The proteasome is the major degradation pathway where the misfolded proteins during protein synthesis and other proteins are proteolyzed. It is present in all eukaryotic cells, archaea, and some bacteria. It is a multicatalytic protease, composed of multiple catalytic and regulatory proteins. It possesses three or four different peptidase activities, including trypsin-like, chymotrypsin-like, and peptidylglutamyl-peptide hydrolyzing activities. Most of proteins are ubiquitinated prior to the proteasome degradation. It is estimated that about 20% of 26S proteasomes are engaged in protein degradation in the absence of proteotoxic stress [1].

Figure 1. Schematic representation of the ubiquitin-proteasome system. The figure is from NIAAA, NIH.

Proteasome inhibitors have found applications as therapeutic drugs against diseases like cancer, and wide application in laboratory research. Bortezomib, also called PS-341, Velcade, and MG-341, is an FDA-approved drug for multiple myeloma and mantle cell lymphoma. It is also used in laboratory experiments to inhibit proteasome activity [2, 3]. Several other proteasome inhibitors are proposed to be useful drugs or are undergoing clinical trials and testing, including disulfiram, epigallocatechin-3-gallate, Salinosporamide A, carfilzomib, ONX 0912, CEP-18770, and MLN9708. Silva MC et al pre-treated neuronal cells with carfilzomib to inhibit proteasome to understand the effect of tau degrader QC-01–175 [4]. GNF6702 inhibits the kinetoplastid proteasome (while possessing no activity against mammalian proteasome) and has been proposed to represent a new class of drugs for Chagas disease, leishmaniasis, and sleeping sickness [5].

Labome surveys the literature for antibodies, instruments, and other reagents. Among the formal publications that contain the explicit references to proteasome inhibitors, MG132 is the predominant choice cited among the articles. Table 1 lists the number of publications for major proteasome inhibitors. Major features of these three proteasome inhibitors are listed in Table 2.

MG132, also called carbobenzoxy-L-leucyl-L-leucyl-L-leucinal, Z-LLL-CHO, is a peptide aldehyde, one of a group of chemicals able to inhibit different types of proteases, including serine proteases, calpain, etc. MG132 and other peptide aldehydes have been shown to strongly inhibit multiple peptidase activities of proteasomes and calpain activity [10].

Some of the MG132 applications in the publications reviewed by Labome are tabulated in Table 3, along with the references. The publications cited usage of 1, 10, 25, 50 uM with treatment periods ranging from 1 to several days.

The proteasome is an integral part of the cellular function. MG132 and all other proteomsome inhibitors, are toxic to cells and tissues, and will cause cell death at high concentrations or after prolonged treatment. It is advisable to titrate the optimal concentration with a significant range (for example, 1000x). The optimal concentration not only depends on the specific cell type, but also depends on cell culture parameters such as cell confluency, serum concentration and media composition.

MG132 should be stored at -20°C, and can be dissolved in DMSO (10 mg/ml) or methanol (1 mg/ml), and aliquoted and stored at -20 or -80°C. If MG132 DMSO solution produces precipitates when adding to the culture medium, the DMSO stock solution can be warmed to 40°C.

Several suppliers provided MG132 in the publications Labome reviewed. MilliporeSigma (catalog number 474790), with brands Sigma-Aldrich, Calbiochem, EMD Biosciences, and Merck, is one of the major MG132 suppliers among the publications. Frottin F et al treated HEK293T cells with 10 uM MG-132 from Cayman Chemical to study nucleolus as a phase-separated protein quality control entity [22]. Yasuda S et al studied phase separation of proteasomes with MG-132 from Peptide Institute [23].

Figure 2. Chemical structure of MG132.

Lactacystin is an antibiotic, isolated from Streptomyces and now can be synthesized chemically. It hydrolyzes in cells and in vivo into clasto-lactacystin beta-lactone, which is likely the active inhibitor that covalently modifies the N-terminal threonine of subunit X in the mammalian 20S proteasome. The covalent binding is highly specific thus it does not affect cysteine or serine proteases, behaving more specifically than peptide aldehydes like MG132.

Figure 3. Chemical structure of lactacystin.

Lactacystin has been used at 10 uM, 25 uM for hours in cell culture.

MilliporeSigma lactacystin was used to study the effect of Bacillus anthracis edema toxin on nuclear glycogen synthase kinase 3beta [25], and to investigate the regulation of LRRK2 ubiquitination and degradation [26].

BIOMOL (now Enzo Life Sciences) clasto-lactacystin beta-lactone was used to study the role of activation function-1 in regulating proteasome-dependent nuclear mobility and E6-associated protein ubiquitin ligase recruitment to the estrogen receptor beta [18].

Lactacystin from Bio-Connect [19] and MilliporeSigma [24, 27] was also cited.

MilliporeSigma proteasome inhibitor I (PSI) was used to incubate transfected cells [28], and Peptide Institute proteasome inhibitor PSI (N-carbobenzoxy-L-isoleucyl-L-gamma-t-butyl-L-glutamyl-L-alanyl-L-leucinal) was used to treat the cells [29].

Velcade (synonym: PS-341, MG-341, Bortezomib), was used in laboratory experiments to inhibit proteasome activity [2, 30]. Ling Q et al used 5 mM bortezomib in protoplasts [12]. Chui AJ et al treated HEK 293T and RAW 264.7 cells with 20 uM Bortezomib from LC laboratories for 3 - 6 hours to block the proteasome activity [17]. Velcade cell toxicity has been examined. HEK-293 cells treated with 50 uM/L Velcade for 8 hours had no significant cell loss. Prolonged incubation (24 hours) with 0.5 to 50 μmol/L Velcade caused about 40% cell death [27]. Biovision PS-341 was used in 0.1 uM to study eukaryotic proteasomal degradation caused by Legionella pneumophila [3].

ADP-ribosylation of PI31 by ADP-ribosyltransferase tankyrase (TNKS) inhibits 20S repression by PI31 and thus promotes 26S proteasome activity. XAV939, a small-molecule inhibitor of TNKS, can block PI31 ADP-ribosylation. MilliporeSigma XAV939 was used to investigate the role of Etv2 in vascular development [31] and Cellagen XAV939 was used to investigate the regulatory mechanism of the stem cell properties of mouse mammary epithelial cells during development [32].

Ling Q et al inhibited proteasome activity in Arabidopsis protoplasts with 1-10 uM epoxomicin [12]. Yasuda S et al used b-AP15 from LifeSensors, DBeQ from Sigma and CX-5461 from ChemScene to inhibit proteasome activity [23]. Nonspecific aminopeptidase inhibitor bestatin methyl ester (Me-Bs) was used as well [30].

One important issue in using proteasome inhibition is to ensure that the proteasome activity is indeed attenuated. Several approaches can be used to verify the inhibition. One easy way is to use Suc-LLVY-AMC (N-succinyl-L-leucyl-L-leucyl-L-leucyl-7-amido-4-methylcoumarin), as a fluorescent proteasome activity indicator [33]. A ubiquitin-fusion degradation substrate, Ub-G76V-GFP, fluorescences upon proteasomal inhibition. Besse A et al transfected AMO-1 cells with the Ub-G76V-GFP vector (Addgene plasmid 11941) and measured proteasomal inhibition through a BD Fortessa flow cytometer [34]. Another way is to examine the ratio between mono- and poly-ubiquitin moiety levels through western blot. There should be a lower level of mono-ubiquitin if the proteasome activity is inhibited.