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1. TrmB Family Transcription Factor as a Thiol-Based Regulator of Oxidative Stress Response

   https://doi.org/10.1128/mbio.00633-22  

   Mondragon, Paula ; Hwang, Sungmin ; Kasirajan, Lakshmi ; Oyetoro, Rebecca ; Nasthas, Angelina ; Winters, Emily ; Couto-Rodriguez, Ricardo L. ; Schmid, Amy ; Maupin-Furlow, Julie A. ( August 2022 , mBio)

   Babitzke, Paul (Ed.)

   ABSTRACT Oxidative stress causes cellular damage, including DNA mutations, protein dysfunction, and loss of membrane integrity. Here, we discovered that a TrmB (transcription regulator of mal operon) family protein (Pfam PF01978) composed of a single winged-helix DNA binding domain (InterPro IPR002831) can function as thiol-based transcriptional regulator of oxidative stress response. Using the archaeon Haloferax volcanii as a model system, we demonstrate that the TrmB-like OxsR is important for recovery of cells from hypochlorite stress. OxsR is shown to bind specific regions of genomic DNA, particularly during hypochlorite stress. OxsR-bound intergenic regions were found proximal to oxidative stress operons, including genes associated with thiol relay and low molecular weight thiol biosynthesis. Further analysis of a subset of these sites revealed OxsR to function during hypochlorite stress as a transcriptional activator and repressor. OxsR was shown to require a conserved cysteine (C24) for function and to use a CG-rich motif upstream of conserved BRE/TATA box promoter elements for transcriptional activation. Protein modeling suggested the C24 is located at a homodimer interface formed by antiparallel α helices, and that oxidation of this cysteine would result in the formation of an intersubunit disulfide bond. This covalent linkage may promote stabilization of an OxsR homodimer with the enhanced DNA binding properties observed in the presence of hypochlorite stress. The phylogenetic distribution TrmB family proteins, like OxsR, that have a single winged-helix DNA binding domain and conserved cysteine residue suggests this type of redox signaling mechanism is widespread in Archaea. IMPORTANCE TrmB-like proteins, while not yet associated with redox stress, are found in bacteria and widespread in archaea. Here, we expand annotation of a large group of TrmB-like single winged-helix DNA binding domain proteins from diverse archaea to function as thiol-based transcriptional regulators of oxidative stress response. Using Haloferax volcanii as a model, we reveal that the TrmB-like OxsR functions during hypochlorite stress as a transcriptional activator and repressor of an extensive gene coexpression network associated with thiol relay and other related activities. A conserved cysteine residue of OxsR serves as the thiol-based sensor for this function and likely forms an intersubunit disulfide bond during hypochlorite stress that stabilizes a homodimeric configuration with enhanced DNA binding properties. A CG-rich DNA motif in the promoter region of a subset of sites identified to be OxsR-bound is required for regulation; however, not all sites have this motif, suggesting added complexity to the regulatory network. 

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2. Shared TIR enzymatic functions regulate cell death and immunity across the tree of life

   https://doi.org/10.1126/science.abo0001  

   Essuman, Kow ; Milbrandt, Jeffrey ; Dangl, Jeffery L. ; Nishimura, Marc T. ( July 2022 , Science)

   BACKGROUND Diverse organisms, from archaea and bacteria to plants and humans, use receptor systems to recognize both pathogens and dangerous self-derived or environmentally derived stimuli. These intricate, well-coordinated immune systems, composed of innate and adaptive components, ensure host survival. In the late 20th century, researchers identified the Toll/interleukin-1/resistance gene (TIR) domain as an evolutionarily conserved component of animal and plant innate immune systems. Today, TIR-domain proteins are known to be broadly distributed across the tree of life. The TIR domain was first recognized as an adaptor for the assembly of macromolecular signaling complexes in mammalian innate immune pathways. Work on axon degeneration in animals—as well as on plant, archaeal, and bacterial immune systems—has uncovered additional enzymatic activities for TIR domains. ADVANCES Mammalian axons initiate a self-destruct program upon injury and during disease that is mediated by the sterile alpha and TIR motif containing 1 (SARM1) protein. The SARM1 TIR domain enzymatically consumes the essential metabolic cofactor nicotinamide adenine dinucleotide (NAD + ) to promote axonal death. Identification of the SARM1 NAD + -consuming enzyme (NADase) revealed that TIR domains can function as enzymes. Given the evolutionary conservation of TIR domains, studies investigated whether the SARM1 TIR NADase was also conserved. Indeed, bacteria, archaea, and plant TIR domains possess NADase activity. In prokaryotes, TIR NADase activity is found in an ancient antiphage immune system. In plants, identification of TIR NADase activity and linkage of TIR enzymatic products to downstream signaling components addressed the question of how nucleotide-binding, leucine-rich repeat (NLR) receptors trigger hypersensitive cell death during an immune response. Studies in plants show that their TIR domains can cleave nucleic acids and possess 2′,3′ cyclic adenosine monophosphate (2′,3′-cAMP) and 2′,3′ cyclic guanosine monophosphate (2′,3′-cGMP) synthetase activity that aids cell death programs in plant innate immunity. Thus, TIR domains constitute an ancient family of enzymes that are activated in immune and cell death pathways. OUTLOOK The discovery of TIR-domain enzyme activities carries implications for innate immunity and neurodegeneration. The identification of the SARM1 NADase defined a drug target for a wide number of neurodegenerative diseases that is being exploited in both preclinical and clinical studies. Hyperactive mutations in the SARM1 NADase have been discovered in amyotrophic lateral sclerosis (ALS) patients. Future work will seek to clarify the contribution of the SARM1 axon degeneration pathway to ALS pathogenesis. NAD + biology influences cellular processes from metabolism to DNA repair to aging. How TIR enzymes influence the NAD + metabolome and its associated pathways in bacteria, archaea, plants, and animals will be an exciting area for upcoming investigation. The discovery of the diversity of TIR enzymatic products is revealing signaling pathways across kingdoms. Discovery of TIR enzymatic function in plants and animals may yet inspire studies of enzymatic functions for Toll-like receptors in animals. We anticipate that cross-kingdom studies of TIR-domain function will guide interventions that will span the tree of life, from treating human neurodegenerative disorders and bacterial infections to preventing plant diseases. Conserved TIR-domain enzymatic activity. TIR-domain proteins from prokaryotes and eukaryotes cleave NAD + into nicotinamide (Nam), ADP-ribose (ADPR), cyclic ADP-ribose (cADPR), isomers of cyclic ADP-ribose (2′ or 3′cADPR), and related molecules [e.g., phosphoribosyl adenosine monophosphate (pRib-AMP)]. Plant TIR domains also possess a nuclease activity, can degrade DNA and RNA, and can function as a 2′,3′-cAMP or 2′,3′-cGMP synthetase. TIR enzymatic activity drives cell death and immune pathways across kingdoms. TIR activity can kill cells directly through NAD + depletion or indirectly using enzymatic products as signal molecules. The representative TIR domain structure shown here is Protein Data Bank ID 6O0Q. EDS1, enhanced disease susceptibility 1; ThsA, Thoeris A. 

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3. Crystal structure and RNA-binding properties of an Hfq homolog from the deep-branching Aquificae: conservation of the lateral RNA-binding mode

   https://doi.org/10.1107/S2059798317000031  

   Stanek, Kimberly A. ; Patterson-West, Jennifer ; Randolph, Peter S. ; Mura, Cameron ( April 2017 , Acta Crystallographica Section D Structural Biology)

   The host factor Hfq, as the bacterial branch of the Sm family, is an RNA-binding protein involved in the post-transcriptional regulation of mRNA expression and turnover. Hfq facilitates pairing between small regulatory RNAs (sRNAs) and their corresponding mRNA targets by binding both RNAs and bringing them into close proximity. Hfq homologs self-assemble into homo-hexameric rings with at least two distinct surfaces that bind RNA. Recently, another binding site, dubbed the `lateral rim', has been implicated in sRNA·mRNA annealing; the RNA-binding properties of this site appear to be rather subtle, and its degree of evolutionary conservation is unknown. An Hfq homolog has been identified in the phylogenetically deep-branching thermophile Aquifex aeolicus ( Aae ), but little is known about the structure and function of Hfq from basal bacterial lineages such as the Aquificae. Therefore, Aae Hfq was cloned, overexpressed, purified, crystallized and biochemically characterized. Structures of Aae Hfq were determined in space groups P 1 and P 6, both to 1.5 Å resolution, and nanomolar-scale binding affinities for uridine- and adenosine-rich RNAs were discovered. Co-crystallization with U 6 RNA reveals that the outer rim of the Aae Hfq hexamer features a well defined binding pocket that is selective for uracil. This Aae Hfq structure, combined with biochemical and biophysical characterization of the homolog, reveals deep evolutionary conservation of the lateral RNA-binding mode, and lays a foundation for further studies of Hfq-associated RNA biology in ancient bacterial phyla. 

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4. The DNA replication protein Orc1 from the yeast Torulaspora delbrueckii is required for heterochromatin formation but not as a silencer-binding protein

   https://doi.org/10.1093/genetics/iyac110  

   Maria, Haniam ; Rusche, Laura N. ; Rando, ed., O. ( July 2022 , Genetics)

   To understand the process by which new protein functions emerge, we examined how the yeast heterochromatin protein Sir3 arose through gene duplication from the conserved DNA replication protein Orc1. Orc1 is a subunit of the origin recognition complex (ORC), which marks origins of DNA replication. In Saccharomyces cerevisiae, Orc1 also promotes heterochromatin assembly by recruiting the structural proteins Sir1-4 to silencer DNA. In contrast, the paralog of Orc1, Sir3, is a nucleosome-binding protein that spreads across heterochromatic loci in conjunction with other Sir proteins. We previously found that a nonduplicated Orc1 from the yeast Kluyveromyces lactis behaved like ScSir3 but did not have a silencer-binding function like ScOrc1. Moreover, K. lactis lacks Sir1, the protein that interacts directly with ScOrc1 at the silencer. Here, we examined whether the emergence of Sir1 coincided with Orc1 acting as a silencer-binding protein. In the nonduplicated species Torulaspora delbrueckii, which has an ortholog of Sir1 (TdKos3), we found that TdOrc1 spreads across heterochromatic loci independently of ORC, as ScSir3 and KlOrc1 do. This spreading is dependent on the nucleosome binding BAH domain of Orc1 and on Sir2 and Kos3. However, TdOrc1 does not have a silencer-binding function: T. delbrueckii silencers do not require ORC-binding sites to function, and Orc1 and Kos3 do not appear to interact. Instead, Orc1 and Kos3 both spread across heterochromatic loci with other Sir proteins. Thus, Orc1 and Sir1/Kos3 originally had different roles in heterochromatin formation than they do now in S. cerevisiae.

    

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5. Resistance-Guided Mining of Bacterial Genotoxins Defines a Family of DNA Glycosylases

   https://doi.org/10.1128/mbio.03297-21  

   Bradley, Noah P. ; Wahl, Katherine L. ; Steenwyk, Jacob L. ; Rokas, Antonis ; Eichman, Brandt F. ( April 2022 , mBio)

   Simmons, Lyle A. ; Bush, Karen (Ed.)

   ABSTRACT Unique DNA repair enzymes that provide self-resistance against therapeutically important, genotoxic natural products have been discovered in bacterial biosynthetic gene clusters (BGCs). Among these, the DNA glycosylase AlkZ is essential for azinomycin B production and belongs to the HTH_42 superfamily of uncharacterized proteins. Despite their widespread existence in antibiotic producers and pathogens, the roles of these proteins in production of other natural products are unknown. Here, we determine the evolutionary relationship and genomic distribution of all HTH_42 proteins from Streptomyces and use a resistance-based genome mining approach to identify homologs associated with known and uncharacterized BGCs. We find that AlkZ-like (AZL) proteins constitute one distinct HTH_42 subfamily and are highly enriched in BGCs and variable in sequence, suggesting each has evolved to protect against a specific secondary metabolite. As a validation of the approach, we show that the AZL protein, HedH4, associated with biosynthesis of the alkylating agent hedamycin, excises hedamycin-DNA adducts with exquisite specificity and provides resistance to the natural product in cells. We also identify a second, phylogenetically and functionally distinct subfamily whose proteins are never associated with BGCs, are highly conserved with respect to sequence and genomic neighborhood, and repair DNA lesions not associated with a particular natural product. This work delineates two related families of DNA repair enzymes—one specific for complex alkyl-DNA lesions and involved in self-resistance to antimicrobials and the other likely involved in protection against an array of genotoxins—and provides a framework for targeted discovery of new genotoxic compounds with therapeutic potential. IMPORTANCE Bacteria are rich sources of secondary metabolites that include DNA-damaging genotoxins with antitumor/antibiotic properties. Although Streptomyces produce a diverse number of therapeutic genotoxins, efforts toward targeted discovery of biosynthetic gene clusters (BGCs) producing DNA-damaging agents is lacking. Moreover, work on toxin-resistance genes has lagged behind our understanding of those involved in natural product synthesis. Here, we identified over 70 uncharacterized BGCs producing potentially novel genotoxins through resistance-based genome mining using the azinomycin B-resistance DNA glycosylase AlkZ. We validate our analysis by characterizing the enzymatic activity and cellular resistance of one AlkZ ortholog in the BGC of hedamycin, a potent DNA alkylating agent. Moreover, we uncover a second, phylogenetically distinct family of proteins related to Escherichia coli YcaQ, a DNA glycosylase capable of unhooking interstrand DNA cross-links, which differs from the AlkZ-like family in sequence, genomic location, proximity to BGCs, and substrate specificity. This work defines two families of DNA glycosylase for specialized repair of complex genotoxic natural products and generalized repair of a broad range of alkyl-DNA adducts and provides a framework for targeted discovery of new compounds with therapeutic potential. 

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