Substrate-Assisted Catalysis Unifies Two Families of Chitinolytic Enzymes

Hen egg-white lysozyme has long been the paradigm for enzymatic glycosyl hydrolysis with retention of configuration, with a protonated carboxylic acid and a deprotonated carboxylate participating in general acid-base catalysis. In marked contrast, the retaining chitin degrading enzymes from glycosyl hydrolase families 18 and 20 all have a single glutamic acid as the catalytic acid but lack a nucleophile on the enzyme. Both families have a catalytic (βα)8-barrel domain in common. X-ray structures of three different chitinolytic enzymes complexed with substrates or inhibitors identify a retaining mechanism involving a protein acid and the carbonyl oxygen atom of the substrate’s C2 N-acetyl group as the nucleophile. These studies unambiguously demonstrate the distortion of the sugar ring toward a sofa conformation, long postulated as being close to that of the transition state in glycosyl hydrolysis.

Homing endonuclease I-TevIII: dimerization as a means to a double-strand break

Homing endonucleases are unusual enzymes, capable of recognizing lengthy DNA sequences and cleaving site-specifically within genomes. Many homing endonucleases are encoded within group I introns, and such enzymes promote the mobility reactions of these introns. Phage T4 has three group I introns, within the td, nrdB and nrdD genes. The td and nrdD introns are mobile, whereas the nrdB intron is not. Phage RB3 is a close relative of T4 and has a lengthier nrdB intron. Here, we describe I-TevIII, the H–N–H endonuclease encoded by the RB3 nrdB intron. In contrast to previous reports, we demonstrate that this intron is mobile, and that this mobility is dependent on I-TevIII, which generates 2-nt 3′ extensions. The enzyme has a distinct catalytic domain, which contains the H–N–H motif, and DNA-binding domain, which contains two zinc fingers required for interaction with the DNA substrate. Most importantly, I-TevIII, unlike the H–N–H endonucleases described so far, makes a double-strand break on the DNA homing site by acting as a dimer. Through deletion analysis, the dimerization interface was mapped to the DNA-binding domain. The unusual propensity of I-TevIII to dimerize to achieve cleavage of both DNA strands underscores the versatility of the H–N–H enzyme family

The eukaryotic linear motif resource ELM: 10 years and counting

The eukaryotic linear motif (ELM http://elm.eu.org) resource is a hub for collecting, classifying and curating information about short linear motifs (SLiMs). For >10 years, this resource has provided the scientific community with a freely accessible guide to the biology and function of linear motifs. The current version of ELM contains ∼200 different motif classes with over 2400 experimentally validated instances manually curated from >2000 scientific publications. Furthermore, detailed information about motif-mediated interactions has been annotated and made available in standard exchange formats. Where appropriate, links are provided to resources such as switches.elm.eu.org and KEGG pathways.Fil: Dinkel, Holder. European Molecular Biology Laboratory; AlemaniaFil: Van Roey, Kim. European Molecular Biology Laboratory; AlemaniaFil: Michael, Sushama. European Molecular Biology Laboratory; AlemaniaFil: Davey, Norman E.. University Of California ; Estados UnidosFil: Weatheritt, Robert J.. MRC. Laboratory of Molecular Biology; Estados UnidosFil: Born, Diana. Ruprecht-Karls-Universität; AlemaniaFil: Speck, Tobias. Ruprecht-Karls-Universität; AlemaniaFil: Kruger, Daniel. Ruprecht-Karls-Universität; AlemaniaFil: Grebnev, Gleb. University College Dublin; IrlandaFil: Kuban, Marta. Maria Sklodowska-Curie Memorial Cancer Center. Laboratory of Bioinformatics and Biostatistics; PoloniaFil: Strumillo, Marta. Maria Sklodowska-Curie Memorial Cancer Center. Laboratory of Bioinformatics and Biostatistics; PoloniaFil: Uyar, Bora. European Molecular Biology Laboratory; AlemaniaFil: Budd, Aidan. European Molecular Biology Laboratory; AlemaniaFil: Altenberg, Brigitte. European Molecular Biology Laboratory; AlemaniaFil: Seiler, Markus. European Molecular Biology Laboratory; AlemaniaFil: Chemes, Lucia Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquimicas de Buenos Aires; Argentina. Fundación Instituto Leloir; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Glavina, Juliana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Sánchez Miguel, Ignacio Enrique. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Diella, Francesca. European Molecular Biology Laboratory; AlemaniaFil: Gibson, Toby J. European Molecular Biology Laboratory; Alemani

The EMT transcription factor ZEB1 governs a fitness-promoting but vulnerable DNA replication stress response

The DNA damage response (DDR) and epithelial-to-mesenchymal transition (EMT) are two crucial cellular programs in cancer biology. While the DDR orchestrates cell cycle progression, DNA repair and cell death, EMT promotes invasiveness, cellular plasticity and intratumor heterogeneity. Therapeutic targeting of EMT transcription factors, such as ZEB1, remains challenging, but tumor-promoting DDR alterations elicit specific vulnerabilities. Using multi-omics, inhibitors and high-content microscopy, we discover a chemoresistant ZEB1 high expressing sub-population (ZEB1hi) with co-rewired cell cycle progression and proficient DDR across tumor entities. ZEB1 stimulates accelerated S-phase entry via CDK6, inflicting endogenous DNA replication stress. However, DDR buildups involving constitutive MRE11-dependent fork resection allow homeostatic cycling and enrichment of ZEB1hi cells during TGFβ-induced EMT and chemotherapy. Thus, ZEB1 promotes G1/S transition to launch a progressive DDR benefitting stress tolerance, which concurrently manifests a targetable vulnerability in chemoresistant ZEB1hi cells. Our study thus highlights the translationally relevant intercept of the DDR and EMT

ELM—the database of eukaryotic linear motifs

Linear motifs are short, evolutionarily plastic components of regulatory proteins and provide low-affinity interaction interfaces. These compact modules play central roles in mediating every aspect of the regulatory functionality of the cell. They are particularly prominent in mediating cell signaling, controlling protein turnover and directing protein localization. Given their importance, our understanding of motifs is surprisingly limited, largely as a result of the difficulty of discovery, both experimentally and computationally. The Eukaryotic Linear Motif (ELM) resource at http://elm.eu.org provides the biological community with a comprehensive database of known experimentally validated motifs, and an exploratory tool to discover putative linear motifs in user-submitted protein sequences. The current update of the ELM database comprises 1800 annotated motif instances representing 170 distinct functional classes, including approximately 500 novel instances and 24 novel classes. Several older motif class entries have been also revisited, improving annotation and adding novel instances. Furthermore, addition of full-text search capabilities, an enhanced interface and simplified batch download has improved the overall accessibility of the ELM data. The motif discovery portion of the ELM resource has added conservation, and structural attributes have been incorporated to aid users to discriminate biologically relevant motifs from stochastically occurring non-functional instance

Encompassing new use cases - level 3.0 of the HUPO-PSI format for molecular interactions.

BACKGROUND: Systems biologists study interaction data to understand the behaviour of whole cell systems, and their environment, at a molecular level. In order to effectively achieve this goal, it is critical that researchers have high quality interaction datasets available to them, in a standard data format, and also a suite of tools with which to analyse such data and form experimentally testable hypotheses from them. The PSI-MI XML standard interchange format was initially published in 2004, and expanded in 2007 to enable the download and interchange of molecular interaction data. PSI-XML2.5 was designed to describe experimental data and to date has fulfilled this basic requirement. However, new use cases have arisen that the format cannot properly accommodate. These include data abstracted from more than one publication such as allosteric/cooperative interactions and protein complexes, dynamic interactions and the need to link kinetic and affinity data to specific mutational changes. RESULTS: The Molecular Interaction workgroup of the HUPO-PSI has extended the existing, well-used XML interchange format for molecular interaction data to meet new use cases and enable the capture of new data types, following extensive community consultation. PSI-MI XML3.0 expands the capabilities of the format beyond simple experimental data, with a concomitant update of the tool suite which serves this format. The format has been implemented by key data producers such as the International Molecular Exchange (IMEx) Consortium of protein interaction databases and the Complex Portal. CONCLUSIONS: PSI-MI XML3.0 has been developed by the data producers, data users, tool developers and database providers who constitute the PSI-MI workgroup. This group now actively supports PSI-MI XML2.5 as the main interchange format for experimental data, PSI-MI XML3.0 which additionally handles more complex data types, and the simpler, tab-delimited MITAB2.5, 2.6 and 2.7 for rapid parsing and download

Commencement Program, December (1998)

https://red.mnstate.edu/commencement/1169/thumbnail.jp

Capturing variation impact on molecular interactions in the IMEx Consortium mutations data set

The current wealth of genomic variation data identified at nucleotide level presents the challenge of understanding by which mechanisms amino acid variation affects cellular processes. These effects may manifest as distinct phenotypic differences between individuals or result in the development of disease. Physical interactions between molecules are the linking steps underlying most, if not all, cellular processes. Understanding the effects that sequence variation has on a molecule's interactions is a key step towards connecting mechanistic characterization of nonsynonymous variation to phenotype. We present an open access resource created over 14 years by IMEx database curators, featuring 28,000 annotations describing the effect of small sequence changes on physical protein interactions. We describe how this resource was built, the formats in which the data is provided and offer a descriptive analysis of the data set. The data set is publicly available through the IntAct website and is enhanced with every monthly release

Sugar-fermenting yeast as an organic source of carbon dioxide to attract the malaria mosquito Anopheles gambiae s.s.

<p>Abstract</p> <p>Background</p> <p>Carbon dioxide (CO₂) plays an important role in the host-seeking process of opportunistic, zoophilic and anthropophilic mosquito species and is, therefore, commonly added to mosquito sampling tools. The African malaria vector <it>Anopheles gambiae sensu stricto </it>is attracted to human volatiles augmented by CO₂. This study investigated whether CO₂, usually supplied from gas cylinders acquired from commercial industry, could be replaced by CO₂derived from fermenting yeast (yeast-produced CO₂).</p> <p>Methods</p> <p>Trapping experiments were conducted in the laboratory, semi-field and field, with <it>An. gambiae s.s</it>. as the target species. MM-X traps were baited with volatiles produced by mixtures of yeast, sugar and water, prepared in 1.5, 5 or 25 L bottles. Catches were compared with traps baited with industrial CO₂. The additional effect of human odours was also examined. In the laboratory and semi-field facility dual-choice experiments were conducted. The effect of traps baited with yeast-produced CO₂on the number of mosquitoes entering an African house was studied in the MalariaSphere. Carbon dioxide baited traps, placed outside human dwellings, were also tested in an African village setting. The laboratory and semi-field data were analysed by a χ²-test, the field data by GLM. In addition, CO₂concentrations produced by yeast-sugar solutions were measured over time.</p> <p>Results</p> <p>Traps baited with yeast-produced CO₂caught significantly more mosquitoes than unbaited traps (up to 34 h post mixing the ingredients) and also significantly more than traps baited with industrial CO₂, both in the laboratory and semi-field. Adding yeast-produced CO₂to traps baited with human odour significantly increased trap catches. In the MalariaSphere, outdoor traps baited with yeast-produced or industrial CO₂+ human odour reduced house entry of mosquitoes with a human host sleeping under a bed net indoors. <it>Anopheles gambiae s.s</it>. was not caught during the field trials. However, traps baited with yeast-produced CO₂caught similar numbers of <it>Anopheles arabiensis </it>as traps baited with industrial CO₂. Addition of human odour increased trap catches.</p> <p>Conclusions</p> <p>Yeast-produced CO₂can effectively replace industrial CO₂for sampling of <it>An. gambiae s.s</it>.. This will significantly reduce costs and allow sustainable mass-application of odour-baited devices for mosquito sampling in remote areas.</p