Caspase 6

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Introduction

Caspases are a class of cysteine proteases, responsible for cleaving substrates after an aspartate residue, and are the main mediators of apoptosis and inflammation. Caspases can be divided into three categories based on their sequence similarities and function: (a) inflammatory caspases, (b) apoptotic initiators, and (c) apoptotic effectors. All caspases are produced in cells as catalytically inactive zymogens, and must undergo proteolytic activation during apoptosis. Apoptotic initiators process and activate apoptotic effectors. Caspase-6, caspase-3 and caspase-7 are classified as apoptotic effectors. The Mch2 gene in humans encodes caspase-6, and caspase-6 is often activated by caspase-3 during apoptosis, rather than by initiator caspases. But even when caspase-3 is inactive, caspase-6 is observed to be activated, and the autoactivation of caspase-6 can occur in vivo and in vitro. In the process of apoptosis, caspase-6 localizes in the nucleus, cleaves the lamin protein and nuclear mitotic apparatus protein (NuMA), and induces nuclear fragmentation. However, unlike caspase-3 and caspase-7, caspase-6 activity does not always induce apoptosis. Caspase-6 activity localizes to the cytoplasm and neurites of Alzheimer's disease (AD) neurons, and does not show the classic characteristics of apoptosis. Caspase-6 is widely expressed in the brain and peripheral tissues. In addition to participating in apoptosis, caspase-6 is also highly involved in axon pruning during development and in adult brain; pathological axon degeneration; and a variety of neurodegenerative diseases’ occurrence and progression, such as Huntington's disease (HD) and AD.

Autoactivation Mechanism of Caspase-6 and the Function of the Prodomain

Caspase-6 is represented as a dimer zymogen consisting of a short prodomain, a large subunit (p20), a small subunit (p10), and an intersubunit linker. The three cleavage sites of caspase-6 are the residues TEDD²³ after the prodomain and the DVVD¹⁷⁹ and TEVD¹⁹³ residues in the intersubunit linker (Figure 1).

Figure 1. Schematic diagram of caspase-6 (Wang, X.J.; et al. 2015)

Activation of the effector caspases requires cleavage at the intersubunit linker to release the N-terminus of the small subunit p10. Subsequently, the end is rotated about 180° to form a loop bundle with the four loops of the adjacent catalytic unit. The cleavage at either or both two intersubunit cleavage sites is sufficient to activate caspase-6, while prodomain inhibits the autoactivation of caspase-6 in vivo, but not in vitro. Caspase-6 can be autoactivated or activated by caspase-3, but the way of autoactivation is different from that of caspase-3. During the autoactivation process, caspase-6 first loses its prodomain (cleaved at TETD²³), then cleaved at TEVD¹⁹³, and finally cleaved at DVVD¹⁷⁹. In contrast, caspase-3 is activated by first cleaving caspase-6 at DVVD¹⁷⁹, and then at TETD23 and TEVD¹⁹³. The zymogen structure of caspase-6 shows that the intersubunit cleavage site TEVD¹⁹³ binds to the active site of the same catalytic unit (Figure 2). The binding conformation of this cleavage site is unique to caspase-6, because it has a long intersubunit linker, while the linker of caspase-3 and caspase-7 is too short to place the cleavage sites between subunits in its own active site.

Figure 2. Structure of caspase-6 zymogen (Wang, X.J.; et al. 2015)

The Activation and Regulation Model of Caspase-6

So far, several caspase-6 structures in different states or in combination with different inhibitors have been published. Through the analysis of these structures and related biochemical studies, scientists have summarized the activation and regulation pathways of caspase-6 (Figure 3). This pathway contains 7 different forms of caspase-6, six of which are seen by crystallographic methods. The seventh structural model represents the proposed transient intermediate, it shows the TEVD¹⁹³ binding state after a small conformational change, and is prepared for intramolecular self-cleavage. Caspase-6 zymogen can be activated through autoactivation or caspase-3-mediated activation: When the concentration of caspase-6 is low, prodomain will inhibit self-cleavage, and the expression of caspase-6 will be directly upregulated by p53. When the concentration of caspase-6 increases, the prodomain is removed first, after which autoactivation commences. The prodomain inhibits the autoactivation of caspase-6. The inhibitory effect is only effective at low caspase-6 concentrations and does not inhibit caspase-3-mediated caspase-6 activation. The presence of TEVD¹⁹³ in the active site inhibits the activation and activity of caspase-6 by competing with the substrate and preventing intermolecular cleavage at this site. Phosphorylation at Ser257 locks the zymogen in the TEVD¹⁹³-bound state and prevents self-cleavage, while dephosphorylation facilitates autoactivation. Autoactivated or caspase-3-activated, unphosphorylated caspase-6 is active and can cleave the substrate. However, this active caspase-6 may still be further regulated by caspase inhibitors.

Figure 3. The activation and regulation map of caspase-6 (Wang, X.J.; et al. 2015)