Proteasome is the main pathway by which misfolded proteins and other proteins are proteolyzed during protein synthesis. It is found in all eukaryotic cells, archaea and some bacteria and is a protease with multiple catalytic activity, consisting of multiple catalytic and regulatory proteins. It has three or four different peptidase activities, including trypsin-like, chymotrypsin-like, and peptidylglutamyl-peptide hydrolytic activity. There are two types of proteases according to the sedimentation coefficient. The relative molecular weight of 26S proteasome was about 2000 kDa, which was dependent on ATP. The relative molecular weight of 20S proteasome is about 750 kDa, independent of ATP. Most proteins require ubiquitination before they can be degraded by the proteasome.
Proteasome inhibitors have been used to treat diseases, such as cancer. They are widely used in laboratory research. Bortezomib, also known as PS-341 (Velcade, or MG-341), is an FDA-approved treatment for multiple myeloma and mantle cell lymphoma. It can also be used in laboratory studies to inhibit the activity of the proteasome. Several other protease inhibitors have also been developed as drugs or are in clinical trials, including disulfiram, epigallocatechin-3-gallate, Salinosporamide A, carfilzomib, ONX0912, CEP-18770, and MLN9708.
MG132
MG132, also known as benzyloxy carbonyl-L-leucinal, Z-Ll-Cho, is a peptide-aldehyde that inhibits the activity of various proteases, including serine proteases, calproteases, and so on. MG132 and other peptide aldehydes can effectively inhibit the protease activity of multiple peptidases of the proteasome, and can inhibit the activity of calsin.
Proteasome is an integral part of cell function. MG132, like all other proteasome inhibitors, is toxic to cells and tissues and can lead to cell death when used in high concentrations or over long periods of time. It is advisable to choose the optimum concentration of dilution in the range of 1000 times. The optimal concentration is not only dependent on cell type, but also depends on cell culture parameters such as cell saturation, serum concentration, and medium composition.
MG132 should be stored at -20℃, soluble in DMSO (10 mg/ mL) and methanol (1 mg/mL), and can be stored at -20℃ or -80℃ respectively after packaging. If MG132 DMSO solution precipitates during medium addition, the original DMSO solution can be heated to 40℃.
Lactacystin
Lactacystin is a microbial metabolite isolated from streptomyces. Chemical synthesis is now possible. It can be hydrolyzed to clasto-lactacystin β-lactone in cells and in vivo, and it may exert its inhibitory effect by covalently modifying the threonine activity of the N-terminal of the X subunit of the mammalian 20S proteasome. This covalent binding is highly specific, so it does not affect cysteine or serine protease activity, but rather acts more like through a peptide aldehyde like MG132.
Other Proteasome Inhibitors
Proteasome Inhibitor I
Calbiochem’s proteasome inhibitor I (PSI) is used to incubate transfected cells, The Peptide Institute’s proteasome inhibitor I was used to treat cells.
Bortezomib
Bortezomib (PS-341) was used in laboratory experiments to inhibit proteasome activity. The cytotoxicity of PS-341 has been studied. Hek-293 cells were treated with 50 umol/L PS-341 for 8 h without obvious cell death. Prolonged culture (24 h) with 0.5 ~ 50μmol/ L PS-341 resulted in about 40% cell death.