Peptidyl-Resin Substrates as a Tool in the Analysis of Caspase Activity

Abstract

Caspases, proteolytic enzymes belonging to the group of cysteine proteases, play a crucial role in apoptosis. Understanding their activity and substrate specificity is extremely important. Fluorescence-based approaches, including fluorogenic substrates, are generally used to confirm cleavage preferences. Here we present a new method of substrate specificity and activity analysis based on the application of fix-charge tagged peptides located on the resin. The proteolysis of peptide bond on the resin, occurring even with low efficiency, results in the formation of N-terminal fragments of model peptide containing ionization enhancers in the form of quaternary ammonium groups, allowing for ultrasensitive and reliable analysis by LC-MS/MS. The possibility of application of the proposed solution was tested through the analysis of substrate specificity and activity of caspase 3 or 7. The obtained results confirm the known substrate specificity of executioner caspases. Our solution also allowed us to observe that caspases can hydrolyze peptides shorter than those presented to date in the scientific literature.

New Oxazolo[5,4-d]pyrimidines as Potential Anticancer Agents: Their Design, Synthesis, and In Vitro Biological Activity Research

“…It is the formation of a complex consisting of the DR death domain with the FADD adapter protein and initiating caspases such as caspase-8 or -10 that leads to the direct activation of the effector caspase-3 and, consequently, the initiation of the irreversible process of cell death [ 56 , 57 ]. Caspase-3 in particular is the proteolytic enzyme responsible for the intracellular induction of an apoptosis-inducing signal [ 58 ]. In contrast, BCL-2 is a pro-survival (i.e., anti-apoptotic) protein that suppresses cell death by inhibiting the release of apoptosis regulators such as cytochrome c [ 59 ].…”

Switch-on Fluorescence Analysis of Protease Activity with the Assistance of a Nickel Ion-Nitrilotriacetic Acid-Conjugated Magnetic Nanoparticle

“…Proteases participate in many important physiological processes, such as protein digestion, blood coagulation, and immune system activation. The occurrence of some diseases is closely related to the abnormal activity of proteases [1][2][3][4][5]. For example, matrix metalloproteinases are overexpressed in some cancers, serine proteases are involved in the closely coordinated coagulation cascade, human immunodeficiency virus (HIV) proteases are overexpressed in the life cycle of HIV patients, SARS-CoV-2 main proteases are related to COVID-19, and β/γ-secretases are responsible for the production of toxic β-amyloid peptides in the brains of patients with Alzheimer's disease.…”