Caspase-9 Activation of Procaspase-3 but Not Procaspase-6 Is Based on the Local Context of Cleavage Site Motifs and on Sequence

Abstract

Studying the interactions between a protease and its protein substrates at a molecular level is crucial for identifying the factors facilitating selection of particular proteolytic substrates and not others. These selection criteria include both the sequence and the local context of the substrate cleavage site where the active site of the protease initially binds and then performs proteolytic cleavage. Caspase-9, an initiator of the intrinsic apoptotic pathway, mediates activation of executioner procaspase-3 by cleavage of the intersubunit linker (ISL) at site 172IETD↓S. Although procaspase-6, another executioner, possesses two ISL cleavage sites (site 1, 176DVVD↓N; site 2, 190TEVD↓A), neither is directly cut by caspase-9. Thus, caspase-9 directly activates procaspase-3 but not procaspase-6. To elucidate this selectivity of caspase-9, we engineered constructs of procaspase-3 (e.g., swapping the ISL site, 172IETD↓S, with DVVDN and TEVDA) and procaspase-6 (e.g., swapping site 1, 176DVVD↓N, and site 2, 190TEVD↓A, with IETDS). Using the substrate digestion data of these constructs, we show here that the P4–P1′ sequence of procaspase-6 ISL site 1 (DVVDN) can be accessed but not cleaved by caspase-9. We also found that caspase-9 can recognize the P4–P1′ sequence of procaspase-6 ISL site 2 (TEVDA); however, the local context of this cleavage site is the critical factor that prevents proteolytic cleavage. Overall, our data have demonstrated that both the sequence and the local context of the ISL cleavage sites play a vital role in preventing the activation of procaspase-6 directly by caspase-9.

Deorphanizing Caspase-3 and Caspase-9 Substrates In and Out of Apoptosis with Deep Substrate Profiling

“…Knowledge of substrates that caspases can cleave is extensive and yet is still incomplete. Some caspases, including caspase-3 and -7, have hundreds of known substrates. − In contrast, little is known about the substrates of caspase-9, other than downstream procaspase-3 and -7 proteolysis. In fact, procaspase-6, which is highly homologous to procaspase-3 and -7, and often likewise classified as an executioner, is not directly activated by caspase-9. − The first non-procaspase substrate of caspase-9 identified was vimentin, which was determined to be cleaved in apoptotic cells.…”

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

“…5A ). Considering that in staurosporine-induced apoptosis, caspase 9 activates procaspase 3 but not procaspase 6, 37 we aimed to investigate whether our selective AIE fluorogens would manage to distinguish the executioner caspase cascade in given time frames. First, we performed western blot analysis to monitor pro-caspase 3 and pro-caspase 6 activation and the processing of caspase substrates, i.e.…”

“…To fully decipher the kinetics of caspase 6 activation, we decided to stimulate Jurkat T cells with staurosporine (STS), a protein kinase inhibitor that induces mitochondrial damage, cytochrome c release and subsequent activation of the caspase 9-dependent apoptosis pathway (Figure 5A). Considering that in staurosporine-induced apoptosis, caspase 9 activates procaspase 3 but not procaspase 6 37 , we aimed to investigate whether our selective AIE fluorogens would manage to distinguish the executioner caspase cascade in given time frames. First, we performed Western blot analysis to monitor pro-caspase 3 and pro-caspase 6 activation and the processing of caspase substrates, i.e., PARP is cleaved by active caspase 3, and lamin A is processed by caspase 6 (Figure 5B, Figure S4).…”

Blockade of ß-Adrenergic Receptors by Nebivolol Enables Tumor Control Potential for Uveal Melanoma in 3D Tumor Spheroids and 2D Cultures

“…It is well known that activation of caspase-3/7 requires the processing of pro-caspase-3, which is dependent on cytochrome c release from impaired mitochondria, other factors, and ATP. Subsequently, caspase-3/7 activity triggers apoptosis and finally cell death [ 51 , 52 , 53 ]. Consistent with this, nebivolol concentrations ≥20–30 µM completely blocked spheroid cell repopulation, allowing tumor control ( Figure 4 ).…”