FLIP(L): the pseudo-caspase

Abstract

Possessing structural homology with their active enzyme counterparts but lacking catalytic activity, pseudoenzymes have been identified for all major enzyme groups. Caspases are a family of cysteine-dependent aspartate-directed proteases that play essential roles in regulating cell death and inflammation. Here, we discuss the only human pseudo-caspase, FLIP(L), a paralog of the apoptosis-initiating caspases, caspase-8 and caspase-10. FLIP(L) has been shown to play a key role in regulating the processing and activity of caspase-8, thereby modulating apoptotic signaling mediated by death receptors (such as TRAIL-R1/R2), TNF receptor-1 (TNFR1), and Toll-like receptors. In this review, these canonical roles of FLIP(L) are discussed. Additionally, a range of nonclassical pseudoenzyme roles are described, in which FLIP(L) functions independently of caspase-8. These nonclassical pseudoenzyme functions enable FLIP(L) to play key roles in the regulation of a wide range of biological processes beyond its canonical roles as a modulator of cell death.

Keywords: DISC; FLIP(L); apoptosis; autophagy; caspase; necroptosis; pseudo-caspase; pseudoenzymes.

Fig. 1

Schematic representation of cellular FLIP splice variants. (A) The genomic organization of the splice variants of CFLAR/c‐FLIP. The 48‐kb CFLAR gene is comprised of 14 exons. While transcribed into 11 splice forms, only FLIP(L), FLIP(S), and FLIP(R) are expressed at the protein level. Translation start and stop sites are indicated by triangles and stars, respectively. 111(B) All splice variants contain tandem DEDs allowing recruitment to the DISC. Only FLIP(L) is a true pseudo‐caspase, possessing a pseudoenzymatic region that can be cleaved by DISC‐bound procaspase‐8 at Asp 376 to form p43‐ and p12‐FLIP(L) cleavage products. The FLIP(L) pseudo‐caspase domain also contains a NLS and NES. FLIP (S/R) lack the pseudo‐caspase domain of FLIP(L). They differ from each other in their C terminus.

Fig. 2

(Upper) Representation of the crystal structure of the caspase‐8 and FLIP(L) heterodimer (PDB 3H13) 33. The formation of the heterodimer between the (pseudo)catalytic domains of FLIP(L) (green) and caspase‐8 (blue) is energetically favorable compared with the caspase‐8 homodimer. This explains why FLIP(L) can promote caspase‐8 activation and apoptosis in certain conditions (see text for details), as when part of a FLIP(L)/caspase‐8 heterodimer, caspase‐8 has restricted enzymatic activity that is capable of efficiently cleaving and processing adjacent procaspase‐8 homodimers, the rate‐limiting step of procaspase‐8 activation. (Lower) The amino acid residues in the active caspase domain of caspase‐8 and the equivalent pseudo‐caspase region of FLIP(L) have been highlighted in red. Notably, the amino acid changes in FLIP(L)’s pseudo‐caspase domain that render it catalytically inactive are evolutionarily conserved, suggestive of important functionality.

Fig. 3

Processing and substrates of procaspase‐8 homodimers and procaspase‐8/FLIP(L) heterodimers. (A) Homodimerization is initially mediated via homotypic DED interactions. Within a homodimer, the caspase‐8 catalytic domains are arranged in an antiparallel fashion. This creates an enzymatic active site, which can then cleave adjacent homodimers in the region between large (p18) and small (p10) catalytic subunits. This cleavage enables the second activation step to take place, which is intradimer cleavage in the linker region between p18 and the DEDs. The p18/p10 heterotetramers that are formed can be released from the complex and activate apoptosis by cleaving procaspases‐3/7 and BID. (B) Formation of a heterodimer between the (pseudo)catalytic regions procaspase‐8 and FLIP(L) is energetically favorable compared with caspase‐8 homodimers. This promotes formation of a FLIP(L)/caspase‐8 heterodimeric enzyme that can efficiently cleave adjacent procaspase‐8 homodimers between their large and small catalytic subunits, thereby promoting processing of these homodimers. The heterodimer also cleaves adjacent heterodimers and RIPK1. The lack of critical cysteine in FLIP(L)’s ‘active site’ and lack of a suitable target site for caspase‐8 in the region between FLIP(L)’s DED2 and p20 subunit prevent intradimer cleavage of the heterodimer, which is therefore retained in the complex.

Fig. 4

Schematic summary of some of the key caspase‐8‐independent roles of FLIP(L). In addition to functions directly related to its active paralog caspase‐8, FLIP(L) also acts independently. These nonclassical roles of FLIP(L), a selection of which are summarized, comprise an array of key regulatory functions across a wide range of biological processes, not limited to the modulation of cell death.