Selective chemical reagents to investigate the role of caspase 6 in apoptosis in acute leukemia T cells.

Activated effector caspases 3, 6 and 7 are responsible for cleaving a number of target substrates, leading to the ultimate destruction of cells via apoptosis. The functions of caspases 3 and 7 in apoptosis execution have been widely studied over the years with multiple chemical probes for both of these enzymes. In contrast, caspase 6 seems to be largely neglected when compared to the heavily studied caspases 3 and 7. Therefore, the development of new small-molecule reagents for the selective detection and visualization of caspase 6 activity can improve our understanding of molecular circuits of apoptosis and shed new light on how they intertwine with other types of programmed cell death. In this study, we profiled caspase 6 substrate specificity at the P5 position and discovered that, similar to caspase 2, caspase 6 prefers pentapeptide substrates over tetrapeptides. Based on these data, we developed a set of chemical reagents for caspase 6 investigation, including coumarin-based fluorescent substrates, irreversible inhibitors and selective aggregation-induced emission luminogens (AIEgens). We showed that AIEgens are able to distinguish between caspase 3 and caspase 6 in vitro. Finally, we validated the efficiency and selectivity of the synthesized reagents by monitoring lamin A and PARP cleavage via mass cytometry and western blot analysis. We propose that our reagents may provide new research prospects for single-cell monitoring of caspase 6 activity to reveal its function in programmed cell death pathways.

Evaluation of the effects of phosphorylation of synthetic peptide substrates on their cleavage by caspase-3 and -7.

Caspases are a family of enzymes that play roles in cell death and inflammation. It has been suggested that in the execution phase of the apoptotic pathway, caspase-3, -6 and -7 are involved. The substrate specificities of two proteases (caspases 3 and 7) are highly similar, which complicates the design of compounds that selectively interact with a single enzyme exclusively. The recognition of residues other than Asp in the P1 position of the substrate by caspase-3/-7 has been reported, promoting interest in the effects of phosphorylation of amino acids in the direct vicinity of the scissile bond. To evaluate conflicting reports on this subject, we synthesized a series of known caspase-3 and -7 substrates and phosphorylated analogs, performed enzyme kinetic assays and mapped the peptide cleavage sites using internally quenched fluorescent peptide substrates. Caspases 3 and 7 will tolerate pSer at the P1 position but only poorly at the P2' position. Our investigation demonstrates the importance of peptide length and composition in interpreting sequence/activity relationships. Based on the results, we conclude that the relationship between caspase-3/-7 and their substrates containing phosphorylated amino acids might depend on the steric conditions and not be directly connected with ionic interactions. Thus, the precise effect of phospho-amino acid residues located in the vicinity of the cleaved bond on the regulation of the substrate specificity of caspases remains difficult to predict. Our observations allow to predict that natural phosphorylated proteins may be cleaved by caspases, but only when extended substrate binding site interactions are satisfied.

Re-emerging Aspartic Protease Targets: Examining Cryptococcus neoformans Major Aspartyl Peptidase 1 as a Target for Antifungal Drug Discovery.

Cryptococcosis is an invasive infection that accounts for 15% of AIDS-related fatalities. Still, treating cryptococcosis remains a significant challenge due to the poor availability of effective antifungal therapies and emergence of drug resistance. Interestingly, protease inhibitor components of antiretroviral therapy regimens have shown some clinical benefits in these opportunistic infections. We investigated Major aspartyl peptidase 1 (May1), a secreted Cryptococcus neoformans protease, as a possible target for the development of drugs that act against both fungal and retroviral aspartyl proteases. Here, we describe the biochemical characterization of May1, present its high-resolution X-ray structure, and provide its substrate specificity analysis. Through combinatorial screening of 11,520 compounds, we identified a potent inhibitor of May1 and HIV protease. This dual-specificity inhibitor exhibits antifungal activity in yeast culture, low cytotoxicity, and low off-target activity against host proteases and could thus serve as a lead compound for further development of May1 and HIV protease inhibitors.