bluuebunny/crossref_metadata_2025 huggingface dataset:
Functional analysis of deoxyhexose sugar utilization inEscherichia colireveals fermentative metabolism under aerobic conditions
Authors: Millard Pierre , Pérochon Julien , Letisse Fabien
Published: April 2021

L-rhamnose and L-fucose are the two main 6-deoxyhexosesEscherichia colican use as carbon and energy sources. Deoxyhexose metabolism leads to the formation of lactaldehyde whose fate depends on oxygen availability. Under anaerobic conditions, lactaldehyde is reduced to 1,2-propanediol whereas under aerobic condition, it should be oxidised into lactate and then channelled into the central metabolism. However, although this all-or-nothing view is accepted in the literature, it seems overly simplistic since propanediol is also reported to be present in the culture medium during aerobic growth on L-fucose. To clarify the functioning of 6-deoxyhexose sugar metabolism, a quantitative metabolic analysis was performed to determine extra- and intracellular fluxes inE. coliK-12 MG1655 (a laboratory strain) and inE. coliNissle 1917 (a human commensal strain) during anaerobic and aerobic growth on L-rhamnose and L-fucose. As expected, lactaldehyde is fully reduced to 1,2-propanediol in anoxic conditions allowing complete reoxidation of the NADH produced by glyceraldehyde-3-phosphate-dehydrogenase. We also found that net ATP synthesis is ensured by acetate production. More surprisingly, lactaldehyde is also primarily reduced into 1,2-propanediol under aerobic conditions. For growth on L-fucose,13C-metabolic flux analysis revealed a large excess of available energy, highlighting the need to better characterize ATP utilization processes. The probioticE. coliNissle 1917 strain exhibits similar metabolic traits, indicating that they are not the result of the K-12 strain’s prolonged laboratory use.

IMPORTANCE

E. coli’s ability to survive, grow and colonize the gastrointestinal tract stems from its use of partially digested food and hydrolysed glycosylated proteins (mucins) from the intestinal mucus layer as substrates. These include L-fucose and L-rhamnose, two 6-deoxyhexose sugars, whose catabolic pathways have been established by genetic and biochemical studies. However, the functioning of these pathways has only partially been elucidated. Our quantitative metabolic analysis provides a comprehensive picture of 6-deoxyhexose sugar metabolism inE. coliunder anaerobic and aerobic conditions. We found that 1,2-propanediol is a major by-product under both conditions, revealing the key role of fermentative pathways in 6-deoxyhexose sugar metabolism. This metabolic trait is shared by bothE. colistrains studied here, a laboratory strain and a probiotic strain. Our findings add to our understanding ofE. coli’s metabolism and of its functioning in the bacterium’s natural environment.
Formins play important role inLeishmaniaphysiology by acting as cytosolic actin bundlers
Authors: Kushwaha Renu , Seth Arunava , Jijumon A.S. , Reshmi P.B. , Dileep Drisya , Datta Rupak , Maiti Sankar
Published: April 2021

Formins are a highly conserved eukaryotic family of proteins that regulate actin dynamics. They play important physiological roles in cell adhesion, motility, vesicular trafficking and cytokinesis. Although sequence analysis of Trypanosomatida genomes predicted multiple formin-encoding genes, none of them are functionally characterized yet. We report here experimental identification and functional characterization of two constitutively expressed formins from the trypanosomatid protozoaLeishmania majorviz. LmForminA and LmForminB. These formins exhibited irregular cytosolic distribution that co-localized with actin patches. Co-sedimentation assay and surface plasmon resonance confirmed that purified LmForminA and B FH2 domains can bind actin, albeit with differential affinity. Interestingly, both LmForminA and B FH2 domains were found to be actin bundlers as revealed by low-speed co-sedimentation assay and TIRF microscopy. LmForminA and B also had actin-nucleating activities, which was abolished by mutating their conserved Ile residue crucial for actin assembly. The Ile-mutant formins, however, retained their actin binding and bundling properties. Treatment ofLeishmaniacells with formin inhibitor SMIFH2 severely perturbed parasite growth and morphology indicating that Lmformins are physiologically important and may be considered as novel drug targets.
A species-specific signature residue in the PB2 subunit of the bat influenza virus polymerase restricts viral RNA synthesis
Authors: Banerjee Saptarshi , De Aratrika , Kedia Nandita , Linfa Wang , Mondal Arindam
Published: April 2021

Bat influenza A viruses (H17N10 and H18N11) are genetically distant from conventional influenza A viruses and replicates poorly in non-bat hosts species. However, the reason behind the lower replication fitness of these viruses are yet to be elucidated. In this work, we have identified species-specific signature residues, present in viral PB2 protein, which is a major determinant of polymerase fitness in human, avian and bat cell lines. Through extensive sequence and structural comparison between the bat and non-bat influenza virus RNA polymerases, we have identified a previously uncharacterized PB2-282 residue, which is serine in bat virus PB2 protein but harbours highly conserved glutamic acid in conventional influenza A viruses. Introduction of these bat specific signatures in the polymerase of a human adapted strain of influenza A/H1N1 virus drastically reduces its polymerase activity and replication efficiency in cell lines of human, bat and canine origin. In contrast, introduction of the human specific signatures in bat virus PB2 (H17N10), significantly enhances its function in the context of a chimeric RNA polymerase. Interestingly, the PB2-282 resides within an evolutionary conserved “S-E-S” motif present across different genera of influenza viruses but is replaced with a “S-S-T” motif in bat influenza viruses, indicating that this E to S transition may serve as a species-specific adaptation signature that modulates the activity of bat virus polymerase in other host species.

Importance

Recent isolation of influenza A like viruses (H17N10 and H18N11) in bats raised concerns about their potential of zoonotic transmission in human. Here we present species-specific signature residues present in the bat influenza virus polymerase, which may act as critical modulators of bat virus propagation in non-bat host species. We utilize bioinformatics based comparative analysis followed by functional screening in order to identify the PB2-282ndposition, which harbors a highly conserved glutamic acid in conventional influenza A viruses, but contains an unusual serine in case of bat influenza viruses. Human adapted polymerase, harboring bat specific signature (PB2-S282) performs poorly, while bat PB2 protein harboring human specific signature (PB2-E282) shows increased fitness in human cells. Together, our data identifies novel species-specific signatures present within the influenza virus polymerase that may serve as a key factor in the adaptation of influenza viruses from bat to non-bat host species and vice versa.
The C-terminus of the prototypical M2 muscarinic receptor localizes to the mitochondria and regulates cell respiration under stress conditions
Authors: Fasciani Irene , Petragnano Francesco , Wang Ziming , Edwards Ruairidh , Telegu Narasimha , Pietrantoni Ilaria , Zabel Ulrike , Zauber Henrik , Grieben Marlies , Terzenidou Maria E. , Di Gregorio Jacopo , Pellegrini Cristina , Santini Silvano , Taddei Anna R. , Pöhl Barbel , Aringhieri Stefano , Carli Marco , Aloisi Gabriella , Marampon Francesco , Charlesworth Eve , Roman Alexandra , Diecke Sebastian , Flati Vincenzo , Giorgi Franco , Amicarelli Fernanda , Tobin Andrew B. , Scarselli Marco , Tokatlidis Kostas , Rossi Mario , Lohse Martin J. , Annibale Paolo , Maggio Roberto
Published: April 2021

Muscarinic acetylcholine receptors are prototypical G protein-coupled receptors (GPCRs), members of a large family of seven transmembrane receptors mediating a wide variety of extracellular signals. We show here, in cultured cells and in a murine model, that the carboxyl terminal fragment of the muscarinic M2receptor, comprising the transmembrane regions 6 and 7 (M2tail), is expressed by virtue of an internal ribosome entry site localized in the third intracellular loop. Single cell imaging and import in isolated yeast mitochondria reveals that M2tail, whose expression is upregulated in cells undergoing integrated stress response, does not follow the normal route to the plasma membrane, but is almost exclusively sorted to the mitochondria inner membrane: here it controls oxygen consumption, cell proliferation and the formation of reactive oxygen species by reducing oxidative phosphorylation. Crispr/Cas9 editing of the key methionine where cap-independent translation begins in human induced pluripotent stem cells, reveals the physiological role of this process in influencing cell proliferation and oxygen consumption at the endogenous level. The expression of the C-terminal domain of a GPCR, capable of regulating mitochondrial function, constitutes a hitherto unknown mechanism notably unrelated to its canonical signalling function as a GPCR at the plasma membrane. This work thus highlights a potential novel mechanism thar cells may use for controlling their metabolism under variable environmental conditions, notably as a negative regulator of cell respiration.
SUFFICIENT ACTIVITY OF THE UBIQUITIN PROTEASOME SYSTEM IN AGED MICE AND DURING RETINAL DEGENERATION SUPPORTS DHFR-BASED CONDITIONAL CONTROL OF PROTEIN ABUNDANCE IN THE RETINA
Authors: Peng Hui , Ramadurgum Prerana , Woodard DaNae R. , Daniel Steffi , Renwick Marian , Aredo Bogale , Datta Shyamtanu , Chen Bo , Ufret-Vincenty Rafael , Hulleman John D.
Published: April 2021

TheEscherichia colidihydrofolate reductase (DHFR) destabilizing domain (DD) serves as a promising approach to conditionally regulate protein abundance in a variety of tissues. In the absence of TMP, a DHFR stabilizer, the DD is degraded by the ubiquitin proteasome system (UPS). To test whether this approach could be effectively applied to a wide variety of aged and disease-related ocular mouse models, which may have a compromised UPS, we evaluated the DHFR DD system in aged mice (up to 24 mo), a light-induced retinal degeneration (LIRD) model, and two genetic models of retinal degeneration (rd2andAbca4−/−mice). Aged, LIRD, andAbca4−/−mice all had similar proteasomal activities and high-molecular weight ubiquitin levels compared to control mice. However,rd2mice displayed compromised chymotrypsin activity compared to control mice. Nonetheless, the DHFR DD was effectively degraded in all model systems, includingrd2mice. Moreover, TMP increased DHFR DD-dependent retinal bioluminescence in all mouse models, however the fold induction was slightly, albeit significantly, lower inAbca4−/−mice. Thus, the destabilized DHFR DD-based approach allows for efficient control of protein abundance in aged mice and retinal degeneration mouse models, laying the foundation to use this strategy in a wide variety of mice for the conditional control of gene therapies to potentially treat multiple eye diseases.
β-RA targets mitochondrial metabolism and adipogenesis, leading to therapeutic benefits against CoQ deficiency and age-related overweight
Authors: Hidalgo-Gutiérrez Agustín , Barriocanal-Casado Eliana , Díaz-Casado María Elena , González-García Pilar , Chiozzi Riccardo Zenezini , Acuña-Castroviejo Darío , López Luis Carlos
Published: April 2021

Primary mitochondrial diseases are caused by mutations in mitochondrial or nuclear genes, leading to abnormal function of specific mitochondrial pathways. Mitochondrial dysfunction is also a secondary event in more common pathophysiological conditions, such as obesity and metabolic syndrome. In both cases, the improvement and management of mitochondrial homeostasis remains challenging. Here, we show that beta-resorcylic acid (β-RA), a natural phenolic compound, competesin vivowith 4-hydroxybenzoic acid, the natural precursor of Coenzyme Q biosynthesis. This leads to a decrease of demethoxyubiquinone, an intermediate metabolite of CoQ biosynthesis that is abnormally accumulated inCoq9R239Xmice. As a consequence, β-RA rescues the phenotype ofCoq9R239Xmice, a model of primary mitochondrial encephalopathy. Moreover, we observe that long-term treatment with β-RA also reduces the size and content of the white adipose tissue (WAT) that is normally accumulated during aging in wild-type mice, leading to a prevention of hepatic steatosis and an increase in survival at the old stage of life. The reduction in WAT content is due to a decrease in adipogenesis, an adaptation of the mitochondrial proteome in the kidneys, and a stimulation of glycolysis and acetyl-CoA metabolism. Therefore, our results demonstrate that β-RA acts through different cellular mechanisms, with effects on mitochondrial metabolism, and it may be used for the treatment of primary Coenzyme Q deficiency, overweight, and hepatic steatosis.
Novel amiloride derivatives that inhibit bacterial motility across multiple strains and stator types
Authors: Islam MI , Bae JH , Ishida T , Ridone P , Lin J , Kelso MJ , Sowa Y , Buckley BJ , Baker MAB
Published: April 2021

The bacterial flagellar motor (BFM) is a protein complex that confers motility to cells and contributes to survival and virulence. The BFM consists of stators that are ion-selective membrane protein complexes and a rotor that directly connects to a large filament, acting as a propeller. The stator complexes couple ion transit across the membrane to torque that drives rotation of the motor. The most common ion gradients that drive BFM rotation are protons (H+) and sodium ions (Na+). The sodium-powered stators, like those in the PomAPomB stator complex of Vibrio spp, can be inhibited by sodium channel inhibitors, in particular, by phenamil, a potent and widely used inhibitor. However, relatively few new sodium-motility inhibitors have been described since the discovery of phenamil. In this study, we characterised two possible motility inhibitors HM2-16F and BB2-50F from a small library of previously reported amiloride derivatives. We used three approaches: effect on rotation of tethered cells, effect on free swimming bacteria and effect on rotation of marker beads. We showed that both HM2-16F and BB2-50F stopped rotation of tethered cells driven by Na+motors comparable to phenamil at matching concentrations, and could also stop rotation of tethered cells driven by H+motors. Bead measurements in presence and absence of stators confirmed that the compounds did not inhibit rotation via direct association with the stator, in contrast to the established mode of action of phenamil. Overall, HM2-16F and BB2-50F stopped swimming in both Na+and H+stator types, and in pathogenic and non-pathogenic strains.

Importance

Here we characterised two novel amiloride derivatives in the search for antimicrobial compounds that target bacterial motility. Our two compounds were shown to inhibit flagellar motility at 10 μM across multiple strains, from non-pathogenicE. coliwith flagellar rotation driven by proton or chimeric sodium-powered stators, to proton-powered pathogenicE. coli(EHEC/UPEC) and lastly in sodium-poweredVibrio alginolyticus. Broad anti-motility compounds such as these are important tools in our efforts control virulence of pathogens in health and agricultural settings.
Towards a comprehensive delineation of white matter tract-related deformation
Authors: Zhou Zhou , Li Xiaogai , Liu Yuzhe , Fahlstedt Madelen , Georgiadis Marios , Zhan Xianghao , Raymond Samuel J. , Grant Gerald , Kleiven Svein , Camarillo David , Zeineh Michael
Published: April 2021

Finite element (FE) models of the human head are valuable instruments to explore the mechanobiological pathway from external loading, localized brain response, and resultant injury risks. The injury predictability of these models depends on the use of effective criteria as injury predictors. The FE-derived normal deformation along white matter (WM) fiber tracts (i.e., tract-oriented strain) has recently been suggested as an appropriate predictor for axonal injury. However, the tract-oriented strain only represents a partial depiction of the WM fiber tract deformation. A comprehensive delineation of tract-related deformation may improve the injury predictability of the FE head model by delivering new tract-related criteria as injury predictors. Thus, the present study performed a theoretical strain analysis to comprehensively characterize the WM fiber tract deformation by relating the strain tensor of the WM element to its embedded fiber tract. Three new tract-related strains with exact analytical solutions were proposed, measuring the normal deformation perpendicular to the fiber tracts (i.e., tract-perpendicular strain), and shear deformation along and perpendicular to the fiber tracts (i.e., axial-shear strain and lateral-shear strain, respectively). The injury predictability of these three newly-proposed strain peaks along with the previously-used tract-oriented strain peak and maximum principal strain (MPS) were evaluated by simulating 151 impacts with known outcome (concussion or non-concussion). The results preliminarily showed that four tract-related strain peaks exhibited superior performance than MPS in discriminating concussion and non-concussion cases. This study presents a comprehensive quantification of WM tract-related deformation and advocates the use of orientation-dependent strains as criteria for injury prediction, which may ultimately contribute to an advanced mechanobiological understanding and enhanced computational predictability of brain injury.
Multifunctional RNA-binding proteins influence mRNA abundance and translational efficiency of distinct sets of target genes
Authors: Schneider-Lunitz Valentin , Ruiz-Orera Jorge , Hubner Norbert , van Heesch Sebastiaan
Published: April 2021

RNA-binding proteins (RBPs) can regulate more than a single aspect of RNA metabolism. We searched for such previously undiscovered multifunctionality within a set of 143 RBPs, by defining the predictive value of RBP abundance for the transcription and translation levels of known RBP target genes across 80 human hearts. This led us to newly associate 27 RBPs with cardiac translational regulationin vivo. Of these, 21 impacted both RNA expression and translation, albeit for virtually independent sets of target genes. We highlight a subset of these, including G3BP1, PUM1, UCHL5, and DDX3X, where dual regulation is achieved through differential affinity for target length, by which separate biological processes are controlled. Similar to the RNA helicase DDX3X, the known splicing factors EFTUD2 and PRPF8 - all identified as multifunctional RBPs by our analysis - selectively influence target translation rates depending on 5’ UTR structure. Our analyses identify dozens of RBPs as being multifunctional and pinpoint potential novel regulators of translation, postulating unanticipated complexity of protein-RNA interactions at consecutive stages of gene expression.
Large-scale Integrative Taxonomy (LIT): resolving the data conundrum for dark taxa
Authors: Hartop Emily , Srivathsan Amrita , Ronquist Fredrik , Meier Rudolf
Published: April 2021

New, rapid, accurate, scalable, and cost-effective species discovery and delimitation methods are needed for tackling “dark taxa”, that we here define as clades for which <10% of all species are described and the estimated diversity exceeds 1000 species. Species delimitation for these taxa should be based on multiple data sources (“integrative taxonomy”) but collecting multiple types of data risks impeding a discovery process that is already too slow. We here develop explicit methods to avoid this by applying Large-scale Integrative Taxonomy (LIT). Preliminary species hypotheses are generated based on inexpensive data that are obtained quickly and cost-effectively in a technical exercise. The validation step is then based on a more expensive type of data that are only obtained for specimens selected based on objective criteria. We here use this approach to sort 18 000 scuttle flies (Diptera: Phoridae) from Sweden into 315 preliminary species hypotheses based on NGS barcode (313bp) clusters. These clusters were subsequently tested with morphology and used to develop quantitative indicators for predicting which barcode clusters are in conflict with morphospecies. For this purpose, we first randomly selected 100 clusters for in-depth validation with morphology. Afterwards, we used a linear model to demonstrate that the best predictors for conflict between barcode clusters and morphology are maximum p-distance within the cluster and cluster stability across different clustering thresholds. A test of these indicators using the 215 remaining clusters reveals that these predictors correctly identify all clusters that conflict with morphology. The morphological validation step in our study involved only 1 039 specimens (5.8% of all specimens), but a newly proposed simplified protocol would only require the study of 915 (5.1%: 2.5 specimens per species), as we show that clusters without signatures of incongruence can be validated by only studying two specimens representing the most divergent haplotypes. To test the generality of our results across different barcode clustering techniques, we establish that the levels of conflict are similar across Objective Clustering (OC), Automatic Barcode Gap Discovery (ABGD), Poisson Tree Processes (PTP) and Refined Single Linkage (RESL) (used by Barcode of Life Data System (BOLD) to assign Barcode Index Numbers (BINs)). OC and ABGD achieved a maximum congruence score with morphology of 89% while PTP was slightly less effective (84%). RESL could only be tested for a subset of the specimens because the algorithm is not public. BINs based on 277 of the original 1 714 haplotypes were 86% congruent with morphology while the values were 89% for OC, 74% for PTP, and 72% for ABGD.
The transcriptomic landscape of the photoperiodic stress response in Arabidopsis thaliana resembles the response to pathogen infection
Authors: Cortleven Anne , Roeber Venja M. , Frank Manuel , Bertels Jonas , Lortzing Vivien , Beemster Gerrit , Schmülling Thomas
Published: April 2021

Plants are exposed to regular diurnal rhythms of light and dark. Changes in the photoperiod by the prolongation of the light period cause photoperiod stress in short day-adapted Arabidopsis thaliana. Here we report on the transcriptional response to photoperiod stress of wild-type A. thaliana and photoperiod stress-sensitive cytokinin signalling and clock mutants. Transcriptomic changes induced by photoperiod stress included numerous changes in reactive oxygen (ROS)-related transcripts and showed a strong overlap with changes occurring in response to ozone stress and pathogen attack, which have in common the induction of an apoplastic oxidative burst. A core set of photoperiod stress-responsive genes has been identified, including salicylic acid (SA) biosynthesis and signalling genes. Genetic analysis revealed a central role for NPR1 in the photoperiod stress response as npr1-1 mutants were stress-insensitive. Photoperiod stress treatment led to a strong increase in camalexin levels which is consistent with shared photoperiod stress and pathogen response pathways. Photoperiod stress induced resistance of Arabidopsis plants to a subsequent infection by Pseudomonas syringae cv. tomato DC3000 indicating priming of the defence response. Together, photoperiod stress causes transcriptional reprogramming resembling plant pathogen defence responses and induces systemic acquired resistance in the absence of a pathogen.

One sentence summary

Photoperiod stress results in significant dynamic transcriptomic changes related to oxidative stress similar to those caused by pathogen attack and primes the defence response against a subsequent pathogen infection.
Melanocore uptake by keratinocytes occurs through phagocytosis and involves Protease-activated receptor-2 activation
Authors: Moreiras Hugo , Neto Matilde V. , Bento-Lopes Liliana , Escrevente Cristina , Ramalho José S. , Seabra Miguel C. , Barral Duarte C.
Published: April 2021

In the skin epidermis, melanin is produced and stored within melanosomes in melanocytes and then transferred to keratinocytes. Different models have been proposed to explain the melanin transfer mechanism, which differ essentially in how melanin is transferred – either in a membrane-bound melanosome or as a melanosome core, i.e. melanocore. Here we investigated the endocytic route followed by melanocores and melanosomes during internalization by keratinocytes, by comparing the uptake of melanocores isolated from the supernatant of melanocyte cultures with melanosomes isolated from melanocytes. We show that inhibition of actin dynamics impairs the uptake of both melanocores and melanosomes. Moreover, depletion of critical proteins involved in actin-dependent uptake mechanisms, namely Rac1 and CtBP1/BARS, together with inhibition of Rac1-dependent signaling pathways or macropinocytosis suggest that melanocores are internalized by phagocytosis, whereas melanosomes are internalized by macropinocytosis. Furthermore, we confirmed that melanocore, but not melanosome uptake is dependent on the Protease-activated receptor-2 (PAR-2) and found that PAR-2 can be specifically activated by melanocores. As skin pigmentation was shown to be regulated by PAR-2 activation, our results further support the melanocore mechanism of melanin transfer and further refine this model, which can now be described as coupled melanocore exo/phagocytosis.
Defense by duplication: The relation between phenotypic glyphosate resistance and EPSPS gene copy number variation inAmaranthus palmeri
Authors: Yakimowski Sarah B. , Teitel Zachary , Caruso Christina M.
Published: April 2021

Gene copy number variation (CNV) has been increasingly associated with organismal responses to environmental stress, but we know little about the quantitative relation between CNV and phenotypic variation. In this study we quantify variation in EPSPS (5-enolpyruvylshikimate-3-phosphate synthase) copy number using digital drop PCR and variation in phenotypic glyphosate resistance in 22 populations ofAmaranthus palmeri(Palmer Amaranth), a range-expanding agricultural weed. Overall, we detected a significant positive relation between population mean copy number and mean resistance. The majority of populations exhibited high glyphosate resistance, yet maintained low-resistance individuals resulting in bimodality in many populations. We investigated linear and threshold models for the relation between copy number and resistance, and found evidence for a threshold of ~15 EPSPS copies: there was a steep increase in resistance before the threshold, followed by a much shallower slope. Moreover, as copy number increases, the range of variation in resistance decreases. This suggests a working hypothesis that as EPSPS copies and dosage increases, negative epistatic interactions may be compensated. We detected a quadratic relation between mean resistance and variation (s.d.) in resistance, consistent with the prediction that as phenotypic resistance increases in populations, stabilizing selection decreases variation in the trait. Finally, patterns of variation across the landscape are consistent with less variation among populations in mean copy number / resistance in Georgia where glyphosate resistance was first detected, and wider variation among populations in resistance and copy number in a more northern state where resistance evolution may be at a younger evolutionary state.
Kinetic and structural characterization of the self-labeling protein tags HaloTag7, SNAP-tag and CLIP-tag
Authors: Wilhelm Jonas , Kühn Stefanie , Tarnawski Miroslaw , Gotthard Guillaume , Tünnermann Jana , Tänzer Timo , Karpenko Julie , Mertes Nicole , Xue Lin , Uhrig Ulrike , Reinstein Jochen , Hiblot Julien , Johnsson Kai
Published: April 2021

The self-labeling protein tags (SLPs) HaloTag7, SNAP-tag and CLIP-tag allow the covalent labeling of fusion proteins with synthetic molecules for applications in bioimaging and biotechnology. To guide the selection of an SLP-substrate pair and provide guidelines for the design of substrates, we report a systematic and comparative study on the labeling kinetics and substrate specificities of HaloTag7, SNAP-tag and CLIP-tag. HaloTag7 reaches almost diffusion-limited labeling rates with certain rhodamine substrates, which are more than two orders of magnitude higher than those of SNAP-tag for the corresponding substrates. SNAP-tag labeling rates however are less affected by the structure of the label than those of HaloTag7, which vary over six orders of magnitude for commonly employed substrates. Solving the crystal structures of HaloTag7 and SNAP-tag labeled with fluorescent substrates allowed us to rationalize their substrate preferences. We also demonstrate how these insights can be exploited to design substrates with improved labeling kinetics.
Subthalamic nucleus stabilizes movements by reducing neural spike variability in monkey basal ganglia: chemogenetic study
Authors: Hasegawa Taku , Chiken Satomi , Kobayashi Kenta , Nambu Atsushi
Published: April 2021

Abstracts

The subthalamic nucleus (STN) projects to the external pallidum (GPe) and internal pallidum (GPi), the relay and output nuclei of the basal ganglia (BG), respectively, and plays an indispensable role in controlling voluntary movements. To elucidate the neural mechanism by which the STN controls GPe/GPi activity and movements, we utilized a chemogenetic method to reversibly suppress the motor subregion of the STN in three macaque monkeys (Macaca fuscata, both sexes) engaged in reaching tasks. Systemic administration of chemogenetic ligands prolonged movement time and increased spike train variability in the GPe/GPi, but only slightly affected firing rate modulations. Across-trial analyses revealed that the irregular discharge activity in the GPe/GPi coincided with prolonged movement time. STN suppression also induced excessive abnormal movements in the contralateral forelimbs, which was preceded by STN and GPe/GPi phasic activity changes. Our results suggest that the STN stabilizes spike trains in the BG and achieves stable movements.
Proteome-scale amino-acid resolution footprinting of protein-binding sites in the intrinsically disordered regions of the human proteome
Authors: Benz Caroline , Ali Muhammad , Krystkowiak Izabella , Simonetti Leandro , Sayadi Ahmed , Mihalic Filip , Kliche Johanna , Andersson Eva , Jemth Per , Davey Norman E. , Ivarsson Ylva
Published: April 2021

Specific protein-protein interactions are central to all processes that underlie cell physiology. Numerous studies using a wide range of experimental approaches have identified tens of thousands of human protein-protein interactions. However, many interactions remain to be discovered, and low affinity, conditional and cell type-specific interactions are likely to be disproportionately under-represented. Moreover, for most known protein-protein interactions the binding regions remain uncharacterized. We previously developed proteomic peptide phage display (ProP-PD), a method for simultaneous proteome-scale identification of short linear motif (SLiM)-mediated interactions and footprinting of the binding region with amino acid resolution. Here, we describe the second-generation human disorderome (HD2), an optimized ProP-PD library that tiles all disordered regions of the human proteome and allows the screening of ~1,000,000 overlapping peptides in a single binding assay. We define guidelines for how to process, filter and rank the results and provide PepTools, a toolkit for annotation and analysis of identified hits. We uncovered 2,161 interaction pairs for 35 known SLiM-binding domains and confirmed a subset of 38 interactions by biophysical or cell-based assays. Finally, we show how the amino acid resolution binding site information can be used to pinpoint functionally important disease mutations and phosphorylation events in intrinsically disordered regions of the human proteome. The HD2 ProP-PD library paired with PepTools represents a powerful pipeline for unbiased proteome-wide discovery of SLiM-based interactions.
Excitation spectral microscopy for highly multiplexed fluorescence imaging and quantitative biosensing
Authors: Chen Kun , Yan Rui , Xiang Limin , Xu Ke
Published: April 2021

The multiplexing capability of fluorescence microscopy is severely limited by the broad fluorescence spectral width. Spectral imaging offers potential solutions, yet typical approaches to disperse the local emission spectra notably impede the attainable throughput. Here we show that using a single, fixed fluorescence emission detection band, through frame-synchronized fast scanning of the excitation wavelength from a white lampviaan acousto-optic tunable filter (AOTF), up to 6 subcellular targets, labeled by common fluorophores of substantial spectral overlap, can be simultaneously imaged in live cells with low (∼1%) crosstalks and high temporal resolutions (down to ∼10 ms). The demonstrated capability to quantify the abundances of different fluorophores in the same sample through unmixing the excitation spectra next enables us to devise novel, quantitative imaging schemes for both bi-state and FRET (Förster resonance energy transfer) fluorescent biosensors in live cells. We thus achieve high sensitivities and spatiotemporal resolutions in quantifying the mitochondrial matrix pH and intracellular macromolecular crowding, and further demonstrate, for the first time, the multiplexing of absolute pH imaging with three additional target organelles/proteins to elucidate the complex, Parkin-mediated mitophagy pathway. Together, excitation spectral microscopy provides exceptional opportunities for highly multiplexed fluorescence imaging. The prospect of acquiring fast spectral images without the need for fluorescence dispersion or care for the spectral response of the detector offers tremendous potential.
Engineering Insulin Cold Chain Resilience to Improve Global Access
Authors: Maikawa Caitlin L. , Mann Joseph L. , Kannan Aadithya , Meis Catherine M. , Grosskopf Abigail K. , Ou Ben S. , Smith Anton A. A. , Fuller Gerald G. , Maahs David M. , Appel Eric A.
Published: April 2021

There are 150 million people with diabetes worldwide who require insulin replacement therapy and the prevalence of diabetes is rising fastest in middle and low-income countries. Current formulations require costly refrigerated transport and storage to prevent loss of insulin integrity. This study shows the development of simple “drop-in” amphiphilic copolymer excipients to maintain formulation integrity, bioactivity, pharmacokinetics and pharmacodynamics for over 6 months when subjected to severe stressed aging conditions that cause current commercial formulation to fail in under 2 weeks. Further, when these copolymers are added to Humulin R (Eli Lilly) in original commercial packaging they prevent insulin aggregation for up to 4 days at 50 °C compared to less than 1 day for Humulin R alone. These copolymers demonstrate promise as simple formulation additives to increase the cold chain resilience of commercial insulin formulations, thereby expanding global access to these critical drugs for treatment of diabetes.
Data-driven and interpretable machine-learning modeling to explore the fine-scale environmental determinants of malaria vectors biting rates in rural Burkina Faso
Authors: Taconet Paul , Porciani Angélique , Soma Dieudonné Diloma , Mouline Karine , Simard Frédéric , Koffi Alphonsine Amanan , Pennetier Cedric , Dabiré Roch Kounbobr , Mangeas Morgan , Moiroux Nicolas
Published: April 2021

Background

Improving the knowledge and understanding of the environmental determinants of malaria vectors abundances at fine spatiotemporal scales is essential to design locally tailored vector control intervention. This work aimed at exploring the environmental tenets of human-biting activity in the main malaria vectors (Anopheles gambiae s.s.,Anopheles coluzzii andAnopheles funestus)in the health district of Diébougou, rural Burkina Faso.

Methods

Anopheleshuman-biting activity was monitored in 27 villages during 15 months (in 2017-2018), and environmental variables (meteorological and landscape) were extracted from high resolution satellite imagery. A two-step data-driven modeling study was then carried-out. Correlation coefficients between the biting rates of each vector species and the environmental variables taken at various temporal lags and spatial distances from the biting events were first calculated. Then, multivariate machine-learning models were generated and interpreted to i) pinpoint primary and secondary environmental drivers of variation in the biting rates of each species and ii) identify complex associations between the environmental conditions and the biting rates.

Results

Meteorological and landscape variables were often significantly correlated with the vectors’ biting rates. Many nonlinear associations and thresholds were unveiled by the multivariate models, both for meteorological and landscape variables. From these results, several aspects of the bio-ecology of the main malaria vectors were precised or hypothesized for the Diébougou area, including breeding sites typologies, development and survival rates in relation to weather, flight ranges from breeding sites, dispersal related to landscape openness.

Conclusions

Using high resolution data in an interpretable machine-learning modeling framework proved to be an efficient way to enhance the knowledge of the complex links between the environment and the malaria vectors at a local scale. More broadly, the emerging field of interpretable machine-learning has significant potential to help improving our understanding of the complex processes leading to malaria transmission.
The ubiquitin ligase HOIL-1L regulates immune responses by interacting with linear ubiquitin chains
Authors: Gomez-Diaz Carlos , Jonsson Gustav , Schodl Katrin , Deszcz Luiza , Bestehorn Annika , Eislmayr Kevin , Almagro Jorge , Kavirayani Anoop , Fennell Lilian M , Hagelkruys Astrid , Kovarik Pavel , Penninger Josef M , Ikeda Fumiyo
Published: April 2021

The Linear Ubiquitin Assembly Complex (LUBAC), composed of HOIP, HOIL-1L and SHARPIN, promotes Tumor Necrosis Factor (TNF)-dependent NF-κB signaling in diverse cell types. HOIL-1L contains an Npl4 Zinc Finger (NZF) domain that specifically recognizes linear ubiquitin chains, but its physiological rolein vivohas remained unclear. Here, we demonstrate that the HOIL-1L NZF domain has important regulatory functions in inflammation and immune responses in mice. We generated knockin mice (Hoil-1lT20;A;R208A/T201A;R208A) expressing a HOIL-1L NZF mutant, and observed attenuated responses to TNF- and LPS-induced shock, including prolonged survival, stabilized body temperature, reduced cytokine production and liver damage markers. Cells derived from the HOIL-1L knockin mice show reduced TNF-dependent NF-κB activation and incomplete recruitment of HOIL-1L into TNF Receptor (TNFR) Complex I. We further show that the HOIL-1L-NZF domain cooperates with SHARPIN to prevent TNFR-dependent skin inflammation. Collectively, our data suggest that linear ubiquitin-chain binding by HOIL-1L regulates immune responses and inflammationin vivo.
Synthetic biology meets proteomics: Construction of a la carte QconCATs for
absolute protein quantification
Authors: Johnson James R , Harman Victoria M , de Matos Ferraz Franco Catarina , Rockliffe Nicola R , Sun Yaqi , Liu Luning , Takemori Ayako , Takemori Nobuaki , Beynon Robert J
Published: April 2021

We report a new approach to the assembly and construction of QconCATs,
quantitative concatamers for proteomic applications that yield stoichiometric
quantities of sets of stable isotope-labelled internal standards. The new
approach is based on synthetic biology precepts of biobricks, making use of loop
assembly to construct larger entities from individual biobricks. It offers a
major gain in flexibility of QconCAT implementation and enables rapid and
efficient editability that permits, for example, substitution of one peptide for
another. The basic building block (a Qbrick) is a segment of DNA that encodes
two or more quantification peptides for a single protein, readily held in a
repository as a library resource. These Qbricks are then assembled in a one tube
ligation reaction that enforces the order of assembly, to yield short QconCATs
that are useable for small quantification products. However, the DNA context of
the short also allows a second cycle of assembly such that five different short
QconCATs can be assembled into a longer QconCAT in a second, single tube
ligation. From a library of Qbricks, a bespoke QconCAT can be assembled quickly
and efficiently in a form suitable for expression and labelling in vivo or in
vitro. We refer to this approach as the ALACAT strategy as it permits a la carte
design of quantification standards.
Pch2 orchestrates the meiotic recombination checkpoint from the cytoplasm
Authors: Herruzo Esther , Lago-Maciel Ana , Baztán Sara , Santos Beatriz , Carballo Jesús A. , San-Segundo Pedro A.
Published: April 2021

During meiosis, defects in critical events trigger checkpoint activation and restrict cell cycle progression. The budding yeast Pch2 AAA+ ATPase orchestrates the checkpoint response launched by synapsis deficiency; deletion ofPCH2or mutation of the ATPase catalytic sites suppress the meiotic block of thezip1Δ mutant lacking the central region of the synaptonemal complex. Pch2 action enables adequate levels of phosphorylation of the Hop1 axial component at threonine 318, which in turn promotes activation of the Mek1 effector kinase and the ensuing checkpoint response. Inzip1Δ chromosomes, Pch2 is exclusively associated to the rDNA region, but this nucleolar fraction is not required for checkpoint activation, implying that another yet uncharacterized Pch2 population must be responsible for this function. Here, we have artificially redirected Pch2 to different subcellular compartments by adding ectopic NES or NLS sequences or by trapping Pch2 in an immobile extranuclear domain, and we have evaluated the effect on Hop1 chromosomal distribution and checkpoint activity. We have also deciphered the spatial and functional impact of Pch2 regulators including Orc1, Dot1 and Nup2. We conclude that the cytoplasmic pool of Pch2 is sufficient to support the meiotic recombination checkpoint involving the subsequent Hop1-Mek1 activation on chromosomes, whereas the nuclear accumulation of Pch2 has pathological consequences. We propose that cytoplasmic Pch2 provokes a conformational change in Hop1 that poises it for its chromosomal incorporation and phosphorylation. Our discoveries shed light into the intricate regulatory network controlling the accurate balance of Pch2 distribution among different cellular compartments, which is essential for proper meiotic outcomes.
Perineuronal nets affect memory and learning after synapse withdrawal
Authors: Ruzicka Jiri , Dalecka Marketa , Safrankova Kristyna , Peretti Diego , Mallucci Giovanna , Jendelova Pavla , Kwok Jessica CF , Fawcett James W
Published: April 2021

Perineuronal nets (PNNs) enwrap mature neurons, playing a role in the control of plasticity and synapse dynamics. PNNs have been shown to have effects on memory formation, retention and extinction in a variety of animal models. It has been proposed that the cavities in PNNs which contain synapses can act as a memory store, which remains stable after events that cause synaptic withdrawal such as anoxia or hibernation. We examine this idea by monitoring positional memory before and after synaptic withdrawal caused by acute hibernation-like state (HLS). Animals lacking hippocampal PNNs due to enzymatic digestion by chondroitinase ABC or knockout of the PNN component aggrecan were compared with wild type controls. HLS-induced synapse withdrawal caused a memory deficit, but not to the level of naïve animals and not worsened by PNN attenuation. After HLS, animals lacking PNNs showed faster relearning. Absence of PNNs affected the restoration of inhibitory and excitatory synapses on PNN-bearing neurons. The results support a role for hippocampal PNNs in learning, but not in long-term memory storage.
A3D 2.0 update for the prediction and optimization of protein solubility
Authors: Pujols Jordi , Iglesias Valentín , Santos Jaime , Kuriata Aleksander , Kmiecik Sebastian , Ventura Salvador
Published: April 2021

Protein aggregation propensity is a property imprinted in protein sequences and structures, being associated with the onset of human diseases and limiting the implementation of protein-based biotherapies. Computational approaches stand as cost-effective alternatives for reducing protein aggregation and increasing protein solubility. AGGRESCAN 3D (A3D) is a structure-based predictor of aggregation that takes into account the conformational context of a protein, aiming to identify aggregation-prone regions exposed in protein surfaces. Here we inspect the updated 2.0 version of the algorithm, which extends the application of A3D to previously inaccessible proteins and incorporates new modules to assist protein redesign. Among these features, the new server includes stability calculations and the possibility to optimize protein solubility using an experimentally validated computational pipeline. Finally, we employ defined examples to navigate the A3D RESTful service, a routine to handle extensive protein collections. Altogether, this work is conceived to train and assist A3D non-experts in the study of aggregation-prone regions and protein solubility redesign.
Rapid auxin-mediated phosphorylation of Myosin regulates trafficking and polarity in Arabidopsis
Authors: Han Huibin , Verstraeten Inge , Roosjen Mark , Mazur Ewa , Rýdza Nikola , Hajný Jakub , Ötvös Krisztina , Weijers Dolf , Friml Jiří
Published: April 2021

The signaling molecule auxin controls plant development through a well-known transcriptional mechanism that regulates many genes. However, auxin also triggers cellular responses within seconds or minutes, and mechanisms mediating such fast responses have remained elusive. Here, we identified an ultrafast auxin-mediated protein phosphorylation response in Arabidopsis roots that is largely independent of the canonical TIR1/AFB receptors. Among targets of this novel response are Myosin XI and its adaptor protein MadB2. We show that their auxin-mediated phosphorylation regulates trafficking and polar, subcellular distribution of PIN auxin transporters. This phosphorylation-based auxin signaling module is indispensable during developmental processes that rely on auxin-mediated PIN repolarization, such as termination of shoot gravitropic bending or vasculature formation and regeneration. Hence, we identified a fast, non-canonical auxin response targeting multiple cellular processes and revealed auxin-triggered phosphorylation of a myosin complex as the mechanism for feedback regulation of directional auxin transport, a central component of auxin canalization, which underlies self-organizing plant development.