1.Three-dimensional cultures modeling premalignant progression of human breast epithelial cells: role of cysteine cathepsins.
Mullins SR1, Sameni M, Blum G, Bogyo M, Sloane BF, Moin K. Biol Chem. 2012 Dec;393(12):1405-16.
The expression of the cysteine protease cathepsin B is increased in early stages of human breast cancer.To assess the potential role of cathepsin B in premalignant progression of breast epithelial cells, we employed a 3D reconstituted basement membrane overlay culture model of MCF10A human breast epithelial cells and isogenic variants that replicate the in vivo phenotypes of hyper plasia(MCF10AneoT) and atypical hyperplasia (MCF10AT1). MCF10A cells developed into polarized acinar structures with central lumens. In contrast, MCF10AneoT and MCF10AT1 cells form larger structures in which the lumens are filled with cells. CA074Me, a cell-permeable inhibitor selective for the cysteine cathepsins B and L,reduced proliferation and increased apoptosis of MCF10A, MCF10AneoT and MCF10AT1 cells in 3D culture. We detected active cysteine cathepsins in the isogenic MCF10 variants in 3D culture with GB111, a cell-permeable activity based probe, and established differential inhibition of cathepsin B in our 3D cultures.
2.Quantitative analysis in LC3-II protein in vitro maturation of porcine oocyte.
Lee S1, Hiradate Y2, Hoshino Y2, Tanemura K2, Sato E2. Zygote. 2014 Aug;22(3):404-10. doi: 10.1017/S0967199413000269. Epub 2013 Jun 12.
Microtubule-associated protein light chain 3 (LC3)-II is a marker of autophagosome. In this study, LC3-II expression was used to identify autophagy, during the in vitro maturation of porcine oocytes. In a time-course experiment, cumulus-oocyte complexes (COCs) were cultured in NCSU23 medium for 0 h, 14 h, 28 h or 42 h. The cumulus cells were removed and denuded oocytes were processed for western blotting or immunostaining. Western blotting showed that the LC3-II levels changed over time, with maximum levels observed at 14 h and minimum levels at 42 h. Immunostaining of LC3 showed the signals with dot shapes and ring shapes in oocytes at every group that probably represent autophagosomes. To ascertain whether autophagic induction and degradation were occurring, we treated the cultures with autophagic inhibitors. Lysosomal protease inhibitor E64d and pepstatin A increased the LC3-II levels and wortmannin, inhibitor of autophagic induction, decreased the LC3-II levels.
3.Excitotoxic glutamate insults block autophagic flux in hippocampal neurons.
Kulbe JR, Mulcahy Levy JM, Coultrap SJ, Thorburn A, Bayer KU. Brain Res. 2014 Jan 13;1542:12-9.
Excitotoxic insults such as cerebral ischemia are thought to enhance neuronal autophagy, which is then thought to promote neuronal cell death. Excitotoxic insults indeed increase autophagy markers. Notably, however, autophagy markers can be increased either by autophagy induction (as this enhances their production) or by late-stage autophagy inhibition (as this prevents their degradation during autophagic flux). By comparing each condition with and without protease inhibitors that prevent autophagic degradation of the autophagy markers, the results of this study show that excitotoxic glutamate increases autophagy markers by a late-stage block of autophagy. Initially, this study set out to test if the CaMKII inhibitor tatCN21 mediates its post-insult neuroprotection by regulating autophagy. While tatCN21 partially inhibited basal autophagy in hippocampal neurons, it had no effects on the already blocked autophagy after excitotoxic glutamate insults, indicating that autophagy inhibition is not its neuroprotective mechanism.
4.Expression, characterization, and cellular localization of knowpains, papain-like cysteine proteases of the Plasmodium knowlesi malaria parasite.
Prasad R1, Atul, Soni A, Puri SK, Sijwali PS. PLoS One. 2012;7(12):e51619. doi: 10.1371/journal.pone.0051619. Epub 2012 Dec 12.
Papain-like cysteine proteases of malaria parasites degrade haemoglobin in an acidic food vacuole to provide amino acids for intraerythrocytic parasites. These proteases are potential drug targets because their inhibitors block parasite development, and efforts are underway to develop chemotherapeutic inhibitors of these proteases as the treatments for malaria. Plasmodium knowlesi has recently been shown to be an important human pathogen in parts of Asia. We report expression and characterization of three P. knowlesi papain-like proteases, termed knowpains (KP2-4). Recombinant knowpains were produced using a bacterial expression system, and tested for various biochemical properties. Antibodies against recombinant knowpains were generated and used to determine their cellular localization in parasites. Inhibitory effects of the cysteine protease inhibitor E64 were assessed on P. knowlesi culture to validate drug target potential of knowpains.