 4

The pharmacologic properties of ajoene, the major sulfur-containing compound purified from garlic, and its possible role in the prevention and treatment of cancer has received increasing attention. Several studies demonstrated that induction of apoptosis and cell cycle blockade are typical biologic effects observed in tumor cells after proteasome inhibition. The proteasome is responsible for the degradation of a variety of intracellular proteins and plays a key role in the regulation of many cellular processes. The aim of the present work was therefore to explore the effects of ajoene on the proteasome activities. In vitro activities of 20S proteasome purified from human erythrocytes on fluorogenic peptide substrates specific for trypsin-like, chymotrypsin-like and peptidylglutamyl peptide hydrolyzing activities revealed that ajoene inhibited the trypsin-like activity in a dose- and time-dependent manner. Further, the ability of 20S proteasome to degrade the OVA(51-71) peptide, a model proteasomal substrate, was partially but significantly inhibited by ajoene. In addition, when human leukemia cell line HL60 was treated with ajoene, both trypsin- and chymotrypsin-like activities were affected, cells arrested in G2/M phase and total amount of cytosolic proteasome increased. All these data clearly indicate that ajoene may affect proteasome function and activity both in vitro and in the living cell. This is a novel aspect in the biologic profile of this garlic compound giving new insights into the understanding of the molecular mechanisms of its potential antitumor action.

Topics: Acetylcysteine; Antineoplastic Agents; Cell Division; Chymotrypsin; Disulfides; Dose-Response Relationship, Drug; Enzyme Inhibitors; G2 Phase; Garlic; HL-60 Cells; Humans; Hybridomas; Ovalbumin; Peptides; Plant Extracts; Plant Stems; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Sulfoxides; Time Factors; Trypsin

Earlier studies have demonstrated that human platelets contain the 20S proteasome, and its protein activator. However, understanding the potential role of the proteasome in human platelets requires a detailed knowledge about its chymotryptic-like activity, a crucial one for protein degradation in all eukaryotic cells. In this communication we have shown that human platelet 20S proteasome exhibited chymotryptic-like activity towards succinyl-Leu-Leu-Val-Tyr-7-amido-4-methylcoumarin as substrate at a broad pH range, with optimum between pH 7.5-8.0 and 5.0-5.5. These two activities were markedly inhibited by a 10 micromol/l concentration of two structurally unrelated proteasome inhibitors: lactacystin/beta-lactone or benzyloxycarbonyl-Ile-Glu(O-tert.-butyl)-Ala-leucinal, but not by ebelactone B, an inhibitor of lysosomal cathepsin A/deamidase. The chymotryptic-like activity of the 20S proteasome against succinyl-Leu-Leu-Val-Tyr-7-amido-4-methylcoumarin was also significantly inhibited in platelets, after exposure of platelet-rich plasma to 10 micromol/l lactacystin and benzyloxycarbonyl-Ile-Glu(O-tert.-butyl)-Ala-leucinal for up to 60 min. This indicates that these inhibitors can enter platelets and selectively inhibit 20S proteasome activity. We also demonstrated for the first time by Western blot analysis that human platelets contain a proteasome activator, PA28, which is known to play a key role in antigen processing by significant stimulation of the proteasomal chymotryptic-like activity. Since the platelet 20S proteasome was also present in a latent form, this suggests that its activity may be regulated in vivo in human platelets. All these results can therefore be beneficial in future studies on the role of the 20S proteasome in platelet biology.

Topics: Acetylcysteine; Blood Platelets; Chymotrypsin; Coumarins; Cysteine Endopeptidases; Enzyme Activation; Enzyme Inhibitors; Humans; Hydrogen-Ion Concentration; Multienzyme Complexes; Oligopeptides; Proteasome Endopeptidase Complex

We have developed a novel LPS probe using a highly purified and homogenous preparation of [(3)H] Escherichia coli LPS from the deep rough mutant, which contains a covalently linked, photoactivable 4-p-(azidosalicylamido)-butylamine group. This cross-linker was used to identify the LPS-binding proteins in membranes of the murine-macrophage-like cell line RAW 264.7. The alpha-subunit (PSMA1 C2, 29.5 kDa) and the beta-subunit (PSMB4 N3, 24.36 kDa) of the 20S proteasome complex were identified as LPS-binding proteins. This is the first report demonstrating LPS binding to enzymes such as the proteasome subunits. Functionally, LPS enhanced the chymotrypsin-like activity of the proteasome to degrade synthetic peptides in vitro and, conversely, the proteasome inhibitor lactacystin completely blocked the LPS-induced proteasome's chymotrypsin activity as well as macrophage TNF-alpha secretion and the expression of multiple inflammatory mediator genes. Lactacystin also completely blocked the LPS-induced expression of Toll-like receptor 2 mRNA. In addition, lactacystin dysregulated mitogen-activated protein kinase phosphorylation in LPS-stimulated macrophages, but failed to inhibit IL-1 receptor-associated kinase-1 activity. Importantly, lactacystin also prevented LPS-induced shock in mice. These data strongly suggest that the proteasome complex regulates the LPS-induced signal transduction and that it may be an important therapeutic target in Gram-negative sepsis.

Topics: Acetylcysteine; Acute-Phase Proteins; Animals; Carrier Proteins; Cell Line; Chymotrypsin; Cross-Linking Reagents; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Down-Regulation; Enzyme Activation; Escherichia coli; Glutamate Synthase; Leupeptins; Lipopolysaccharides; Macrophages; Macrophages, Peritoneal; Membrane Glycoproteins; Membrane Proteins; Methanosarcina; Mice; Mice, Inbred C3H; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Multienzyme Complexes; Phosphorylation; Proteasome Endopeptidase Complex; Shock, Septic; Signal Transduction; Tritium

Although proteasomes are mainly located in the cytosol, it is known that significant amounts are also associated with endoplasmic reticulum (ER) membranes where they may play a role in the degradation of specific ER membrane proteins. The present studies were undertaken to compare ER and cytosolic proteasomal activities in WB rat liver cells. N-Heptyl-beta-thioglucopyranoside (HTG) extracts of membrane or cytosol fractions were chromatographed in glycerol/ATP buffers on size-exclusion and ion-exchange columns and the elution profiles of proteasomal peptidase activity and immunoreactive components of the 20S complex, 19S complex, and PA28 were compared. Cytosol fractions showed a single peak of chymotrypsin-like peptidase activity (Cht-L), which was inhibited completely by 5 microM lactacystin (LC) or SDS (0.03%) and corresponded to 26S proteasomes based upon the presence of both 20S and 19S components. By comparison, membrane fractions contained two major peaks of Cht-L activity. The first peak shared the same properties as the peak activity observed in cytosol fractions. However, the second peak was stimulated by SDS and was LC-insensitive (5 microM) and contained trypsin-like (T-L) and peptide-glutamyl peptidase (PGPH) but no cathepsin or calcium-activated protease activities. PA28 activator protein was present in both membrane and cytosol fractions. Thus, the principal difference between cytosolic and membrane activity was that the latter fractions contained a novel membrane-associated LC-insensitive protease(s) catalyzing three of the major peptidase activities of the proteasome.

Topics: Acetylcysteine; Animals; Anion Exchange Resins; Blotting, Western; Calcium; Cathepsins; Cell Cycle Proteins; Cell Membrane; Cells, Cultured; Chromatography; Chromatography, Ion Exchange; Chymotrypsin; Cysteine Endopeptidases; Cytosol; Detergents; Dose-Response Relationship, Drug; Endopeptidases; Endoplasmic Reticulum; Epithelium; Liver; Multienzyme Complexes; Peptide Hydrolases; Precipitin Tests; Proteasome Endopeptidase Complex; Proteins; Rats; Resins, Synthetic; Sodium Dodecyl Sulfate

Constitutive activation of NF-kappaB in WEHI 231 early mature B cells resembles the persistent activation of NF-kappaB that is observed upon prolonged stimulation of other cells. In both cases, NF-kappaB DNA binding complexes are found in the nucleus, despite the abundance of cytosolic IkappaB alpha. Recently, we have shown that prolonged activation of 70Z/3 cells with lipopolysaccharide results in the degradation of IkappaB beta, followed by its subsequent resynthesis as a hypophosphorylated protein. This protein was shown to facilitate transport of a portion of NF-kappaB to the nucleus in a manner that protects it from cytosolic IkappaB alpha. We now demonstrate that the most abundant form of IkappaB beta in WEHI 231 cells is a hypophosphorylated protein. This hypophosphorylated IkappaB beta is found in a stable complex with NF-kappaB in the cytosol and is also detected in NF-kappaB DNA binding complexes in the nucleus. It is likely that hypophosphorylated IkappaB beta in WEHI 231 cells also protects NF-kappaB from IkappaB alpha, thus leading to the continuous nuclear import of this transcription factor.

Topics: Acetylcysteine; Antioxidants; B-Lymphocytes; Biological Transport; Cell Line; Cell Nucleus; Chymotrypsin; Cysteine Proteinase Inhibitors; Cytosol; DNA; Enzyme Inhibitors; Feedback; Glycoproteins; Lipopolysaccharides; NF-kappa B; Phosphorylation; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-rel; Pyrrolidines; RNA, Messenger; Thiocarbamates; Tosylphenylalanyl Chloromethyl Ketone; Transcription Factor RelB; Transcription Factors

Lactacystin inhibits cell proliferation and induces a distinctive, predominantly bipolar (two-neurite-bearing) morphology in Neuro 2A murine neuroblastoma cells. It binds with high specificity to the multicatalytic 20S proteasome and inhibits at least three of its peptidase activities (chymotrypsin-like, trypsin-like and peptidylglutamyl-peptide hydrolyzing), each at a different rate, without inhibiting other known proteases. The chymotrypsin-like and trypsin-like activities of the proteasome are inhibited most rapidly, and irreversibly. In an effort to determine which of the peptidase activities needs to be inhibited for neurite outgrowth to occur, we treated Neuro 2A cells with peptide aldehydes that selectively inhibit different proteasome activities.. Treatment with peptide aldehydes ending in a hydrophobic residue, all of which inhibit the chymotrypsin-like activity, results in a bipolar morphology in Neuro 2A cells, whereas treatment with a peptide aldehyde inhibitor of the trypsin-like activity does not lead to a detectable change in morphology. One of the inhibitors that induces neurite outgrowth has been previously shown to inhibit the chymotrypsin-like activity of the proteasome without inhibiting the other apparently distinct peptidase activities that cleave after neutral residues, the so-called 'branched chain amino acid preferring' (BrAAP) and 'small neutral amino acid preferring' (SNAAP) activities, or the peptidylglutamyl-peptide hydrolyzing (PGPH) activity.. The chymotrypsin-like activity appears to antagonize bipolar-type neurite outgrowth in Neuro 2A cells, while the trypsin-like, PGPH, BrAAP and SNAAP appear not to do so. Selective inhibition of a single peptidase activity, as opposed to general inhibition of the proteasome, appears sufficient to induce a specific cellular process. Selective inhibition might be useful in managing diseases where only one activity is involved without completely inhibiting the proteasome. It is also possible that endogenous regulators of the proteasome could affect cellular processes and that certain peptidase activities of the proteasome may have roles in specifying a given cell fate.

Topics: Acetylcysteine; Chymotrypsin; Cysteine Endopeptidases; Enzyme Inhibitors; Multienzyme Complexes; Neuroblastoma; Proteasome Endopeptidase Complex; Protein Binding; Substrate Specificity; Tumor Cells, Cultured