bluuebunny/crossref_metadata_2025_split huggingface dataset:
Structure of the integrin receptor αMβ2 headpiece in complex with a function-modulating nanobody
Authors: Jensen Rasmus K. , Pedersen Henrik , Lorentzen Josefine , Laursen Nick Stub , Vorup-Jensen Thomas , Andersen Gregers Rom 
Published: July 2021

AbstractThe integrin receptor αMβ2 mediates phagocytosis of complement-opsonized objects, adhesion to the extracellular matrix and trans-endothelial migration of leukocytes. Here we present the first atomic structure of the human αMβ2 headpiece fragment in complex with the nanobody hCD11bNb1 determined at a resolution of 3.2 Å. The receptor headpiece adopts the closed conformation expected to have low ligand affinity. The crystal structure advocates that in the R77H αM variant associated with systemic lupus erythematosus, the modified allosteric coupling between ligand coupling and integrin outside-inside signalling is due to subtle conformational effects transmitted over 40 Å. The nanobody binds to the αI domain of the αM subunit in an Mg2+ independent manner with low nanomolar affinity. Biochemical and biophysical experiments with purified proteins argue that the nanobody acts as a competitive inhibitor through steric hindrance exerted on the thioester domain of iC3b attempting to bind the αM subunit. Surprisingly, the nanobody stimulates the interaction of cell-bound αMβ2 with iC3b suggesting that it represents a novel high-affinity proteinaceous αMβ2 specific agonist. We propose a model based on the conformational spectrum of the receptor to reconcile these conflicting observations regarding the functional consequences of hCD11bNb1 binding to αMβ2. Furthermore, our data suggest that the iC3b-αMβ2 complex may be more dynamic than predicted from the crystal structure of the core complex.
EpicPCR-Directed Cultivation of a Candidatus Saccharibacteria Symbiont Reveals a Type IV Pili-dependent Epibiotic Lifestyle
Authors: Xie Bingliang , Wang Jian , Nie Yong , Chen Dongwei , Hu Beiyu , Wu Xiaolei , Du Wenbin 
Published: July 2021

AbstractCandidate phyla radiations (CPR), accounting for a major microbial supergroup with remarkably small genomes and reduced sizes, are widely distributed yet mostly uncultured. Limited culture and its obligate reliance upon other bacteria hindered investigation of their lifestyles. In this work we isolated a CPR bacterium, TM7i, with its host Leucobacter aridocollis J1, by combination of Emulsion, Paired Isolation and Concatenation PCR (epicPCR) detection and filtrate co-culture. Genomic profiling of TM7 genomes and microscopic investigation of TM7i-J1 symbiosis suggest the conservation of type IV pili and a pili-dependent lifestyle of TM7. Further, we observed twitching motility of TM7i mediated by pili and its role played in the interaction with its host. Our results shed a light on the lifestyle about this enigmatic bacterial radiation, which may also be adopted by other CPR organisms. The epicPCR-directed isolation method underlines high efficiency of CPR bacteria isolation and thus may be used in other symbiotic or epibiotic microorganisms.
Kinetic characterization and thermostability ofC. eleganscytoplasmic and mitochondrial malate dehydrogenases
Authors: Thomas Matthew J. , Cassidy Emma R. , Robinson Devin S. , Walstrom Katherine M. 
Published: July 2021

AbstractMalate dehydrogenase (MDH) catalyzes the conversion of NAD+and malate to NADH and oxaloacetate in the last step of the citric acid cycle. Eukaryotes have at least two MDH isozymes, one that is imported into the mitochondria and one that remains in the cytoplasm. We overexpressed and purifiedCaenorhabditis eleganscytoplasmic MDH-1 (F46E10.10) and mitochondrial MDH-2 (F20H11.3) inE. coli. Our goal was to compare the kinetic and structural properties of these enzymes becauseC. eleganscan survive adverse environmental conditions, such as lack of food and elevated temperatures. In steady-state enzyme kinetics assays, we determined that theKMvalues for oxaloacetate were 54 and 52 μM, and theKMvalues for NADH were 61 and 107 μM, for MDH-1 and MDH-2, respectively. We partially purified endogenous MDH from a mixed population of worms and separated MDH-1 from MDH-2 using anion exchange chromatography. Both endogenous enzymes had aKMfor oxaloacetate similar to that of the corresponding recombinant enzyme. The reaction velocities of the recombinant enzymes had slightly different temperature-dependencies: MDH-1 and MDH-2 had maximum activity at 40 °C and 35 °C, respectively. In a thermotolerance assay, MDH-1 was much more thermostable than MDH-2. Molecular homology modeling predicted that MDH-1 had more salt-bridges between the subunits than mammalian MDH1 enzymes, and these ionic interactions may contribute to its thermostability. In contrast, the MDH-2 homology model predicted fewer ionic interaction between the subunits compared to mammalian MDH2 enzymes. These results suggest that the increased structural stability of MDH-1 may facilitate its ability to remain active in adverse environmental conditions. In contrast, MDH-2 may use other strategies, such as protein binding partners, to function under similar conditions.
Gating mechanism of human N-type voltage-gated calcium channel
Authors: Dong Yanli , Gao Yiwei , Xu Shuai , Wang Yuhang , Yu Zhuoya , Li Yue , Li Bin , Yang Bei , Zhang Xuejun Cai , Jiang Daohua , Huang Zhuo , Zhao Yan 
Published: July 2021

AbstractN-type voltage-gated calcium (CaV) channels mediate Ca2+ influx at the presynaptic terminals in response to action potential and play vital roles in synaptogenesis, neurotransmitter releasing, and nociceptive transmission. Here we elucidate a cryo-electron microscopy (cryo-EM) structure of the human CaV2.2 complex at resolution of 2.8 Å. This complex structure reveals how the CaV2.2, β1, and α2δ1 subunits are assembled. In our structure, the second voltage-sensing domain (VSD) is stabilized at a resting-state conformation, which is distinct from the other three VSDs of CaV2.2 as well as activated VSDs observed in previous structures of CaV channels. The structure also shows that the intracellular gate formed by S6 helices is closed, and a W-helix from the DII-III linker is determined to act as a blocking-ball that causes closed-state inactivation in CaV2.2. Collectively, our structure provides previously unseen structural insights into fundamental gating mechanisms of CaV channels.
Myths and mechanisms: RecBCD and Chi hotspots as determinants of self vs. non-self
Authors: Subramaniam Suriyen , Smith Gerald R. 
Published: July 2021

AbstractBacteria face a challenge when DNA enters their cells by transformation, mating, or phage infection. Should they treat this DNA as an invasive foreigner and destroy it, or consider it one of their own and potentially benefit from incorporating new genes or alleles to gain useful functions? It is frequently stated that the short nucleotide sequence Chi (5’ GCTGGTGG 3’) recognized by RecBCD helicase-nuclease allows Escherichia coli to distinguish self (i.e., E. coli) DNA from non-self (i.e., any other) DNA and to destroy non-self DNA, and that Chi is “over-represented” in the E. coli genome. We show here that these dogmas are incorrect and apparently based on false assumptions. We note Chi’s wide-spread occurrence and activity in distantly related species. We illustrate multiple, highly non-random features of the genomes of coli and coliphage P1 that account for Chi’s high frequency and genomic position, leading us to propose that P1 selects for Chi’s enhancement of recombination, whereas E. coli selects for the preferred codons in Chi. We discuss other, substantiated mechanisms for self vs. non-self determination involving RecBCD and for RecBCD’s destruction of DNA that cannot recombine, whether foreign or domestic.
Long-range single-molecule mapping of chromatin modification in eukaryotes
Authors: Weng Zhe , Ruan Fengying , Chen Weitian , Xie Zhe , Xie Yeming , Zhang Chen , Chen Zhichao , Wang Juan , Sun Yuxin , Fang Yitong , Guo Mei , Tong Yiqin , Li Yaning , Tang Chong 
Published: July 2021

AbstractThe epigenetic modifications of histones are essential markers related to the development and pathogenesis of diseases, including human cancers. Mapping histone modification has emerged as the widely used tool for studying epigenetic regulation. However, existing approaches are limited by fragmentation and short-read sequencing represent the average chromatin status in samples and cannot provide information about the long-range chromatin states. We leveraged the advantage of long read sequencing to develop a method “BIND&MODIFY” for profiling the histone modification of individual DNA fibers. Our approach is based on the recombinant fused protein A-M.EcoGII, which tethers the methyltransferase M.EcoGII to the protein binding sites and locally labels the neighboring DNA regions through artificial methylations. We demonstrated that the aggregated BIND&MODIFY signal matches the bulk-level ChIP-seq and CUT&TAG, verify the single-molecule heterogenous histone modification status, and quantify the correlation between distal elements. This method could be an essential tool in future third-generation sequencing ages.
Therapeutic targeting of SETD2-deficient cancer cells with the small-molecule compound RITA
Authors: Lopez Kirsten A. , Sarkar Sovan , Seraia Elena , Toffanin Chiara , Cooper Christian , Challoumas Michalis , Okonjo Fiona A. , Jones George D. D. , Buffa Francesca , Ebner Daniel , Humphrey Timothy C. 
Published: July 2021

AbstractThe histone methyltransferase SETD2 and its associated histone mark H3 lysine 36 trimethylation (H3K36me3) are frequently lost in certain cancer types, identifying SETD2 as an important therapeutic target. Here we show that SETD2-deficient cancer cells are profoundly sensitive to the compound RITA, resulting in significant p53 induction and apoptosis. This is further associated with defects in DNA replication, leading to delays in S-phase progression, increased recruitment of replication stress markers, and reduced replication fork progression. RITA sensitivity is linked to the phenol sulphotransferase SULT1A1, which we find to be highly upregulated in cells that lack SETD2. Depletion of SULT1A1 or addition of the phenol sulphotransferase inhibitor DCNP abolishes these phenotypes and suppresses the sensitivity of SETD2-deficient cancer cells, identifying SULT1A1 activity to be critical in mediating the potent cytotoxicity of RITA against SETD2-deficient cells. These findings define a novel therapeutic strategy for targeting the loss of SETD2 in cancer.Significance StatementThe histone-modifying enzyme SETD2 has emerged as an important tumour suppressor in a number of different cancer types, identifying it as a promising therapeutic target. The concept of synthetic lethality, a genetic interaction in which the simultaneous loss of two genes or pathways that regulate a common essential process renders the cell nonviable, is a valuable tool for killing cancer cells that have known mutations. In this study, we conducted a synthetic lethality screen for compounds that specifically target SETD2-deficient cancer cells. The top hit, a compound called RITA, reduces cell viability and induces cell death only in the context of SETD2 loss, thereby highlighting a potential novel therapeutic strategy for treating SETD2-deficient cancers.
CLUH interactome reveals an association to SPAG5 and a proximity to the translation of mitochondrial protein
Authors: Hémono Mickaële , Haller Alexandre , Chicher Johana , Duchêne Anne-Marie , Ngondo Richard Patryk 
Published: July 2021

ABSTRACTMitochondria require thousands of proteins to fulfil their essential function in energy production and other fundamental biological processes. These proteins are mostly encoded by the nuclear genome, translated in the cytoplasm before being imported into the organelle. RNA binding proteins (RBPs) are central players in the regulation of this process by affecting mRNA translation, stability or localization. CLUH is an RBP recognizing specifically mRNAs coding for mitochondrial proteins, but its precise molecular function and interacting partners remain undiscovered in mammals. Here we reveal for the first time CLUH interactome in mammalian cells. Using both co-IP and BioID proximity-labeling approaches, we identify novel molecular partners interacting stably or transiently with CLUH in HCT116 cells and mouse embryonic stem cells. We reveal a stable RNA-independent interaction of CLUH with itself and with SPAG5 in cytosolic granular structures. More importantly, we uncover an unexpected proximity of CLUH to mitochondrial proteins and their cognate mRNAs in the cytosol. Additionally, our data highlight the importance of CLUH TPR domain for its interactions with both proteins and mRNAs. Overall, through the analysis of CLUH interactome, our study sheds a new light on CLUH molecular function by highlighting its association to the translation and subcellular localization of some mRNAs coding for mitochondrial proteins.
Cryo-EM structure of alpha-synuclein fibrils amplified by PMCA from PD and MSA patient brains
Authors: Burger Domenic , Fenyi Alexis , Bousset Luc , Stahlberg Henning , Melki Ronald 
Published: July 2021

AbstractSynucleinopathies are neurodegenerative diseases related to the aggregation of the protein alpha-synuclein (aSyn). Among these diseases, Parkinson’s disease (PD) and multiple system atrophy (MSA) are most prevalent. aSyn can readily form different fibrillar polymorphs, if exposed to an air-water interface or by templating with pre-existing fibrils. We here report the structures of three fibrillar polymorphs that were obtained after seeding monomeric aSyn with PD and MSA patients brain homogenates using protein misfolding cyclic amplification (PMCA). Seeding with a control brain homogenate did not produce fibrils, and seeding with other in vitro generated fibrillar polymorphs as a control faithfully produced polymorphs of a different type. The here determined fibril structures from PD and MSA brain tissue represent new folds, which partly resemble that of previously reported in vitro generated fibrils from Y39 phosphorylated aSyn protein. The relevance of these fibrils for synucleinopathies in humans remains to be further investigated.Impact StatementNeurodegenerative diseases such as Parkinson’s disease (PD) and Multiple System Atrophy (MSA) are suspected to be causatively related to the prion-like propagation of aggregates of the protein alpha-synuclein (aSyn). The fibril structures reported here were obtained after seeding from diseased human brain homogenate and differ from all previously published aSyn fibril arrangements. In case these fibrils would turn out to be the long sought causative agents of these diseases, their structures might lead to the development of therapeutic strategies to modify these diseases and to a better understanding of the mechanistic processes that lead to neurodegeneration and spreading of the diseases.
A recombinant protein containing influenza viral conserved epitopes and superantigen induces broad-spectrum protection
Authors: Li Yansheng , Xu Mingkai , Li Yongqiang , Gu Wu , Halimu Gulinare , Li Yuqi , Zhang Zhichun , Zhou Libao , Liao Hui , Yao Songyuan , Zhang Huiwen , Zhang Chenggang 
Published: July 2021

AbstractInfluenza pandemic poses public health threats annually for lacking vaccine which provides cross-protection against novel and emerging influenza viruses. Combining conserved antigens inducing cross-protective antibody response with epitopes activating cross-protective cytotoxic T-cells would offer an attractive strategy for developing universal vaccine. In this study, we constructed a recombinant protein NMHC consisting of influenza viral conserved epitopes and superantigen fragment. NMHC promoted the mature of bone marrow-derived dendritic cells and induced CD4+T cells to differentiate into Th1, Th2 and Th17 subtypes. Mice vaccinated with NMHC produced high level of immunoglobulins which cross-bound to HA fragments from six influenza virus subtypes with high antibody titers. Anti-NMHC serum showed potent hemagglutinin inhibition effects to highly divergent group 1 (H1 subtypes) and group 2 (H3 subtype) influenza virus strains. And purified anti-NMHC antibodies could bind to multiple HAs with high affinities. NMHC vaccination effectively protected the mice from infection and lung damage challenged by two subtypes of H1N1 influenza virus. Moreover, NMHC vaccination elicited CD4+and CD8+T-cell responses to clear the virus from infected tissue and prevent virus spreading. In conclusion, this study provided proof of concept for triggering both B cells and T cells immune responses against multiple influenza virus infection, and NMHC may be a potential candidate of universal broad-spectrum vaccine for various influenza virus prevention and therapy.
Protein kinase signalling at the Leishmania kinetochore captured by XL-BioID
Authors: Geoghegan Vincent , Jones Nathaniel G. , Dowle Adam , Mottram Jeremy C. 
Published: July 2021

AbstractElucidating protein kinase signaling pathways is an important but challenging problem in cell biology. Phosphoproteomics has been used to identify many phosphorylation sites, however the spatial context of these sites within the cell is mostly unknown, making it difficult to reconstruct signalling pathways. To address this problem an in vivo proximity capturing workflow was developed, consisting of proximity biotinylation followed by protein cross-linking (XL-BioID). This was applied to protein kinases of the Leishmania kinetochore, leading to the discovery of a novel essential kinetochore protein, KKT26. XL-BioID enabled the quantification of proximal phosphosites at the kinetochore through the cell cycle, allowing the phosphorylation state of the kinetochore to be followed during assembly. A specific inhibitor of kinetochore protein kinases KKT10/KKT19 was used to show that XL-BioID provides a spatially focussed view of protein kinase inhibition, identifying 16 inhibitor-responsive proximal phosphosites, including 3 on KKT2, demonstrating the potential of this approach for discovery of in vivo kinase signalling pathways.
Mosaic cis-regulatory evolution drives transcriptional partitioning of HERVH endogenous retrovirus in the human embryo
Authors: Carter Thomas A. , Singh Manvendra , Dumbović Gabrijela , Chobirko Jason D. , Rinn John L. , Feschotte Cédric 
Published: July 2021

AbstractThe human endogenous retrovirus type-H (HERVH) family is expressed in the preimplantation embryo. A subset of these elements are specifically transcribed in pluripotent stem cells where they appear to exert regulatory activities promoting self-renewal and pluripotency. How HERVH elements achieve such transcriptional specificity remains poorly understood. To uncover the sequence features underlying HERVH transcriptional activity, we performed a phyloregulatory analysis of the long terminal repeats (LTR7) of the HERVH family, which harbor its promoter, using a wealth of regulatory genomics data. We found that the family includes at least 8 previously unrecognized subfamilies that have been active at different timepoints in primate evolution and display distinct expression patterns during human embryonic development. Notably, nearly all HERVH elements transcribed in ESCs belong to one of the youngest subfamilies we dubbed LTR7up. LTR7 sequence evolution was driven by complex mutational processes, including multiple recombination events between subfamilies, that led to transcription factor binding motif modules characteristic of each subfamily. Using a reporter assay, we show that one such motif, a predicted SOX2/3 binding site unique to LTR7up, is essential for robust promoter activity in induced pluripotent stem cells. Together these findings illuminate the mechanisms by which HERVH diversified its expression pattern during evolution to colonize distinct cellular niches within the human embryo.
Positive selection and enhancer evolution shaped lifespan and body mass in great apes
Authors: Tejada-Martinez Daniela , Avelar Roberto A. , Lopes Inês , Zhang Bruce , Novoa Guy , de Magalhães João Pedro , Trizzino Marco 
Published: July 2021

AbstractWithin primates, the great apes are outliers both in terms of body size and lifespan, since they include the largest and longest-lived species in the order. Yet, the molecular bases underlying such features are poorly understood. Here, we leveraged an integrated approach to investigate multiple sources of molecular variation across primates, focusing on ~1,550 genes previously described as tumor suppressors, oncogenes, ageing genes in addition to a novel Build of the CellAge database of cell-senescence genes (version 2), herein presented for the first time. Specifically, we analyzed dN/dS rates, positive selection, gene expression (RNA-seq) and gene regulation (ChIP-seq). By analyzing the correlation between dN/dS, maximum lifespan and body mass we identified 67 genes that in primates co-evolved with those traits. Further, we identified 6 genes, important for immunity, neurodevelopment and telomere maintenance (includingTERF2), under positive selection in the great ape ancestor. RNA-seq data, generated from the liver of six species representing all the primate lineages, revealed that ~8% of the longevity genes are differentially expressed in apes relative to other primates. Importantly, by integrating RNA-seq with ChIP-seq for H3K27ac (which marks active enhancers), we show that the differentially expressed longevity genes are significantly more likely than expected to be located near a novel “ape-specific” enhancer. Moreover, these particular ape-specific enhancers are enriched for young transposable elements, and specifically SINE-Vntr-Alus (SVAs). In summary, we demonstrate that multiple evolutionary forces have contributed to the evolution of lifespan and body size in primates.