Caspase substrates won't be defined by a four-letter code

Abstract

Inflammatory cell death can be mediated by the murine caspase-1 and -11. Genetic and cell biological data point to conflicting conclusions whether these caspases cleave the same substrates or use distinct mechanisms to mediate inflammation and cell death. Peptide screening and biochemical analysis by Gonzales Ramirez et al. now suggest caspase-11 specificity may be determined outside the known substrate motif and identifies substrates cleaved preferentially by caspase-1, providing new opportunities to uniquely target inflammatory caspases.

Introduction

The caspase family of proteases regulates processes related to cell death and inflammation. The apoptotic caspases, including the initiator caspase-8, -9, and -10 and their direct substrates, the effector caspase-3, -6, and -7, are essential for immunologically silent, apoptotic cell death. Conversely, the inflammatory caspase-1, -4, -5, and -11 are executioners of a lytic form of cell death known as pyroptosis, which is accompanied by release of the potent proinflammatory cytokines interleukin-1β (IL-1β)² and IL-18. Both caspase-1 and caspase-11, the murine homolog of human caspase-4 and -5, regulate pyroptosis through cleavage of the terminal effector protein gasdermin D (1, 2). Following gasdermin D cleavage, oligomers of the N-terminal product insert into the plasma membrane, resulting in cell rupture and pyroptotic death (3). Caspase-1 is also known to directly cleave the inactive zymogens of the two cytokines (pro-IL-1β and pro-IL-18) to release their active forms. Although genetic evidence suggests that these cytokines are not directly cleaved by caspase-11, the two caspases are 53% identical in their catalytic domains and their substrate specificities, based on prior peptide screening, are highly overlapping (4, 5). The current Editors' Pick by Gonzalez Ramirez et al. (6) provides intriguing data that may explain this discrepancy and identifies new peptide sequences that could be developed to interrogate pyroptosis pathways.

Caspases are cysteine proteases that cleave their substrates after an aspartic acid at position 1 (P₁), and the P₁–P₄ amino acids are believed to create a recognition motif that confers both efficacy and a degree of specificity for particular caspases (7, 8). The motif is flexible in composition, except for the mandatory aspartic acid immediately N-terminal to the cleavage site. Peptide screening tools have been extensively used to identify tetrapeptide substrate motifs for each caspase and the optimal sequences for caspase-1 and -11 have been reported as Trp-Glu-His-Asp (WEHD) and Leu-Glu-His-Asp (LEHD) respectively (4, 5). Gonzalez Ramirez et al. (6) hypothesized that the inability of caspase-11 to efficiently cleave the prointerleukins may be due to differences in either the preferences of the enzyme active sites or in the substrate tetrapeptide motifs (6).

To test the former hypothesis, the authors used massive hybrid combinatorial substrate library (HyCoSuL) peptide screening to determine the relative enzymatic activity of caspase-1 and caspase-11 when incubated with a library of tetrapeptides containing the mandatory Asp at P₁, randomized residues at any two of P₂, P₃, or P₄ and a set amino acid at the remaining of these three positions (6, 9). These libraries employ 19 naturally occurring amino acids and 110 amino acids not found in natural proteins in an effort to more stringently scrutinize optimal binding peptides. The authors found that, if only natural amino acids were used, the previously reported optimal motifs were indeed accurate. However, substitutions with unnatural amino acids at most positions could increase enzymatic activity for both caspases. The authors also tested the specificity of some of these sequences across both enzymes; although they could identify sequences such as the WQPD peptide that were highly selective for caspase-1, they did not find any selective for caspase-11.

Gonzalez Ramirez et al. (6) then examined the tetrapeptide motifs of murine pro-IL-1β, pro-IL-18, and gasdermin D (Fig. 1). Interestingly, the only difference between the motifs of IL-18 and gasdermin D is a glutamic acid (Glu, E) at P₃ of the pro-IL-18 tetrapeptide, which is replaced by a leucine (Leu, L) in the gasdermin D motif. Consistent with the previously reported, optimal LEHD tetrapeptide for caspase-11 cleavage (5), the enzymatic activity of caspase-11 toward peptides with Glu at P₃ was not reduced compared with its activity on peptides with Leu at P₃. The authors did however experimentally confirm that caspase-11 does not cleave the prointerleukins. These data suggest that the tetrapeptide motif alone is not responsible for the inability of caspase-11 to efficiently target the prointerleukins. This is perhaps not surprising, as, especially for the inflammatory caspases, these optimal tetrapeptides do not commonly match the motifs found upstream of the cleavage site in natural substrates (4).

Figure 1.

Substrate tetrapeptide motifs alone do not determine the specificity of murine inflammatory caspases. HyCoSuL peptide screening suggests that the efficiency of cleaving optimal substrate tetrapeptide motifs is similar between murine inflammatory caspases. However, Caspase-1 cleaves three natural substrates with equivalent efficiency, while Caspase-11 has a remarkable preference to cleave gasdermin D. Therefore, Gonzalez Ramirez et al. posited that distal regions of the substrates could allosterically regulate caspase-11 activity. Ac: acetyl group, ACC: 7-amino-4-carbamoylmethylcoumarin fluorescent tag.

The existence of other factors responsible for this difference in specificity, either in concert with the tetrapeptide motif or independently of it, remains to be determined. Caspase-11 was able to cleave gasdermin D in a cell-free system in the absence of any potential co-factors or chaperones. This led the authors to speculate that an exosite on caspase-11 may be the cause of its perceived specificity for gasdermin D over pro-IL-1β and pro-IL-18 (6). Exosites are allosteric regions of an enzyme that must be occupied as a prerequisite for substrate cleavage. The specific interaction of a distal portion of gasdermin D with a putative exosite is unlikely to be required for protease activity, however. This is because the P₃ Leu tetrapeptides used in the HyCoSuL screen, which are similar to the gasdermin D tetrapeptide motif but do not possess any additional structural elements of that protein, permitted ∼75% caspase activity relative to the maximal caspase-11 activity seen when using a P₃ methionine (Met, M) tetrapeptide. This means that P₃ Leu peptides confer the fourth highest enzymatic activity of all tetrapeptides containing naturally occurring amino acids at this position. Instead, it is more likely that there are sections of the poorly targeted inflammatory cytokines that inhibit caspase-11 activity. Indeed, Gonzalez Ramirez et al. (6) hypothesize that strings of negatively charged amino acids present N-terminal to the tetrapeptide motifs of the cytokine zymogens may be responsible for repulsion or inhibition of caspase-11 through interaction with the hypothetical allosteric site (Fig. 1).

Alternative hypotheses are possible, such as that steric hindrance may simply prevent an interaction between the prointerleukins and caspase-11; however, a specific allosteric site on caspase-11 that regulates the substrates it is able to recognize is an attractive prospect. This is particularly true in the context of developing highly specific caspase inhibitors as tools for molecular biology, as the approach of using competitive peptide–based inhibitors for this purpose has been met with only limited success historically (10). Many pyroptotic phenotypes observed in primary human cells are difficult to recapitulate in cell lines or animal models, and the ability to cleanly remove only one inflammatory caspase from a system through chemical intervention would greatly expand our ability to investigate IL-1β or IL-18-driven biology and disease. Although a specific caspase-11 inhibitor may not itself be beneficial in this regard, it may lead to a clearer understanding of how to inhibit the human orthologues of this protein, caspase-4 and -5, to fully characterize their roles in infection and inflammatory diseases.