Trace incorporation of heavy water reveals slow and heterogeneous pathogen growth rates in cystic fibrosis sputum
Sebastian H. Kopf, Alex L. Sessions, Elise S. Cowley, Carmen Reyes, Lindsey Van Sambeek, Yang Hu, Victoria J. Orphan, Roberta Kato, and Dianne K. Newman
A major challenge in treating chronic infections is the lack of insight into microbial survival mechanisms in vivo. Many drugs require cells to be doubling rapidly to have their greatest effect, yet the in vivo pathogen growth rate is largely unknown. By labeling freshly expectorated mucus from cystic fibrosis patients with heavy water, we found that the effective growth rates of Staphylococcus aureus are at least two orders of magnitude slower, on average, than typically studied in the laboratory, and are extremely heterogeneous at the single-cell level. These findings underscore the need to study slow growth physiology to gain insight into pathogen survival mechanisms, motivated by the hope that such insight will ultimately help improve drug design and clinical outcomes. (See pp. E110–E116.)
An inhibitor of HIV-1 protease modulates constitutive eIF2α dephosphorylation to trigger a specific integrated stress response
Aude De Gassart, Bojan Bujisic, Léa Zaffalon, Laurent A. Decosterd, Antonia Di Micco, Gianluca Frera, Rémy Tallant, and Fabio Martinon
The integrated stress response (ISR) is an adaptation pathway that integrates multiple stress signals to decrease translation rates and promote specific transcriptional programs. Modulation of this pathway is emerging as a possible therapeutic strategy in pathologies associated with defects in protein homeostasis. In this paper, the antiviral drug Nelfinavir is found to be a strong and unconventional inducer of the ISR both in vitro and in vivo. This study uncovers an atypical mechanism that can initiate this pathway and provides insights on possible mechanisms underlying the metabolic deregulations observed in Nelfinavir-treated patients. Nelfinavir has been used for years to treat HIV; its repositioning in diseases that would benefit from decreased translation speed could therefore be of interest. (See pp. E117–E126.)
Protein structural and surface water rearrangement constitute major events in the earliest aggregation stages of tau
Anna Pavlova, Chi-Yuan Cheng, Maia Kinnebrew, John Lew, Frederick W. Dahlquist, and Songi Han
Amyloid fibril formation is a key process accompanying many neurodegenerative diseases. Oligomers formed in the early stages of aggregation have been thought to play a key role in disease effects, but their studies are challenging. We use site-specific measurements of surface water diffusion, protein segmental dynamics, and interstrand packing to track early tau protein aggregation processes in situ. Our study reveals that tau aggregation is accompanied by a dramatic structural transformation within minutes of initiating aggregation, followed by the formation of partially structured aggregation intermediates and their rearrangement into stable aggregate species with β-sheet signatures and fibrils. Our findings suggest that therapeutic intervention may focus on disrupting the earliest aggregation events occurring in solution. (See pp. E127–E136.)
Understanding TRPV1 activation by ligands: Insights from the binding modes of capsaicin and resiniferatoxin
Khaled Elokely, Phanindra Velisetty, Lucie Delemotte, Eugene Palovcak, Michael L. Klein, Tibor Rohacs, and Vincenzo Carnevale
Using computational methodologies, we refined the binding modes of the transient receptor potential cation channel subfamily V member 1 (TRPV1) modulators, capsaicin and resiniferatoxin, provided by the recent experimental cryo-electron microscopy electron density. The resulting insights enable us to predict the binding pose of 96 additional TRPV1 agonists, which we compare with reported mutagenesis studies. Specifically, we characterize the response of five previously unidentified mutants to capsaicin and resiniferatoxin. Analysis of the amino acids engaged in favorable ligand–channel interactions defines the key structural determinants of the TRPV1 vanilloid binding site. (See pp. E137–E145.)
Chromosome position determines the success of double-strand break repair
Cheng-Sheng Lee, Ruoxi W. Wang, Hsiao-Han Chang, Daniel Capurso, Mark R. Segal, and James E. Haber
Based on published chromosome conformation capture data, we investigated the effect of 3D nuclear architecture in budding yeast on repair of a broken chromosome by homologous recombination. When a nuclease-induced double-strand break (DSB) is created at one locus, efficiency of repair depends on the contact frequency of the donor locus with the DSB site. When the location of the DSB is shifted to another location, the recombination rate of a donor can change by almost 20-fold. The rate of repair of a given locus is strongly influenced by four key factors: the size of donor homology, the rate of 5′ to 3′ resection of DSB ends, and the abundance of single-strand DNA binding protein complex, replication protein factor A (RPA), as well as a cis-acting recombination enhancer. (See pp. E146–E154.)
Critical role of RAGE and HMGB1 in inflammatory heart disease
Anna Bangert, Martin Andrassy, Anna-Maria Müller, Mariella Bockstahler, Andrea Fischer, Christian H. Volz, Christoph Leib, Stefan Göser, Sevil Korkmaz-Icöz, Stefan Zittrich, Andreas Jungmann, Felix Lasitschka, Gabriele Pfitzer, Oliver J. Müller, Hugo A. Katus, and Ziya Kaya
Myocardial inflammation leads in many cases to cardiomyopathy and contributes to progressive heart failure. The exact pathological mechanism of disease induction and progression in the setting of heart failure is unknown. High-mobility group box 1 (HMGB1), an evolutionarily abundant and highly conserved protein, promotes cardiac inflammation, and in turn immunity, as a damage-associated molecular pattern. HMGB1 stimulates immunity, at least in part, through interaction with its principal binding partner RAGE (receptor for advanced glycation end products). Here we show that HMGB1 and RAGE appear to be important components in cardiac troponin I-induced experimental autoimmune myocarditis as well as in patients with myocarditis. Both molecules represent potential drug targets and show significant potential in heart failure treatment. (See pp. E155–E164.)
Deubiquitinase CYLD acts as a negative regulator for bacterium NTHi-induced inflammation by suppressing K63-linked ubiquitination of MyD88
Byung-Cheol Lee, Masanori Miyata, Jae Hyang Lim, and Jian-Dong Li
Despite the critical role for myeloid differentiation factor 88 (MyD88) in mediating pathogen-induced innate and adaptive immune responses, how functional activity of MyD88 is tightly controlled remains unknown. Among various types of posttranslational modification, ubiquitination and deubiquitination of host signaling molecules play an important role in tightly regulating immune response. However, inducible ubiquitination, in particular proteolysis-independent polyubiquitination as well as deubiquitination of MyD88, remains largely unclear. Here, we demonstrate that deubiquitinase CYLD negatively regulates MyD88-mediated inflammation by directly interacting with MyD88 and deubiquitinating nontypeable Haemophilus influenzae-induced lysine 63-linked polyubiquitination of MyD88. Thus, our studies may not only bring insights into the negative regulation of Toll-like receptor–MyD88-dependent signaling but may also lead to the development of a previously unidentified therapeutic strategy for uncontrolled inflammation. (See pp. E165–E171.)
Functional screen identifies kinases driving prostate cancer visceral and bone metastasis
Claire M. Faltermeier, Justin M. Drake, Peter M. Clark, Bryan A. Smith, Yang Zong, Carmen Volpe, Colleen Mathis, Colm Morrissey, Brandon Castor, Jiaoti Huang, and Owen N. Witte
Therapies are urgently needed to treat metastatic prostate cancer. Mutationally activated and wild-type kinases such as BCR-ABL and BTK are effective therapeutic targets in multiple cancers. Genetically altered kinases are rare in prostate cancer. Wild-type kinases may be implicated in prostate cancer progression, but their therapeutic potential in metastatic prostate cancer remains unknown. Using phosphoproteomics and gene expression datasets, we selected 125 wild-type kinases implicated in human prostate cancer metastasis to screen for metastatic ability in vivo. The RAF family, MERTK, and NTRK2 drove prostate cancer bone and visceral metastasis and were highly expressed in human metastatic prostate cancer tissues. These studies reveal that wild-type kinases can drive metastasis and that the RAF family, MERTK, and NTRK2 may represent important therapeutic targets. (See pp. E172–E181.)
Biochemical evidence of a role for matrix trimerization in HIV-1 envelope glycoprotein incorporation
Philip R. Tedbury, Mariia Novikova, Sherimay D. Ablan, and Eric O. Freed
The HIV matrix domain of Gag is known to play a key role in the incorporation of the envelope glycoprotein into viral particles. Here we provide evidence that matrix is organized as a trimer in HIV-1 particles, and that the ability of matrix to form trimers is essential for the packaging of the envelope glycoprotein. The requirement for matrix trimerization depends on steric constraints between matrix and the cytoplasmic tail of envelope and matrix. This represents a novel model of envelope incorporation in this family of viruses, and suggests that the matrix trimer interface may represent a novel drug target to inhibit the production of infectious viral particles and combat the spread of AIDS. (See pp. E182–E190.)
Structurally conserved erythrocyte-binding domain in Plasmodium provides a versatile scaffold for alternate receptor engagement
Jakub Gruszczyk, Nicholas T. Y. Lim, Alicia Arnott, Wen-Qiang He, Wang Nguitragool, Wanlapa Roobsoong, Yee-Foong Mok, James M. Murphy, Katherine R. Smith, Stuart Lee, Melanie Bahlo, Ivo Mueller, Alyssa E. Barry, and Wai-Hong Tham
Plasmodium vivax is responsible for the most widely distributed recurring human malaria infections whereas Plasmodium falciparum inflicts the most mortality and morbidity in human populations. Malaria parasites enter our blood cells by making proteins that recognize and bind to their cognate receptors on the red blood cell surface. Our research describes, to our knowledge, the first crystal structure of PvRBP2a, an erythrocyte-binding protein from P. vivax, which revealed a structural scaffold similar to that of PfRh5, the essential erythrocyte-binding protein in P. falciparum. Structural comparisons between PvRBP2a and PfRh5 provide an important foundation toward understanding how P. vivax and P. falciparum parasites use a homologous erythrocyte-binding protein family to engage alternate erythrocyte receptors and ultimately govern host cell specificity. (See pp. E191–E200.)
HIV-1 RNA genome dimerizes on the plasma membrane in the presence of Gag protein
Jianbo Chen, Sheikh Abdul Rahman, Olga A. Nikolaitchik, David Grunwald, Luca Sardo, Ryan C. Burdick, Sergey Plisov, Edward Liang, Sheldon Tai, Vinay K. Pathak, and Wei-Shau Hu
Dimerization of the RNA genome is a key event in HIV-1 virion assembly and has a strong impact in viral replication and evolution. Packaging the dimeric genome allows frequent recombination to rescue genetic information in damaged RNAs and to generate variants that can evade the host immune response or resist antiviral treatments. Furthermore, genome packaging is regulated by recognition of dimeric RNA. Our studies demonstrate that HIV-1 RNAs dimerize not in the cytoplasm but on the plasma membrane, often early during the assembly process, and that Gag protein is required for maintenance of the RNA dimer. These studies address the timing, location, and partners involved in RNA dimerization, an important process for HIV-1 replication. (See pp. E201–E208.)
Mechanistic insights into c-di-GMP–dependent control of the biofilm regulator FleQ from Pseudomonas aeruginosa
Bruno Y. Matsuyama, Petya V. Krasteva, Claudine Baraquet, Caroline S. Harwood, Holger Sondermann, and Marcos V. A. S. Navarro
Pseudomonas aeruginosa, an opportunistic pathogen that can cause fatal chronic infections, relies on the intracellular second-messenger c-di-GMP to form robust multicellular biofilms during host tissue colonization. c-di-GMP is sensed directly by the transcription regulator FleQ, which inversely regulates flagellar motility and exopolysaccharide secretion to secure a planktonic to sessile life-form transition. FleQ belongs to the diverse family of AAA+ ATPase enhancer-binding proteins, but how its noncanonical function on transcriptional regulation is controlled by c-di-GMP remains enigmatic. Here, we report structural and functional data that identify an unusual mode of c-di-GMP recognition accompanied by a major quaternary structure reorganization. Our analyses offer a consensus to previous studies and unique insights into the mechanism of action of FleQ and FleQ-like proteins. (See pp. E209–E218.)
Functional hierarchy underlies preferential connectivity disturbances in schizophrenia
Genevieve J. Yang, John D. Murray, Xiao-Jing Wang, David C. Glahn, Godfrey D. Pearlson, Grega Repovs, John H. Krystal, and Alan Anticevic
Schizophrenia is linked to widespread neuronal-level changes causing cortical excitation-inhibition imbalance. However, functional neuroimaging reveals preferential association network dysconnectivity. Therefore, a tension exists between two competing frameworks: global versus localized neural dysfunction in schizophrenia. To link these levels of analysis, this study initially simulated cellular-level glutamatergic deficits, generating network-level predictions of cortical imbalance. Schizophrenia results revealed widespread hyperconnectivity in line with model predictions, yet effects were most profound within association networks. These clinical effects were computationally captured by considering a pre-existing functional cortical hierarchy between association and sensory regions. This study reveals that widespread cellular deficits in schizophrenia can give rise to network-preferential disruptions observed in neuroimaging as an emergent property of pre-existing functional differences between cortical areas. (See pp. E219–E228.)
The K+ channel KIR2.1 functions in tandem with proton influx to mediate sour taste transduction
Wenlei Ye, Rui B. Chang, Jeremy D. Bushman, Yu-Hsiang Tu, Eric M. Mulhall, Courtney E. Wilson, Alexander J. Cooper, Wallace S. Chick, David C. Hill-Eubanks, Mark T. Nelson, Sue C. Kinnamon, and Emily R. Liman
Among the five basic tastes, sour is one of the least understood. Notably, the sour receptor remains to be identified, and molecular mechanisms by which sour stimuli are detected are largely not known. Previous work has shown that H+ ions can directly enter sour taste cells, eliciting a change in membrane potential and acidification of the cell cytosol. In the present work, we identify a second element of sensory transduction, a K+ channel, KIR2.1, which is inhibited by intracellular acidification. The presence of an acid-sensitive K+ channel in sour taste cells allows for amplification of the sensory response and may explain why weak acids that produce intracellular acidification, such as acetic acid, taste more sour than strong acids. (See pp. E229–E238.)
In vitro reconstruction and analysis of evolutionary variation of the tomato acylsucrose metabolic network
Pengxiang Fan, Abigail M. Miller, Anthony L. Schilmiller, Xiaoxiao Liu, Itai Ofner, A. Daniel Jones, Dani Zamir, and Robert L. Last
Throughout the course of human history, plant-derived natural products have been used in medicines, in cooking, as pest control agents, and in rituals of cultural importance. Plants produce rapidly diversifying specialized metabolites as protective agents and to mediate interactions with beneficial organisms. In vitro reconstruction of the cultivated tomato insect protective acylsucrose biosynthetic network showed that four acyltransferase enzymes are sufficient to produce the full set of naturally occurring compounds. This system enabled identification of simple changes in enzyme structure leading to much of the acylsucrose diversity produced in epidermal trichomes of wild tomato. These findings will enable analysis of trichome specialized metabolites throughout the Solanaceae and demonstrate the feasibility of engineering these metabolites in plants and microorganisms. (See pp. E239–E248.)
Progressive forest canopy water loss during the 2012–2015 California drought
Gregory P. Asner, Philip G. Brodrick, Christopher B. Anderson, Nicholas Vaughn, David E. Knapp, and Roberta E. Martin
The state of California has a globally important economy and a population exceeding 38 million. The state relies on its forested watersheds to support numerous services, such as water provisioning, carbon storage, timber products, ecotourism, and recreation. However, secular changes in air temperature, combined with periodic and prolonged drought, pose a compounding challenge to forest health. Here we use new remote-sensing and modeling techniques to assess changes in the canopy water content of California’s forests from 2011 to 2015. Our resulting maps of progressive canopy water stress identify at-risk forest landscapes and watersheds at fine resolution, and offer geographically explicit information to support innovative forest management and policies in preparation for climate change. (See pp. E249–E255.)