Z leu leu glu amc

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Z-Leu-Leu-Glu-AMC is a fluorogenic substrate that can be used to detect and measure the activity of protease enzymes, specifically caspase-3 and caspase-7. This substrate is composed of the peptide sequence Z-Leu-Leu-Glu linked to the fluorescent reporter molecule 7-amino-4-methylcoumarin (AMC).

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13 protocols using z leu leu glu amc

Proteasome Activity Assay Protocol

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Catalytic assays were performed in triplicate for chymotrypsin-like, trypsin-like, and caspase-like activity using Suc-LLVY-AMC, Bz-Val-Gly-Arg-AMC, and Z-Leu-Leu-Glu-AMC substrates (Enzo Life Sciences, USA), respectively, at final concentrations of 100 μM (see supplementary material for details).

Esfahanian N., Nelson M., Autenried R., Pattison J.S., Callegari E, & Rezvani K. (2021). Comprehensive Analysis of Proteasomal Complexes in Mouse Brain Regions Detects ENO2 as a Potential Partner of the Proteasome in the Striatum. Cellular and molecular neurobiology, 42(7), 2305-2319.

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Proteasome Activity Assay Protocol

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Proteasome activity assays were performed as described previously [31 (link), 45 , 46 (link)]. Normalized samples (nuclear = 2μg; cytoplasmic = 20μg) were diluted in MilliQ H₂O and mixed with reaction buffer (250mM HEPES, pH 7.5, 5mM EDTA, 0.5% NP-40, 0.01% SDS, 5mM ATP). Fluorogenic peptides Suc-leu-leu-val-thy-AMC (#BML-P802-0005, Enzo Life Sciences), Bz-val-gly-arg-AMC (#BML-BW9375-0005, Enzo Life Sciences) and z-leu-leu-glu-AMC (#BML-ZW9345-0005; Enzo Life Sciences) were then added to the samples at a concentration of 10μM to assess proteasome chymotrypsin-like, trypsin-like and peptidylglutamyl-peptide hydrolyzing-like activities, respectively. The reaction was incubated at 37°C for 2 hours and fluorescence monitored at 360 (excitation)/460 (emission) on a monochromatic plate reader (Synergy H1; Biotek, Winooski, VT). Protein free blanks were used and an AMC standard curve was produced. The scan with the peak level of AMC was used for statistical analyses. Data are presented as the percentage change in relative fluorescent units (RFU) relative to the Control group.

McFadden T., Musaus M., Nelsen J.L., Martin K., Jones N., Smith P., Kugler H, & Jarome T.J. (2020). Dysregulation of protein degradation in the hippocampus is associated with impaired spatial memory during the development of obesity. Behavioural brain research, 393, 112787.

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Proteasome Regulation and Autophagy Modulation

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Bortezomib and carfilzomib were purchased from selleck chemicals. Other reagents were purchased from puromycin (Sigma), cycloheximide (Sigma), SUC-LLVY-AMC (Enzo Life Science), Z-Leu-Leu-Glu-AMC (Enzo Life Science), Boc-Leu-Arg-Arg-AMC (Enzo Life Science). Antibodies used were as follows: PA28α (Cell Signaling), PSMA2 (Cell Signaling), S5a (Cell signaling), PA28β (Cell Signaling), Phospho-eIF2α (Ser51) (Cell signaling), eIF2α (Cell signaling), α-tubulin (Genetex), PA28γ (Genetex), PSMB5 (Genetex), PSMB6 (Enzo life science), PSMB7 (Genetex), PSMB8 (Genetex), PSMB9 (R&D systems), PSMB10 (R&D systems), Rpt5 (Enzo life science), Ubiquitin (Cell Signaling), β-actin (Santa Cruz Technology), TCF11/NRF1 (Cell Signaling), LC3B (Cell Signaling), p62/SQSTM1 (MBL International) human Ig lambda light chain (R&D systems), actin (Sigma). pLKO.1 empty vector, shRNA vector targeting human PA28α, NRF1 siRNA (ON-TARGETplus SMARTpool), PA28α siRNA (Accell SMARTpool), and control siRNA were purchased from Dharmacon. Native Mark unstained protein standard was purchased from life technologies.

Gu Y., Barwick B.G., Shanmugam M., Hofmeister C.C., Kaufman J., Nooka A., Gupta V., Dhodapkar M., Boise L.H, & Lonial S. (2020). Downregulation of PA28α induces proteasome remodeling and results in resistance to proteasome inhibitors in multiple myeloma. Blood Cancer Journal, 10(12), 125.

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Proteasomal Activity in Hypoxic Lung

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Chymotrypsin-like, trypsin-like, and caspase-like proteasome activities were
measured in mouse lung and mouse pulmonary artery after exposure to 3 weeks of
normoxia or hypoxia. Lung tissue from a single animal, or pulmonary arteries
pooled from four animals within the same treatment group, were prepared in
homogenization buffer (25 mM Tris pH 7.5, 100 mM NaCl, 5 mM ATP, 0.2% glycerol).
Samples were then centrifuged for 10 min at 5000 rpm at 4 ℃, the supernatant was
collected, and protein concentrations were determined by bicinchoninic acid
(BCA) Protein Assay Kit (Thermo Scientific, Rockford, IL). To measure
proteasomal activity, samples were then incubated for 1 hour with assay buffer
[50 mM Tris pH 7.5, 2 mM dithiothreitol (DTT), 5 mM MgCl₂, 2 mM ATP]
containing the fluorogenic substrate [40 µM Suc-Leu-Leu-Val-Tyr-
7-amino-4-methylcoumarin (AMC), chymotrypsin-like; Boc-Leu-Arg-Arg-AMC,
trypsin-like; or Z-Leu-Leu-Glu-AMC, caspase-like; Enzo Life Sciences,
Farmingdale, NY]. Samples treated with either 1 µg purified human 20S Proteasome
(Enzo Life Sciences) or 40 nM MG132 were used as positive and negative controls,
respectively. Proteolytic activities were measured by observing the fluorescence
created by release of the AMC fluorescent group with a Wallac Victor 3
fluorimeter (PerkinElmer, Waltham, MA) every 10 minutes over 1 hour
(excitation = 390 nm and emission = 460 nm).

Wade B.E., Zhao J., Ma J., Hart C.M, & Sutliff R.L. (2018). Hypoxia-induced alterations in the lung ubiquitin proteasome system during pulmonary hypertension pathogenesis. Pulmonary Circulation, 8(3), 2045894018788267.

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Proteasomal Activity Measurement in Erythroblasts

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UPS activity was measured as previously described (Brehm et al., 2015 (link)). Briefly, erythroblasts were gently lysed in TSDG buffer (10 mM Tris-HCl, 1.1 mM MgCl2, 10 mM NaCl, 0.1 mM EDTA, 1 mM DTT, 2 mM ATP, 10% (v/v) glycerol, pH 7.0) with 3 freeze-thaw cycles to prevent UPS denaturation. 10 μg cell of lysates were assayed for protease activity in assay buffer (50 mM Tris-HCl, pH 7.5, 5 mM MgCl₂, 2 mM ATP) with either 100 μM Suc-Leu-Leu-Val-Tyr-aminomethyl-coumarin (Suc-Leu-Leu-Val-Tyr-AMC, Enzo Life Sciences) for chymotryptic-like activity, BZ-Val-Gly-Arg-AMC (Enzo Life Sciences) for tryptic-like activity, or Z-Leu-Leu-Glu-AMC (Enzo Life Sciences) for caspase-like activity. UPS activity was determined by measuring, over time, the fluorescence of AMC liberated from peptides substrate (Ex/Em: 380nm/460nm). For purified UPS activity, 2 μg of Human 26S Proteasome Protein (R&D Systems) were incubated, in TSDG buffer, with the indicated concentrations of sodium acetate, sodium succinate, sodium malonate, sodium L-lactate, L-lactic acid or with epoxomicin (10 μM; Sigma–Aldrich) for 30 min at room temperature. The chymotryptic-like activity was then measured as described above.

Caielli S., Cardenas J., de Jesus A.A., Baisch J., Walters L., Blanck J.P., Balasubramanian P., Stagnar C., Ohouo M., Hong S., Nassi L., Stewart K., Fuller J., Gu J., Banchereau J.F., Wright T., Goldbach-Mansky R, & Pascual V. (2021). ERYTHROID MITOCHONDRIAL RETENTION TRIGGERS MYELOID-DEPENDENT TYPE I INTERFERON IN HUMAN SLE. Cell, 184(17), 4464-4479.e19.

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Proteasome Peptidase Activity Assay

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Proteasome peptidase assays were performed as previously described by us Aghdam et al. (2013 (link)). The cells were collected and lysed using the lysis buffer containing 50 mmol/L Tris‐HCl pH 7.4, 1 mmol/L ATP, 10% glycerol, 0.1% NP40, 2 mmol/L MgCl₂, 1.5 mmol/L DTT, and 0.03% SDS. BCA protein assay (Bio‐Rad) was used to determine protein concentrations. The protein lysates (50 μg/well) were added into a dark 96‐well microplate and probed with the fluorogenic peptide substrates: Z‐Leu‐Leu‐Glu‐AMC for caspase‐like, Ac‐Arg‐Leu‐Arg‐ AMC for trypsin‐like, and Suc‐Leu‐Leu‐Val‐Tyr‐AMC for chymotrypsin‐like (Enzo Life Sciences, Farmingdale, NY) at the final concentration of 100 μmol/L in a total volume of 0.1 mL. The plates were incubated in the dark for 30 min at 37°C. Following incubation, the reaction was stopped using 100% ethanol (100 μL/well). The fluorescent intensity was measured using a plate reader (PerkinElmer, Wellesley, MA). The excitation and emission wavelengths were 355 and 460 nm, respectively.

Farnoodian M., Kinter J.B., Yadranji Aghdam S., Zaitoun I., Sorenson C.M, & Sheibani N. (2015). Expression of pigment epithelium‐derived factor and thrombospondin‐1 regulate proliferation and migration of retinal pigment epithelial cells. Physiological Reports, 3(1), e12266.

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In-Gel Proteasome Activity Assay

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In‐gel activity/assembly assay was performed as previously described (Vernace et al, 2007). Briefly, individual tissues were dissected in PBS and homogenized in proteasome buffer (50 mM Tris–HCl, pH 7.4, 5 mM MgCl₂, 1 mM ATP, 1 mM DTT and 10% glycerol) on ice, centrifuged at 16,000 g (4°C, 10 min) and equal amounts of proteins loaded on a Bio‐Rad TGX 7.5% precast gel. Gels were incubated in proteasome buffer containing 0.4 mM Z‐Leu‐Leu‐Glu‐AMC (Enzo Life Sciences) for 15 min (37°C). Proteasome bands were visualized with UV light (360 nm) on a ChemiDoc station (Bio‐Rad). Differential activity was estimate by densitometry using ImageJ.

Tain L.S., Sehlke R., Jain C., Chokkalingam M., Nagaraj N., Essers P., Rassner M., Grönke S., Froelich J., Dieterich C., Mann M., Alic N., Beyer A, & Partridge L. (2017). A proteomic atlas of insulin signalling reveals tissue‐specific mechanisms of longevity assurance. Molecular Systems Biology, 13(9), 939.

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Proteasome Activity Measurement in Fibroblasts

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Measurement of proteasome proteolytic activities was performed as previously described [22 (link),56 (link)]. Briefly, human fibroblasts were plated in 60-mm dishes and were treated for 24 h with a concentration of 5 μM for each compound and 1 μg/mL and 10 μg/mL of concentration for each extract. After the treatment cells were lysed with a buffer suitable for the isolation of 26S proteasome (0.2% Nonidet P-40, 5 mM ATP, 10% glycerol, 20 mM KCl, 1 mM EDTA, 1 mM dithiothreitol and 20 mM Tris, pH 7.6). Lysates were cleared with centrifugation at 19,000g (4 °C) and 20 μg of proteins were immediately used to determine the main proteasome proteolytic activities [chymotrypsin-like (CT-L/LLVY) and caspase-like (C-L/LLE)]. Activities were assayed by recording the hydrolysis of the fluorogenic peptides Suc–Leu–Leu–Val–Tyr–AMC and Z-Leu–Leu–Glu–AMC (Enzo Life Sciences), at 37 °C for 30 min. The fluorescence was measured at a VersaFluorTM Fluorometer System (Bio-Rad laboratories) at excitation and emission wavelengths of 350 and 440 nm, respectively. Each sample was prepared in duplicate.

Georgousaki K., Tsafantakis N., Gumeni S., Lambrinidis G., González-Menéndez V., Tormo J.R., Genilloud O., Trougakos I.P, & Fokialakis N. (2020). Biological Evaluation and In Silico Study of Benzoic Acid Derivatives from Bjerkandera adusta Targeting Proteostasis Network Modules. Molecules, 25(3), 666.

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Proteasome Activity Assays

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Proteasome activity assays were performed as described previously [15 (link), 28 ]. Normalized samples (nuclear = 2μg; cytoplasmic = 20μg) were diluted in MilliQ H₂O and mixed with reaction buffer (250mM HEPES, pH 7.5, 5mM EDTA, 0.5% NP-40, 0.01% SDS, 5mM ATP). Fluorogenic peptides Suc-leu-leu-val-thy-AMC (#BML-P802-0005, Enzo Life Sciences), Bz-val-gly-arg-AMC (#BML-BW9375-0005, Enzo Life Sciences) and z-leu-leu-glu-AMC (#BML-ZW9345-0005; Enzo Life Sciences) were then added to the samples at a concentration of 10μM to assess proteasome chymotrypsin-like, trypsin-like and peptidylglutamyl-peptide hydrolyzing-like activities, respectively. The reaction was incubated at 37°C for 2 hours and fluorescence monitored at 360 (excitation)/460 (emission) on a monochromatic plate reader (Synergy H1; Biotek, Winooski, VT). Protein free blanks were used and an AMC standard curve was produced. The scan with the peak level of AMC was used for statistical analyses. Data are presented as the percentage change in relative fluorescent units (RFU) relative to the Control group.

McFadden T., Farrell K., Martin K., Musaus M, & Jarome T.J. (2022). Short-term exposure to an obesogenic diet causes dynamic dysregulation of proteasome-mediated protein degradation in the hypothalamus of female rats. Nutritional neuroscience, 26(4), 290-302.

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Proteasome Enzymatic Activity Assay

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Cells were lysed on ice by using a buffer suitable for the isolation of 26S proteasome (0.2% Nonidet P-40, 5 mM ATP, 10% glycerol, 20 mM KCl, 1 mM EDTA, 1 mM dithiothreitol, and 20 mM Tris, pH 7.6). Lysates were cleared with centrifugation at 19,000g (4 °C), and, after protein content adjustment with Bradford, supernatants were immediately used to determine the three proteasome proteolytic activities as described previously [14,25] . Briefly, the chymotrypsin-like (CT-L/LLVY), caspase-like (C-L/LLE) and trypsin-like (T-L/LRR) activities were assayed by recording the hydrolysis of the fluorogenic peptides Suc–Leu–Leu–Val–Tyr–AMC, Z-Leu–Leu–Glu–AMC, and Boc–Leu Arg–Arg–AMC (Enzo Life Sciences, Farmingdale, NY, USA), respectively, at 37 °C for 3 min. The fluorescence was measured at a VersaFluorTM Fluorometer System (Bio-Rad laboratories, Hercules, CA, USA) at excitation and emission wavelengths of 350 and 440 nm, respectively.

Sklirou A.D., Ralli M., Dominguez M., Papassideri I., Skaltsounis A.L, & Trougakos I.P. (2015). Hexapeptide-11 is a novel modulator of the proteostasis network in human diploid fibroblasts. Redox Biology, 5, 205-215.

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