Publications by authors named "L Aravind"

382 Publications

GREB1: An evolutionarily conserved protein with a glycosyltransferase domain links ERα glycosylation and stability to cancer.

Sci Adv 2021 Mar 17;7(12). Epub 2021 Mar 17.

Laboratory of NFκB Signalling, Institute of Molecular and Cell Biology (IMCB), A*STAR (Agency for Science, Technology and Research, Singapore 138673, Singapore.

What covalent modifications control the temporal ubiquitination of ERα and hence the duration of its transcriptional activity remain poorly understood. We show that GREB1, an ERα-inducible enzyme, catalyzes O-GlcNAcylation of ERα at residues T553/S554, which stabilizes ERα protein by inhibiting association with the ubiquitin ligase ZNF598. Loss of GREB1-mediated glycosylation of ERα results in reduced cellular ERα levels and insensitivity to estrogen. Higher expression in ERα breast cancer is associated with greater survival in response to tamoxifen, an ERα agonist. Mice lacking exhibit growth and fertility defects reminiscent of phenotypes in ERα-null mice. In summary, this study identifies GREB1, a protein with an evolutionarily conserved domain related to DNA-modifying glycosyltransferases of bacteriophages and kinetoplastids, as the first inducible and the only other (apart from OGT) O-GlcNAc glycosyltransferase in mammalian cytoplasm and ERα as its first substrate.
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http://dx.doi.org/10.1126/sciadv.abe2470DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7968844PMC
March 2021

Reformulation of an extant ATPase active site to mimic ancestral GTPase activity reveals a nucleotide base requirement for function.

Elife 2021 Mar 11;10. Epub 2021 Mar 11.

Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, United States.

Hydrolysis of nucleoside triphosphates releases similar amounts of energy. However, ATP hydrolysis is typically used for energy-intensive reactions, whereas GTP hydrolysis typically functions as a switch. SpoIVA is a bacterial cytoskeletal protein that hydrolyzes ATP to polymerize irreversibly during sporulation. SpoIVA evolved from a TRAFAC class of P-loop GTPases, but the evolutionary pressure that drove this change in nucleotide specificity is unclear. We therefore reengineered the nucleotide-binding pocket of SpoIVA to mimic its ancestral GTPase activity. SpoIVA functioned properly as a GTPase but failed to polymerize because it did not form an NDP-bound intermediate that we report is required for polymerization. Further, incubation of SpoIVA with limiting ATP did not promote efficient polymerization. This approach revealed that the nucleotide base, in addition to the energy released from hydrolysis, can be critical in specific biological functions. We also present data suggesting that increased levels of ATP relative to GTP at the end of sporulation was the evolutionary pressure that drove the change in nucleotide preference in SpoIVA.
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http://dx.doi.org/10.7554/eLife.65845DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7952092PMC
March 2021

Unification and extensive diversification of M/Orf3-related ion channel proteins in coronaviruses and other nidoviruses.

Virus Evol 2021 Jan 16;7(1):veab014. Epub 2021 Feb 16.

Department of Biology, College of Arts and Sciences, Saint Louis University, St. Louis, MO 63103, USA.

The coronavirus, Severe Acute Respiratory Syndrome (SARS)-CoV-2, responsible for the ongoing coronavirus disease 2019 (COVID-19) pandemic, has emphasized the need for a better understanding of the evolution of virus-host interactions. ORF3a in both SARS-CoV-1 and SARS-CoV-2 are ion channels (viroporins) implicated in virion assembly and membrane budding. Using sensitive profile-based homology detection methods, we unify the SARS-CoV ORF3a family with several families of viral proteins, including ORF5 from MERS-CoVs, proteins from beta-CoVs (ORF3c), alpha-CoVs (ORF3b), most importantly, the Matrix (M) proteins from CoVs, and more distant homologs from other nidoviruses. We present computational evidence that these viral families might utilize specific conserved polar residues to constitute an aqueous pore within the membrane-spanning region. We reconstruct an evolutionary history of these families and objectively establish the common origin of the M proteins of CoVs and Toroviruses. We also show that the divergent ORF3 clade (ORF3a/ORF3b/ORF3c/ORF5 families) represents a duplication stemming from the M protein in alpha- and beta-CoVs. By phyletic profiling of major structural components of primary nidoviruses, we present a hypothesis for their role in virion assembly of CoVs, ToroVs, and Arteriviruses. The unification of diverse M/ORF3 ion channel families in a wide range of nidoviruses, especially the typical M protein in CoVs, reveal a conserved, previously under-appreciated role of ion channels in virion assembly and membrane budding. We show that M and ORF3 are under different evolutionary pressures; in contrast to the slow evolution of M as core structural component, the ORF3 clade is under selection for diversification, which suggests it might act at the interface with host molecules and/or immune attack.
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http://dx.doi.org/10.1093/ve/veab014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7928690PMC
January 2021

Jumbo Phages: A Comparative Genomic Overview of Core Functions and Adaptions for Biological Conflicts.

Viruses 2021 Jan 5;13(1). Epub 2021 Jan 5.

National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, MD 20894, USA.

Jumbo phages have attracted much attention by virtue of their extraordinary genome size and unusual aspects of biology. By performing a comparative genomics analysis of 224 jumbo phages, we suggest an objective inclusion criterion based on genome size distributions and present a synthetic overview of their manifold adaptations across major biological systems. By means of clustering and principal component analysis of the phyletic patterns of conserved genes, all known jumbo phages can be classified into three higher-order groups, which include both myoviral and siphoviral morphologies indicating multiple independent origins from smaller predecessors. Our study uncovers several under-appreciated or unreported aspects of the DNA replication, recombination, transcription and virion maturation systems. Leveraging sensitive sequence analysis methods, we identify novel protein-modifying enzymes that might help hijack the host-machinery. Focusing on host-virus conflicts, we detect strategies used to counter different wings of the bacterial immune system, such as cyclic nucleotide- and NAD-dependent effector-activation, and prevention of superinfection during pseudolysogeny. We reconstruct the RNA-repair systems of jumbo phages that counter the consequences of RNA-targeting host effectors. These findings also suggest that several jumbo phage proteins provide a snapshot of the systems found in ancient replicons preceding the last universal ancestor of cellular life.
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http://dx.doi.org/10.3390/v13010063DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7824862PMC
January 2021

An RNA Repair Operon Regulated by Damaged tRNAs.

Cell Rep 2020 Dec;33(12):108527

RNA Biology Laboratory, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, MD 21702, USA. Electronic address:

Many bacteria contain an RNA repair operon, encoding the RtcB RNA ligase and the RtcA RNA cyclase, that is regulated by the RtcR transcriptional activator. Although RtcR contains a divergent version of the CARF (CRISPR-associated Rossman fold) oligonucleotide-binding regulatory domain, both the specific signal that regulates operon expression and the substrates of the encoded enzymes are unknown. We report that tRNA fragments activate operon expression. Using a genetic screen in Salmonella enterica serovar Typhimurium, we find that the operon is expressed in the presence of mutations that cause tRNA fragments to accumulate. RtcA, which converts RNA phosphate ends to 2', 3'-cyclic phosphate, is also required. Operon expression and tRNA fragment accumulation also occur upon DNA damage. The CARF domain binds 5' tRNA fragments ending in cyclic phosphate, and RtcR oligomerizes upon binding these ligands, a prerequisite for operon activation. Our studies reveal a signaling pathway involving broken tRNAs and implicate the operon in tRNA repair.
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http://dx.doi.org/10.1016/j.celrep.2020.108527DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7790460PMC
December 2020

Unification of the M/ORF3-related proteins points to a diversified role for ion conductance in pathogenesis of coronaviruses and other nidoviruses.

bioRxiv 2020 Nov 11. Epub 2020 Nov 11.

The new coronavirus, SARS-CoV-2, responsible for the COVID-19 pandemic has emphasized the need for a better understanding of the evolution of virus-host conflicts. ORF3a in both SARS-CoV-1 and SARS-CoV-2 are ion channels (viroporins) and involved in virion assembly and membrane budding. Using sensitive profile-based homology detection methods, we unify the SARS-CoV ORF3a family with several families of viral proteins, including ORF5 from MERS-CoVs, proteins from beta-CoVs (ORF3c), alpha-CoVs (ORF3b), most importantly, the Matrix (M) proteins from CoVs, and more distant homologs from other nidoviruses. By sequence analysis and structural modeling, we show that these viral families utilize specific conserved polar residues to constitute an ion-conducting pore in the membrane. We reconstruct the evolutionary history of these families, objectively establish the common origin of the M proteins of CoVs and Toroviruses. We show that the divergent ORF3a/ORF3b/ORF5 families represent a duplication stemming from the M protein in alpha- and beta-CoVs. By phyletic profiling of major structural components of primary nidoviruses, we present a model for their role in virion assembly of CoVs, ToroVs and Arteriviruses. The unification of diverse M/ORF3 ion channel families in a wide range of nidoviruses, especially the typical M protein in CoVs, reveal a conserved, previously under-appreciated role of ion channels in virion assembly, membrane fusion and budding. We show that the M and ORF3 are under differential evolutionary pressures; in contrast to the slow evolution of M as core structural component, the CoV-ORF3 clade is under selection for diversification, which indicates it is likely at the interface with host molecules and/or immune attack.

Importance: Coronaviruses (CoVs) have become a major threat to human welfare as the causative agents of several severe infectious diseases, namely Severe Acute Respiratory Syndrome (SARS), Middle Eastern Respiratory Syndrome (MERS), and the recently emerging human coronavirus disease 2019 (COVID-19). The rapid spread, severity of these diseases, as well as the potential re-emergence of other CoV-associated diseases have imposed a strong need for a thorough understanding of function and evolution of these CoVs. By utilizing robust domain-centric computational strategies, we have established homologous relationships between many divergent families of CoV proteins, including SARS-CoV/SARS-CoV-2 ORF3a, MERS-CoV ORF5, proteins from both beta-CoVs (ORF3c) and alpha-CoVs (ORF3b), the typical CoV Matrix proteins, and many distant homologs from other nidoviruses. We present evidence that they are active ion channel proteins, and the Cov-specific ORF3 clade proteins are under selection for rapid diversification, suggesting they might have been involved in interfering host molecules and/or immune attack.
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http://dx.doi.org/10.1101/2020.11.10.377366DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7668736PMC
November 2020

A Membrane-Tethered Ubiquitination Pathway Regulates Hedgehog Signaling and Heart Development.

Dev Cell 2020 11 22;55(4):432-449.e12. Epub 2020 Sep 22.

Departments of Biochemistry and Medicine, Stanford University School of Medicine, Stanford, CA 94305, USA. Electronic address:

The etiology of congenital heart defects (CHDs), which are among the most common human birth defects, is poorly understood because of its complex genetic architecture. Here, we show that two genes implicated in CHDs, Megf8 and Mgrn1, interact genetically and biochemically to regulate the strength of Hedgehog signaling in target cells. MEGF8, a transmembrane protein, and MGRN1, a RING superfamily E3 ligase, assemble to form a receptor-like ubiquitin ligase complex that catalyzes the ubiquitination and degradation of the Hedgehog pathway transducer Smoothened. Homozygous Megf8 and Mgrn1 mutations increased Smoothened abundance and elevated sensitivity to Hedgehog ligands. While mice heterozygous for loss-of-function Megf8 or Mgrn1 mutations were normal, double heterozygous embryos exhibited an incompletely penetrant syndrome of CHDs with heterotaxy. Thus, genetic interactions can arise from biochemical mechanisms that calibrate morphogen signaling strength, a conclusion broadly relevant for the many human diseases in which oligogenic inheritance is emerging as a mechanism for heritability.
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http://dx.doi.org/10.1016/j.devcel.2020.08.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7686252PMC
November 2020

Comprehensive classification of ABC ATPases and their functional radiation in nucleoprotein dynamics and biological conflict systems.

Nucleic Acids Res 2020 10;48(18):10045-10075

National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, MD 20894, USA.

ABC ATPases form one of the largest clades of P-loop NTPase fold enzymes that catalyze ATP-hydrolysis and utilize its free energy for a staggering range of functions from transport to nucleoprotein dynamics. Using sensitive sequence and structure analysis with comparative genomics, for the first time we provide a comprehensive classification of the ABC ATPase superfamily. ABC ATPases developed structural hallmarks that unambiguously distinguish them from other P-loop NTPases such as an alternative to arginine-finger-based catalysis. At least five and up to eight distinct clades of ABC ATPases are reconstructed as being present in the last universal common ancestor. They underwent distinct phases of structural innovation with the emergence of inserts constituting conserved binding interfaces for proteins or nucleic acids and the adoption of a unique dimeric toroidal configuration for DNA-threading. Specifically, several clades have also extensively radiated in counter-invader conflict systems where they serve as nodal nucleotide-dependent sensory and energetic components regulating a diversity of effectors (including some previously unrecognized) acting independently or together with restriction-modification systems. We present a unified mechanism for ABC ATPase function across disparate systems like RNA editing, translation, metabolism, DNA repair, and biological conflicts, and some unexpected recruitments, such as MutS ATPases in secondary metabolism.
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http://dx.doi.org/10.1093/nar/gkaa726DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7544218PMC
October 2020

Identification of Uncharacterized Components of Prokaryotic Immune Systems and Their Diverse Eukaryotic Reformulations.

J Bacteriol 2020 11 19;202(24). Epub 2020 Nov 19.

Computational Biology Branch, National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, Maryland, USA

Nucleotide-activated effector deployment, prototyped by interferon-dependent immunity, is a common mechanistic theme shared by immune systems of several animals and prokaryotes. Prokaryotic versions include CRISPR-Cas with the CRISPR polymerase domain, their minimal variants, and systems with second messenger oligonucleotide or dinucleotide synthetase (SMODS). Cyclic or linear oligonucleotide signals in these systems help set a threshold for the activation of potentially deleterious downstream effectors in response to invader detection. We establish such a regulatory mechanism to be a more general principle of immune systems, which can also operate independently of such messengers. Using sensitive sequence analysis and comparative genomics, we identify 12 new prokaryotic immune systems, which we unify by this principle of threshold-dependent effector activation. These display regulatory mechanisms paralleling physiological signaling based on 3'-5' cyclic mononucleotides, NAD-derived messengers, two- and one-component signaling that includes histidine kinase-based signaling, and proteolytic activation. Furthermore, these systems allowed the identification of multiple new sensory signal sensory components, such as a tetratricopeptide repeat (TPR) scaffold predicted to recognize NAD-derived signals, unreported versions of the STING domain, prokaryotic YEATS domains, and a predicted nucleotide sensor related to receiver domains. We also identify previously unrecognized invader detection components and effector components, such as prokaryotic versions of the Wnt domain. Finally, we show that there have been multiple acquisitions of unidentified STING domains in eukaryotes, while the TPR scaffold was incorporated into the animal immunity/apoptosis signal-regulating kinase (ASK) signalosome. Both prokaryotic and eukaryotic immune systems face the dangers of premature activation of effectors and degradation of self-molecules in the absence of an invader. To mitigate this, they have evolved threshold-setting regulatory mechanisms for the triggering of effectors only upon the detection of a sufficiently strong invader signal. This work defines general templates for such regulation in effector-based immune systems. Using this, we identify several previously uncharacterized prokaryotic immune mechanisms that accomplish the regulation of downstream effector deployment by using nucleotide, NAD-derived, two-component, and one-component signals paralleling physiological homeostasis. This study has also helped identify several previously unknown sensor and effector modules in these systems. Our findings also augment the growing evidence for the emergence of key animal immunity and chromatin regulatory components from prokaryotic progenitors.
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http://dx.doi.org/10.1128/JB.00365-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7685563PMC
November 2020

Mycobacterium tuberculosis Rv0991c Is a Redox-Regulated Molecular Chaperone.

mBio 2020 08 25;11(4). Epub 2020 Aug 25.

Department of Microbiology, New York University School of Medicine, New York, New York, USA

The bacterial pathogen is the leading cause of death by an infectious disease among humans. Here, we describe a previously uncharacterized protein, Rv0991c, as a molecular chaperone that is activated by oxidation. Rv0991c has homologs in most bacterial lineages and appears to function analogously to the well-characterized redox-regulated chaperone Hsp33, despite a dissimilar protein sequence. Rv0991c is transcriptionally coregulated with and chaperone genes in , suggesting that Rv0991c functions with these chaperones in maintaining protein quality control. Supporting this hypothesis, we found that, like oxidized Hsp33, oxidized Rv0991c prevents the aggregation of a model unfolded protein and promotes its refolding by the Hsp70 chaperone system. Furthermore, Rv0991c interacts with DnaK and can associate with many other proteins. We therefore propose that Rv0991c, which we named "Ruc" (redox-regulated protein with unstructured C terminus), represents a founding member of a new chaperone family that protects and other species from proteotoxicity during oxidative stress. infections are responsible for more than 1 million deaths per year. Developing effective strategies to combat this disease requires a greater understanding of biology. As in all cells, protein quality control is essential for the viability of , which likely faces proteotoxic stress within a host. Here, we identify an protein, Ruc, that gains chaperone activity upon oxidation. Ruc represents a previously unrecognized family of redox-regulated chaperones found throughout the bacterial superkingdom. Additionally, we found that oxidized Ruc promotes the protein-folding activity of the essential Hsp70 chaperone system. This work contributes to a growing body of evidence that oxidative stress provides a particular strain on cellular protein stability.
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http://dx.doi.org/10.1128/mBio.01545-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7448276PMC
August 2020

Antigen Discovery, Bioinformatics and Biological Characterization of Novel Immunodominant Babesia microti Antigens.

Sci Rep 2020 06 12;10(1):9598. Epub 2020 Jun 12.

Laboratory of Emerging Pathogens, Division of Emerging and Transfusion Transmitted Diseases, Office of Blood Research and Review, Center for Biologics Evaluation and Research, Food and Drug Administration, Silver Spring, MD, 20993, USA.

Babesia microti is an intraerythrocytic parasite and the primary causative agent of human babesiosis. It is transmitted by Ixodes ticks, transfusion of blood and blood products, organ donation, and perinatally. Despite its global public health impact, limited progress has been made to identify and characterize immunodominant B. microti antigens for diagnostic and vaccine use. Using genome-wide immunoscreening, we identified 56 B. microti antigens, including some previously uncharacterized antigens. Thirty of the most immunodominant B. microti antigens were expressed as recombinant proteins in E. coli. Among these, the combined use of two novel antigens and one previously described antigen provided 96% sensitivity and 100% specificity in identifying B. microti antibody containing sera in an ELISA. Using extensive computational sequence and bioinformatics analyses and cellular localization studies, we have clarified the domain architectures, potential biological functions, and evolutionary relationships of the most immunodominant B. microti antigens. Notably, we found that the BMN-family antigens are not monophyletic as currently annotated, but rather can be categorized into two evolutionary unrelated groups of BMN proteins respectively defined by two structurally distinct classes of extracellular domains. Our studies have enhanced the repertoire of immunodominant B. microti antigens, and assigned potential biological function to these antigens, which can be evaluated to develop novel assays and candidate vaccines.
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http://dx.doi.org/10.1038/s41598-020-66273-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7293334PMC
June 2020

Novel Immunoglobulin Domain Proteins Provide Insights into Evolution and Pathogenesis Mechanisms of SARS-Related Coronaviruses.

bioRxiv 2020 Mar 7. Epub 2020 Mar 7.

Department of Biology, College of Arts and Sciences, Saint Louis University, MO 63110.

A novel coronavirus (SARS-CoV-2) is the causative agent of an emergent severe respiratory disease (COVID-19) in humans that is threatening to result in a global health crisis. By using genomic, sequence, structural and evolutionary analysis, we show that Alpha- and Beta-CoVs possess several novel families of immunoglobulin (Ig) domain proteins, including ORF8 and ORF7a from SARS-related coronaviruses and two protein groups from certain Alpha-CoVs. Among them, ORF8 is distinguished in being rapidly evolving, possessing a unique insert and a hypervariable position among SARS-CoV-2 genomes in its predicted ligand-binding groove. We also uncover many Ig proteins from several metazoan viruses which are distinct in sequence and structure but share an architecture comparable to that of CoV Ig domain proteins. Hence, we propose that deployment of Ig domain proteins is a widely-used strategy by viruses, and SARS-CoV-2 ORF8 is a potential pathogenicity factor which evolves rapidly to counter the immune response and facilitate the transmission between hosts.
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http://dx.doi.org/10.1101/2020.03.04.977736DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7217140PMC
March 2020

Novel Immunoglobulin Domain Proteins Provide Insights into Evolution and Pathogenesis of SARS-CoV-2-Related Viruses.

mBio 2020 05 29;11(3). Epub 2020 May 29.

Department of Biology, College of Arts and Sciences, Saint Louis University, St. Louis, Missouri, USA

A novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was recently identified as the causative agent for the coronavirus disease 2019 (COVID-19) outbreak that has generated a global health crisis. We use a combination of genomic analysis and sensitive profile-based sequence and structure analysis to understand the potential pathogenesis determinants of this virus. As a result, we identify several fast-evolving genomic regions that might be at the interface of virus-host interactions, corresponding to the receptor binding domain of the Spike protein, the three tandem Macro fold domains in ORF1a, and the uncharacterized protein ORF8. Further, we show that ORF8 and several other proteins from alpha- and beta-CoVs belong to novel families of immunoglobulin (Ig) proteins. Among them, ORF8 is distinguished by being rapidly evolving, possessing a unique insert, and having a hypervariable position among SARS-CoV-2 genomes in its predicted ligand-binding groove. We also uncover numerous Ig domain proteins from several unrelated metazoan viruses, which are distinct in sequence and structure but share comparable architectures to those of the CoV Ig domain proteins. Hence, we propose that SARS-CoV-2 ORF8 and other previously unidentified CoV Ig domain proteins fall under the umbrella of a widespread strategy of deployment of Ig domain proteins in animal viruses as pathogenicity factors that modulate host immunity. The rapid evolution of the ORF8 Ig domain proteins points to a potential evolutionary arms race between viruses and hosts, likely arising from immune pressure, and suggests a role in transmission between distinct host species. The ongoing COVID-19 pandemic strongly emphasizes the need for a more complete understanding of the biology and pathogenesis of its causative agent SARS-CoV-2. Despite intense scrutiny, several proteins encoded by the genomes of SARS-CoV-2 and other SARS-like coronaviruses remain enigmatic. Moreover, the high infectivity and severity of SARS-CoV-2 in certain individuals make wet-lab studies currently challenging. In this study, we used a series of computational strategies to identify several fast-evolving regions of SARS-CoV-2 proteins which are potentially under host immune pressure. Most notably, the hitherto-uncharacterized protein encoded by ORF8 is one of them. Using sensitive sequence and structural analysis methods, we show that ORF8 and several other proteins from alpha- and beta-coronavirus comprise novel families of immunoglobulin domain proteins, which might function as potential immune modulators to delay or attenuate the host immune response against the viruses.
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http://dx.doi.org/10.1128/mBio.00760-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7267882PMC
May 2020

Evolutionarily ancient BAH-PHD protein mediates Polycomb silencing.

Proc Natl Acad Sci U S A 2020 05 11;117(21):11614-11623. Epub 2020 May 11.

Institute of Molecular Biology, University of Oregon, Eugene, OR 97403;

Methylation of histone H3 lysine 27 (H3K27) is widely recognized as a transcriptionally repressive chromatin modification but the mechanism of repression remains unclear. We devised and implemented a forward genetic scheme to identify factors required for H3K27 methylation-mediated silencing in the filamentous fungus and identified a bromo-adjacent homology (BAH)-plant homeodomain (PHD)-containing protein, EPR-1 (effector of polycomb repression 1; NCU07505). EPR-1 associates with H3K27-methylated chromatin, and loss of EPR-1 de-represses H3K27-methylated genes without loss of H3K27 methylation. EPR-1 is not fungal-specific; orthologs of EPR-1 are present in a diverse array of eukaryotic lineages, suggesting an ancestral EPR-1 was a component of a primitive Polycomb repression pathway.
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http://dx.doi.org/10.1073/pnas.1918776117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7261115PMC
May 2020

NONU-1 Encodes a Conserved Endonuclease Required for mRNA Translation Surveillance.

Cell Rep 2020 03;30(13):4321-4331.e4

Department of Molecular, Cell, and Developmental Biology, University of California at Santa Cruz, Santa Cruz, CA, USA. Electronic address:

Cellular translation surveillance rescues ribosomes that stall on problematic mRNAs. During translation surveillance, endonucleolytic cleavage of the problematic mRNA is a critical step in rescuing stalled ribosomes. Here we identify NONU-1 as a factor required for translation surveillance pathways including no-go and nonstop mRNA decay. We show that (1) NONU-1 reduces nonstop and no-go mRNA levels; (2) NONU-1 contains an Smr RNase domain required for mRNA decay; (3) the domain architecture and catalytic residues of NONU-1 are conserved throughout metazoans and eukaryotes, respectively; and (4) NONU-1 is required for the formation of mRNA cleavage fragments in the vicinity of stalled ribosomes. We extend our results in C. elegans to homologous factors in S. cerevisiae, showing the evolutionarily conserved function of NONU-1. Our work establishes the identity of a factor critical to translation surveillance and will inform mechanistic studies at the intersection of translation and mRNA decay.
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http://dx.doi.org/10.1016/j.celrep.2020.03.023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7184879PMC
March 2020

Functional Innovation in the Evolution of the Calcium-Dependent System of the Eukaryotic Endoplasmic Reticulum.

Front Genet 2020 6;11:34. Epub 2020 Feb 6.

National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, MD, United States.

The origin of eukaryotes was marked by the emergence of several novel subcellular systems. One such is the calcium (Ca)-stores system of the endoplasmic reticulum, which profoundly influences diverse aspects of cellular function including signal transduction, motility, division, and biomineralization. We use comparative genomics and sensitive sequence and structure analyses to investigate the evolution of this system. Our findings reconstruct the core form of the Ca-stores system in the last eukaryotic common ancestor as having at least 15 proteins that constituted a basic system for facilitating both Ca flux across endomembranes and Ca-dependent signaling. We present evidence that the key EF-hand Ca-binding components had their origins in a likely bacterial symbiont other than the mitochondrial progenitor, whereas the protein phosphatase subunit of the ancestral calcineurin complex was likely inherited from the asgard archaeal progenitor of the stem eukaryote. This further points to the potential origin of the eukaryotes in a Ca-rich biomineralized environment such as stromatolites. We further show that throughout eukaryotic evolution there were several acquisitions from bacteria of key components of the Ca-stores system, even though no prokaryotic lineage possesses a comparable system. Further, using quantitative measures derived from comparative genomics we show that there were several rounds of lineage-specific gene expansions, innovations of novel gene families, and gene losses correlated with biological innovation such as the biomineralized molluscan shells, coccolithophores, and animal motility. The burst of innovation of new genes in animals included the wolframin protein associated with Wolfram syndrome in humans. We show for the first time that it contains previously unidentified Sel1, EF-hand, and OB-fold domains, which might have key roles in its biochemistry.
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http://dx.doi.org/10.3389/fgene.2020.00034DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7016017PMC
February 2020

Highly regulated, diversifying NTP-dependent biological conflict systems with implications for the emergence of multicellularity.

Elife 2020 02 26;9. Epub 2020 Feb 26.

Computational Biology Branch, National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, United States.

Social cellular aggregation or multicellular organization pose increased risk of transmission of infections through the system upon infection of a single cell. The generality of the evolutionary responses to this outside of Metazoa remains unclear. We report the discovery of several thematically unified, remarkable biological conflict systems preponderantly present in multicellular prokaryotes. These combine thresholding mechanisms utilizing NTPase chaperones (the MoxR-vWA couple), GTPases and proteolytic cascades with hypervariable effectors, which vary either by using a reverse transcriptase-dependent diversity-generating system or through a system of acquisition of diverse protein modules, typically in inactive form, from various cellular subsystems. Conciliant lines of evidence indicate their deployment against invasive entities, like viruses, to limit their spread in multicellular/social contexts via physical containment, dominant-negative interactions or apoptosis. These findings argue for both a similar operational 'grammar' and shared protein domains in the sensing and limiting of infections during the multiple emergences of multicellularity.
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http://dx.doi.org/10.7554/eLife.52696DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7159879PMC
February 2020

The Genes Reveal the Biosynthetic and Evolutionary Origins of the Group B Hemolytic Lipid, Granadaene.

Front Microbiol 2019 21;10:3123. Epub 2020 Jan 21.

Department of Global Health, University of Washington, Seattle, WA, United States.

Group B (GBS) is a β-hemolytic, Gram-positive bacterium that commonly colonizes the female lower genital tract and is associated with fetal injury, preterm birth, spontaneous abortion, and neonatal infections. A major factor promoting GBS virulence is the β-hemolysin/cytolysin, which is cytotoxic to several host cells. We recently showed that the ornithine rhamnolipid pigment, Granadaene, produced by the gene products of the operon, is hemolytic. Here, we demonstrate that heterologous expression of the GBS operon conferred hemolysis, pigmentation, and cytoxicity to , a model non-hemolytic Gram-positive bacterium. Similarly, pigment purified from is hemolytic, cytolytic, and identical in structure to Granadaene extracted from GBS, indicating the operon is sufficient for Granadaene production in a heterologous host. Using a systematic survey of phyletic patterns and contextual associations of the genes, we identify homologs of the operon in physiologically diverse Gram-positive bacteria and propose undescribed functions of gene products. Together, these findings bring greater understanding to the biosynthesis and evolutionary foundations of a key GBS virulence factor and suggest that such potentially toxic lipids may be encoded by other bacteria.
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http://dx.doi.org/10.3389/fmicb.2019.03123DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6985545PMC
January 2020

TET methylcytosine oxidases: new insights from a decade of research.

J Biosci 2020 ;45

Division of Signaling and Gene Expression, La Jolla Institute for Immunology, La Jolla, CA 92037, USA.

In mammals, DNA methyltransferases transfer a methyl group from S-adenosylmethionine to the 5 position of cytosine in DNA. The product of this reaction, 5-methylcytosine (5mC), has many roles, particularly in suppressing transposable and repeat elements in DNA. Moreover, in many cellular systems, cell lineage specification is accompanied by DNA demethylation at the promoters of genes expressed at high levels in the differentiated cells. However, since direct cleavage of the C-C bond connecting the methyl group to the 5 position of cytosine is thermodynamically disfavoured, the question of whether DNA methylation was reversible remained unclear for many decades. This puzzle was solved by our discovery of the TET (Ten- Eleven Translocation) family of 5-methylcytosine oxidases, which use reduced iron, molecular oxygen and the tricarboxylic acid cycle metabolite 2-oxoglutarate (also known as a-ketoglutarate) to oxidise the methyl group of 5mC to 5-hydroxymethylcytosine (5hmC) and beyond. TET-generated oxidised methylcytosines are intermediates in at least two pathways of DNA demethylation, which differ in their dependence on DNA replication. In the decade since their discovery, TET enzymes have been shown to have important roles in embryonic development, cell lineage specification, neuronal function and cancer. We review these findings and discuss their implications here.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7216820PMC
October 2020

HMCES Functions in the Alternative End-Joining Pathway of the DNA DSB Repair during Class Switch Recombination in B Cells.

Mol Cell 2020 01 2;77(2):384-394.e4. Epub 2019 Dec 2.

National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, MD 20894, USA. Electronic address:

HMCES (5hmC binding, embryonic stem cell-specific-protein), originally identified as a protein capable of binding 5-hydroxymethylcytosine (5hmC), an epigenetic modification generated by TET proteins, was previously reported to covalently crosslink to DNA at abasic sites via a conserved cysteine. We show here that Hmces-deficient mice display normal hematopoiesis without global alterations in 5hmC. HMCES specifically enables DNA double-strand break repair through the microhomology-mediated alternative-end-joining (Alt-EJ) pathway during class switch recombination (CSR) in B cells, and HMCES deficiency leads to a significant defect in CSR. HMCES mediates Alt-EJ through its SOS-response-associated-peptidase domain (SRAPd), a function that requires DNA binding but is independent of its autopeptidase and DNA-crosslinking activities. We show that HMCES is recruited to switch regions of the immunoglobulin locus and provide a potential structural basis for the interaction of HMCES with long DNA overhangs generated by Alt-EJ during CSR. Our studies provide further evidence for a specialized role for HMCES in DNA repair.
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http://dx.doi.org/10.1016/j.molcel.2019.10.031DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6980713PMC
January 2020

Unusual Activity of a TET/JBP Family Enzyme.

Biochemistry 2019 09 22;58(35):3627-3629. Epub 2019 Aug 22.

La Jolla Institute for Immunology and Sanford Consortium for Regenerative Medicine , La Jolla , California 92037 , United States.

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http://dx.doi.org/10.1021/acs.biochem.9b00609DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7416651PMC
September 2019

The catalytic core of DEMETER guides active DNA demethylation in .

Proc Natl Acad Sci U S A 2019 08 13;116(35):17563-17571. Epub 2019 Aug 13.

Department of Plant and Microbial Biology, North Carolina State University, Raleigh, NC 27695;

The DEMETER (DME) DNA glycosylase demethylates the maternal genome in the central cell prior to fertilization and is essential for seed viability. DME preferentially targets small transposons that flank coding genes, influencing their expression and initiating plant gene imprinting. DME also targets intergenic and heterochromatic regions, but how it is recruited to these differing chromatin landscapes is unknown. The C-terminal half of DME consists of 3 conserved regions required for catalysis in vitro. We show that this catalytic core guides active demethylation at endogenous targets, rescuing developmental and genomic hypermethylation phenotypes. However, without the N terminus, heterochromatin demethylation is significantly impeded, and abundant CG-methylated genic sequences are ectopically demethylated. Comparative analysis revealed that the conserved DME N-terminal domains are present only in flowering plants, whereas the domain architecture of DME-like proteins in nonvascular plants mainly resembles the catalytic core, suggesting that it might represent the ancestral form of the 5mC DNA glycosylase found in plant lineages. We propose a bipartite model for DME protein action and suggest that the DME N terminus was acquired late during land plant evolution to improve specificity and facilitate demethylation at heterochromatin targets.
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http://dx.doi.org/10.1073/pnas.1907290116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6717269PMC
August 2019

Identification of the mAm Methyltransferase PCIF1 Reveals the Location and Functions of mAm in the Transcriptome.

Mol Cell 2019 08 3;75(3):631-643.e8. Epub 2019 Jul 3.

Division of Newborn Medicine, Boston Children's Hospital, 300 Longwood Avenue, Boston, MA 02115, USA; Department of Pediatrics, Harvard Medical School, Boston, MA 02115, USA. Electronic address:

mRNAs are regulated by nucleotide modifications that influence their cellular fate. Two of the most abundant modified nucleotides are N-methyladenosine (mA), found within mRNAs, and N,2'-O-dimethyladenosine (mAm), which is found at the first transcribed nucleotide. Distinguishing these modifications in mapping studies has been difficult. Here, we identify and biochemically characterize PCIF1, the methyltransferase that generates mAm. We find that PCIF1 binds and is dependent on the mG cap. By depleting PCIF1, we generated transcriptome-wide maps that distinguish mAm and mA. We find that mA and mAm misannotations arise from mRNA isoforms with alternative transcription start sites (TSSs). These isoforms contain mAm that maps to "internal" sites, increasing the likelihood of misannotation. We find that depleting PCIF1 does not substantially affect mRNA translation but is associated with reduced stability of a subset of mAm-annotated mRNAs. The discovery of PCIF1 and our accurate mapping technique will facilitate future studies to characterize mAm's function.
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http://dx.doi.org/10.1016/j.molcel.2019.06.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6703822PMC
August 2019

Structural basis of HMCES interactions with abasic DNA and multivalent substrate recognition.

Nat Struct Mol Biol 2019 07 24;26(7):607-612. Epub 2019 Jun 24.

Structural Genomics Consortium, University of Toronto, Toronto, Ontario, Canada.

Embryonic stem cell-specific 5-hydroxymethylcytosine-binding protein (HMCES) can covalently cross-link to abasic sites in single-stranded DNA at stalled replication forks to prevent genome instability. Here, we report crystal structures of the human HMCES SOS response-associated peptidase (SRAP) domain in complex with DNA-damage substrates, including HMCES cross-linked with an abasic site within a 3' overhang DNA. HMCES interacts with both single-strand and duplex segments of DNA, with two independent duplex DNA interaction sites identified in the SRAP domain. The HMCES DNA-protein cross-link structure provides structural insights into a novel thiazolidine covalent interaction between the DNA abasic site and conserved Cys 2 of HMCES. Collectively, our structures demonstrate the capacity for the SRAP domain to interact with a variety of single-strand- and double-strand-containing DNA structures found in DNA-damage sites, including 5' and 3' overhang DNAs and gapped DNAs with short single-strand segments.
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http://dx.doi.org/10.1038/s41594-019-0246-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6609482PMC
July 2019

Evidence based interventions and implementation gaps in control of tuberculosis: A systematic review in low and middle-income countries with special focus on India.

Indian J Tuberc 2019 Apr 11;66(2):268-278. Epub 2019 Apr 11.

Health System Research India Initiative (HSRII), S-10, Vrindavan Gardens, Pattom P.O, Thiruvananthapuram, 695004, India.

We synthesised the findings of intervention studies on Tuberculosis control (TC) in low- and middle-income countries with specific reference to India through a systematic review during the period 2000-2017 in order to identify the implementation gap. The research questions were framed using PICOS (population, intervention, comparison, outcomes and study design) framework and PRISMA (preferred reporting items for systematic reviews and meta-analyses) guidelines were used for study selection. The search was mainly carried out in MEDLINE/PubMed, Web of Knowledge and Cochrane libraries. DOTS was found to be the most effective intervention program for control of Tuberculosis. Lack of utilization of the capacity of various level health staff, accessibility in utilizing health facilities and insufficient community involvement was identified as the major gaps for TC. In the case of India, each state has its own priority and applicability for different TC interventions. Most of the studies on implementation of the TC program supported the encouraging effect of the intervention in the control of Tuberculosis. The specific need of each country is clearly reflected in many of the selected studies. In order to establish the association of intervention and its implementation gaps on TB control, more rigorous evaluation methods are needed including meta-analysis. REGISTRATION: PROSPERO registration number: CRD42018070406.
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http://dx.doi.org/10.1016/j.ijtb.2019.04.006DOI Listing
April 2019

Oxidative opening of the aromatic ring: Tracing the natural history of a large superfamily of dioxygenase domains and their relatives.

J Biol Chem 2019 06 15;294(26):10211-10235. Epub 2019 May 15.

From the Computational Biology Branch, NCBI, National Library of Medicine, National Institutes of Health, Bethesda, Maryland 20894,

A diverse collection of enzymes comprising the protocatechuate dioxygenases (PCADs) has been characterized in several extradiol aromatic compound degradation pathways. Structural studies have shown a relationship between PCADs and the more broadly-distributed, functionally enigmatic Memo domain linked to several human diseases. To better understand the evolution of this PCAD-Memo protein superfamily, we explored their structural and functional determinants to establish a unified evolutionary framework, identifying 15 clearly-delineable families, including a previously-underappreciated diversity in five Memo clade families. We place the superfamily's origin within the greater radiation of the nucleoside phosphorylase/hydrolase-peptide/amidohydrolase fold prior to the last universal common ancestor of all extant organisms. In addition to identifying active-site residues across the superfamily, we describe three distinct, structurally-variable regions emanating from the core scaffold often housing conserved residues specific to individual families. These were predicted to contribute to the active-site pocket, potentially in substrate specificity and allosteric regulation. We also identified several previously-undescribed conserved genome contexts, providing insight into potentially novel substrates in PCAD clade families. We extend known conserved contextual associations for the Memo clade beyond previously-described associations with the AMMECR1 domain and a radical -adenosylmethionine family domain. These observations point to two distinct yet potentially overlapping contexts wherein the elusive molecular function of the Memo domain could be finally resolved, thereby linking it to nucleotide base and aliphatic isoprenoid modification. In total, this report throws light on the functions of large swaths of the experimentally-uncharacterized PCAD-Memo families.
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http://dx.doi.org/10.1074/jbc.RA119.007595DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6664185PMC
June 2019

Antimicrobial Peptides, Polymorphic Toxins, and Self-Nonself Recognition Systems in Archaea: an Untapped Armory for Intermicrobial Conflicts.

mBio 2019 05 7;10(3). Epub 2019 May 7.

National Center for Biotechnology Information, National Library of Medicine, Bethesda, Maryland, USA.

Numerous, diverse, highly variable defense and offense genetic systems are encoded in most bacterial genomes and are involved in various forms of conflict among competing microbes or their eukaryotic hosts. Here we focus on the offense and self-versus-nonself discrimination systems encoded by archaeal genomes that so far have remained largely uncharacterized and unannotated. Specifically, we analyze archaeal genomic loci encoding polymorphic and related toxin systems and ribosomally synthesized antimicrobial peptides. Using sensitive methods for sequence comparison and the "guilt by association" approach, we identified such systems in 141 archaeal genomes. These toxins can be classified into four major groups based on the structure of the components involved in the toxin delivery. The toxin domains are often shared between and within each system. We revisit halocin families and substantially expand the halocin C8 family, which was identified in diverse archaeal genomes and also certain bacteria. Finally, we employ features of protein sequences and genomic locus organization characteristic of archaeocins and polymorphic toxins to identify candidates for analogous but not necessarily homologous systems among uncharacterized protein families. This work confidently predicts that more than 1,600 archaeal proteins, currently annotated as "hypothetical" in public databases, are components of conflict and self-versus-nonself discrimination systems. Diverse and highly variable systems involved in biological conflicts and self-versus-nonself discrimination are ubiquitous in bacteria but much less studied in archaea. We performed comprehensive comparative genomic analyses of the archaeal systems that share components with analogous bacterial systems and propose an approach to identify new systems that could be involved in these functions. We predict polymorphic toxin systems in 141 archaeal genomes and identify new, archaea-specific toxin and immunity protein families. These systems are widely represented in archaea and are predicted to play major roles in interactions between species and in intermicrobial conflicts. This work is expected to stimulate experimental research to advance the understanding of poorly characterized major aspects of archaeal biology.
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http://dx.doi.org/10.1128/mBio.00715-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6509191PMC
May 2019

Deciphering the Role of a SLOG Superfamily Protein YpsA in Gram-Positive Bacteria.

Front Microbiol 2019 5;10:623. Epub 2019 Apr 5.

Department of Cell Biology, Microbiology and Molecular Biology, University of South Florida, Tampa, FL, United States.

Bacteria adapt to different environments by regulating cell division and several conditions that modulate cell division have been documented. Understanding how bacteria transduce environmental signals to control cell division is critical in understanding the global network of cell division regulation. In this article we describe a role for YpsA, an uncharacterized protein of the SLOG superfamily of nucleotide and ligand-binding proteins, in cell division. We observed that YpsA provides protection against oxidative stress as cells lacking show increased susceptibility to hydrogen peroxide treatment. We found that the increased expression of leads to filamentation and disruption of the assembly of FtsZ, the tubulin-like essential protein that marks the sites of cell division in . We also showed that YpsA-mediated filamentation is linked to the growth rate. Using site-directed mutagenesis, we targeted several conserved residues and generated YpsA variants that are no longer able to inhibit cell division. Finally, we show that the role of YpsA is possibly conserved in Firmicutes, as overproduction of YpsA in also impairs cell division.
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http://dx.doi.org/10.3389/fmicb.2019.00623DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6459960PMC
April 2019