The Molecular Evolution of C14 Peptidases in Microbial Eukaryotes

Abstract

This thesis examines C14 peptidases within sets of eukaryotic predicted proteins, incorporating genomic and transcriptomic data from a wide range of microbial eukaryotes. It aims to elucidate aspects of this enzyme family that have been neglected in the literature, which focuses on animal caspases and plant and fungal metacaspases. The computational approach adopted here enables an exploration of proteomes across the eukaryote tree of life, presenting a novel perspective on the diversity within the C14 peptidase family. Chapter 1 introduces current knowledge about the eukaryote tree of life (eToL) and paracaspases, metacaspases and caspases (PMCs) of the C14 peptidase family, tracing the history of their discovery and classification. Chapter 1 further examines methods for identifying the taxonomic origins of proteins within a set, aiming to determine which protein nodes may represent ancestral forms. Chapter 2 presents two main conclusions from sequence similarity network (SSN) analyses. The first SSN confirms that C14 peptidases form part of the hub of the caspase-haemoglobinase fold superfamily. Structural analysis across the network reveals the presence of the p10 region of the protein in all examined sequences in the superfamily, in contrast with the view that it is absent in ancestral metacaspases. Analysis of a C14 peptidase SSN identifies bacterial generic PMCs as the likely ancestral versions, thereby challenging the existing paradigm of metacaspases as the most ancient members of the C14 family. In addition, the distribution of paracaspases in eukaryotes is far broader and more ancient than previously thought. Chapter 3 focuses on the region of the C14 peptidase network composed of eukaryote metacaspases. The evidence suggests that both type II and type III metacaspases likely appeared before the evolution of photosynthetic clades, rather than being confined to them. It is also apparent that there are many other metacaspase subtypes that have not been formally identified. Chapter 4 explores the presence of metacaspases in Nucleocytoviricota, a phylum of double-stranded large DNA viruses that infect protist hosts, further demonstrating the utility of SSNs in tracing the evolutionary history of these enzymes. In summary, this thesis contributes to the broader understanding of the C14 peptidase family. It offers insights that invite reconsideration of accepted theories and indicates an expanded diversity and evolutionary context for these enzymes. It underscores the value of integrating sequence similarity networks with structural and functional information for proteomic analysis.