The class III ribonucleotide reductase from Neisseria bacilliformis can utilize thioredoxin as a reductant
Yifeng Wei, Michael A. Funk, Leonardo A. Rosado, Jiyeon Baek, Catherine L. Drennan, and JoAnne Stubbe
Ribonucleotide reductases (RNRs) catalyze nucleotide reduction via complex radical chemistry, providing deoxynucleotides for DNA synthesis in all domains of life. Many anaerobic bacteria and archaea contain the class III O2-sensitive RNR, and those that have been studied to date couple nucleotide reduction to formate oxidation. Here (pp. E3756–E3765) we report the characterization of a second class III RNR subtype that couples nucleotide reduction to the oxidation of thioredoxin. Because of the central role of formate and thiols in many anaerobic processes, the distribution of class III RNRs among different organisms may shed light on aspects of anaerobic biochemistry.
The Cap1–claudin-4 regulatory pathway is important for renal chloride reabsorption and blood pressure regulation
Yongfeng Gong, Miao Yu, Jing Yang, Ernie Gonzales, Ronaldo Perez, Mingli Hou, Piyush Tripathi, Kathleen S. Hering-Smith, L. Lee Hamm, and Jianghui Hou
How the kidney handles chloride reabsorption has long been a mystery. Here, we have discovered a pathway in the kidney that utilizes channel-activating protease 1 and claudin-4 to physiologically regulate tight junction permeability to chloride. Such a pathway not only is important in renal regulation of chloride but also may play key roles in chloride transport across many other epithelia such as the lung, the salivary gland, and the skin. This discovery (pp. E3766–E3774) also attests to a concept of “druggable” tight junction. Proteases or protease-dependent mechanisms may be developed as pharmacological tools to transiently regulate tight junction permeability in the kidney, the intestine, and the blood–brain barrier.
Transcription factor PREP1 induces EMT and metastasis by controlling the TGF-β–SMAD3 pathway in non-small cell lung adenocarcinoma
Maurizio Risolino, Nadia Mandia, Francescopaolo Iavarone, Leila Dardaei, Elena Longobardi, Serena Fernandez, Francesco Talotta, Fabrizio Bianchi, Federica Pisati, Lorenzo Spaggiari, Patrick N. Harter, Michel Mittelbronn, Dorothea Schulte, Mariarosaria Incoronato, Pier Paolo Di Fiore, Francesco Blasi, and Pasquale Verde
Epithelial–mesenchymal transition (EMT) is a transdifferentiation program implicated in tumor cell dissemination, controlled by networks of transcription complexes responsive to paracrine factors, such as TGF-β. Pre–B-cell leukemia homeobox (Pbx)-regulating protein-1 (PREP1) is a ubiquitous homeodomain transcription factor involved in early development, genomic stability, insulin sensitivity, and hematopoiesis. PREP1 is a haploinsufficient oncosuppressor in mouse tumorigenesis. By characterizing PREP1 as a novel regulator of EMT in human lung adenocarcinoma, we show (pp. E3775–E3784) that PREP1 also harbors prometastatic properties. While autosustaining its activity by stabilizing its transcriptional partner PBX1, PREP1 modulates the responsiveness of lung cancer cells to TGF-β by controlling the expression of two proinvasive transcription factors (SMAD3 and Fos-related antigen 1) implicated in metastasis mechanisms. Thus, PREP1 represents a novel, promising therapeutic target in non-small cell lung cancer.
Src promotes GTPase activity of Ras via tyrosine 32 phosphorylation
Severa Bunda, Pardeep Heir, Tharan Srikumar, Jonathan D. Cook, Kelly Burrell, Yoshihito Kano, Jeffrey E. Lee, Gelareh Zadeh, Brian Raught, and Michael Ohh
Despite the well-established connection between Ras and Src, there currently is no evidence of direct interaction between these two proteins. We show here (pp. E3785–E3794) that Src binds to and phosphorylates GTP-loaded Ras on a conserved Y32 residue within the switch I region. It has been shown that Raf binds to Ras with an affinity 1,000-fold greater than that of GAP. However, it has remained unclear how GAP is able to outcompete Raf for Ras upon Raf displacement. We show here that Y32 phosphorylation inhibits Raf binding to Ras and concomitantly promotes GAP association and GTP hydrolysis, thereby ensuring unidirectionality to the Ras GTPase cycle. These findings reveal new fundamental mechanistic insight into how Src negatively regulates Ras.
Efficient replication of a paramyxovirus independent of full zippering of the fusion protein six-helix bundle domain
Melinda A. Brindley, Philippe Plattet, and Richard Karl Plemper
Protein-mediated membrane fusion is essential for eukaryotic cell biology. In several major human viral pathogens the full closure of a six-helix bundle (6HB) fusion core structure is considered imperative for the opening of a productive fusion pore. We tested this paradigm by engineering covalently restricted paramyxovirus fusion proteins that are incapable of full 6HB zippering. These proteins remained fusion competent without disruption of the restricting bonds, although fusion activity was reduced. Recombinant virions harboring the engineered fusion proteins confirmed efficient, 6HB closure-independent paramyxovirus entry. We demonstrate (pp. E3795–E3804) that full 6HB zippering does not govern paramyxovirus entry. Our results may extend to other viruses using related fusiogenic glycoproteins and affect the design of antiparamyxovirus strategies targeting virus entry.
Ultrafast tissue staining with chemical tags
Johannes Kohl, Julian Ng, Sebastian Cachero, Ernesto Ciabatti, Michael-John Dolan, Ben Sutcliffe, Adam Tozer, Sabine Ruehle, Daniel Krueger, Shahar Frechter, Tiago Branco, Marco Tripodi, and Gregory S. X. E. Jefferis
Cellular and subcellular structures in thick biological samples typically are visualized either by genetically encoded fluorescent proteins or by antibody staining against proteins of interest. However, both approaches have drawbacks. Fluorescent proteins do not survive treatments for tissue preservation well, are available in only a few colors, and often emit weak signals. Antibody stainings are slow, do not penetrate thick samples well, and often result in considerable background staining. We have overcome these limitations by using genetically encoded chemical tags that result in rapid, even staining of thick biological samples with high-signal and low-background labeling. We introduce (pp. E3805–E3814) tools for flies and mice that drastically improve the speed and specificity for labeling genetically marked cells in biological tissues.
Temporally defined neocortical translation and polysome assembly are determined by the RNA-binding protein Hu antigen R
Matthew L. Kraushar, Kevin Thompson, H. R. Sagara Wijeratne, Barbara Viljetic, Kristina Sakers, Justin W. Marson, Dimitris L. Kontoyiannis, Steven Buyske, Ronald P. Hart, and Mladen-Roko Rasin
The neocortex is an intricate and diverse cellular network in the brain, generating complex thought and voluntary motor behavior. Although recent attention has focused on the genome and transcriptome, our goal is to study the role of posttranscriptional processing and mRNA translation in neocortical development. In this work (pp. E3815–E3824), we show that the protein components of actively translating ribosomes and their mRNA cargo in the developing neocortex depend on the temporally specific action of an RNA-binding protein, Hu antigen R (HuR). We further show that HuR is required for the development of neocortical neurons and structure. This study contributes to our overall understanding of how the regulation of functional gene expression influences neocortical development.
Design of donecopride, a dual serotonin subtype 4 receptor agonist/acetylcholinesterase inhibitor with potential interest for Alzheimer's disease treatment
Cédric Lecoutey, Damien Hedou, Thomas Freret, Patrizia Giannoni, Florence Gaven, Marc Since, Valentine Bouet, Céline Ballandonne, Sophie Corvaisier, Aurélie Malzert Fréon, Serge Mignani, Thierry Cresteil, Michel Boulouard, Sylvie Claeysen, Christophe Rochais, Patrick Dallemagne
Targeting more than one molecular cause implied in the pathogenesis of Alzheimer’s disease (AD) with a sole drug is considered a promising challenge, because it may address the numerous failures that recently occurred during clinical trials that were conducted in this area. Donecopride has been designed by us as a multitarget-directed ligand, targeting both serotonin subtype 4 receptor and acetylcholinesterase with excellent in vitro activities (pp. E3825–E3830). The latter seems able to not only restore the cholinergic neurotransmission altered in AD but also, promote the secretion of a neurotrophic protein that is detrimental to the neurotoxic amyloid-β peptide. With its excellent drugability, donecopride further displayed significant procognitive effects in mice and generated a promising lead for a previously unidentified approach in AD treatment.