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In this study, oxidative stress and lipid peroxidation in honey bee larvae, pupae and the midguts of adult bees were investigated during a one-year exposure to radiofrequency electromagnetic fields (RF-EMFs) at a frequency of 900 MHz under field conditions. The experiment was carried out on honey bee colonies at three locations with electric field levels of 30 mV m−1, 70 mV m−1 and 1000 mV m−1. Antioxidant enzymes, glutathione-S-transferase (GST), catalase (CAT) and superoxide dismutase (SOD) and thiobarbituric acid reactive substances (TBARS) as indicators of lipid peroxidation were measured spectrophotometrically. The GST activity within the same developmental stage showed no significant differences regardless of electric field level or sampling time. The highest GST activity was found in the pupae, followed by activity in the larvae and midguts. Both CAT activity and TBARS concentration were the highest in the midguts, regardless of field level and sampling time. The larvae showed a significantly higher TBARS concentration at the location with an electric field level of 1000 mV m−1 compared to the locations with lower levels. Our results show that RF-EMFs at a frequency of 900 MHz can cause oxidative stress in honey bees, with the larval stage being more sensitive than the pupal stage, but there was no linear relationship between electric field level and effect in any of the developmental stages.

Since genome sequencing became accessible, determining how specific differences in genotypes lead to complex phenotypes such as disease has become one of the key goals in biomedicine. Predicting effects of sequence variants on cellular or organismal phenotype faces several challenges. First, variants simultaneously affect multiple protein properties and predicting their combined effect is complex. Second, effects of changes in a single protein propagate through the cellular network, which we only partially understand. In this review, we emphasize the importance of both biochemistry and genetics in addressing these challenges. Moreover, we highlight work that blurs the distinction between biochemistry and genetics fields to provide new insights into the genotype-to-phenotype relationships., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Allosteric regulation is central to protein function in cellular networks. A fundamental open question is whether cellular regulation of allosteric proteins occurs only at a few defined positions or at many sites distributed throughout the structure. Here, we probe the regulation of GTPases-protein switches that control signaling through regulated conformational cycling-at residue-level resolution by deep mutagenesis in the native biological network. For the GTPase Gsp1/Ran, we find that 28% of the 4,315 assayed mutations show pronounced gain-of-function responses. Twenty of the sixty positions enriched for gain-of-function mutations are outside the canonical GTPase active site switch regions. Kinetic analysis shows that these distal sites are allosterically coupled to the active site. We conclude that the GTPase switch mechanism is broadly sensitive to cellular allosteric regulation. Our systematic discovery of new regulatory sites provides a functional map to interrogate and target GTPases controlling many essential biological processes., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Molecular switch proteins whose cycling between states is controlled by opposing regulators 1,2 are central to biological signal transduction. As switch proteins function within highly connected interaction networks 3 , the fundamental question arises of how functional specificity is achieved when different processes share common regulators. Here we show that functional specificity of the small GTPase switch protein Gsp1 in Saccharomyces cerevisiae (the homologue of the human protein RAN) 4 is linked to differential sensitivity of biological processes to different kinetics of the Gsp1 (RAN) switch cycle. We make 55 targeted point mutations to individual protein interaction interfaces of Gsp1 (RAN) and show through quantitative genetic 5 and physical interaction mapping that Gsp1 (RAN) interface perturbations have widespread cellular consequences. Contrary to expectation, the cellular effects of the interface mutations group by their biophysical effects on kinetic parameters of the GTPase switch cycle and not by the targeted interfaces. Instead, we show that interface mutations allosterically tune the GTPase cycle kinetics. These results suggest a model in which protein partner binding, or post-translational modifications at distal sites, could act as allosteric regulators of GTPase switching. Similar mechanisms may underlie regulation by other GTPases, and other biological switches. Furthermore, our integrative platform to determine the quantitative consequences of molecular perturbations may help to explain the effects of disease mutations that target central molecular switches., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

A newly described coronavirus named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is the causative agent of coronavirus disease 2019 (COVID-19), has infected over 2.3 million people, led to the death of more than 160,000 individuals and caused worldwide social and economic disruption 1,2 . There are no antiviral drugs with proven clinical efficacy for the treatment of COVID-19, nor are there any vaccines that prevent infection with SARS-CoV-2, and efforts to develop drugs and vaccines are hampered by the limited knowledge of the molecular details of how SARS-CoV-2 infects cells. Here we cloned, tagged and expressed 26 of the 29 SARS-CoV-2 proteins in human cells and identified the human proteins that physically associated with each of the SARS-CoV-2 proteins using affinity-purification mass spectrometry, identifying 332 high-confidence protein-protein interactions between SARS-CoV-2 and human proteins. Among these, we identify 66 druggable human proteins or host factors targeted by 69 compounds (of which, 29 drugs are approved by the US Food and Drug Administration, 12 are in clinical trials and 28 are preclinical compounds). We screened a subset of these in multiple viral assays and found two sets of pharmacological agents that displayed antiviral activity: inhibitors of mRNA translation and predicted regulators of the sigma-1 and sigma-2 receptors. Further studies of these host-factor-targeting agents, including their combination with drugs that directly target viral enzymes, could lead to a therapeutic regimen to treat COVID-19.

An outbreak of the novel coronavirus SARS-CoV-2, the causative agent of COVID-19 respiratory disease, has infected over 290,000 people since the end of 2019, killed over 12,000, and caused worldwide social and economic disruption 1,2 . There are currently no antiviral drugs with proven efficacy nor are there vaccines for its prevention. Unfortunately, the scientific community has little knowledge of the molecular details of SARS-CoV-2 infection. To illuminate this, we cloned, tagged and expressed 26 of the 29 viral proteins in human cells and identified the human proteins physically associated with each using affinity-purification mass spectrometry (AP-MS), which identified 332 high confidence SARS-CoV-2-human protein-protein interactions (PPIs). Among these, we identify 67 druggable human proteins or host factors targeted by 69 existing FDA-approved drugs, drugs in clinical trials and/or preclinical compounds, that we are currently evaluating for efficacy in live SARS-CoV-2 infection assays. The identification of host dependency factors mediating virus infection may provide key insights into effective molecular targets for developing broadly acting antiviral therapeutics against SARS-CoV-2 and other deadly coronavirus strains., Competing Interests: Conflicts: The Krogan Laboratory has received research support from Vir Biotechnology and F. Hoffmann-La Roche. Kevan Shokat has consulting agreements for the following companies involving cash and/or stock compensation: Black Diamond Therapeutics, BridGene Biosciences, Denali Therapeutics, Dice Molecules, eFFECTOR Therapeutics, Erasca, Genentech/Roche, Janssen Pharmaceuticals, Kumquat Biosciences, Kura Oncology, Merck, Mitokinin, Petra Pharma, Qulab Inc. Revolution Medicines, Type6 Therapeutics, Venthera, Wellspring Biosciences (Araxes Pharma). Jack Taunton is a cofounder and shareholder of Global Blood Therapeutics, Principia Biopharma, Kezar Life Sciences, and Cedilla Therapeutics. Jack Taunton and Phillip P. Sharp are listed as inventors on a provisional patent application describing PS3061.

Evolution and design of protein complexes are almost always viewed through the lens of amino acid mutations at protein interfaces. We showed previously that residues not involved in the physical interaction between proteins make important contributions to oligomerization by acting indirectly or allosterically. In this work, we sought to investigate the mechanism by which allosteric mutations act, using the example of the PyrR family of pyrimidine operon attenuators. In this family, a perfectly sequence-conserved helix that forms a tetrameric interface is exposed as solvent-accessible surface in dimeric orthologs. This means that mutations must be acting from a distance to destabilize the interface. We identified 11 key mutations controlling oligomeric state, all distant from the interfaces and outside ligand-binding pockets. Finally, we show that the key mutations introduce conformational changes equivalent to the conformational shift between the free versus nucleotide-bound conformations of the proteins., (Copyright © 2014, American Association for the Advancement of Science.)

Here we review mechanisms of protein evolution leading to structural changes in protein complexes. These mechanisms include mutations directly within protein interfaces, as well as the effects of mutations that propagate from distant regions of the protein. We also discuss the constraints protein complex structures impose on sequence evolution. We interpret, wherever possible, these mechanisms using amino acid residue contact networks. Many insights into protein evolution come from studies of monomers, and these results facilitate our understanding of evolution of protein complexes. Finally, we highlight the potential of formalizing a phylogenetic framework to integrate residue evolution, structure evolution, and to quantify changes in residue contact networks in protein families., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Microbial communities represent the largest portion of the Earth's biomass. Metagenomics projects use high-throughput sequencing to survey these communities and shed light on genetic capabilities that enable microbes to inhabit every corner of the biosphere. Metagenome studies are generally based on (i) classifying and ranking functions of identified genes; and (ii) estimating the phyletic distribution of constituent microbial species. To understand microbial communities at the systems level, it is necessary to extend these studies beyond the species' boundaries and capture higher levels of metabolic complexity. We evaluated 11 metagenome samples and demonstrated that microbes inhabiting the same ecological niche share common preferences for synonymous codons, regardless of their phylogeny. By exploring concepts of translational optimization through codon usage adaptation, we demonstrated that community-wide bias in codon usage can be used as a prediction tool for lifestyle-specific genes across the entire microbial community, effectively considering microbial communities as meta-genomes. These findings set up a 'functional metagenomics' platform for the identification of genes relevant for adaptations of entire microbial communities to environments. Our results provide valuable arguments in defining the concept of microbial species through the context of their interactions within the community.

Is the order in which proteins assemble into complexes important for biological function? Here, we seek to address this by searching for evidence of evolutionary selection for ordered protein complex assembly. First, we experimentally characterize the assembly pathways of several heteromeric complexes and show that they can be simply predicted from their three-dimensional structures. Then, by mapping gene fusion events identified from fully sequenced genomes onto protein complex assembly pathways, we demonstrate evolutionary selection for conservation of assembly order. Furthermore, using structural and high-throughput interaction data, we show that fusion tends to optimize assembly by simplifying protein complex topologies. Finally, we observe protein structural constraints on the gene order of fusion that impact the potential for fusion to affect assembly. Together, these results reveal the intimate relationships among protein assembly, quaternary structure, and evolution and demonstrate on a genome-wide scale the biological importance of ordered assembly pathways., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Abstract The interface of protein structural biology, protein biophysics, molecular evolution, and molecular population genetics forms the foundations for a mechanistic understanding of many aspects of protein biochemistry. Current efforts in interdisciplinary protein modeling are in their infancy and the state-of-the art of such models is described. Beyond the relationship between amino acid substitution and static protein structure, protein function, and corresponding organismal fitness, other considerations are also discussed. More complex mutational processes such as insertion and deletion and domain rearrangements and even circular permutations should be evaluated. The role of intrinsically disordered proteins is still controversial, but may be increasingly important to consider. Protein geometry and protein dynamics as a deviation from static considerations of protein structure are also important. Protein expression level is known to be a major determinant of evolutionary rate and several considerations including selection at the mRNA level and the role of interaction specificity are discussed. Lastly, the relationship between modeling and needed high-throughput experimental data as well as experimental examination of protein evolution using ancestral sequence resurrection and in vitro biochemistry are presented, towards an aim of ultimately generating better models for biological inference and prediction., (Copyright © 2012 The Protein Society.)

Oligomerization plays an important role in the function of many proteins. Thus, understanding, predicting, and, ultimately, engineering oligomerization presents a long-standing interest. From the perspective of structural biology, protein-protein interactions have mainly been analyzed in terms of the biophysical nature and evolution of protein interfaces. Here, our aim is to quantify the importance of the larger structural context of protein interfaces in protein interaction evolution. Specifically, we ask to what extent intersubunit geometry affects oligomerization state. We define a set of structural parameters describing the overall geometry and relative positions of interfaces of homomeric complexes with different oligomeric states. This allows us to quantify the contribution of direct sequence changes in interfaces versus indirect changes outside the interface that affect intersubunit geometry. We find that such indirect, or allosteric mutations affecting intersubunit geometry via indirect mechanisms are as important as interface sequence changes for evolution of oligomeric states.

The role of ubiquitin in many of the known cellular processes, not just protein degradation, is based on its unique ability to bind a range of proteins that are structurally and functionally different. To understand how ubiquitin can bind to proteins with different structures, we review the extent of the conservation and variation that occur in the structures of two free ubiquitins and ubiquitins in 16 complexes that have been determined at high resolution (1.2-2A). Around 80% of the atomic groups in these structures have positions that differ less than 1A. This conserved core provides a rigid platform for flexible loop regions, 39 residues with side chains that can take up different conformations, and a flexible six-residue region at the C-terminus. In most cases the ability of ubiquitin to bind different structures is limited in part by a central set of residues that largely conserve their conformations. The accommodation of differences in binding proteins is enabled by changes in the flexible surface side chains, loop movements, different specific interactions, water molecules in the interface and the flexible C-terminus., (Copyright 2010 Elsevier Ltd. All rights reserved.)

Wrnip1 (Werner helicase-interacting protein 1) has been implicated in the bypass of stalled replication forks in bakers' yeast. However, the function(s) of human Wrnip1 has remained elusive so far. Here we report that Wrnip1 is distributed inside heterogeneous structures detectable in nondamaged cells throughout the cell cycle. In an attempt to characterize these structures, we found that Wrnip1 resides in DNA replication factories. Upon treatments that stall replication forks, such as UVC light, the amount of chromatin-bound Wrnip1 and the number of foci significantly increase, further implicating Wrnip1 in DNA replication. Interestingly, the nuclear pattern of Wrnip1 appears to extend to a broader landscape, as it can be detected in promyelocytic leukemia nuclear bodies. The presence of Wrnip1 into these heterogeneous subnuclear structures requires its ubiquitin-binding zinc finger (UBZ) domain, which is able to interact with different ubiquitin (Ub) signals, including mono-Ub and chains linked via lysine 48 and 63. Moreover, the oligomerization of Wrnip1 mediated by its C terminus is also important for proper subnuclear localization. Our study is the first to reveal the composite and regulated topography of Wrnip1 in the human nucleus, highlighting its potential role in replication and other nuclear transactions.