DNA duplex recognition activates Exo1 nuclease activity [DNA and Chromosomes]

June 10th, 2019 by Yuxi Li, Jiangchuan Shen, Hengyao Niu

Exonuclease 1 (Exo1) is an evolutionarily conserved eukaryotic nuclease that plays a multifaceted role in maintaining genome stability. The biochemical attributes of Exo1 have been extensively characterized via conventional assays. However, the key step governing its activation remains elusive. Extending upon a previous finding that Exo1 can digest a randomly selected ssDNA, but not a poly dT oligonucleotide and using purified recombinant Exo1 and nuclease and electrophoretic mobility shift assays, here we determined that DNA hairpins with a stem size of 4 bp or longer are able to activate Exo1-mediated digestion of ssDNA. We further provide evidence suggesting that Exo1 uses an evolutionarily conserved residue, Lys-185. This residue interacted with the phosphate group bridging the third and fourth nucleotide on the digestion strand of the substrate DNA for duplex recognition, critical for Exo1 activation on not only ssDNA, but also dsDNA. Additionally, the defect of an exo1-K185A mutant in duplex digestion was partially rescued by longer overhanging DNA. However, we noted that the enhanced Exo1 nuclease activity by longer overhanging DNA is largely eliminated by replication protein A (RPA), likely due to the previously reported RPA activity that strips Exo1 off the ssDNA. We conclude that duplex DNA contact by Exo1 is a general mechanism that controls its activation and that this mechanism is particularly important for digestion of duplex DNA whose nascent ssDNA is bound by RPA.

Unraveling the role of the transcriptional regulator VirS in low pH-induced responses of Mycobacterium tuberculosis and identification of VirS inhibitors [Microbiology]

May 24th, 2019 by Swati Singh, Nikita Goswami, Anil K. Tyagi, Garima Khare

The ability of Mycobacterium tuberculosis to respond and adapt to various stresses such as oxygen/nitrogen radicals and low pH inside macrophages is critical for the persistence of this human pathogen inside its host. We have previously shown that an AraC/XylS-type transcriptional regulator, VirS, which is induced in low pH, is involved in remodeling the architecture of the bacterial cell envelope. However, how VirS influences gene expression to coordinate these pH responses remains unclear. Here, using a genetic biosensor of cytoplasmic pH, we demonstrate that VirS is required for the intracellular pH maintenance in response to acidic stress and inside acidified macrophages. Furthermore, we observed that VirS plays an important role in blocking phagosomal–lysosomal fusions. Transcriptomics experiments revealed that VirS affects the expression of genes encoding metabolic enzymes, cell-wall envelope proteins, efflux pumps, ion transporters, detoxification enzymes, and transcriptional regulators expressed under low-pH stress. Employing electrophoretic mobility-shift assays, DNA footprinting, and in silico analysis, we identified a DNA sequence to which VirS binds and key residues in VirS required for its interaction with DNA. A significant role of VirS in M. tuberculosis survival in adverse conditions suggested it as a potential anti-mycobacterial drug target. To that end, we identified VirS inhibitors in a virtual screen; the top hit compounds inhibited its DNA-binding activity and also M. tuberculosis growth in vitro and inside macrophages. Our findings establish that VirS mediates M. tuberculosis responses to acidic stress and identify VirS-inhibiting compounds that may form the basis for developing more effective anti-mycobacterial agents.
  • Posted in Journal of Biological Chemistry, Publications
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Face-time with TAR: Portraits of an HIV-1 RNA with diverse modes of effector recognition relevant for drug discovery [RNA]

May 12th, 2019 by Sai Shashank Chavali, Rachel Bonn-Breach, Joseph E. Wedekind

Small molecules and short peptides that potently and selectively bind RNA are rare, making the molecular structures of these complexes highly exceptional. Accordingly, several recent investigations have provided unprecedented structural insights into how peptides and proteins recognize the HIV-1 trans-activation response (TAR) element, a 59-nucleotide long, noncoding RNA segment in the 5'-long terminal repeat region of viral transcripts. Here we offer an integrated perspective on these advances by describing earlier progress on TAR-binding to small molecules, and by drawing parallels to recent successes in the identification of compounds that target the hepatitis C virus internal ribosome entry site (IRES) and the flavin-mononucleotide riboswitch. We relate this work to recent progress that pinpoints specific determinants of TAR recognition by: (i) viral Tat proteins, (ii) an innovative lab-evolved TAR-binding protein and (iii) an ultrahigh-affinity cyclic peptide. New structural details are used to model the TAR-Tat-super elongation complex (SEC) that is essential for efficient viral transcription and represents a focal point for antiviral drug design. A key prediction is that the Tat transactivation domain makes modest contacts with the TAR apical loop, whereas its arginine-rich motif (ARM) spans the entire length of the TAR major groove. This expansive interface has significant implications for drug discovery and design, and further suggests that future lab-evolved proteins could be deployed to discover steric restriction points that block Tat-mediated recruitment of the host SEC to HIV-1 TAR.

Phosphorylation of Tyr950 in the proteasome scaffolding protein RPN2 modulates its interaction with theubiquitin receptor RPN13 [Protein Synthesis and Degradation]

May 7th, 2019 by Casey W. Hemmis, Stephanie C. Heard, Christopher P. Hill

Protein substrates are targeted to the 26S proteasome through several ubiquitin receptors. One of these receptors, RPN13, is recruited to the proteasome by binding of its N-terminal pleckstrin-like receptor of ubiquitin (PRU) domain to C-terminal residues of the scaffolding protein RPN2. The RPN13 PRU domain is followed by a flexible linker and a C-terminal deubiquitylase adaptor (DEUBAD) domain, which recruits and activates the deubiquitylase UCH37. Both RPN13 and UCH37 have been implicated in human cancers, and inhibitors of the RPN2–RPN13 interaction are being developed as potential therapeutic anti-cancer agents. Our current study builds on the recognition that a residue central to the RPN2–RPN13 interaction, RPN2 Tyr950, is phosphorylated in Jurkat cells. We found that the Tyr950 phosphorylation enhances binding to RPN13. The crystal structure of the RPN2–RPN13 pTyr950-ubiquitin complex was determined at 1.76 Å resolution, and reveals specific interactions with positively charged side chains in RPN13 that explain how phosphorylation increases binding affinity without inducing conformational change. Mutagenesis and quantitative binding assays were then used to validate the crystallographic interface. Our findings support a model in which RPN13 recruitment to the proteasome is enhanced by phosphorylation of RPN2 Tyr950, have important implications for efforts to develop specific inhibitors of the RPN2–RPN13 interaction, and suggest the existence of a previously unknown stressrresponse pathway.
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The FtsK-like motor TraB is a DNA-dependent ATPase that forms higher-order assemblies [Microbiology]

February 5th, 2019 by Eric Amado, Gunther Muth, Ignacio Arechaga, Elena Cabezon

TraB is an FtsK-like DNA translocase responsible for conjugative plasmid transfer in mycelial Streptomyces. Unlike other conjugative systems, which depend on a type IV secretion system, Streptomyces requires only TraB protein to transfer the plasmid as dsDNA. The γ-domain of this protein specifically binds to repeated 8-bp motifs on the plasmid sequence, following a mechanism that is reminiscent of the FtsK/SpoIIIE chromosome segregation system. In this work, we purified and characterized the enzymatic activity of TraB, revealing that it is a DNA-dependent ATPase that is highly stimulated by dsDNA substrates. Interestingly, we found that unlike the SpoIIIE protein, the γ-domain of TraB does not confer sequence-specific ATPase stimulation. We also found that TraB binds G-quadruplex DNA structures with higher affinity than TraB-recognition sequences (TRSs). An EM-based structural analysis revealed that TraB tends to assemble as large complexes comprising four TraB hexamers, which might be a prerequisite for DNA translocation across cell membranes. In summary, our findings shed light on the molecular mechanism used by the DNA-translocating motor TraB, which may be shared by other membrane-associated machineries involved in DNA binding and translocation.

15-Deoxy-{Delta}12,14-prostaglandin J2 promotes phosphorylation of eukaryotic initiation factor 2{alpha} and activates the integrated stress response [Neurobiology]

February 5th, 2019 by Devin Tauber, Roy Parker

Stress granules (SGs) are cytoplasmic RNA-protein aggregates formed in response to inhibition of translation initiation. SGs contribute to the stress response and are implicated in a variety of diseases including cancer and some forms of neurodegeneration. Neurodegenerative diseases often involve chronic phosphorylation of eukaryotic initiation factor 2α (eIF2α), with deletions of eIF2α kinases or treatment with eiF2α kinase inhibitors being protective in some animal models of disease. However, how and why the integrated stress response (ISR) is activated in different forms of neurodegeneration remains unclear. Since neuroinflammation is common to many neurodegenerative diseases, we hypothesized that inflammatory factors contribute to ISR activation in a cell non-autonomous manner. Using fluorescence microscopy and immunoblotting, we show here that the endogenously produced product of inflammation, 15-Deoxy-Δ12,14-prostaglandin J2 (15-d-PGJ2), triggers eIF2α phosphorylation thereby activating the ISR, repressing bulk translation, and triggering stress granule formation. Our findings define a mechanism by which inflammation activates the ISR in a cell non-autonomous manner and suggest that inhibition of 15-d-PGJ2 production might be a useful therapeutic strategy in some neuroinflammatory contexts.
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A stable tetramer is not the only oligomeric state that mitochondrial single-stranded DNA binding proteins can adopt. [DNA and Chromosomes]

January 7th, 2019 by Saurabh P Singh, Vandna Kukshal, Roberto Galletto

Mitochondrial single-stranded DNA binding proteins (mtSSBs) are required for mitochondrial DNA replication and stability and are generally assumed to form homo-tetramers, and this species is proposed to be the one active for ssDNA binding. However, we recently reported that the mtSSB from Saccharomyces cerevisiae (ScRim1) forms homo-tetramers at high protein concentrations, whereas at low protein concentrations, it dissociates into dimers that bind ssDNA with high affinity . In this work, using a combination of analytical ultracentrifugation techniques and DNA binding experiments with fluorescently labeled DNA oligonucleotides, we tested whether the ability of ScRim1 to form dimers is unique among mtSSBs. Whereas human mtSSBs and those from Schizosaccharomyces pombe, Xenopus laevis and Xenopus tropicalis formed stable homo-tetramers, the mtSSBs from Candida albicans and Candida parapsilosis formed stable homo-dimers. Moreover, the mtSSBs from Candida nivariensis and Candida castellii formed tetramers at high protein concentrations, whereas at low protein concentrations they formed dimers, as did ScRim1. Mutational studies revealed that the ability to form either stable tetramers or dimers depended on a complex interplay of more than one amino acid at the dimer dimer interface and the C-terminal unstructured tail. In conclusion, our findings indicate that mtSSBs can adopt different oligomeric states, ranging from stable tetramers to stable dimers, and suggest that a dimer of mtSSB may be a physiologically relevant species that binds to ssDNA in some yeast species.

Post-transcriptional regulation of the Pseudomonas aeruginosa heme assimilation system (Has) fine-tunes extracellular heme sensing [Gene Regulation]

December 28th, 2018 by Alecia T. Dent, Susana Mourino, Weiliang Huang, Angela Wilks

Pseudomonas aeruginosa is an opportunistic pathogen that utilizes heme as a primary iron source within the host. Extracellular heme is sensed via a heme assimilation system (has) that encodes an extra cytoplasmic function (ECF) σ factor system. Herein, using has deletion mutants, qPCR analyses, and immunoblotting, we show that the activation of the σ factor HasI requires heme release from the hemophore HasAp to the outer membrane receptor HasR. Using RT-PCR and 5’-RACE, we observed that following transcriptional activation of the co-transcribed hasRAp, it is further processed into specific mRNAs varying in stability. We noted that the processing and variation in stability of the hasAp and hasR mRNAs in response to heme provides a mechanism for differential expression from co-transcribed genes. The multiple layers of post-transcriptional regulation of the ECF signaling cascade, including the previously reported post-transcriptional regulation of HasAp by the heme metabolites biliverdin IXβ and IXδ, allows fine tuning of the cell surface signaling system in response to extracellular heme levels. We hypothesize that the complex post-transcriptional regulation of the Has system provides P. aeruginosa an advantage in colonizing a variety of physiological niches in the host.
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Friend or foe — post-translational modifications as regulators of phase separation and RNP granule dynamics [Molecular Bases of Disease]

December 26th, 2018 by Mario Hofweber, Dorothee Dormann

Ribonucleoprotein (RNP) granules are membrane-less organelles consisting of RNA-binding proteins (RBPs) and RNA. RNA granules form through liquid–liquid phase separation (LLPS), whereby weak promiscuous interactions among RBPs and/or RNAs create a dense network of interacting macromolecules and drive the phase separation. Post-translational modifications (PTMs) of RBPs have emerged as important regulators of LLPS and RNP granule dynamics, as they can directly weaken or enhance the multivalent interactions between phase-separating macromolecules or can recruit or exclude certain macromolecules into or from condensates. Here, we review recent insights into how PTMs regulate phase separation and RNP granule dynamics, in particular arginine (R)-methylation and phosphorylation. We discuss how these PTMs regulate the phase behavior of prototypical RBPs and how, as “friend or foe”, they might influence the assembly, disassembly, or material properties of cellular RNP granules, such as stress granules or amyloid-like condensates. We particularly highlight how PTMs control the phase separation and aggregation behavior of disease-linked RBPs. We also review how disruptions of PTMs might be involved in aberrant phase transitions and the formation of amyloid-like protein aggregates as observed in neurodegenerative diseases.

Nrf1-mediated transcriptional regulation of the proteasome requires a functional TIP60 complex [Protein Synthesis and Degradation]

December 17th, 2018 by Janakiram R Vangala, Senthil K Radhakrishnan

Inhibition of the proteasome leads to proteotoxic stress which is characterized by the buildup of ubiquitinated proteins that cannot be degraded properly. The transcription factor Nrf1 (also called NFE2L1) counteracts proteotoxic stress by inducing transcription of proteasome subunit genes resulting in the restoration of proteasome activity. Further understanding of the Nrf1 pathway is therefore of interest in both neurodegeneration, where proteasome activity could be enhanced, and cancer, where suppression of this pathway could potentiate the cell-killing effect mediated by proteasome inhibitor drugs. Here, to identify novel regulators of Nrf1, we performed an RNA interference screen in an engineered cell line reporting on Nrf1 transcriptional activity. In addition to validating known regulators, we discovered that the AAA+ ATPase RUVBL1 is necessary for Nrf1’s transcriptional activity. Given that RUVBL1 is part of different multi-subunit complexes that play key roles in transcription, we dissected this phenomenon further and found that the TIP60 chromatin regulatory complex is essential for Nrf1-dependent transcription of proteasome genes. Consistent with these observations, Nrf1, RUVBL1, and TIP60 proteins were co-recruited to the promoter regions of proteasome genes after proteasome inhibitor treatments. More importantly, depletion of RUVBL1 or TIP60 in various cancer cells sensitized them to cell death induced by proteasome inhibition. Overall, our study provides a framework for manipulating the TIP60-Nrf1 axis to alter proteasome function in various human diseases including cancer.