Genetically encoded fluorescent biosensors illuminate kinase signaling in cancer [Cell Biology]

August 21st, 2019 by Wei Lin, Sohum Mehta, Jin Zhang

Protein kinase signaling networks stringently regulate cellular processes, such as proliferation, motility, and cell survival. These networks are also central to the evolution and progression of cancer. Accordingly, genetically encoded fluorescent biosensors capable of directly illuminating the spatiotemporal dynamics of kinase signaling in live cells are being increasingly used to investigate kinase signaling in cancer cells and tumor tissue sections. These biosensors enable visualization of biological processes and events directly in situ, preserving the native biological context and providing detailed insight into their localization and dynamics in cells. Herein, we first review common design strategies for kinase activity biosensors, including signaling targets, biosensor components and fluorescent proteins involved. Subsequently, we discuss applications of biosensors to study the biology and management of cancer. These versatile molecular tools have been deployed to study oncogenic kinase signaling in living cells, image kinase activities in tumors, or decipher the mechanisms of anticancer drugs. We anticipate that the diversity and precision of genetically encoded biosensors will expand their use to further unravel the dysregulation of kinase signaling in cancer and the modes of actions of cancer-targeting drugs.

The N-terminal domain of an archaeal multidrug and toxin extrusion (MATE) transporter mediates proton coupling required for prokaryotic drug resistance [Molecular Biophysics]

July 9th, 2019 by Kevin L. Jagessar, Hassane S. Mchaourab, Derek P. Claxton

As a contributor to multidrug resistance, the family of multidrug and toxin extrusion (MATE) transporters couples the efflux of chemically dissimilar compounds to electrochemical ion gradients. Although divergent transport mechanisms have been proposed for these transporters, previous structural and functional analyses of members of the MATE subfamily DinF suggest that the N-terminal domain (NTD) supports substrate and ion binding. In this report, we investigated the relationship of ligand binding within the NTD to the drug resistance mechanism of the H+-dependent MATE from the hyperthermophilic archaeon Pyrococcus furiosus (PfMATE). To facilitate this study, we developed a cell growth assay in Escherichia coli to characterize the resistance conferred by PfMATE to toxic concentrations of the antimicrobial compound rhodamine 6G. Expression of wild type PfMATE promoted cell growth in the presence of drug, but amino acid substitutions of conserved NTD residues compromised drug resistance. Steady-state binding analysis with purified PfMATE indicated that substrate affinity was unperturbed in these NTD variants. However, exploiting Trp fluorescence as an intrinsic reporter of conformational changes, we found that these variants impaired formation of a unique H+-stabilized structural intermediate. These results imply that disruption of H+ coupling is the origin of compromised toxin resistance in PfMATE variants. These findings support a model mechanism wherein the NTD mediates allosteric coupling to ion gradients through conformational changes to drive substrate transport in PfMATE. Furthermore, the results provide evidence for diverging transport mechanisms within a prokaryotic MATE subfamily.
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Fast photochemical oxidation of proteins(FPOP): A powerful mass spectrometry based structural proteomics tool [Methods and Resources]

July 1st, 2019 by Dante T Johnson, Luciano H Di Stefano, Lisa M Jones

Fast photochemical oxidation of proteins (FPOP) is a MS-based method that has proved useful in studies of protein structures, interactions, conformations, and protein folding. The success of this method relies on the irreversible labeling of solvent-exposed amino acid side chains by hydroxyl radicals. FPOP generates these radicals through laser-induced photolysis of hydrogen peroxide. The data obtained provide residue-level resolution of protein structures and interactions on the microsecond timescale, enabling investigations of fast processes such as protein folding and weak protein-protein interactions. An extensive comparison between FPOP and other footprinting techniques gives insight on their complementarity as well as the robustness of FPOP to provide unique structural information once unattainable. The versatility of this method is evidenced by both the heterogeneity of samples that can be analyzed by FPOP and the myriad of applications for which the method has been successfully used: from proteins of varying size to intact cells. This review discusses the wide applications of this technique and highlights its high potential. Applications including, but not limited to protein folding, membrane proteins, structure elucidation, and epitope mapping are showcased. Furthermore, the use of FPOP has been extended to probing proteins in-cell and in vivo. These promising developments are also presented herein.

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.
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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.