Monitoring the biomolecular interactions and the activity of Zn-containing enzymes involved in conformational diseases: experimental methods for therapeutic purposes.

Zinc metalloproteases (ZnMPs) participate in diverse biological reactions, encompassing the synthesis and degradation of all the major metabolites in living organisms. In particular, ZnMPs have been recognized to play a very important role in controlling the concentration level of several peptides and/or proteins whose homeostasis has to be finely regulated for the correct physiology of cells. Dyshomeostasis of aggregation-prone proteins causes pathological conditions and the development of several different diseases. For this reason, in recent years, many analytical approaches have been applied for studying the interaction between ZnMPs and their substrates/inhibitors and how environmental factors can affect enzyme activities. In this scenario, nuclear magnetic resonance, X-ray diffraction, mass spectrometric (MS), and optical methods occupy a very important role in elucidating different aspects of the ZnMPs-substrates/inhibitors interaction, ranging from identification of cleavage sites to quantitation of kinetic parameters and inhibition constants. Here, an overview of all the main achievements in the application of different experimental approaches with special attention to MS methods to the investigation of ZnMPs-substrates/inhibitors interaction is given. A general MS experimental protocol which has been proved to be useful to study such interactions is also described.