Death regulator Nedd2-like caspase (Nc, Nedd2-like caspase or Dronc) was firstly identified and characterised in Drosophila in 1999 as a cysteine protease containing an amino-terminal caspase recruitment domain. At first, it was thought of as an effector caspase involved in apoptosis, but subsequent findings have proved that it is, in fact, an initiator caspase with a crucial role in said type of programmed cell death.
Structure Caspase Dronc is a Drosophila melanogaster protein codified by the Dronc gene. It belongs to the cysteine-aspartic proteases family, as it is a protease enzyme that takes part in programmed cell death processes. It is composed of beta-sheet structures surrounded by alpha-helices, which is a common feature in the protein family. Caspases are formed by ensembled or single active heterodimers that come from the intra-chain proteolytic cleavage of inactive zymogens. This cleavage is coordinated by a specific initiator caspase (which undergoes adaptor-assisted self-activation) for each effector caspase. A common feature among initiator caspases is the presence of a long amino-terminal domain with intermolecular interaction motifs such as the caspase recruiting domain (CARD).
Five caspases have been identified in Drosophila. Two of these present long prodomains (structural domains of the caspases) and are considered to be initiator caspases (Dronc and DCP-2 / DREDD). The other three (DCP-1, DrICE and DECAY), which have short prodomains, act as effector caspases activated by initiator caspases. The uncleaved Dronc zymogen is only presented as a monomer, while the autocleaved protein forms a homodimer. It is presumed that its autocatalytic cleavage activates the protein by generating a dimerisation in which both monomers stabilise their active sites mutually. Caspase Dronc's active site is located between positions 271 and 318 of the protein sequence.
Genetics The Dronc gene is located in the 3L chromosome of the fly, between the 9,968,479 bp and the 9,971,002 bp. It transcripts one single polypeptide and there are 66 reported alleles of the gene. Two mutations have been found in the positions 150 and 184, respectively. The Dronc protein has a length of 450 amino acids and a mass of 51,141 Da. +COMPLETE DRONC SEQUENCE11213141MQPPELEIGMPKRHREHIRKNLNILVEWTNYERLAMECVQQGILTVQMLR5161718191NTQDLNGKPFNMDEKDVRVEQHRRLLLKITQRGPTAYNLLINALRNINCL101111121131141DAAVLLESVDESDSRPPFISLNERRTSRKSADIVDTPSPEASEGPCVSKL151161171181191RNEPLGALTPYVGVVDGPEVKKSKKIHGGDSAILGTYKMQSRFNRGVLLM201211221231241VNIMDYPDQNRRRIGAEKDSKSLIHLFQELNFTIFPYGNVNQDQFFKLLT251261271281291MVTSSSYVQNTECFVMVLMTHGNSVEGKEKVEFCDGSVVDMQKIKDHFQT301311321331341AKCPYLVNKPKVLMFPFCRGDEYDLGHPKNQGNLMEPVYTAQEEKWPDTQ351361371381391TEGIPSPSTNVPSLADTLVCYANTPGYVTHRDLDTGSWYIQKFCQVMADH401411421431441AHDTDLEDILKKTSEAVGNKRTKKGSMQTGAYDNLGFNKKLYFNPGFFNE
Human orthologs Although most human caspases are considered orthologs of caspase Dronc, the one that resembles it the most is Caspase-2. However, Nedd2-like caspase is the functional homolog of mammalian Caspase-9.
Location Due to its function and its mostly hydrophilic character, Dronc can be located in the apoptosome, the plasma membrane and the nucleus of the cell. Cells containing this protein have been found in the following structures of the Drosophila melanogaster: extended germ band embryo; eye disc; foregut primordium; germline cyst; and gut section.
Activation
The activation of caspases is a fundamental step required to execute apoptosis. Initiator caspases, such as Dronc, which is the only initiator caspase in Drosophila melanogaster, are activated through different mechanisms, the main one being autocleavage. Understanding how caspases are activated is a crucial element in order to come up with therapeutic treatments triggering specific caspases. For instance, it has been shown that mice missing or having inactivated caspase-9 present several neurological impairments and have a faulty response to cell damage. Just as caspase 9 in mammals, caspase Dronc is a protein that has a caspase activation and recruitment domain (CARD). It is the only Drosophila caspase presenting this domain: this can represent the possibility of establishing a valuable comparison between their activation or inhibition mechanisms.
Activation
Autocleavage To activate zymogen at first, caspase Dronc autocleaves at residue E352 (Glu352, a glutamic acid residue). Autocleavage at this residue induces dimerization and stabilization of the active site. Autocleavage at E352 results in Pr1, which weighs 40kDa. One study showed that, in Drosophila melanogaster S2 cells, Dronc autocleavage is essential for the zymogen activation. Even though caspase Dronc goes through other processes which will be discussed later, Dronc autocleavage was found to be absolutely necessary to induce apoptosis, whereas other cell cofactors seemed to not be enough to induce activation of Dronc if it had not previously been autocleaved at residue E352. After autocleavage, catalytic activity of Dronc is drastically higher than that of the zymogen. However, these results seem to be contradictory with those of another study conducted in BG2 cells. This study concluded that autocleavage at residue E352, just as autocleavage at other sites such as E143, is not needed for Dronc to activate and, consequently, to induce apoptosis.
Processing by DrICE Dronc can also be cleaved by the effector caspase DrICE, which is activated by caspase Dronc itself after autocleavage. However, it has also been suggested that caspases other than Dronc could activate DrICE. Even though uncleavable mutations of Dronc may have the ability to process caspase DrICE, it is autocleaved Dronc that processes caspase DrICE in Drosophila melanogaster cells. Cleavage of Dronc by DrICE occurs at residue D135 (Asp135, an aspartic acid residue). At the beginning of apoptosis, full-length Dronc and autocleaved Dronc, Pr1, are present and can be used as a substrate for DrICE processing. Cleavage at residue D135 can therefore result in two products: cleavage of Dronc Pr1 results in fully processed Dronc, whereas cleavage of full Dronc results in Pr2, which weights 36kDa. However, since Dronc Pr1 seems to be the most abundant during early stages of apoptosis, most caspase Dronc ends up being fully processed.
Interaction with apoptosome In humans, initiator caspases such as Caspase-2 and Caspase-9 have a prodomain that cleaves caspases to a holoenzyme complex in order to activate the protein. These caspases have a caspase recruitment domain (CARD) that allows them to interact with Apaf-1. The Drosophila melanogaster Apaf-1 homolog, Dark, forms an oligomer with eight Dark chains when dATP is present. This complex is called the apoptosome. Dark, just as Apaf-1, has an N-terminal CARD domain that interacts with caspase Dronc, which is incorporated into this protein complex that will induce cell death.
Recruitment of Dronc by Dark seems to facilitate Dronc autocleavage at residue E352. Dark might not be enough on its own to activate caspase Dronc; it has been suggested that other factors could increase the activation of Dronc (and DrICE) through interaction with the apoptosome. However, in Drosophila melanogaster cells, Dark activity is necessary for normal execution of apoptosis.
Inhibition
Ubiquitylation
Activation of Dronc is also monitored by ubiquitylation. (see figure 2) Ubiquitylation is a modification that happens after protein translation: in this process, protein ubiquitin conjugates to a lysine residue. However, the exact mechanism of Dronc ubiquitylation remains partially unknown to this day. In Drosophila melanogaster cells, mono-ubiquitylation on residue K78 (located in the CARD domain) is carried in caspase Dronc. Ubiquitylation of this residue of the CARD domain, which interacts with the apoptosome during apoptosis, keeps Dronc from activating in the apoptosome and prevents apoptosis of the cell. Ubiquitylation by E3 ubiquitin ligase activity of Diap-1 negatively regulates Dronc activity. Even a partial decrease in ubiquitylation is already enough to importantly rise Dronc activity. Moreover, it has also been shown that K78 mono-ubiquitylation assumes an inhibitory role in Dronc's non-apoptotic capacities, which may not need its catalytic movement, but still being significant for the endurance of the fruit fly. In Drosophila melanogaster cells, caspase Dronc is ubiquitylated by Diap-1. Similarly, effector caspases Caspase-3 and Caspase-7 are monoubiquitylated by cIAP2 in vitro.
Diap-1 Inhibitor of Apoptosis Proteins (IAPs) are a family of proteins that act as endogenous inhibitors of apoptosis: they inhibit caspases. Diap-1 is the Drosophila melanogaster IAP that interacts with both Dronc and DrICE through IAP domains. Some studies have found that Diap-1 inhibits and degrades caspase Dronc, and impairment of Diap-1 interaction with Dronc would prevent the caspase from degrading.
Diap-1 regulates caspase activity in Drosophila melanogaster, thus making Dronc activation dependent on removing Diap-1. Diap-1 is the protein that inhibits both Dronc and DrICE, and prevents apoptosis from being executed. Removal of Diap-1 RNAi triggers caspase activation and, thus, apoptosis. Moreover, during apoptosis, removal of Diap-1 facilitates interaction between Dronc and Dark, which supports the fact that Diap-1 is charged of regulating and inhibiting caspase Dronc. In fact, during apoptosis, proteosomal degradation of Diap-1 takes place (and is necessary) right before cleavage and activation of caspases Dronc and DrICE. Finally, binding of Diap-1 seems to not be sufficient for Dronc inhibition; it seems that ubiquitylation the RING domain of Diap-1 is necessary for complete inhibition of Dronc.
Functions Caspase Dronc has an essential catalytic activity. It is defined as a cysteine protease (or thiol protease), which means that a nucleophilic cysteine thiol forms a catalytic triad (Cys–His–Asn) at the active site of the enzyme that intercedes in the catalysis. The main function of this kind of endopeptidases is to catalyze the hydrolysis of peptide bonds in order to cleave proteins into smaller fragments (see figure 3). For that reason, Nedd2-like caspase is responsible for the activation of effector caspases. On the other hand, as a caspase, Dronc is fully involved in programmed cell death (PCD) processes, which have repercussions in regulative and reproductive functions of the Drosophila melanogaster.
Apoptotic functions Apoptosis is an essential process for the development of multicellular organisms. Its role is to remove excess cells during development (e.g. sculpting the digits of vertebrates), as well as being responsible for detaching damaged, potentially dangerous, cells. Because of its extreme importance, this pathway has been shown to be vastly conserved throughout evolution. One event that is capable of triggering this type of programmed cell death is the activation of the JNK (c-Jun N-terminal kinase) signalling pathway, due to stress caused by chromosomal instability (CIN). The apoptosis pathway is regulated by caspases, a family of proteases that lead to the disassembling of the cell by cleaving protein targets following an aspartate residue. In response to certain apoptotic stimuli, the inactive caspases zymogens turn into active enzymes that start a cascade of caspases-induced proteolytic cleavage processes which culminate with the DNA decomposition and the cell death. Next, neighbouring cells or macrophages engulf whatever is left of the dwindled cell (often called apoptotic bodies) in order to minimize its effect on the surrounding ones, and at the same time to avoid inducing an immune response in the body. Studies link failure of apoptosis activation with the development of some types of cancer.