Crystal Structure of Procaspase-1 Zymogen Domain Reveals Insight into Inflammatory Caspase Autoactivation

Abstract

One key event in inflammatory signaling is the activation of the initiator caspase, procaspase-1. Presented here is the crystal structure of the procaspase-1 zymogen without its caspase recruitment domain solved to 2.05 Å . Although the isolated domain is monomeric in solution, the protein appeared dimeric in crystals. The loop arrangements in the dimer provide insight into the first autoproteolytic events that occur during activation by oligomerization. Additionally, in contrast to other caspases, we demonstrate that autoproteolysis at the second cleavage site, Asp 316 , is necessary for conversion to a stable dimer in solution. Critical elements of secondary structure are revealed in the crystal structure that explain why a dimeric protein is favored after proteolysis at this aspartic acid. Dimer stabilization is concurrent with a 130-fold increase in k cat , the sole contributing kinetic factor to an activated and efficient mediator of inflammation.Caspases are a family of aspartic acid-specific cysteine proteases that are involved in inflammation and apoptosis. This enzyme family is generally categorized into three groups as follows: inflammatory initiators (caspases-1, -4, and -5), apoptotic initiators (caspases-2, -8, -9, and -10), and apoptotic effectors (caspases-3, -6, and -7). Because of its role as the protease responsible for processing prointerleukin-1␤ (pro-IL 2 -1␤) to an active 17-kDa form, caspase-1 is a major driver of inflammation and innate immunity (1-3). The enzyme also participates in the activation of prointerleukin-18 and prointerleukin-33 (4, 5). Caspase-1 activation and the subsequent release of IL-1␤, IL-18, and IL-33 are one of the first lines of defense against invading microbes and viruses. Excessive caspase-1 activity, however, can lead to pathologies associated with several autoimmune and inflammatory diseases such as septic shock, inflammatory bowel disease, familial cold autoinflammatory syndrome, rheumatoid arthritis, osteoarthritis, and gout (6 -8).Caspase-1 knock-out mice have strongly supported this assertion, demonstrating an absence of processed IL-1␤ and IL-18 and subsequent decreases in production of cytokines IL-1␣, IL-6, interferon-␥, and tumor necrosis factor-␣ when challenged with lipopolysaccharide or Listeria monocytogenes (6, 9, 10). More recent studies have established the involvement of caspase-1 in neurodegenerative disorders such as trauma and ischemic brain injury, Huntington disease, and amyotrophic lateral sclerosis (7).Pathways leading to the activation of the enzyme are triggered by diverse pathogen and damage-associated molecular patterns such as lipopolysaccharide, flagellin, DNA, RNA, uric acid crystals, bacterial toxins, and UVB damage (11). These patterns are often recognized by Toll-like receptors on the cell surface or by nod-like receptors inside the cell. After pattern recognition, intracellular events result in the conversion of the inactive procaspase-1 zymogen to a fully processed, active enzyme.Procaspase-1 conversion is induced by macromole...

Caspase-1 autoproteolysis is differentially required for NLRP1b and NLRP3 inflammasome function

“…Local increase in caspase-1 zymogen through dimerization increases the catalytic activity required to initiate its own activation by autoproteolysis. Once proteolysed, the caspase-1 p20 and p10 subunits assemble into enzymatically active p20/p10 dimers that cleave caspase-1 substrates such as pro-IL-1β and pro-IL-18 (9,10). Inflammasomes form the molecular platforms that allow caspase-1 dimerization, activation, and autoproteolysis (11).…”

“…Most inflammasomes are composed of specific cytosolic pathogen recognition receptors (PRRs), as well as the apoptosis-associated speck-like protein containing a caspase activation and recruitment domain (CARD) (ASC) adaptor protein that enables the recruitment and activation of the caspase-1 protease. Once caspase-1 is oligomerized within an inflammasome platform, the enzyme undergoes autoproteolysis to form heterodimers of active caspase-1 (9)(10)(11)(12). In the mouse, at least five distinct inflammasomes have been described, distinguished by the PRR that induces the complex formation.…”

Studies of the molecular mechanism of caspase-8 activation by solution NMR

“…And although our understanding of the underlying molecular processes has greatly advanced with the development of important concepts like the induced-proximity model 13,14, the exact molecular details are still not fully understood. Only a few procaspase structures are available such as for Drosophila melanogaster Dronc, 20 procaspase-7, 18 procaspase-1 21 and procaspase-8. 22 Much more structural information is available on active caspases in the presence of bound inhibitors, whereas at present no structural information is available for inhibitor-free apoptotic initiator caspases.…”

“…17 Additional crystal structures of inflammatory caspase-1 and executioner caspase-7 in the absence of inhibitors report on structural changes upon inhibitor binding, suggesting an induced-fit mechanism of substrate binding. 18,19 Furthermore, a few zymogen structures are available, for example for Drosophila melanogaster Dronc, 20 for executioner procaspase-7, 18 for inflammatory procaspase-1 21 and for procaspase-8. 22 The latter was solved by solution NMR and for the first time revealed the position of the inter-subunit linker in a monomeric zymogen.…”

Quantitative Characterization of the Activation Steps of Mannan-binding Lectin (MBL)-associated Serine Proteases (MASPs) Points to the Central Role of MASP-1 in the Initiation of the Complement Lectin Pathway

“…Autoactivation is central to several proteolytic cascade systems, such as the coagulation cascade (1,2), the complement system (3)(4)(5), the system of digestive enzymes (trypsinogen activation) (6), and the caspase system (7,8). "Autocatalytic activation" in these systems is quite common, where an activated protease cleaves its own zymogen form, serving as positive feedback for the activation process.…”