Caspase-7 uses RNA to enhance proteolysis of poly(ADP-ribose) polymerase 1 and other RNA-binding proteins

Abstract

To achieve swift cell demise during apoptosis, caspases cleave essential proteins for cell survival and removal. In addition to the binding of preferred amino acid sequences to its substrate-binding pocket, caspase-7 also uses exosites to select specific substrates. 4 lysine residues (K38KKK) located in the N-terminal domain of caspase-7 form such an exosite and promote the rapid proteolysis of the poly(ADP-ribose) polymerase 1 (PARP-1), but the mechanism of recognition remains mostly unknown. In this study, we show that the overall positive charge of the exosite is the critical feature of this evolutionarily conserved binding site. Additionally, interaction with the caspase-7 exosite involves both the Zn3 and BRCT domains of PARP-1 and is mediated by RNA. Indeed, PARP-1 proteolysis efficacy is sensitive to RNase A and promoted by added RNA. Moreover, using affinity chromatography and gel shift assays, we demonstrate that caspase-7, but not caspase-3 or a caspase-7 with a mutated exosite, binds nucleic acids. Finally, we show that caspase-7 prefers RNA-binding proteins (RNA-BPs) as substrates compared to caspase-3 and that RNA enhances proteolysis by caspase-7 of many of these RNA-BPs. Thus, we have uncovered an unusual way by which caspase-7 selects and cleaves specific substrates.

The multifaceted role of PARP1 in RNA biogenesis

“…Our results showing that PARP1's Zn3 is important for its in‐vitro, is consistent with other studies that use deletion mutants to detail PARP1's RNA binding characteristics (Desroches & Denault, 2019; Guetg et al, 2012; Huambachano et al, 2011). However, despite the importance of Zn3, in its absence, PARP1 is still able to bind RNA (Melikishvili, Chariker, et al, 2017), suggesting that other regions of PARP1 also might be contributing to its RNA binding function.…”

“…However, despite the importance of Zn3, in its absence, PARP1 is still able to bind RNA (Melikishvili, Chariker, et al, 2017), suggesting that other regions of PARP1 also might be contributing to its RNA binding function. Indeed, the WGR domain (Huambachano et al, 2011) and the BRCA C‐terminus (BRCT) domain (Desroches & Denault, 2019) of PARP1 have also been implicated in RNA binding. Other PARP family members have been shown to bind RNA using different domains.…”

“…It is possible that RNA activation of PARylation could be dependent on the particular PARP isoform, since several other PARPs (PARP10, PARP11, PARP15, TRPT1) were recently shown to MARylate phosphorylated ends of RNA (Munnur, Somers, et al, 2019). On the other hand, since PARP1 can act as a scaffold between RNA and effector proteins (Desroches & Denault, 2019), it is possible that activation is through these effector proteins. More detailed studies will be needed to unravel the exact mechanisms and functional outcomes of PARP‐RNA interactions in vivo.…”

PARP1 (poly(ADP-ribose) polymerase 1)

“…As mentioned before, PARP1 is known to be cleaved and inactivated by active caspases 3 and 7 and this cleavage is accepted as a 'hallmark of apoptosis' (Castri et al, 2014;Desroches & Denault, 2019). The cleavage causes the formation of 24 kDa and 89 kDa fragments.…”

Crosstalk of the Caspase Family and Mammalian Target of Rapamycin Signaling

“…With the finding of the amplified identified substrates of caspases, this shows that other than the specific sequences, the structure and extended sequence of caspase substrates can explain the cleaved preferences of caspases [ 10 ]. For instance, caspase-7 has higher potential for poly ADP-ribose polymerase cleavage than caspase-3 using exosites [ 118 ].…”