1. A new heterobifunctional cross-linking reagent for the study of biological interactions between proteins. II. Application to the troponin C-troponin I interaction
P C Chong, R S Hodges J Biol Chem. 1981 May 25;256(10):5071-6.
A simple chromatographic procedure using DEAE-Sephadex has been established to isolate the troponin I-troponin C complex from unbound troponin I (TnI) and troponin C (TnC). A 1:1 complex can be formed between bovine cardiac carboxamidomethylated troponin I and rabbit skeletal troponin C. The formation of the complex is calcium dependent. It is stable to DEAE-chromatography in 6 M urea, 3 mM Ca2+ and can be dissociated on DEAE-chromatography in the presence of 6 M urea, 1 mM ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid. TnC was modified with the photoaffinity probe AGTC (N-(4-azidobenzoyl-[2-3H]glycyl)-S-(2-thiopyridyl)-cysteine) at its single cysteine residue (position 98). Photolysis of the CM (carboxamidomethylated)-TnI-AGC-TnC complex resulted in the formation of a covalently linked 1:1 complex. The isolated covalently linked complex could be treated with dithiothreitol to reduce the disulfide bond between N-(4-azidobenzoyl-[2-3H]glycyl)-cysteine (AGC) and TnC to complete the transfer of the radiolabeled AGC from cysteine 98 on TnC to CM-TnI. The CM-TnI-AGC was isolated from TnC on DEAE-chromatography in 6 M urea, 1 mM EGTA, 1 mM dithiothreitol buffer. The formation of the covalent bond between the photoaffinity probe and TnI indicates the close proximity of TnI to cysteine 98 on the TnC. These results demonstrate the general utility of the new heterobifunctional cross-linking reagent to study protein interactions.
2. Redox Mechanisms in Cisplatin Resistance of Cancer Cells: The Twofold Role of Gamma-Glutamyltransferase 1 (GGT1)
Alfonso Pompella, Alessandro Corti, Athanase Visvikis Front Oncol. 2022 May 20;12:920316. doi: 10.3389/fonc.2022.920316. eCollection 2022.
Cisplatin (CDDP) is currently employed for the treatment of several solid tumors, but cellular heterogeneity and the onset of drug resistance dictate that suitable biomarkers of CDDP sensitivity are established. Studies on triple-negative breast cancer (TNBC) have recently confirmed the involvement of gamma-glutamyltransferase 1 (GGT1), whose enzyme activity expressed at the cell surface favors the cellular resupply of antioxidant glutathione (GSH) thus offering cancer cells protection against the prooxidant effects of CDDP. However, an additional well-established mechanism depends on GGT1-mediated matabolism of extracellular GSH. It was in fact shown that glycyl-cysteine - the dipeptide originated by GGT1-mediated GSH metabolism at the cell surface - can promptly form adducts with exogenous CDDP, thus hindering its access to the cell, interactions with DNA and overall cytotoxicity. Both mechanisms: mainainance of intracellular GSH levels plus extracellular CDDP detoxication are likely concurring to determine GGT1-dependent CDDP resistance.
