bluuebunny/crossref_metadata_2025 huggingface dataset:
Cell-free mutant analysis combined with structure prediction of a lasso peptide biosynthetic protein B2
Authors: Alfi Almasul , Popov Aleksandr , Kumar Ashutosh , Zhang Kam Y. J. , Dubiley Svetlana , Severinov Konstantin , Tagami Shunsuke
Published: April 2022

Biochemical and structural analyses of purified proteins are essential for the understanding of their properties. However, many proteins are unstable and difficult to purify, hindering their characterization. The B2 proteins of the lasso peptide biosynthetic pathways are cysteine proteases that cleave precursor peptides during the maturation process. The B2 proteins are poorly soluble and no experimentally-solved structures are available. Here, we performed a rabid semi-comprehensive mutational analysis of the B2 protein from the thermophilic actinobacterium, Thermobifida fusca (TfuB2) using a cell-free transcription/translation system, and compared the results with the structure prediction by AlphaFold2. Analysis of 34 TfuB2 mutants with substitutions of hydrophobic residues confirmed the accuracy of the predicted structure, and revealed a hydrophobic patch on the protein surface, which likely serves as the binding site of the partner protein, TfuB1. Our results suggest that the combination of rapid cell-free mutant analyses with precise structure predictions can greatly accelerate structure-function research of proteins for which no structures are available.
The purified recombinant FAT domain of the Focal Adhesion Kinase does not bind directly to recombinant Talin or MBD2 in vitro
Authors: Naser Rayan , Momin Afaque A. , Ali Amal J. , Merzaban Jasmeen , Arold Stefan T.
Published: April 2022

Controlled localization and activation of the focal adhesion kinase (FAK) functionally links adhesion, migration and survival of the cell. The C-terminal focal adhesion targeting (FAT) domain of FAK is an important regulator of the localization, activation and molecular associations of FAK. Here, we aimed to investigate the structural basis for how FAK FAT binds to Talin and MBD2, which were previously reported to be cytoplasmic and nuclear ligands, respectively. Using several biophysical methods with purified recombinantly expressed protein constructs, we failed to observe measurable interactions between FAT and either the Talin FERM domain or MBD2. We conclude that the association of FAT with these proteins requires additional factors or post-translational modifications not present in bacterially produced purified proteins.
Bi-phasic regulation of AIMP2 and its splice variant in PARP-1-dependent neurodegeneration
Authors: Lee Min Hak , Byun Mi Ran , Lee Seok Won , Lee Eui Jin , Jo Young Ok , Kim Sung Hyun , Seol Wongi , Baek Kyunghwa , Choi Jin Woo
Published: April 2022

Parthanatos is a significant molecular cause of Parkinson’s disease, in which AIMP2 aberrantly activates PARP-1 through a physical interaction. Interestingly, AIMP2 has an antagonistic splice variant, named DX2, which compromises AIMP2-induced apoptosis via p53 or inflammatory pathway. Here we suggested that DX2 binds to PARP-1 with a higher affinity than AIMP2, deactivating it and improving synaptic physiology. To deliver DX2 into deep brain areas, miR142 target sequence-embedded adeno-associated virus was designed to avoid unexpected expression in hematopoietic cells. RNAseq analysis revealed that DX2 selectively suppressed cell death-associated pathways, such as p53 and neuroinflammation. Upon a single intracranial injection, both behaviour and motility were mitigated in three animal models of Parkinsonism, induced by MPTP, rotenone, or 6-OHDA. Efficacy was observed in therapeutic model as well as preventive ones. Thus, AIMP2 and DX2 are suggested to act as an ‘ON/OFF’ switch for PARP-1. In particular, as cell survival properties of DX2 was exerted only when AIMP2 is accumulated abnormally, without its own additional tumorigenicity, DX2 could be a unique therapeutic tool for treating patients with Parkinson’s disease.
Prediction of quality-control degradation signals in yeast proteins
Authors: Johansson Kristoffer E. , Mashahreh Bayan , Hartmann-Petersen Rasmus , Ravid Tommer , Lindorff-Larsen Kresten
Published: April 2022

Effective proteome homeostasis is key to cellular and organismal survival, and cells therefore contain efficient quality control systems to monitor and remove potentially toxic misfolded proteins. Such general protein quality control to a large extent relies on the efficient and robust delivery of misfolded or unfolded proteins to the ubiquitin-proteasome system. This is achieved via recognition of so-called degradation motifs—degrons—that are assumed to become exposed as a result of protein misfolding. Despite their importance, the nature and sequence properties of quality-control degrons remain elusive. Here, we have used data from a yeast-based screen of 23,600 17-residue peptides to build a predictor of quality-control degrons. The resulting model, QCDPred (Quality Control Degron Prediction), achieves good accuracy using only the sequence composition of the peptides as input. Our analysis reveals that strong degrons are enriched in hydrophobic amino acids and depleted in negatively charged amino acids, in line with the expectation that they are buried in natively folded proteins. We applied QCDPred to the yeast proteome, enabling us to analyse more widely the potential effects of degrons. As an example, we show a correlation between cellular abundance and degron potential in disordered regions of proteins. Together with recent results on membrane proteins, our work suggest that the recognition of exposed hydrophobic residues is a key and generic mechanism for proteome homeostasis. QCDPred is freely available as open source code and via a web interface.
Using Culture-Enriched Phenotypic Metagenomics for Targeted High-Throughput Monitoring of Clinically-Important Fraction of Beta-Lactam Resistome
Authors: Zhang Zhiguo , Zhang Guoqing , Ju Feng
Published: April 2022

The high bacterial community complexity greatly hinders the monitoring of clinically prevalent extended-spectrum beta-lactam resistant bacteria, which are usually present as rare but key populations involving in the environmental dissemination of clinical resistance. Here, we introduce culture-enriched phenotypic metagenomics that integrates culture enrichment, phenotypic screening and metagenomic analyses as an emerging methodology to profile beta-lactam resistome in a municipal wastewater treatment plant (WWTP) and its receiving river. The results showed that clinically prevalent carbapenemase genes (e.g., NDM and KPC family) and extended-spectrum beta-lactamase genes (e.g., CTX-M, TEM and OXA family) considerably existed in the WWTP and showed prominent potential in horizontal dissemination. Strikingly, carbapenem and polymyxin resistance genes co-occurred in highly virulent Enterobacter kobei (MCR-1 and NDM-6) and Citrobacter freundii (ArnA and NDM-16) genomes. Overall, this study exemplifies phenotypic metagenomics for high-throughput surveillance of targeted fraction of clinically-important resistome and substantially expands current knowledge on extended-spectrum beta-lactam resistance in WWTPs.
Detecting macroevolutionary genotype-phenotype associations using error-corrected rates of protein convergence
Authors: Fukushima Kenji , Pollock David D.
Published: April 2022

On macroevolutionary timescales, extensive mutations and phylogenetic uncertainty mask the signals of genotype-phenotype associations underlying convergent evolution. To overcome this problem, we extended the widely used framework of nonsynonymous-to-synonymous substitution rate ratios and developed the novel metricωC, which measures the error-corrected convergence rate of protein evolution. WhileωCdistinguishes natural selection from genetic noise and phylogenetic errors in simulation and real examples, its accuracy allows an exploratory genome-wide search of adaptive molecular convergence without phenotypic hypothesis or candidate genes. Using gene expression data, we explored over 20 million branch combinations in vertebrate genes and identified the joint convergence of expression patterns and protein sequences with amino acid substitutions in functionally important sites, providing hypotheses on undiscovered phenotypes. We further extended our method with a heuristic algorithm to detect highly repetitive convergence among computationally nontrivial higher-order phylogenetic combinations. Our approach allows bidirectional searches for genotype-phenotype associations, even in lineages that diverged for hundreds of millions of years.
Agricultural margins could enhance landscape connectivity for pollinating insects across the Central Valley of California, U.S.A
Authors: Dilts Thomas E. , Black Scott , Hoyle Sarah , Jepsen Sarina , May Emily , Forister Matthew L.
Published: April 2022

One of the defining features of the Anthropocene is eroding ecosystem services as a function of decreases in biodiversity and overall reductions in the abundance of once-common organisms, including many insects that play innumerable roles in natural communities and agricultural systems that support human society. It is now clear that the preservation of insects cannot rely solely on the legal protection of natural areas far removed from the densest areas of human habitation. Instead, a critical challenge moving forward is to intelligently manage areas that include intensively farmed landscapes, such as the Central Valley of California. Here we attempt to meet this challenge with a tool for modeling landscape connectivity for insects (with pollinators in particular in mind) that builds on available information including lethality of pesticides and expert opinion on insect movement. Despite the massive fragmentation of the Central Valley, we find that connectivity is possible, especially utilizing the restoration or improvement of agricultural margins which (in their summed area) exceed natural areas. Finally, we highlight steps moving forward and the great many knowledge gaps that could be addressed in the field to improve future iterations of our modeling approach.
Novel Antibody Interfaces Revealed Through Structural Mining
Authors: Yin Yizhou , Romei Matthew , Sankar Kannan , Pal Lipika R. , Hoi Kam Hon , Yang Yanli , Leonard Brandon , De Leon Boenig Gladys , Kumar Nikit , Matsumoto Marissa , Payandeh Jian , Harris Seth F. , Moult John , Lazar Greg A.
Published: April 2022

Antibodies are fundamental effectors of humoral immunity, and have become a highly successful class of therapeutics. There is increasing evidence that antibodies utilize transient homotypic interactions to enhance function, and elucidation of such interactions can provide insights into their biology and new opportunities for their optimization as drugs. Yet the transitory nature of weak interactions makes them difficult to investigate. Capitalizing on their rich structural data and high conservation, we have characterized all the ways that antibody Fab regions interact crystallographically. This approach led to the discovery of previously unrealized interfaces between antibodies. While diverse interactions exist, β-sheet dimers and variable-constant elbow dimers are recurrent motifs. Disulfide engineering enabled interactions to be trapped and investigated structurally and functionally, providing experimental validation of the interfaces and illustrating their potential for optimization. This work provides first insight into previously undiscovered oligomeric interactions between antibodies, and enables new opportunities for their biotherapeutic optimization.
Exploring the effect of mechanical anisotropy of protein structures in the unfoldase mechanism of AAA+ molecular machines
Authors: Varikoti Rohith Anand , Fonseka Hewafonsekage Yasan Y. , Kelly Maria S. , Javidi Alex , Damre Mangesh , Mullen Sarah , Nugent Jimmie L. , Gonzales Christopher M. , Stan George , Dima Ruxandra I.
Published: April 2022

Essential cellular processes of microtubule disassembly and protein degradation, which span lengths from tens of μm to nm, are mediated by specialized molecular machines with similar hexameric structure and function. Our molecular simulations at atomistic and coarse-grained scales show that both the microtubule severing protein spastin and the caseinolytic protease ClpY, accomplish spectacular unfolding of their diverse substrates, a microtubule lattice and dihydrofolate reductase (DHFR), by taking advantage of mechanical anisotropy in these proteins. By considering wild-type and variants of DHFR, we found that optimal ClpY-mediated action probes favorable orientations of the substrate relative to the machine. Unfolding of wild-type DHFR involves strong mechanical interfaces near each terminal and occurs along branched pathways, whereas unfolding of DHFR variants involves softer mechanical interfaces and occurs through single pathways, but translocation hindrance can arise from internal mechanical resistance. For spastin, optimum severing action initiated by pulling on a tubulin subunit is achieved through the orientation of the machine versus the substrate (microtubule lattice). Moreover, changes in the strength of the interactions between spastin and a microtubule filament, which can be driven by the tubulin code, lead to drastically different outcomes for the integrity of the hexameric structure of the machine.
Mechanical morphotype switching as an adaptive response in mycobacteria
Authors: Eskandarian Haig Alexander , Chen Yu-Xiang , Toniolo Chiara , Belardinelli Juan M. , Palcekova Zuzana , Ashby Paul , Fantner Georg E. , Jackson Mary , McKinney John D. , Javid Babak
Published: April 2022

Invading microbes face a myriad of cidal mechanisms of phagocytes that inflict physical damage to microbial structures. How intracellular bacterial pathogens adapt to these stresses is not fully understood. Here, we report a new virulence mechanism by which mycobacteria alter the mechanical stiffness of their cell surface to become refractory to killing during infection. Long-Term Time-Lapse Atomic Force Microscopy was used to reveal a process of “mechanical morphotype switching” in mycobacteria exposed to host intracellular stress. A “soft” mechanical morphotype switch enhances tolerance to intracellular macrophage stress, including cathelicidin. Genetic manipulation, by deletion of uvrA, or pharmacological treatment, with bedaquiline, locked mycobacteria into a “soft” mechanical morphotype state, enhancing survival in macrophages. Our study proposes microbial mechanical adaptation as a new axis for surviving host-mediated stressors.

One-Sentence Summary

Bacteria alter their cell surface mechanical properties to increase survival during macrophage infection.
Characterizing a New Fluorescent Protein for Low Limit of Detection Sensing in the Cell-Free System
Authors: Copeland Caroline E. , Kim Jeehye , Copeland Pearce L. , Heitmeier Chloe J. , Kwon Yong-Chan
Published: April 2022

Cell-free protein synthesis-based biosensors have been developed as highly accurate, low- cost biosensors. However, since most biomarkers exist at low concentrations in various types of biopsies, the biosensor’s dynamic range must be increased in the system to achieve the low limits of detection necessary while deciphering from higher background signals. Many attempts to increase the dynamic range have relied on amplifying the input signal from the analyte, which can lead to complications of false positives. In this study, we aimed to increase the protein synthesis capability of the cell-free protein synthesis system and the output signal of the reporter protein to achieve a lower limit of detection. We utilized a new fluorescent protein - mNeonGreen, which produces a higher output than those commonly used in cell-free biosensors. Optimizations of DNA sequence and the subsequent cell-free protein synthesis reaction conditions allowed characterizing protein expression variability by given DNA template types, reaction environment, and storage additives that cause the greatest time constraint on designing the cell-free biosensor. Finally, we characterized the fluorescence kinetics of mNeonGreen compared to the commonly used reporter protein, superfolder Green Fluorescent Protein. We expect that this finely tuned cell-free protein synthesis platform with the new reporter protein can be used with sophisticated synthetic gene circuitry networks to increase the dynamic range of a cell-free biosensor to reach lower detection limits and reduce false positives proportion.
A Lipid Atlas of the Human Kidney
Authors: Farrow Melissa A. , Tideman Léonore E.M. , Neumann Elizabeth K. , Patterson Nathan Heath , Migas Lukasz G. , Colley Madeline E. , Allen Jamie L. , Rivera Emilio S. , Romer Carrie E. , Yang Haichun , Brewer Maya , Pingry Ellie , Dufresne Martin , Djambazova Katerina , Sharman Kavya , Kruse Angela R.S. , Gutierrez Danielle B. , Harris Raymond C. , Fogo Agnes B. , de Caestecker Mark P. , Caprioli Richard M. , Van de Plas Raf , Spraggins Jeffrey M.
Published: April 2022

Tissue atlases provide foundational knowledge on the cellular organization and molecular distributions across molecular classes and spatial scales. Here, we construct a comprehensive spatio-molecular lipid atlas of the human kidney from 29 donor tissues using integrated multimodal molecular imaging. Our approach leverages high spatial resolution matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) for untargeted lipid mapping, stained microscopy for histopathological assessment, and tissue segmentation using autofluorescence microscopy. With a combination of unsupervised, supervised, and interpretive machine learning, the atlas provides multivariate lipid profiles of specific multicellular functional tissue units (FTUs) of the nephron, including the glomerulus, proximal tubules, thick ascending limb, distal tubules, and collecting ducts. In total, the atlas consists of tens of thousands of FTUs and millions of mass spectrometry measurements. Detailed patient, clinical, and histopathologic information allowed molecular data to be mined based on these features. As examples, we highlight the discovery of how lipid profiles are altered with sex and differences in body mass index.
Neuronopathic GBA1 L444P mutation accelerates Glucosylsphingosine levels and formation of hippocampal alpha-synuclein inclusions
Authors: Mahoney-Crane Casey L. , Viswanathan Megha , Russell Dreson , Curtiss Rachel A.C. , Freire Jennifer , Bobba Sai Sumedha , Coyle Sean D. , Kandebo Monika , Yao Lihang , Wan Bang-Lin , Hatcher Nathan G. , Smith Sean M. , Marcus Jacob N. , Volpicelli-Daley Laura A.
Published: April 2022

The most common genetic risk factor for Parkinson’s disease (PD) is heterozygous mutations in the GBA1 gene which encodes for the lysosomal enzyme, glucocerebrosidase (GCase). GCase impairments are associated with an accumulation of abnormal α-synuclein (α-syn) called Lewy pathology, which characterizes PD. PD patients heterozygous for the GBA1 L444P mutation (GBA1+/L444P) have a 5.6-fold increased risk of cognitive impairments. In this study, we used GBA1+/L444Pmice to determine the effects of this severe GBA1 mutation on lipid metabolism, expression of synaptic proteins, behavior, and α-syn inclusion formation. GBA1+/L444Pmice showed reduced GCase activity in limbic brain regions and expressed lower levels of hippocampal vGLUT1 compared to wildtype (GBA1+/+) mice. GBA+/L444Pmice also demonstrated impaired fear conditioning, but no motor deficits. We show, using mass spectrometry, that mutant GCase and age increased levels of glucosylsphingosine (GlcSph), but not glucosylceramide (GlcCer), in the brains and serum of GBA1+/L444Pmice. Aged GBA1+/+mice also showed increased levels of GlcSph, and decreased GlcCer. To model disease pathology, templated α-syn pathology was used. α-Syn inclusions were increased in the hippocampus of GBA1+/L444Pmice compared to GBA1+/+mice, but not in the cortex, or substantia nigra pars compacta (SNc). Pathologic α-syn did not cause a loss of dopamine neurons in the SNc. Treatment with a GlcCer synthase inhibitor prevented loss of cortical α-syn inclusions, but not loss of dopamine neurons. Overall, these data suggest the critical importance to evaluate the contribution of hippocampal pathologic α-syn and brain and serum glucosylsphingosine in synucleinopathies.

SIGNIFICANCE STATEMENT

Synucleinopathies, such as Parkinson’s disease (PD) and Dementia with Lewy bodies (DLB), are both pathologically characterized by abnormal α-synuclein (α-syn). Mutant GBA1 is a risk factor for both PD and DLB where a reduction of glucocerebrosidase (GCase) activity is seen. Collectively, this indicates the significance of evaluating mutant GCase in synucleinopathies. Our data suggest the critical importance to evaluate the contribution of hippocampal pathologic α-syn and brain and serum glucosylsphingosine (GlcSph) accumulation in synucleinopathies. Moreover, these pathologic outcomes may contribute to the nonmotor symptoms clinically observed in PD and DLB. Our findings highlight the importance of GlcSph as a relevant biomarker for future therapeutics.
Antibiotic persistence of Brucella abortus in its protective intracellular niche
Authors: Mode Selma , Ketterer Maren , Québatte Maxime , Dehio Christoph
Published: April 2022

Background

Human Brucellosis caused by the facultative intracellular pathogen Brucella spp. is an endemic bacterial zoonosis manifesting as acute or chronic infections associated with high morbidity. Treatment typically involves a combination therapy of two antibiotics administered for several weeks to month, but despite this harsh treatment relapses occur at a rate of 5-15%. Although poor compliance and reinfection may account for a fraction of the observed relapse cases, it is apparent that the properties of the infectious agent itself may play a decisive role in this phenomenon.

Methodology/Principal findings

We used B. abortus carrying a dual reporter in a macrophage infection model to gain a better understanding of the efficacy of recommended therapies in cellulo. For this we used automated fluorescent microscopy as a prime read-out and developed specific CellProfiler pipelines to score infected macrophages at the population and the single cell level. Combining microscopy of constitutive and induced reporters with classical CFU determination, we quantified the protective nature of the Brucella intracellular niche to various antibiotics and the ability of B. abortus to persist in cellulo despite harsh antibiotic treatments.

Conclusion/Significance

We demonstrate that treatment of infected macrophages with antibiotics at recommended concentrations fails to fully prevent growth and persistence of B. abortus in cellulo, which may be explained by a protective nature of the intracellular niche. Moreover, we show the presence of bona fide intracellular persisters upon antibiotic treatment, which are metabolically active and retain the full infectious potential, therefore constituting a plausible reservoir for reinfection and relapse. In conclusion, our results highlight the need to extend the spectrum of models to test new antimicrobial therapies for Brucellosis to better reflect the in vivo infection environment, and to develop therapeutic approaches targeting the persister subpopulation.

Authors Summary

Brucellosis is a zoonosis endemic to many low- and middle-income countries around the world. Recommended therapies by the WHO are comprised of at least two antibiotics for several weeks, sometimes month. Despite these harsh treatments relapses are frequent. The underlying reasons for these relapses, besides reinfection and non-compliance to treatment, are unknown. Our study shows that Brucella abortus can form so called “persisters” under recommended treatments in rich broth but also inside macrophages. This small bacterial subpopulation survives antibiotic treatment and resumes growth after removal of the antibiotics and could therefore serve as a reservoir for relapses in human Brucellosis. Furthermore, we show that the macrophage intracellular niche of Brucella has protective properties against recommended antibiotics as observed for other intracellular pathogens, highlighting the necessity to develop new infection models to assess antibiotic efficacy.
The Statistical Trends of Protein Evolution: A Lesson from AlphaFold Database
Authors: Tang Qian-Yuan , Ren Weitong , Wang Jun , Kaneko Kunihiko
Published: April 2022

The recent development of artificial intelligence provides us with new and powerful tools for studying the mysterious relationship between organism evolution and protein evolution. In this work, based on the AlphaFold Protein Structure Database (AlphaFold DB), we perform comparative analyses of the proteins of different organisms. The statistics of AlphaFold-predicted structures show that, for organisms with higher complexity, their constituent proteins will have larger radii of gyration, higher coil fractions, and slower vibrations, statistically. By conducting normal mode analysis and scaling analyses, we demonstrate that higher organismal complexity correlates with lower fractal dimensions in both the structure and dynamics of the constituent proteins, suggesting that higher functional specialization is associated with higher organismal complexity. We also uncover the topology and sequence bases of these correlations. As the organismal complexity increases, the residue contact networks of the constituent proteins will be more assortative, and these proteins will have a higher degree of hydrophilic-hydrophobic segregation in the sequences. Furthermore, by comparing the statistical structural proximity across the proteomes with the phylogenetic tree of homologous proteins, we show that, statistical structural proximity across the proteomes may indirectly reflect the phylogenetic proximity, indicating a statistical trend of protein evolution in parallel with organism evolution. This study provides new insights into how the diversity in the functionality of proteins increases and how the dimensionality of the manifold of protein dynamics reduces during evolution, contributing to the understanding of the origin and evolution of lives.
Identification of plasma proteins associated with oesophageal cancer chemotherapeutic treatment outcomes using SWATH-MS
Authors: Guo Naici , Minas Giorgos , Synowsky Silvia A. , Dunne Margaret R. , Ahmed Hasnain , McShane Rhiannon , Bhardwaj Anshul , Donlon Noel E. , Lorton Cliona , O’Sullivan Jacintha , Reynolds John V. , Caie Peter D. , Shirran Sally L. , Lynch Andy G. , Stewart Alan J. , Arya Swati
Published: April 2022

Oesophageal adenocarcinoma (OAC) is an aggressive cancer with a five-year survival of <15%. Current chemotherapeutic strategies only benefit a minority (20-30%) of patients and there are no methods available to differentiate between responders and non-responders. We performed quantitative proteomics using Sequential Window Acquisition of all THeoretical fragment-ion spectra-Mass Spectrometry (SWATH-MS) on albumin/IgG-depleted and non-depleted plasma samples from 23 patients with locally advanced OAC prior to treatment. Individuals were grouped based on tumour regression (TRG) score (TRG1/2/3vsTRG4/5) after chemotherapy, and differentially abundant proteins were compared. Protein depletion of highly abundant proteins led to the identification of around twice as many proteins. SWATH-MS revealed significant quantitative differences in the abundance of several proteins between the two groups. These included complement c1q subunit proteins, C1QA, C1QB and C1QC, which were of higher abundance in the low TRG group. Of those that were found to be of higher abundance in the high TRG group, GSTP1 was found to exhibit the lowest p-value and highest classification accuracy and Cohen’s kappa value. Concentrations of these proteins were further examined using ELISA-based assays. This study provides quantitative information relating to differences in the plasma proteome that underpin response to chemotherapeutic treatment in oesophageal cancers.
Chemical conversion of human conventional Pluripotent Stem Cells to Trophoblast Stem Cells
Authors: Zorzan Irene , Betto Riccardo Massimiliano , Rossignoli Giada , Arboit Mattia , Drusin Andrea , Martini Paolo , Martello Graziano
Published: April 2022

In human embryos, naive pluripotent cells of the inner cell mass generate epiblast, primitive endoderm and Trophectoderm (TE) lineage, whence trophoblast cells derive. In vitro, naive pluripotent stem cells (PSCs) retain this potential and can generate trophoblast stem cells (TSCs), while conventional PSCs form amnion-like cells and lack the competence to generate TSCs. Transient histone deacetylase and MEK inhibitions with LIF stimulation can be used to chemically reset conventional to naive PSCs. Here we report that chemical resetting induced expression of both naive and TSC markers and of placental imprinted genes. A modified chemical resetting protocol allowed for the fast and efficient conversion of conventional PSCs into TSCs, entailing shutdown of pluripotency genes and full activation of the trophoblast master regulators, without induction of amnion markers. Chemical resetting generates a responsive intermediate state, in which conventional PSCs rapidly acquire competence to form TSCs without the need of stabilisation and expansion in a naive state. The efficiency and rapidity of our system will be useful for the study of cell fate transitions, and to generate models of placental disorders.
Discovery of a non-canonical prototype long-chain monoacylglycerol lipase through a structure-based endogenous reaction intermediate complex
Authors: Pinotsis Nikos , Krüger Anna , Tomas Nicolas , Chatziefthymiou Spyros D , Litz Claudia , Mortensen Simon Arnold , Daffé Mamadou , Marrakchi Hedia , Antranikian Garabed , Wilmanns Matthias
Published: April 2022

The identification and characterization of enzyme function is largely lacking behind the rapidly increasing availability of large numbers of sequences and associated high-resolution structures. This is often hampered by lack of knowledge onin vivorelevant substrates. Here, we present a case study of a high-resolution structure of an unusual orphan lipase in complex with an endogenous C18 monoacylglycerol ester reaction intermediate from the expression host, which is insoluble under aqueous conditions and thus not accessible for studies in solution. The data allowed its functional characterization as a prototypic long-chain monoacylglycerol lipase, which uses a minimal lid domain to position the substrate through a hydrophobic tunnel directly to the enzyme’s active site. Knowledge about the molecular details of the substrate binding site allowed us to modulate the enzymatic activity by adjusting protein/substrate interactions, demonstrating the potential of our findings for future biotechnology applications.
A consensus view on the folding mechanism of protein G, L and their mutants
Authors: Chang Liwei , Perez Alberto
Published: April 2022

Much of our understanding of folding mechanisms comes from interpretations of experimental ϕ and ψ value analysis – relating the differences in stability of the transition state ensemble (TSE) and folded state. We introduce a unified approach combining simulations and Bayesian inference to provide atomistic detail for the folding mechanism of protein G, L and their mutants. Protein G and L fold to similar topologies despite low sequence similarity, but differ in their folding pathways. A fast folding redesign of protein G, NuG2, switches folding pathways and folds through a similar pathway with protein L. A redesign of protein L also leads to faster folding, respecting the original folding pathway. Our Bayesian inference approach starts from the sameprioron all systems and correctly identifies the folding mechanism for each of the four proteins – a success of the force field and sampling strategy. The approach is computationally efficient and correctly identifies the TSE and intermediate structures along the folding pathway in good agreement with experiments. We complement our findings by using two orthogonal approaches that differ in computational cost and interpretability. Adaptive sampling MD combined with Markov State Model provide a kinetic model that confirms the more complex folding mechanism of protein G and its mutant. Finally, a novel fragment decomposition approach using AlphaFold identifies preferences for secondary structure element combinations that follows the order of events observed in the folding pathways.
The NLRP3 inflammasome selectively drives IL-1β secretion byPseudomonas aeruginosainfected neutrophils and regulates bacterial killingin vivo
Authors: Minns Martin S. , Liboro Karl , Lima Tatiane S. , Abbondante Serena , Miller Brandon A. , Marshall Michaela E. , Tran-Chau Jolynn , Rietsch Arne , Dubyak George R. , Pearlman Eric
Published: April 2022

Macrophages infected with Gram-negative bacteria expressing Type III secretion system (T3SS) activate the NLRC4 inflammasome, resulting in Gasdermin D (GSDMD)-mediated IL-1β secretion and pyroptosis. Here we examined inflammasome signaling in neutrophils infected withPseudomonas aeruginosastrain PAO1 that expresses the T3SS effectors ExoS and ExoT. IL-1β secretion by neutrophils required the T3SS needle and translocon proteins and GSDMD. In macrophages, PAO1 and mutants lacking ExoS and ExoT (ΔexoST) stimulated NLRC4 for IL-1β secretion. While IL-1β release fromΔexoSTinfected neutrophils was also NLRC4-dependent, this was redirected to NLRP3-dependence by PAO1 infection via the ADP ribosyl transferase activity of ExoS. Genetic and pharmacologic approaches revealed that NLRP3, but not NLRC4, was essential for bacterial killing and limiting disease severity in a murine model ofP. aeruginosacorneal infection. This reveals a novel role for ExoS ADPRT in regulating inflammasome subtype usage by neutrophils versus macrophages and an unexpected role for NLRP3 inP. aeruginosakeratitis.
Divergent MEK/ERK and AMPK signaling dictate lipogenic plasticity and dependence on fatty acid synthesis in Glioblastoma
Authors: Eyme Katharina M. , Sammarco Alessandro , Jha Roshani , Mnatsakanyan Hayk , Neustadt Rudolph , Moses Charlotte , Alnasser Ahmad , Tardiff Daniel , Su Baolong , Williams Kevin J , Bensinger Steven J. , Chung Chee Yeun , Badr Christian E.
Published: April 2022

Deregulated de novo lipid synthesis (DNLS) is a potential druggable vulnerability in Glioblastoma (GBM), a highly lethal and incurable cancer. Yet the molecular mechanisms that determine susceptibility to DNLS-targeted therapies remain unknown, and the lack of brain-penetrant inhibitors of DNLS has prevented their clinical evaluation as GBM therapeutics. Here, we report that YTX-7739, a clinical-stage, brain-penetrant inhibitor of stearoyl CoA desaturase (SCD), triggers lipotoxicity in patient-derived GBM stem-like cells (GSCs) and inhibits fatty acid desaturation in GSCs orthotopically implanted in mice. When administered as a single agent, or particularly in combination with the first line GBM chemotherapy, Temozolomide (TMZ), YTX-7739 showed therapeutic efficacy in orthotopic GSC mouse models owing to its lipotoxicity and its ability to impair DNA damage repair. Leveraging genetic, pharmacological, and physiological manipulation of key signaling nodes in gliomagenesis, we uncover that aberrant MEK/ERK signaling and its repression of the energy sensor AMP-activated protein kinase (AMPK) primarily drives therapeutic vulnerability to SCD and other DNLS inhibitors. Conversely, AMPK activation mitigates lipotoxicity and renders GSCs impervious to the loss of DNLS, both in culture and in vivo, by decreasing the saturation state of phospholipids and diverting toxic lipids into lipid droplets. Altogether, our findings reveal mechanisms of metabolic plasticity in GSCs and provide a framework for rational integration of DNLS-targeted therapies for the treatment of GBM.
Intestinal microbiota dysbiosis and liver metabolomic changes during brain death
Authors: Tao R. , Guo W. , Li T. , Wang Y. , Wang P.
Published: April 2022

Background

The causative link between brain death and intestinal microbiota dysbiosis is unclear, and the distortion in liver metabolism caused by brain death requires further exploration.

Material and Methods

A rat model of brain death was constructed and sustained for 9 hours. Intestinal contents and portal vein plasma were collected for microbiota sequencing and microbial metabolite detection. Liver tissue was resected to investigate metabolic alterations, and the results were compared with those of a sham group.

Results

Microbiota dysbiosis occurred at the family and genus levels after 9 hours of brain death. Microbial metabolites remained unchanged in both the intestinal contents and portal vein plasma. Liver metabolic function deteriorated, and the levels of 80% of the differential metabolites decreased in the brain-dead rats. Most of the differential metabolites were related to energy metabolism.

Conclusions

Brain death resulted in microbiota dysbiosis in rats; however, this dysbiosis did not alter microbial metabolites. Deterioration in liver metabolic function during extended periods of brain death may reflect a continuous worsening in energy deficiency.
The concordance of signals based on irregular incremental lines in the human tooth cementum with documented pregnancies: Results from a systematic approach
Authors: Mani-Caplazi Gabriela , Vach Werner , Wittwer-Backofen Ursula , Hotz Gerhard
Published: April 2022

Background and Objective

There is evidence from previous studies that pregnancies and diseases are recorded in the tooth cementum. This study aims to assess the degree of concordance between signals based on irregular incremental lines (ILs) and reported pregnancies.

Material and Methods

23 recent and 24 archaeological human teeth with known birth history were included in this investigation. 129 histological sections of tooth roots were assessed for irregularities in appearance and width using a standardized protocol. Similarity of observed irregularities at the section level allowed us to define signals at the tooth level. The sensitivity of signals to detect pregnancies was determined and related to the signal prevalence.

Results

Pregnancy signals were frequently visually observed. However, applying a standardized process we could only reach signal sensitivities to identify pregnancies up to 20 percentage points above chance level.

Conclusions

Based on a standardized and reproducible method it could be confirmed that some pregnancies leave visible signals in the tooth cementum. The results show the potential of the tooth cementum to support reconstruction of life courses in paleopathology. However, it seems that not all pregnancies affect the cementogenesis in such a way that irregular ILs are identifiable. Further research is needed to better understand which type of pregnancies and other conditions are recorded in the tooth cementum.
A method for stabilising the XX karyotype in female mESC cultures
Authors: Keniry Andrew , Jansz Natasha , Hickey Peter F. , Breslin Kelsey A. , Iminitoff Megan , Beck Tamara , Ritchie Matthew E. , Blewitt Marnie E.
Published: April 2022

Female mouse embryonic stem cells (mESCs) present differently to male mESCs in several fundamental ways, however complications with their in vitro culture have resulted in an underrepresentation of female mESCs in the literature. Recent studies show that the second X chromosome in female, and more specifically the transcriptional activity from both of these chromosomes due to absent X chromosome inactivation, sets female mESCs apart from males. To avoid this undesirable state, female mESCs in culture preferentially adopt an XO karyotype, with this adaption leading to loss of their unique properties in favour of a state near indistinguishable from male. If female pluripotency is to be studied effectively in this system, it is critical that high quality cultures of XX mESCs are available. Here we report a method for better maintaining XX female mESCs in culture, which also stabilises the male karyotype, and makes study of female specific pluripotency more feasible.
Chromatin accessibility landscape and active transcription factors in primary human invasive lobular and ductal breast carcinomas
Authors: Lee Sanghoon , Osmanbeyoglu Hatice Ulku
Published: April 2022

Background

Invasive lobular breast carcinoma (ILC), the second most prevalent histological subtype of breast cancer, exhibits unique molecular features compared with the more common invasive ductal carcinoma (IDC). While genomic and transcriptomic features of ILC and IDC have been characterized, genome-wide chromatin accessibility pattern differences between ILC and IDC remain largely unexplored.

Methods

Here, we characterized tumor-intrinsic chromatin accessibility differences between ILC and IDC using primary tumors from The Cancer Genome Atlas (TCGA) breast cancer assay for transposase-accessible chromatin with sequencing (ATAC-seq) dataset.

Results

We identified distinct patterns of genome-wide chromatin accessibility in ILC and IDC. Inferred patient-specific transcription factor (TF) motif activities revealed regulatory differences between and within ILC and IDC tumors. EGR1, RUNX3, TP63, STAT6, SOX family, and TEAD family TFs were higher in ILC, while ATF4, PBX3, SPDEF, PITX family, and FOX family TFs were higher in IDC.

Conclusions

This study reveals the distinct epigenomic features of ILC and IDC and the active TFs driving cancer progression that may provide valuable information on patient prognosis.
DOT1L activity affects cell lineage progression in the developing brain by controlling metabolic programs
Authors: Appiah Bismark , Fullio Camilla L. , Haffner Christiane , Zeis Patrice , Treppner Martin , Bovio Patrick , Cheffer Arquimedes , Bertani Ilaria , Binder Harald , Grün Dominic , Kalebic Nereo , Taverna Elena , Vogel Tanja
Published: April 2022

Cortical neurogenesis depends on the tight balance between self-renewal and differentiation of apical progenitors (APs), the key progenitor type generating all other neural cells including neocortical neurons. We here report the activity of the histone methyltransferase DOT1L as a gatekeeper for AP cell identity. Combining lineage tracing with single-cell RNA sequencing of clonally related cells, we explore consequences of DOT1L inhibition on AP lineage progression during neurogenesis in the embryonic mouse neocortex. At the cellular level, DOT1L inhibition led to increased neurogenesis driven by a shift from asymmetric self-renewing to symmetric neurogenic divisions of APs. At the molecular level, we show that DOT1L activity preserved AP identity by promoting transcription of a gene set involved in AP metabolism. On a mechanistic level, DOT1L inhibition increased expression of metabolic genes, including microcephaly-associated Asparagine synthetase (Asns) and overexpression of ASNS in APs resulted in increased neuronal differentiation.Asnsexpression was predicted to be controlled through EZH2 and we show that DOT1L activity allows PRC2-mediated repression ofAsnsexpression. Importantly, inhibition of ASNS activity rescued increased AP differentiation upon DOT1L inhibition. Our data show that DOT1L activity/PRC2 crosstalk controls AP lineage progression by regulating AP metabolism, and they provide a mechanistic view on how DOT1L activity might affect neocortical neurogenesis.
Hydrogens and hydrogen-bond networks in macromolecular MicroED data
Authors: Clabbers Max T.B. , Martynowycz Michael W. , Hattne Johan , Gonen Tamir
Published: April 2022

Microcrystal electron diffraction (MicroED) is a powerful technique utilizing electron cryo-microscopy (cryo-EM) for protein structure determination of crystalline samples too small for X-ray crystallography. Electrons interact with the electrostatic potential of the sample, which means that scattered electrons carry informing about the charged state of atoms and can provide strong contrast for visualizing hydrogen atoms. Accurately identifying the positions of hydrogen atoms, and by extension the hydrogen bonding networks, is of importance for drug discovery and electron microscopy can enable such visualization. Using subatomic resolution MicroED data obtained from triclinic hen egg-white lysozyme, we identified hundreds of individual hydrogen atom positions and directly visualize hydrogen bonding interactions and the charged states of residues. Over a third of all hydrogen atoms are identified from strong difference peaks, the most complete view of a macromolecular hydrogen network visualized by electron diffraction to date. These results show that MicroED can provide accurate structural information on hydrogen atoms and non-covalent hydrogen bonding interactions in macromolecules. Furthermore, we find that the hydrogen bond lengths are more accurately described by the inter-nuclei distances than the centers of mass of the corresponding electron clouds. We anticipate that MicroED, coupled with ongoing advances in data collection and refinement, can open further avenues for structural biology by uncovering and understanding the hydrogen bonding interactions underlying protein structure and function.
Dictyostelium discoideum cells sense their local density and retain nutrients when the cells are about to overgrow their food source
Authors: Rijal Ramesh , Kirolos Sara A. , Rahman Ryan J. , Gomer Richard H.
Published: April 2022

Dictyostelium discoideum is a unicellular eukaryote that eats bacteria, and eventually overgrows the bacteria. D. discoideum cells accumulate extracellular polyphosphate (polyP), and the polyP concentration increases as the local cell density increases. At high cell densities, the correspondingly high extracellular polyP concentrations allow cells to sense that they are about to overgrow their food supply and starve, causing the D. discoideum cells to inhibt their proliferation. In this report, we show that high extracellular polyP inhibits exocytosis of undigested or partially digested nutrients. PolyP decreases cell membrane fluidity and plasma membrane recycling, and this requires the G protein-coupled polyP receptor GrlD, the polyphosphate kinase Ppk1, and the inositol hexakisphosphate kinase I6kA. PolyP did not affect random cell motility, cell speed, or F-actin levels. PolyP decreased membrane saturated fatty acids and altered lipid and protein contents in detergent-insoluble lipid microdomains. Together, these data suggest that D. discoideum cells use polyP as a signal to sense their local cell density and reduce cell membrane fluidity and membrane recycling, perhaps as a mechanism to retain ingested food when the cells are about to starve.
A hidden layer of structural variation in transposable elements reveals potential genetic modifiers in human disease-risk loci
Authors: van Bree Elisabeth J. , Guimarães Rita L.F.P. , Lundberg Mischa , Blujdea Elena R. , Rosenkrantz Jimi L. , White Fred T.G. , Poppinga Josse , Ferrer-Raventós Paula , Schneider Anne-Fleur E. , Clayton Isabella , Haussler David , Reinders Marcel J.T. , Holstege Henne , Ewing Adam D. , Moses Colette , Jacobs Frank M.J.
Published: April 2022

Genome-wide association studies (GWAS) have been highly informative in discovering disease-associated loci but are not designed to capture all structural variations in the human genome. Using long-read sequencing data, we discovered widespread structural variation within SINE-VNTR-Alu (SVA) elements, a class of great ape-specific transposable elements with gene-regulatory roles, which represents a major source of structural variability in the human population. We highlight the presence of structurally variable SVAs (SV-SVAs) in neurological disease–associated loci, and we further associate SV-SVAs to disease-associated SNPs and differential gene expression using luciferase assays and expression quantitative trait loci data. Finally, we genetically deleted SV-SVAs in the BIN1 and CD2AP Alzheimer's disease–associated risk loci and in the BCKDK Parkinson's disease–associated risk locus and assessed multiple aspects of their gene-regulatory influence in a human neuronal context. Together, this study reveals a novel layer of genetic variation in transposable elements that may contribute to identification of the structural variants that are the actual drivers of disease associations of GWAS loci.