Caspases in Cell Death, Inflammation, and Disease

Abstract

Caspases are an evolutionary conserved family of cysteine proteases that are centrally involved in cell death and inflammation responses. A wealth of foundational insight into the molecular mechanisms that control caspase activation has emerged in recent years. Important advancements include the identification of additional inflammasome platforms and pathways that regulate activation of inflammatory caspases; the discovery of gasdermin D as the effector of pyroptosis and interleukin (IL)-1 and IL-18 secretion; and the existence of substantial crosstalk between inflammatory and apoptotic initiator caspases. A better understanding of the mechanisms regulating caspase activation has supported initial efforts to modulate dysfunctional cell death and inflammation pathways in a suite of communicable, inflammatory, malignant, metabolic, and neurodegenerative diseases. Here, we review current understanding of caspase biology with a prime focus on the inflammatory caspases and outline important topics for future experimentation.

Keywords: apoptosis; caspase; gasdermin; inflammasome; interleukin; pyroptosis.

Figure 1. Functional classification and domain architecture of murine and human caspases.

The members of the caspase-family are classified as inflammatory or apoptotic based on the described functions and domain architecture. The caspases-1, 4, -5 and -11 are grouped as inflammatory caspases and share a CARD-domain at the N-terminal end. The apoptotic caspases can be further sub-categorized in initiator- and executioner caspases. The caspases-8, -9 and -10 have domain architecture akin to the inflammatory caspases, however, their function is to initiate apoptosis through the activation of the executioner caspases-3, -6 and -7. In addition, caspase-2 shares domain structures with the inflammatory caspases, however, its function is described to be cell cycle related. Structurally, murine caspase-12 clusters with the inflammatory caspases, however, until today its function remains undefined. Alternatively, for human caspase-12, it is generally accepted that 75% of the population is knock-out, while the remaining 25% carry a proteolytically inactive pseudoprotease (h*). The function of caspase-14 is linked to cell differentiation. m, murine and h, human.

Figure 2. Overview of canonical and non-canonical inflammasomes driving pyroptosis and release of IL-1β and IL-18.

Recognition of pathogen-associated molecular patterns (PAMPs) or danger-associated molecular patterns (DAMPs) by their respective inflammasome-sensors NLRP1b, NLRP3, NLRC4, AIM2 and Pyrin leads to the assembly of a multi-protein complex termed the inflammasome. Consequently, caspase-1 undergoes autoproteolytic processing to lock the protease in its active form. Caspase-1 directly cleaves its substrates gasdermin D and the pro-inflammatory cytokines pro-IL-1β and pro-IL-18. The 31kDa N-terminal cleavage fragment of Gsdmd forms pores in the host cell membrane thereby mediating the release of cytoplasmic content, the mature IL-1β and IL-18 and the other DAMPs IL-1α, HMGB1 and ATP. Alternatively, detection of cytosolic LPS coming from Gram-negative bacteria by the murine caspase-11 or its human orthologues caspases-4 and -5 initiates activation of the proteolytic activity thereby cleaving gasdermin D resulting in pyroptosis. Consequently, the NLRP3 inflammasome is activated leading to the caspase-1-mediated cleavage of the pro-inflammatory cytokines pro-IL-1β and pro-IL-18. T3SS, Type III Secretion System.

Figure 3. Caspase crosstalk pathways.

Schematic representation of the death receptor signalling pathways. Upon ligation of TNF ligand to its receptor (TNFR1), several molecules are required to the cytoplasmic domain of the receptor (TRAF, TRADD, RIPK1 and IAP). The cIAPs recruit the LUBAC complex causing ubiquitination of RIPK1 ultimately leading to the activation of NF-κB and induction of inflammation. Dysregulation of this pathway leads to the formation of complex IIa by recruitment of caspase-8 resulting in the induction of apoptosis. Under the conditions that also the activation of caspase-8 is hampered (complex IIb), RIPK1 will recruit RIPK3 resulting in the phosphorylation of MLKL and the induction of necroptosis. Next to the extrinsic apoptosis pathway, the intrinsic apoptosis pathway is described as the activation of the Bax-Bak-dependent mitochondrial outer membrane permeabilization (MOMP). Consequently, cytochrome C is released from the mitochondria thereby stimulating the activation of caspase-9 and downstream executioners caspases-3 and -7 to initiate apoptosis. Chemotherapy or cytotoxic metabolites enable, through MOMP, activation of a RIPK1-caspase-8-driven NLRP3 activation. Also, ZBP1 is a cytoplasmic sensor for viral RNA of Influenza A virus resulting in the activation of the same RIPK1/caspase-8 pathway. Moreover, caspase-8 by itself can cleave pro-IL-1β. Additionally, caspase-8 and FADD have been found to associate with the NRLP3 inflammasome resulting in the gasdermin D-mediated pyroptotic form of cell death.

Figure 4. Therapeutic targets in the inflammasome pathways.

To date, several inhibitors have been described for the NLRP3 inflammasome. Parthenolide, BAY 11-7082 and 3,4-methylenedioxy-β-nitrostyrene (MNS) are known to inhibit both NF-κB signalling and the NLRP3 inflammasome. Additionally, specific inhibition of NLRP3 has been reported by studies using glyburide and its derivative CRID3/MCC950. Also, CY-09 has been described as a NLRP3 specific inhibitor. More downstream, both caspase-1 and gasdermin D have been targeted by several compounds. Belnacasan (VX-765) and Pralnacasan (VX-740) are bioavailable prodrugs of a potent inhibitor for caspase-1. Alternatively, Antabuse and necrosulfonamide (NSA) were reported to block cleavage and/or oligomerisation of Gsdmd thereby preventing pyroptosis. Once released, the mature IL-1β and IL-18 signal through its appropriate receptors to initiate inflammation in neighbouring cells. Also, anti-cytokine therapies have been generated. Anakinra is recombinant IL-1 receptor antagonist (IL-1Ra) thereby preventing IL-1 signalling. Canakinumab is a fully human monoclonal anti-IL-1β antibody directed against human IL-1β. Finally, the extracellular domains of both receptors (IL-1R1 and IL-1RAcP) were made into a fusion protein as a soluble decoy receptor (Rilonacept). Alternatively, two IL-18 targeted therapeutics have been described. GSK1070806 is a neutralizing humanized monoclonal antibody and Tadekinig alfa is recombinant human IL-18BP both involved in captured bio-active IL-18 away from its receptor.