The tale of caspase homologues and their evolutionary outlook: deciphering programmed cell death in cyanobacteria

Abstract

Programmed cell death (PCD), a genetically orchestrated mechanism of cellular demise, is paradoxically required to support life. As in lower eukaryotes and bacteria, PCD in cyanobacteria is poorly appreciated, despite recent biochemical and molecular evidence that supports its existence. Cyanobacterial PCD is an altruistic reaction to stressful conditions that significantly enhances genetic diversity and inclusive fitness of the population. Recent bioinformatic analysis has revealed an abundance of death-related proteases, i.e. orthocaspases (OCAs) and their mutated variants, in cyanobacteria, with the larger genomes of morphologically complex strains harbouring most of them. Sequence analysis has depicted crucial accessory domains along with the proteolytic p20-like sub-domain in OCAs, predicting their functional versatility. However, the cascades involved in sensing death signals, their transduction, and the downstream expression and activation of OCAs remain to be elucidated. Here, we provide a comprehensive description of the attempts to identify mechanisms of PCD and the existence and importance of OCAs based on in silico approaches. We also review the evolutionary and ecological significance of PCD in cyanobacteria. In the future, the analysis of cyanobacterial PCD will identify novel proteins that have varied functional roles in signalling cascades and also help in understanding the incipient mechanism of PCD morphotype(s) from where eukaryotic PCD might have originated.

Keywords: Altruistic adaptation; PCD morphotypes; caspase homologues; horizontal gene transfer; orthocaspases; programmed cell death.

Fig. 1.

Timeline representation of major studies in the field of programmed cell death research in cyanobacteria and related organisms.

Fig. 2.

Progression of a cell through various characteristic events and two putative checkpoints (I and II) during programmed cell death (PCD) in cyanobacteria. Under nutrient starvation, vacuolation (stage I), due to disintegration of cellular components like cyanophycean granules, polyphosphate bodies, thylakoids, etc., initiates the mechanism of cell death; however, nutrient supplementation at an early stage (before passing through putative checkpoint I) may inhibit further progression into the death cascade. In the case of non-replenishment of nutrients, the DNA damage (stage II) in the cell would eventually occur as a consequence of intracellular reactive oxygen species formation due to various factors including thylakoid disintegration, although DNA repair mechanisms (before putative checkpoint II) can resist further progression of the cell towards death. Nevertheless, excessive DNA damage induces a putative wild-type orthocaspase (OCA) proteolytic cascade, identical to eukaryotic apoptosis, resulting in cleavage of crucial cellular proteins (stage III) including cytoskeletal proteins, metabolic enzymes, transcription factors, etc. Activation of an OCA proteolytic cascade leads to irreversible progression of the cell towards death largely due to the loss of important cellular proteins, which finally continues into PCD (stage IV). It is possible that under different environmental constraints initiation of PCD may vary, yet the basic scheme of programmed death should remain identical for cyanobacteria.

Fig. 3.

Distribution and abundance of orthocaspase subtypes among cyanobacteria (OCA per 100 proteins). (A) Percentage distribution of true OCAs and δOCAs among 29 analysed cyanobacterial strains showing that about 56% of all the OCAs are wild-type with conserved HC dyad and 44% are mutated at the active site. (B) Distribution and mean abundance of wild-type and mutated OCAs (δOCAs) among unicellular, filamentous, and heterocytous strains showing that the unicellular strains have lower and the filamentous and heterocytous strains have higher mean abundance of wild-type OCAs than their mutated variants. (C) The abundance of wild-type and mutated OCAs among 28 analysed cyanobacterial strains showing that morphological complexities, better physiological capacities, along with a larger genome favour the presence of wild-type OCAs, as heterocytous strains seemed to have more of them than other strains.

Fig. 4.

Habitat distribution of cyanobacterial strains harbouring wild-type orthocaspases (A) and mutated orthocaspases (δOCAs) (B).

Fig. 5.

Distribution of p20-like sub-domain and accessory domains among wild-type and mutated orthocaspases in 29 analysed cyanobacterial strains (True OCA denotes wild-type orthocaspase and δOCAs denotes mutated orthocaspase). Domains are identified using the Conserved Domains Database of NCBI (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) and PHMMER (https://www.ebi.ac.uk/Tools/hmmer/search/phmmer). It was observed that wild-type OCAs (true OCAs) have more domain variability than mutated OCAs (δOCAs). Moreover, the heterocytous strains also harbour greater domain variability than the other strains.

Fig. 6.

Putative mechanism of orthocaspase (OCA)-mediated programmed cell death (PCD) in cyanobacteria (here, OCAs denote only the wild-type OCAs involve in the proteolytic cascade). (1) Death-inducing peptides, ligands, and other factors bind to WD40, ANF, and CHASE2 domains, respectively, at the surface of target cells. These domains are a part of the membrane-bound OCAs having cytosolic localization of p20-like sub-domains. (2) Binding of these factors to the receptors induces autocatalysis and activation of the p20-like sub-domain, (3) resulting in downstream activation of cytosolic OCAs by proteolysis. The interaction of membrane-bound p20-like sub-domain with cytosolic OCAs may occur via the WD40 domain of the latter. However, the activation of cytosolic OCAs may be due to (4, 5, 6) ROS generation resulting in an oxidative burst and (7) subsequent DNA damage. Such activation of cytosolic OCAs either by extracellular or by intracellular signals leads to (8) downstream activation of a proteolytic cascasde and (9) cleavage of target proteins ultimately leading to cell death. Further, (10) several death-inducing factors can be released by the cell upon death. (11) It is also possible that cyanophages also induce a similar proteolytic cascade leading to PCD during phage infection in cyanobacteria. To avoid confusion created by diverse types of accessory domain in the OCA, due to their diverse functionalities and scope to participate in an array of cellular process, only ANF receptor, CHASE2, and WD40 along with the p20-like catalytic sub-domain have been shown, and all other domains are non-specifically represented by ‘Any Domain’.

Fig. 7.

Mutations at specificity pockets and active sites of orthocaspses (OCAs). (A) Percentage occurrence HC dyad and its mutated variants at the active site of OCAs in 98 OCA sub-types. (B) Distribution of HC dyad and its mutated variants among unicellular, filamentous, and heterocytous strains. (C, D) Mutations at of H-specificity pocket (C) and C-specificity pocket (D).

Fig. 8.

Phylogenetic relationship of 98 p20-like catalytic sub-domains of OCAs obtained from 29 cyanobacterial strains. The phylogenetic tree was constructed using the maximum likelihood method of MEGA 7 with metacaspase-like protein from Thalassiosira pseudomana CCMP1335 as outgroup. Bootstrap values of more than 50 are indicated (1000 replicates). Cyanobacterial strains and protein IDs are shown in the tree. Mutated p20-like sub-domains are represented by a cross and mutated active site amino acid dyads are shaded. Two clades representing mutated p20-like sub-domains are shown, i.e. the YN clade (I) and YS clade (II), with the latter divided into two sub-clusters, IIA and IIB. While clade I and IIB all have YN and YS dyads at mutated active sites, IIA have a majority of YS dyads with few variants. Other forms of mutated active sites were distributed along the tree. Moreover, cluster I and sub-cluster IIB mostly have heterocytous strains, except Crinalium epipsammum PCC 9333, whereas sub-cluster IIA has heterocytous, filamentous, and unicellular strains.