bluuebunny/crossref_metadata_2025_split huggingface dataset:
De novoProtein Sequence Design Based on Deep Learning and Validation on CalB Hydrolase
Authors: Mu Junxi , Li Zhenxin , Zhang Bo , Zhang Qi , Iqbal Jamshed , Wadood Abdul , Wei Ting , Feng Yan , Chen Haifeng 
Published: August 2023

AbstractProtein design is central to nearly all protein engineering problems, as it can enable the creation of proteins with new biological function, such as improving the catalytic efficiency of enzymes. As one of the key tasks of protein design, fixed-backbone protein sequence design aims to design novel sequence that would fold into a given protein backbone structure. However, current sequence design methods have limitations in terms of low sequence diversity and experimental validation of designed protein function, which cannot meet the needs of functional protein design. We firstly constructed Graphormer-based Protein Design (GPD) model that directly applies Transformer to graph-based representation of 3D protein structure, and added Gaussian noise and sequence random mask to node features to improve the sequence recovery and diversity. Additionally, functional filtering based on the structure folding, solubility, and function were performed to improve the success rate in experiments. The process of “sequence design-functional filtering -functional experiment” was carried out for CalB hydrolase. The experimental results showed that the specify activity of designed protein improved 1.7 times than CalB wild type. This design and filtering platform will be a valuable tool for generating industrial enzymes and protein drugs with specific functions.
The potential of young vegetative quinoa (Chenopodium quinoa) as a new sustainable protein-rich winter leafy crop under Mediterranean climate
Authors: Rubinovich Lior , Dagan Reut , Lugasi Yaron , Galili Shmuel , Asher Aviv 
Published: August 2023

AbstractThe demand for protein products has significantly risen in the last few years. In western countries, animals are the primary source of protein; however, plants could take a share of this market due to lower production costs, among other advantages. Quinoa (Chenopodium quinoaWilld.) is a well- known but under-utilized protein-rich crop, commonly cultivated for grain production. These plants were recently evaluated for their use as a non-traditional, green leafy crop. Here we assessed the potential of young vegetative quinoa as a new sustainable winter leafy crop in Israel—serving as a model for Mediterranean semi-arid regions, by evaluating yield, protein content and quality. Five quinoa accessions were sown on three winter sowing dates over two consecutive years. Plants were harvested when they reached 10–13% dry matter (DM). DM yield ranged between 574 and 1,982 kg ha-1and was generally higher in the second year. Protein content ranged from 14.4–34% and was generally higher in the first year. Protein yield ranged from 111–471 kg ha-1and was greatest on the December sowing date. DM and protein yields were positively correlated with plant density. Protein content was negatively correlated with plant density and DM yield. Our findings show that 200 g DM of young vegetative quinoa can meet the protein and most essential amino acid requirements for a 70 kg human adult. Prospects for cultivating young vegetative quinoa in Mediterranean countries as a new sustainable, protein-rich winter leafy crop are therefore high, as supported by its high protein yields and quality, and its requirement for only scant irrigation. Further studies should examine economic and other agrotechnical parameters toward the geographical distribution and expansion of young vegetative quinoa cultivation.
Improvement ofNeisseria gonorrhoeaeculture media to enable growth without CO2
Authors: Menkiti Chukwuma Jude , Snyder Lori A. S. 
Published: August 2023

AbstractBacterial culture is the crucial step in diagnosingNeisseria gonorrhoeaeinfections and is the gold standard for determining their antimicrobial resistance profile. However, culture ofNeisseriaspp. can be challenging in resource poor areas, relying on specialist incubators supplying 5% CO2for growth of the bacteria. Even when such incubators are available, the CO2to run them may be scarce; there were CO2shortages during the COVID-19 pandemic, for example. Although culture jars with gas packs or candles can be used, these are inefficient in terms of use of incubator space and researcher time. To achieve simplicity in culturing ofN. gonorrhoeae, the standard Oxoid GC base medium, made with the Kellogg’s glucose and iron supplements was improved with the addition of 0.75 g/L sodium bicarbonate (NaHCO3), which is inexpensive and really available. This improved media was able to sustain gonococcal growth as well as standard GC media in 5% CO2. Chocolate agar and Thayer-Martin agar with sodium bicarbonate was also developed, with all showing good growth ofN. gonorrhoeaewithout the need for atmospheric CO2.
Charge-Patterned Disordered Peptides Tune Intracellular Phase Separation in Bacteria
Authors: Liao Jane , Yeong Vivian , Obermeyer Allie C. 
Published: August 2023

Subcellular phase separated compartments, known as biomolecular condensates, play an important role in the spatiotemporal organization of cells. To understand the sequence-determinants of phase separation in bacteria, we engineered protein-based condensates inEscherichia coliby utilizing electrostatic interactions as the main driving force. Minimal cationic disordered peptides were used to supercharge negative, neutral, and positive globular model proteins, enabling their phase separation with anionic biomacromolecules in the cell. The phase behavior was governed by the interaction strength between the cationic proteins and anionic biopolymers in addition to the protein concentration. The interaction strength primarily depended on the overall net charge of the protein, but the distribution of charge between the globular and disordered domains also had an impact. Notably, the protein charge distribution between domains could tune mesoscale attributes such as the size, number, and subcellular localization of condensates withinE. colicells. The length and charge density of the disordered peptides had significant effects on protein expression levels, ultimately influencing the formation of condensates. Taken together, charge-patterned disordered peptides provide a platform for understanding the molecular grammar underlying phase separation in bacteria.HighlightsMinimal disordered cationic peptides of varying charge densities can promote protein phase separation in bacterial cells.Protein net charge and charge-patterning are distinct determinants of phase behavior.Protein charge distribution can be used to tune the size, number, position, and reversibility of condensates.Multiple proteins can be selectively recruited to synthetic condensates with disordered cationic peptides.
A patchwork pathway of apparently recent origin enables degradation of the synthetic buffer compound TRIS in bacteria
Authors: Holert Johannes , Borker Aron , Nübel Laura , Daniel Rolf , Poehlein Anja , Philipp Bodo 
Published: August 2023

AbstractThe widely used synthetic buffer compound TRIS was long considered to be biologically inert. Herein, we describe the discovery of a complete bacterial degradation pathway for TRIS. By serendipity, aPseudomonasstrain was isolated from sewage sludge that was able to grow with TRIS as only carbon and nitrogen source. Genome and transcriptome analyses revealed two adjacent gene clusters embedded in a mobile genetic element on a conjugative plasmid to be involved in TRIS degradation. Conjugational transfer of this plasmid intoP. putidaKT2440 enabled this strain to grow with TRIS, demonstrating that the complete TRIS degradation pathway can be transmitted by horizontal gene transfer. Heterologous gene expression revealed cluster I to encode a TRIS uptake protein, a TRIS alcohol dehydrogenase, and a TRIS aldehyde dehydrogenase, catalyzing the oxidation of TRIS into 2-hydroxymethylserine. Gene cluster II encodes a methylserine hydroxymethyltransferase and a D-serine dehydratase which plausibly catalyze the conversion of 2-hydroxymethylserine into pyruvate. Subsequent enrichments from wastewater purification systems led to the isolation of further TRIS-degrading bacteria from thePseudomonasandShinellagenera carrying highly similar TRIS degradation gene clusters.Our data indicate that TRIS degradation evolved recently via gene recruitment and enzyme adaptation from multiple independent metabolic pathways and database searches suggest that the TRIS degradation pathway is now globally distributed. Our study illustrates how engineered environments can enhance the emergence of new microbial metabolic pathways in short evolutionary time scales. This knowledge is valuable for developing new water purification processes in times of increasing water scarcity.
A feedback control mechanism governs the synthesis of lipid-linked precursors of the bacterial cell wall
Authors: Marmont Lindsey S. , Orta Anna K. , Corey Robin A. , Sychantha David , Galliano Ana Fernández , Li Yancheng E. , Baileeves Becca W.A. , Greene Neil G. , Stansfeld Phillip J. , Clemons William M. , Bernhardt Thomas G. 
Published: August 2023

SUMMARYMany bacterial surface glycans such as the peptidoglycan (PG) cell wall, O-antigens, and capsules are built from monomeric units linked to a polyprenyl lipid carrier. How this limiting lipid carrier is effectively distributed among competing pathways has remained unclear for some time. Here, we describe the isolation and characterization of hyperactive variants ofPseudomonas aeruginosaMraY, the essential and conserved enzyme catalyzing the formation of the first lipid-linked PG precursor called lipid I. These variants result in the elevated production of the final PG precursor lipid II in cells and are hyperactive in a purified system. Amino acid substitutions within the activated MraY variants unexpectedly map to a cavity on the extracellular side of the dimer interface, far from the active site. Our structural evidence and molecular dynamics simulations suggest that the cavity is a binding site for lipid II molecules that have been transported to the outer leaflet of the membrane. Overall, our results support a model in which excess externalized lipid II allosterically inhibits MraY, providing a feedback mechanism to prevent the sequestration of lipid carrier in the PG biogenesis pathway. MraY belongs to the broadly distributed polyprenyl-phosphate N-acetylhexosamine 1-phosphate transferase (PNPT) superfamily of enzymes. We therefore propose that similar feedback mechanisms may be widely employed to coordinate precursor supply with demand by polymerases, thereby optimizing the partitioning of lipid carriers between competing glycan biogenesis pathways.
Major urinary protein (Mup) gene family deletion drives sex-specific alterations on the house mouse gut microbiota
Authors: Real Madalena V. F. , Colvin Melanie S. , Sheehan Michael J. , Moeller Andrew H. 
Published: August 2023

AbstractThe gut microbiota is shaped by host metabolism. In house mice (Mus musculus), major urinary protein (MUP) pheromone production represents a considerable energy investment, particularly in sexually mature males. Deletion of theMupgene family shifts mouse metabolism towards an anabolic state, marked by lipogenesis, lipid accumulation, and body mass increases. Given the metabolic implications of MUPs, they may also influence the gut microbiota. Here, we investigated the effect of deletion of theMupgene family on the gut microbiota of sexually mature mice. Shotgun metagenomics revealed distinct taxonomic and functional profiles between wildtype and knockout males, but not females. Deletion of theMupgene cluster significantly reduced diversity in microbial families and functions in male mice. Additionally, specific taxa of the Ruminococcaceae family, which is associated with gut health and reduced risk of developing metabolic syndrome, and several microbial functions, such as transporters involved in vitamin B5 acquisition, were significantly depleted in the microbiota ofMup-knockout males. Altogether these results show that major urinary proteins significantly affect the gut microbiota of house mouse in a sex-specific manner.ImportanceThe community of microorganisms that inhabit the gastrointestinal track of animals, known as the gut microbiota, can have profound effects on host phenotypes. The gut microbiota is in turn shaped by host genes, including those involved with host metabolism. In adult male house mice, expression of the major urinary protein (Mup) gene cluster represents a substantial energy investment, and deletion ofMupgene family leads to fat accumulation and weight gain in males. We show for the first time that deletingMupgenes also alters the gut microbiota of male, but not female, mice in terms of both taxonomic and functional composition. Male mice withoutMupgenes harbored fewer gut bacterial families and reduced abundances of several species, including bacteria previously shown to reduce obesity risk. Studying the impact of theMupgenes on the gut microbiota will help us understand how these genes influence host phenotype more broadly.
In vivocharacterization of a secologanin transporter fromCatharanthus roseus
Authors: Li Fanfan , Shahsavarani Mohammadamin , Handy-Hart Cody-Jordan , Montgomery Victoria , Beech Robin N. , Liu Lan , Bayen Stéphane , Qu Yang , De Luca Vincenzo , Dastmalchi Mehran 
Published: August 2023

SUMMARYMonoterpenoid indole alkaloid (MIA) biosynthesis inCatharanthus roseusis a paragon of the spatiotemporal complexity achievable by plant specialized metabolism. Spanning a range of tissues, four cell types, and five cellular organelles, MIA metabolism is intricately regulated and organized. This high degree of metabolic differentiation requires inter-cellular and organellar transport, which remains understudied. Here, we have fully characterized a vacuolar importer of secologanin belonging to the multidrug and toxic compound extrusion (MATE) family, named CrMATE1/SLTr. Phylogenetic analyses of MATEs suggested a role in alkaloid transport for CrMATE1, andin plantasilencing in two varieties ofC. roseusresulted in a shift in the secoiridoid and MIA profiles. Subcellular localization of CrMATE1 confirmed tonoplast localization. A full panel ofin vivobiochemical characterization using theXenopus laevisoocyte expression system was used to determine substrate range, directionality, and rate. We can confirm that CrMATE1 is a vacuolar importer of secologanin, rapidly transporting 1 mM of secologanin within 25 min. Notably, the absence of CrMATE1 leads to a transport bottleneck, resulting in the conversion of secologanin to its reduced form, secologanol, bothin plantaand in theX. laevissystem. The unique substrate-specific activity of CrMATE1 showcases the utility of transporters as gatekeepers of metabolic flux, mediating the balance between anti-herbivory potency and cell homeostasisin planta. MIA and secoiridoid transporters could also be deployed in heterologous systems to guide biosynthetic pathways and improve titers of valuable and life-saving MIAs.SIGNIFICANCEWe have fully characterized CrMATE1, a multidrug and toxic compound extrusion (MATE) family transporter inCatharanthus roseus,as a vacuolar importer of secologanin. The translocation of secologanin into the vacuole is necessary for the first committed step of monoterpenoid indole alkaloid (MIA) biosynthesis.
ZmPILS6 is an auxin efflux carrier required for maize root morphogenesis
Authors: Cowling Craig L. , Homayouni Arielle L. , Callwood Jodi B. , McReynolds Maxwell R. , Khor Jasper , Ke Haiyan , Draves Melissa A. , Dehesh Katayoon , Walley Justin W. , Strader Lucia C. , Kelley Dior R. 
Published: August 2023

AbstractPlant root systems play a pivotal role in plant physiology and exhibit diverse phenotypic traits. Understanding the genetic mechanisms governing root growth and development in model plants like maize is crucial for enhancing crop resilience to drought and nutrient limitations. This study focused on identifying and characterizing ZmPILS6, an annotated auxin efflux carrier, as a key regulator of various crown root traits in maize. ZmPILS6-modified roots displayed reduced network area and suppressed lateral root formation, desirable traits during drought and low phosphate conditions. The research revealed that ZmPILS6 localizes to the endoplasmic reticulum and plays a vital role in controlling the spatial distribution of indole-3-acetic acid (IAA or “auxin”) in primary roots. The study also demonstrated that ZmPILS6 can actively efflux IAA when expressed in yeast. Furthermore, the loss of ZmPILS6 resulted in significant proteome remodeling in maize roots, particularly affecting hormone signaling pathways. To identify potential interacting partners of ZmPILS6, a weighted gene co-expression analysis (WGNA) was performed. Altogether, this research contributes to the growing knowledge of essential genetic determinants governing maize root morphogenesis, which is crucial for guiding agricultural improvement strategies.Significance StatementCrop yield and stress resilience are significantly influenced by crown root architecture. A reverse genetic screen aimed at identifying novel regulators of maize root morphogenesis led to the discovery of ZmPILS6, an auxin efflux carrier. The loss of ZmPILS6 negatively impacts numerous root traits that are linked to plant physiology and function. Proteomic characterization ofpils6-1roots revealed that this evolutionarily conserved transporter is required for the proper expression of numerous phytohormone pathways, including abscisic acid, gibberellins, and jasmonic acid. Notably, ZmPILS6 appears to have a contrasting role in regulating root morphogenesis compared to its Arabidopsis ortholog, PILS6. This finding emphasizes the need for functional characterization of candidate genes directly within key crops of interest, which cannot always be correctly inferred from other model plants.
Flexibility and distributive synthesis regulate RNA priming and handoff in human DNA polymerase α-primase
Authors: Cordoba John J. , Mullins Elwood A. , Salay Lauren E. , Eichman Brandt F. , Chazin Walter J. 
Published: August 2023

AbstractDNA replication in eukaryotes relies on the synthesis of a ∼30-nucleotide RNA/DNA primer strand through the dual action of the heterotetrameric polymerase α-primase (pol-prim) enzyme. Synthesis of the 7-10-nucleotide RNA primer is regulated by the C-terminal domain of the primase regulatory subunit (PRIM2C) and is followed by intramolecular handoff of the primer to pol α for extension by ∼20 nucleotides of DNA. Here we provide evidence that RNA primer synthesis is governed by a combination of the high affinity and flexible linkage of the PRIM2C domain and the low affinity of the primase catalytic domain (PRIM1) for substrate. Using a combination of small angle X-ray scattering and electron microscopy, we found significant variability in the organization of PRIM2C and PRIM1 in the absence and presence of substrate, and that the population of structures with both PRIM2C and PRIM1 in a configuration aligned for synthesis is low. Crosslinking was used to visualize the orientation of PRIM2C and PRIM1 when engaged by substrate as observed by electron microscopy. Microscale thermophoresis was used to measure substrate affinities for a series of pol-prim constructs, which showed that the PRIM1 catalytic domain does not bind the template or emergent RNA-primed templates with appreciable affinity. Together, these findings support a model of RNA primer synthesis in which generation of the nascent RNA strand and handoff of the RNA-primed template from primase to polymerase α is mediated by the high degree of inter-domain flexibility of pol-prim, the ready dissociation of PRIM1 from its substrate, and the much higher affinity of the POLA1cat domain of polymerase α for full-length RNA-primed templates.
Protein Ensemble Generation through Variational Autoencoder Latent Space Sampling
Authors: Mansoor Sanaa , Baek Minkyung , Park Hahnbeom , Lee Gyu Rie , Baker David 
Published: August 2023

AbstractMapping the ensemble of protein conformations that contribute to function and can be targeted by small molecule drugs remains an outstanding challenge. Here we explore the use of soft-introspective variational autoencoders for reducing the challenge of dimensionality in the protein structure ensemble generation problem. We convert high-dimensional protein structural data into a continuous, low-dimensional representation, carry out search in this space guided by a structure quality metric, then use RoseTTAFold to generate 3D structures. We use this approach to generate ensembles for the cancer relevant protein K-Ras, training the VAE on a subset of the available K-Ras crystal structures and MD simulation snapshots, and assessing the extent of sampling close to crystal structures withheld from training. We find that our latent space sampling procedure rapidly generates ensembles with high structural quality and is able to sample within 1 angstrom of held out crystal structures, with a consistency higher than MD simulation or AlphaFold2 prediction. The sampled structures sufficiently recapitulate the cryptic pockets in the held-out K-Ras structures to allow for small molecule docking.