Evolutionary Analysis and Comparative Genomics of Protein Superfamilies

National Library Of Medicine

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Abstract

The systematic analysis and discovery of biological conflict systems has been a major focus of research in the Aravind lab. Biological replicators, from genes within a genome to whole organisms, are locked in conflicts. Comparative genomics has revealed a staggering diversity of molecular armaments and mechanisms regulating their deployment, collectively termed biological conflict systems. These encompass toxins used in inter- and intraspecific interactions, self/nonself discrimination, antiviral immune mechanisms, and counter-host effectors deployed by viruses and intragenomic selfish elements. These systems possess shared syntactical features in their organizational logic and a set of effectors targeting genetic information flow through the Central Dogma, certain membranes, and key molecules like NAD+. These principles can be exploited to discover new conflict systems through sensitive computational analyses. This has led to significant advances in our understanding of the biology of these systems and furnished new biotechnological reagents for genome editing, sequencing, and beyond. Dr. Aravind and his team applied this to discover novel toxin-, restriction-modification, apoptosis, CRISPR/second messenger-regulated systems, and other enigmatic nucleic acid-targeting systems. One major focus of Dr. Aravind's work was on the systems of viruses that interact with the key cellular metabolite NAD+. Such interactions have emerged as a major feature of viral pathogenesis, e.g., that of the SARS-CoV-2 virus, the age of the COVID-19 pandemic. NAD+ and ADP-ribose (ADPr)-containing molecules are at the interface of virus-host conflicts across life, encompassing RNA processing, restriction, lysogeny/dormancy and functional hijacking. Dr. Aravind's lab objectively defined the central components of the NAD+-ADPr networks involved in these conflicts and systematically surveyed 21,191 completely sequenced viral proteomes representative of all publicly available branches of the viral world to reconstruct a comprehensive picture of the viral NAD+-ADPr systems. These systems have been widely and repeatedly exploited by positive-strand RNA and DNA viruses, especially those with larger genomes and more intricate life-history strategies. His work presented evidence that ADP-ribosyltransferases (ARTs), ADPr-targeting Macro, NADAR and Nudix proteins are frequently packaged into virions, particularly in phages with contractile tails (Myoviruses), and are deployed during infection to modify host macromolecules and counter NAD+-derived signals involved in viral restriction. Genes encoding NAD+-ADPr-utilizing domains were repeatedly exchanged between distantly related viruses, hosts and endo-parasites/symbionts, suggesting selection for them across the virus world. Contextual analysis indicated that the bacteriophage versions of ADPr-targeting domains are more likely to counter soluble ADPr derivatives, while the eukaryotic RNA viral versions, such as those of SARS and related viruses, might prefer macromolecular ADPr adducts. Finally, they also used comparative genomics to predict host systems involved in countering viral ADP ribosylation of host molecules. Dr. Aravind has also systematically investigated major protein superfamilies with the objective of gleaning evidence regarding the early events in the emergence of functional diversity in proteins. On the other end of the evolutionary spectrum, he has also been interested in the emergence of novel biological functions in protein superfamilies at more recent time points. With this objective he carried out a major investigation of the Rhodanese-Phosphatase superfamily. The protein-tyrosine/dual-specificity phosphatases and rhodanese domains constitute a sprawling superfamily of Rossmannoid domains that use a conserved active site with a cysteine to catalyze a range of phosphate-transfer, thiotransfer, selenotransfer and redox activities. While these enzymes have been extensively studied in the context of protein/lipid head group dephosphorylation and various thiotransfer reactions, their overall diversity and catalytic potential remain poorly understood. Using comparative genomics and sequence/structure analysis, Dr. Aravind and his lab comprehensively investigated and developed a natural classification for this superfamily. As a result, they identified several novel clades, both those which retain the catalytic cysteine and those where a distinct active site has emerged in the same location (e.g., diphthine synthase-like methylases and RNA 2' OH ribosyl phosphate transferases). They also presented evidence that the superfamily has a wider range of catalytic capabilities than previously known, including a set of parallel activities operating on various sugar/sugar alcohol groups in the context of NAD+-derivatives and RNA termini, and potential phosphate transfer activities involving sugars and nucleotides. They showed that such activities are particularly expanded in the RapZ-C-DUF488-DUF4326 clade, defined in their work for the first time. Some enzymes from this clade were predicted to catalyze novel DNA-end processing activities as part of nucleic-acid-modifying systems that are likely to function in biological conflicts between viruses and their hosts. Dr. Aravind was also involved in several collaborative projects with wet lab investigators during this period. These helped test and finetune the computational hypothesis emerging from his work. One of the major results was on the origins of innate immunity in collaboration with Aaron Whiteley at the University of Colorado, Boulder. Bacteria use a wide range of immune pathways to counter phage infection. Dr. Aravind's earlier work had shown that a subset of these genes shares homology with components of eukaryotic immune systems, suggesting that eukaryotes horizontally acquired certain innate immune genes from bacteria. His work had shown that proteins containing a NACHT module, the central elements of the animal nucleotide-binding domain and leucine-rich repeat-containing gene family (NLRs), are found in bacteria and defend against phages. With the Whiteley lab, they showed that the NACHT proteins of bacteria provide immunity against both DNA and RNA phages and display the characteristic C-terminal sensor, central NACHT, and N-terminal effector modules. Some bacterial NACHT proteins have domain architectures similar to the human NLRs that are critical components of inflammasomes. Human disease-associated NLR mutations that cause stimulus-independent activation of the inflammasome also activated bacterial NACHT proteins, supporting a shared signaling mechanism. This work established that NACHT module-containing proteins are ancient mediators of innate immunity across the tree of life. Dr. Aravind is working on a collaborative project with the labs of Gisela Storz at NICHD on the transport of Magnesium across the membranes. This work has resulted in the identification of a Magnesium pump that is unique to bacteria and certain basal eukaryotes. Characterization of this P-type ATPase pump by Dr. Aravind has helped understand its potential mode of action. The hypotheses generated in this from his analysis are now being tested in the Storz lab. Dr. Aravind is also working in collaboration with Philip Adam's lab in characterizing a novel flagellar component he identified in the Lyme disease pathogen, Borellia burgdorferi.