scholarly journals Prioritization of antimicrobial targets by CRISPR-based oligo recombineering

2021 ◽  
Author(s):  
HJ. Benns ◽  
M. Storch ◽  
J. Falco ◽  
FR. Fisher ◽  
E. Alves ◽  
...  
Keyword(s):  
Amino Acids ◽  
Protein Function ◽  
Proof Of Concept ◽  
Fitness Effect ◽  
Diverse Range ◽  
Functional Sites ◽  
Wide Range ◽  
And Function ◽  
Intrinsic Reactivity ◽  
Subsequent Target

SummaryNucleophilic amino acids are important in covalent drug development yet underutilized as antimicrobial targets. Over recent years, several chemoproteomic technologies have been developed to mine chemically-accessible residues via their intrinsic reactivity toward electrophilic probes. However, these approaches cannot discern which reactive sites contribute to protein function and should therefore be prioritized for drug discovery. To address this, we have developed a CRISPR-based Oligo Recombineering (CORe) platform to systematically prioritize reactive amino acids according to their contribution to protein function. Our approach directly couples protein sequence and function with biological fitness. Here, we profile the reactivity of >1,000 cysteines on ~700 proteins in the eukaryotic pathogen Toxoplasma gondii and prioritize functional sites using CORe. We competitively compared the fitness effect of 370 codon switches at 74 cysteines and identify functional sites in a diverse range of proteins. In our proof of concept, CORe performed >800 times faster than a standard genetic workflow. Reactive cysteines decorating the ribosome were found to be critical for parasite growth, with subsequent target-based screening validating the apicomplexan translation machinery as a target for covalent ligand development. CORe is system-agnostic, and supports expedient identification, functional prioritization, and rational targeting of reactive sites in a wide range of organisms and diseases.

scholarly journals iProteinDB: an integrative database of Drosophila post-translational modifications

2018 ◽  
Author(s):  
Yanhui Hu ◽  
Richelle Sopko ◽  
Verena Chung ◽  
Romain A. Studer ◽  
Sean D. Landry ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Protein Function ◽  
Large Scale ◽  
Model Organisms ◽  
General Strategy ◽  
Post Translational Modification ◽  
Post Translational Modifications ◽  
Functional Sites ◽  
Evolutionarily Conserved ◽  
And Function

AbstractPost-translational modification (PTM) serves as a regulatory mechanism for protein function, influencing stability, protein interactions, activity and localization, and is critical in many signaling pathways. The best characterized PTM is phosphorylation, whereby a phosphate is added to an acceptor residue, commonly serine, threonine and tyrosine. As proteins are often phosphorylated at multiple sites, identifying those sites that are important for function is a challenging problem. Considering that many phosphorylation sites may be non-functional, prioritizing evolutionarily conserved phosphosites provides a general strategy to identify the putative functional sites with regards to regulation and function. To facilitate the identification of conserved phosphosites, we generated a large-scale phosphoproteomics dataset from Drosophila embryos collected from six closely-related species. We built iProteinDB (https://www.flyrnai.org/tools/iproteindb/), a resource integrating these data with other high-throughput PTM datasets, including vertebrates, and manually curated information for Drosophila. At iProteinDB, scientists can view the PTM landscape for any Drosophila protein and identify predicted functional phosphosites based on a comparative analysis of data from closely-related Drosophila species. Further, iProteinDB enables comparison of PTM data from Drosophila to that of orthologous proteins from other model organisms, including human, mouse, rat, Xenopus laevis, Danio rerio, and Caenorhabditis elegans.

scholarly journals Two Distantly Homologous DnaG Primases from Thermoanaerobacter tengcongensis Exhibit Distinct Initiation Specificities and Priming Activities

2010 ◽  
Vol 192 (11) ◽  
pp. 2670-2681 ◽  
Author(s):  
Jie Li ◽  
Jingfang Liu ◽  
Ligang Zhou ◽  
Huadong Pei ◽  
Jian Zhou ◽  
...  
Keyword(s):  
Amino Acids ◽  
De Novo ◽  
Thermophilic Bacterium ◽  
Structure And Function ◽  
Thermoanaerobacter Tengcongensis ◽  
Wide Range ◽  
Dna Binding Affinity ◽  
Template Specificity ◽  
And Function

ABSTRACT Primase, encoded by dnaG in bacteria, is a specialized DNA-dependent RNA polymerase that synthesizes RNA primers de novo for elongation by DNA polymerase. Genome sequence analysis has revealed two distantly related dnaG genes, TtdnaG and TtdnaG 2, in the thermophilic bacterium Thermoanaerobacter tengcongensis. Both TtDnaG (600 amino acids) and TtDnaG2 (358 amino acids) exhibit primase activities in vitro at a wide range of temperatures. Interestingly, the template recognition specificities of these two primases are quite distinctive. When trinucleotide-specific templates were tested, TtDnaG initiated RNA primer synthesis efficiently only on templates containing the trinucleotide 5′-CCC-3′, not on the other 63 possible trinucleotides. When the 5′-CCC-3′ sequence was flanked by additional cytosines or guanines, the initiation efficiency of TtDnaG increased remarkably. Significantly, TtDnaG could specifically and efficiently initiate RNA primer synthesis on a limited set of tetranucleotides composed entirely of cytosines and guanines, indicating that TtDnaG initiated RNA primer synthesis more preferably on GC-containing tetranucleotides. In contrast, it seemed that TtDnaG2 had no specific initiation nucleotides, as it could efficiently initiate RNA primer synthesis on all templates tested. The DNA binding affinity of TtDnaG2 was usually 10-fold higher than that of TtDnaG, which might correlate with its high activity but low template specificity. These distinct priming activities and specificities of TtDnaG and TtDnaG2 might shed new light on the diversity in the structure and function of the primases.

scholarly journals Universal protein misfolding intermediates can bypass the proteostasis network and remain soluble and non-functional

2021 ◽  
Author(s):  
Daniel Allen Nissley ◽  
Yang Jiang ◽  
Fabio Trovato ◽  
Ian Sitarik ◽  
Karthik Narayan ◽  
...  
Keyword(s):  
Protein Function ◽  
Protein Misfolding ◽  
Misfolded Proteins ◽  
Control Mechanisms ◽  
Functional Sites ◽  
Synonymous Mutations ◽  
Dynamics Simulations ◽  
And Function ◽  
Kinetic Traps

Misfolded protein conformations with decreased functionality can bypass the proteostasis machinery and remain soluble in vivo. This is an unexpected phenomenon as several cellular quality control mechanisms have evolved to rid cells of misfolded proteins. Three questions, then, are: how is it structurally possible for long-lived, soluble, misfolded proteins to bypass the proteostasis machinery and processes? How widespread are these soluble, misfolded states across the proteome? And how long do they persist for? Here, we address these questions using coarse-grain molecular dynamics simulations of the synthesis, termination, and post-translational dynamics of a representative set of cytosolic E. coli proteins. We find that half of all proteins exhibit subpopulations of misfolded conformations that are likely to bypass molecular chaperones, avoid aggregation, and not be degraded. These misfolded states can persist for months or longer for some proteins. Structurally characterizing these misfolded states, we observe they have a large amount of native structure, but also contain localized misfolded regions from non-native changes in entanglement, in which a protein segment threads through a loop formed by another portion of the protein that is not found in the native state. The surface properties of these misfolded states are native like, allowing them to bypass the proteostasis machinery and processes to remain soluble, while their entanglements make these states long-lived kinetic traps, as disentanglement requires unfolding of already folded portions of the protein. In terms of function, one-third of proteins have subpopulations that misfold into less-functional states that have structurally perturbed functional sites yet remain soluble. These results explain how proteins misfold into soluble, non-functional conformations that bypass cellular quality controls, and indicate that, unexpectedly, this is a wide-spread cellular phenomenon that can lead to reduced protein function across the cytosolic proteome. Such entanglements are observed in many native structures, suggesting the non-native entanglements we observe are plausible. More broadly, these near-native entangled structures suggest a hypothesis for how synonymous mutations can modulate downstream protein structure and function, with these mutations partitioning nascent proteins between these kinetically trapped states.

Writing Nature in Cold War American Literature

2018 ◽  
Author(s):  
Sarah Daw
Keyword(s):  
Cold War ◽  
American Literature ◽  
Environmental History ◽  
Zen Buddhism ◽  
Diverse Range ◽  
Paul Bowles ◽  
Wide Range ◽  
Silent Spring ◽  
Recent Developments ◽  
And Function

Writing Nature is the first full-length ecocritical study of Cold War American literature. The book analyses the function and representation of Nature in a wide range of Cold War texts, and reveals the prevalence of portrayals of Nature as an infinite, interdependent ecological system in American literature written between 1945 and 1971. It also highlights the Cold War’s often overlooked role in environmental history, and argues for the repositioning of Rachel Carson’s Silent Spring (1962) within what is shown to be a developing trend of ecological presentations of Nature in literature written after 1945. Ecocritical analysis is combined with historicist research to expose the unacknowledged role of a globally diverse range of non-Western and non-Anglocentric philosophies in shaping Cold War writers’ ecological presentations of Nature, including Sufism, Taoism and Zen Buddhism. The book contains chapters on J. D. Salinger, Jack Kerouac and Allen Ginsberg, Paul Bowles and Mary McCarthy. It also introduces the regional writer Peggy Pond Church, exploring the synergies between the depictions of Nature in her writings and in those of her neighbour and correspondent, the atomic scientist J. Robert Oppenheimer. The place and function of Nature in each writer’s work is assessed in relation to the most recent developments in the field of ecocriticism, and each of the book’s six author case studies is investigated through a combination of textual analysis and detailed archival and historicist research.

CONAN: a web application to detect specificity determinants and functional sites by amino acids co-variation network analysis

2020 ◽  
Author(s):  
N J Fonseca ◽  
M Q L Afonso ◽  
L Carrijo ◽  
L Bleicher
Keyword(s):  
Amino Acids ◽  
Network Analysis ◽  
Web Application ◽  
Source Code ◽  
Functional Sites ◽  
Networks Analysis ◽  
And Function ◽  
Go Terms ◽  
Specificity Determinants

Abstract Summary CONAN is a web application developed to detect specificity determinants and function-related sites by amino acids co-variation networks analysis, emphasizing local coevolutionary constraints. The software allows the characterization of structurally and functionally relevant groups of residues and their relationship with subsets of sequences by automatic cross-referencing with GO terms, UniprotKb annotations and INTERPRO. Availability and implementation CONAN is free and open-source, being distributed in the terms of the GPLV3 license. The software is available as a web application and python script versions and can be accessed at http://bioinfo.icb.ufmg.br/conan. We also provide running instructions, the source code and a user guide.

scholarly journals Mycosporine-Like Amino Acids for Skin Photoprotection

2019 ◽  
Vol 25 (40) ◽  
pp. 5512-5527 ◽  
Author(s):  
Karl P. Lawrence ◽  
Paul F. Long ◽  
Antony R. Young
Keyword(s):  
Amino Acids ◽  
Human Skin ◽  
Marine Species ◽  
Current Data ◽  
Health Concern ◽  
Protective Measure ◽  
Solar Ultraviolet Radiation ◽  
Diverse Range ◽  
Wide Range

Background: Excessive human exposure to solar ultraviolet radiation (UVR) continues to be a major public health concern, with skin cancer rates increasing year on year. The major protective measure is the use of synthetic UVR filters formulated into sunscreens, but there is a growing concern that some of these chemicals cause damage to delicate marine ecosystems. One alternative is the use of biocompatible mycosporine-like amino acids (MAA), which occur naturally in a wide range of marine species. Their role within nature is mainly thought to be photoprotective. However, their potential for human photoprotection is largely understudied. Objective: To review the role of MAA in nature and assess their potential as natural sunscreens for human skin photoprotection. Method: A literature review of all relevant papers was conducted. Conclusion: MAA are natural photostable compounds that are thought to offer photoprotection to marine species. Initially thought of as protective based on their absorption properties in the solar UVR spectrum, it is clear that MAA are multifunctional photoprotective compounds acting as chemical and biological anti-oxidants. This suggests that MAA may offer a novel eco-friendly approach to human skin photoprotection. Most studies have been carried out in vitro and current data strongly suggest that MAA have potential for development as natural biocompatible sunscreens that protect against a diverse range of solar UVR induced adverse effects on human health.

scholarly journals DNA Nanodevices as Mechanical Probes of Protein Structure and Function

2021 ◽  
Vol 11 (6) ◽  
pp. 2802
Author(s):  
Nicholas Stephanopoulos ◽  
Petr Šulc
Keyword(s):  
Protein Structure ◽  
Protein Function ◽  
Cell Activation ◽  
Dna Nanotechnology ◽  
Future Directions ◽  
Precise Control ◽  
Wide Range ◽  
Promising Application ◽  
And Function

DNA nanotechnology has reported a wide range of structurally tunable scaffolds with precise control over their size, shape and mechanical properties. One promising application of these nanodevices is as probes for protein function or determination of protein structure. In this perspective we cover several recent examples in this field, including determining the effect of ligand spacing and multivalency on cell activation, applying forces at the nanoscale, and helping to solve protein structure by cryo-EM. We also highlight some future directions in the chemistry necessary for integrating proteins with DNA nanoscaffolds, as well as opportunities for computational modeling of hybrid protein-DNA nanomaterials.

scholarly journals Rapid Identification of Functional Pyrrolysyl-tRNA Synthetases via Fluorescence-Activated Cell Sorting

2018 ◽  
Vol 20 (1) ◽  
pp. 29
Author(s):  
Andrew Lin ◽  
Qing Lin
Keyword(s):  
Amino Acids ◽  
Cell Sorting ◽  
Fluorescent Protein ◽  
Rapid Identification ◽  
Trna Synthetase ◽  
Fluorescence Activated Cell Sorting ◽  
Wide Range ◽  
Natural Amino Acids ◽  
And Function ◽  
Library Selection

The orthogonal pyrrolysyl-tRNA synthetase/tRNACUA pair and their variants have provided powerful tools for expanding the genetic code to allow for engineering of proteins with augmented structure and function not present in Nature. To expedite the discovery of novel pyrrolysyl-tRNA synthetase (PylRS) variants that can charge non-natural amino acids into proteins site-specifically, herein we report a streamlined protocol for rapid construction of the pyrrolysyl-tRNA synthetase library, selection of the functional PylRS mutants using fluorescence-activated cell sorting, and subsequent validation of the selected PylRS mutants through direct expression of the fluorescent protein reporter using a single bacterial strain. We expect that this protocol should be generally applicable to rapid identification of the functional PylRS mutants for charging a wide range of non-natural amino acids into proteins.

Nine Centuries of Man

2017 ◽  
Keyword(s):  
Twentieth Century ◽  
Diverse Range ◽  
Gender Expectations ◽  
Court Records ◽  
The Past ◽  
Wide Range ◽  
Police Reports ◽  
Late Twentieth ◽  
Scottish History ◽  
Late Twentieth Century

What did it mean to be a man in Scotland over the past nine centuries? Scotland, with its stereotypes of the kilted warrior and the industrial ‘hard man’, has long been characterised in masculine terms, but there has been little historical exploration of masculinity in a wider context. This interdisciplinary collection examines a diverse range of the multiple and changing forms of masculinities from the late eleventh to the late twentieth century, exploring the ways in which Scottish society through the ages defined expectations for men and their behaviour. How men reacted to those expectations is examined through sources such as documentary materials, medieval seals, romances, poetry, begging letters, police reports and court records, charity records, oral histories and personal correspondence. Focusing upon the wide range of activities and roles undertaken by men – work, fatherhood and play, violence and war, sex and commerce – the book also illustrates the range of masculinities that affected or were internalised by men. Together, the chapters illustrate some of the ways Scotland’s gender expectations have changed over the centuries and how, more generally, masculinities have informed the path of Scottish history

Diversity and Integration in Private International Law

2019 ◽  
Keyword(s):  
South America ◽  
International Law ◽  
Value Systems ◽  
Private International Law ◽  
Diverse Range ◽  
Cross Border ◽  
Wide Range ◽  
Depth Analysis ◽  
And Shifts ◽  
Substantive Law

This book opens a cross-regional dialogue and shifts the Eurocentric discussion on diversity and integration to a more inclusive engagement with South America in private international law issues. It promotes a contemporary vision of private international law as a discipline enabling legal interconnectivity, with the potential to transcend its disciplinary boundaries to further promote the reality of cross-border integration, with its focus on the ever-increasing cross-border mobility of individuals. Private international law embraces legal diversity and pluralism. Different legal traditions continue to meet, interact and integrate in different forms, at the national, regional and international levels. Different systems of substantive law couple with divergent systems of private international law (designed to accommodate the former in cross-border situations). This complex legal landscape impacts individuals and families in cross-border scenarios, and international commerce broadly conceived. Private international law methodologies and techniques offer means for the coordination of this constellation of legal orders and value systems in cross-border situations. Bringing together world-renowned academics and experienced private international lawyers from a wide range of jurisdictions in Europe and South America, this edited collection focuses on the connective capabilities of private international law in bridging and balancing legal diversity as a corollary for the development of integration. The book provides in-depth analysis of the role of private international law in dealing with legal diversity across a diverse range of topics and jurisdictions.

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