bluuebunny/crossref_metadata_2025_split huggingface dataset:
Ana1/CEP295 is an essential player in the centrosome maintenance program regulated by Polo kinase
Authors: Pimenta-Marques Ana , Perestrelo Tania , Rodrigues Patricia , Duarte Paulo , Lince-Faria Mariana , Bettencourt-Dias Mónica 
Published: April 2022

AbstractCentrioles play critical roles in our cells, being part of centrosomes and cilia, which are important microtubule organising centers (MTOC) with a variety of roles. While centrioles are very stable structures, they disappear in certain cell types upon differentiation, such as in oocytes. Little is known about the regulation of centriole structural integrity. We previously uncovered that the pericentriolar material (PCM), and its recruiter Polo kinase, are required for both the maintenance of centriole structural integrity and centrosome MTOC activity. Using an hypothesis driven RNAi screen, we show that both the cartwheel and the centriole wall play an important role in centrosome integrity. In particular, we uncovered that the centriole wall protein ANA1 is critical for the integrity of both new and mature centrioles, in Drosophila oogenesis as well as in cultured cells. Moreover, our results show that the activity of both Polo and the PCM in centriole integrity depends on ANA1. Our work suggests that the structural integrity of centrioles, once thought to be very stable organelles, depends on the turnover of key components, suggesting new perspectives for understanding the dysfunction of those structures in disease.
A novel Pyk2-derived peptide inhibits invadopodia-mediated breast cancer metastasis
Authors: Twafra Shams , Sokolik Chana G. , Sneh Tal , Srikanth Kolluru D. , Meirson Tomer , Genna Alessandro , Chill Jordan H. , Gil-Henn Hava 
Published: April 2022

ABSTRACTThe non-receptor tyrosine kinase Pyk2 is highly expressed in breast cancer, where it mediates invadopodia formation and function via interaction with the actin-nucleation promoting factor cortactin. Here, we designed a cell-permeable peptide inhibitor that contains the second proline-rich region (PRR2) sequence of Pyk2, which binds to the SH3 domain of cortactin and blocks spontaneous lung metastasis in immune-competent mice by inhibiting invadopodia maturation and function. The native structure of the Pyk2-PRR2:cortactin-SH3 complex was determined using nuclear magnetic resonance (NMR), revealing an extended class II interaction surface spanning the canonical binding groove and a second hydrophobic surface which significantly contributes to ligand affinity. Using structure-guided design, we created a mutant peptide lacking critical residues involved in binding that failed to inhibit invadopodia maturation and function and consequent metastatic dissemination in mice. Our findings shed light on the specific molecular interactions between Pyk2 and cortactin and suggest that their inhibition may be used as a novel strategy for blocking breast cancer metastasis.
CyTOF-Lec: Single-cell Glycomics Analysis Reveals Glycan Features Defining Cells Differentially Susceptible to HIV
Authors: Ma Tongcui , McGregor Matthew , Giron Leila B. , Xie Guorui , George Ashley F. , Abdel-Mohsen Mohamed , Roan Nadia R. 
Published: April 2022

ABSTRACTHigh-parameter single-cell phenotyping has enabled in-depth classification and interrogation of immune cells, but to date has not allowed for glycan characterization. Here, we develop CyTOF-Lec as an approach to simultaneously characterize many protein and glycan features of human immune cells at the single-cell level. We implemented CyTOF-Lec to compare glycan features between different immune subsets from blood and multiple tissue compartments, and to characterize HIV-infected cell cultures. Using bioinformatics approaches to distinguish preferential infection of cellular subsets from viral-induced remodeling, we demonstrate that HIV upregulates the levels of cell surface fucose and sialic acid in a cell- intrinsic manner, and that memory CD4+ T cells co-expressing high levels of fucose and sialic acid are highly susceptible to HIV infection. Sialic acid levels were found to distinguish memory CD4+ T cell subsets expressing different amounts of viral entry receptors, pro-survival factors, homing receptors, and activation markers, and to play a direct role in memory CD4+ T cells’ susceptibility to HIV infection. The ability of sialic acid to distinguish memory CD4+ T cells with different susceptibilities to HIV infection was experimentally validated through sorting experiments. Together, these results suggest that HIV remodels not only cellular proteins but also glycans, and that glycan expression can differentiate memory CD4+ T cells with vastly different susceptibility to HIV infection.
Unexpected Complexity of the Ammonia Monooxygenase in Archaea
Authors: Hodgskiss Logan H. , Melcher Michael , Kerou Melina , Chen Weiqiang , Ponce-Toledo Rafael I. , Savvides Savvas N. , Wienkoop Stefanie , Hartl Markus , Schleper Christa 
Published: April 2022

AbstractAmmonia oxidation as the first step of nitrification constitutes a critical process in the global nitrogen cycle. However, fundamental knowledge of its key enzyme, the copper-dependent ammonia monooxygenase is lacking, in particular for the environmentally abundant ammonia oxidizing archaea (AOA). Here, the structure of the enzyme is investigated by blue-native gel electrophoresis and proteomics from native membrane complexes of two AOA. Beside the known AmoABC subunits and the earlier predicted AmoX, two new protein subunits, AmoY and AmoZ, were identified. They are unique to AOA, highly conserved and co-regulated, and their genes are linked to other AMO subunit genes in streamlined AOA genomes. Modelling and in gel cross-link approaches support an overall protomer structure similar to the distantly related bacterial particulate methane monooxygenase indicating that AmoY and AmoZ serve an important structural and functional role. These data open avenues for further structure-function studies of this ecologically important key nitrification complex.
Listeria monocytogenes Co-opts Caveolin-Mediated E-cadherin Trafficking and Macropinocytosis for Epithelial Cell-to-Cell Spread
Authors: Radhakrishnan Prathima , Sathe Mugdha , Theriot Julie A. 
Published: April 2022

SUMMARYListeria monocytogenes is an intracellular bacterial pathogen that spreads directly between adjacent host cells without exposure to the extracellular space. Recent studies have identified several host cell factors that promote L. monocytogenes cell-to-cell spread in epithelial monolayers, but details of the mechanism remain unclear. We find that the adherens junction protein, E-cadherin, promotes L. monocytogenes cell-to-cell spread at the recipient side of cell contacts. In particular, two point mutations in E-cadherin’s cytoplasmic domain that prevent its ubiquitination hinder bacterial cell-to-cell spread efficiency without reducing the extent of contact between neighboring cells. As ubiquitination induces E-cadherin endocytosis, we hypothesize that E-cadherin promotes protrusion engulfment, where donor cell protrusions containing L. monocytogenes are taken up by the recipient cell concurrently with E-cadherin internalization. In support of this hypothesis, we show that inhibiting caveolin-mediated membrane trafficking reduces L. monocytogenes cell-to-cell spread only under conditions where E-cadherin can be ubiquitinated. Additionally, we demonstrate that macropinocytosis also contributes to dissemination of L. monocytogenes through an epithelial monolayer.
Systems modelling of TGF-β/Hippo signalling crosstalk uncovers molecular switches that coordinate YAP transcriptional complexes (submitted toiScience)
Authors: Ghomlaghi Milad , Theocharous Mandy , Shin Sung-Young , O’ Neill Eric , Nguyen Lan K. 
Published: April 2022

AbstractThe Hippo pathway is an evolutionarily conserved signaling network that integrates diverse cues to regulate cell fate and organ homeostasis. The central downstream pathway protein is the transcriptional co-activator Yes-associated protein (YAP). Although capable of inducing gene transcription, YAP cannot bind DNA directly. Instead, it mediates transcriptional activity through interaction with distinct DNA-binding transcriptional factors (TFs), including TEAD, SMAD, and p73, to form active and functionally opposing transcriptional complexes. Growing evidence in mammals demonstrates that YAP has a dual role and can either promote cell proliferation or apoptosis, which underpin its ability to function as both an oncogene or a tumour suppressor depending on the specific context. However, the mechanisms by which YAP coordinates its distinct transcriptional complexes and mediates context-dependent function remain poorly defined. This is in part due to the lack of systems-level studies that can decrypt the complexities of upstream signalling pathways and their crosstalk, which together dictate the transcriptional regulation at the YAP level. Here, we undertake an integrative systems-based approach combining computational network modelling and experimental studies to interrogate the dynamic formation of and transition between the YAP-SMAD and YAP-p73 transcriptional complexes, which control proliferative and apoptotic gene expression, respectively. We developed a new experimentally-validated mathematical model of the TGF-β/Hippo signalling crosstalk and used this model to elucidate dynamic network behaviour. Our integrative studies uncovered previously unknown molecular switches that control the YAP-SMAD/p73 complexes in an on/off, switch-like manner. RASSF1A and ITCH were identified as major regulators of the switches, whereby a graded increase in ITCH expression can trigger YAP to abruptly switch from binding p73 to SMAD, swiftly promoting proliferative gene expression. Further, adjusting the model to reflect cell type-specific protein expression profiles using both in-house and publicly available experimental data enabled us to study the YAP switches under diverse and varied cellular contexts. Overall, our studies provide a new quantitative and systems-level understanding of the dynamic regulation of functionally opposing YAP transcriptional complexes in mammalian cells.
The phosphatase Bph and peptidyl-prolyl isomerase PrsA are required for gelatinase expression and activity in Enterococcus faecalis
Authors: Willett Julia L. E. , Robertson Ethan B. , Dunny Gary M. 
Published: April 2022

AbstractEnterococcus faecalis is a common commensal bacterium in the gastrointestinal tract as well as a frequent nosocomial pathogen. The secreted metalloprotease gelatinase (GelE) is an important E. faecalis virulence factor that contributes to numerous cellular activities such as autolysis, biofilm formation, and biofilm-associated antibiotic resistance. Expression of gelE has been extensively studied and is regulated by the Fsr quorum-sensing system. Here, we identify two additional factors regulating gelatinase expression and activity in E. faecalis OG1RF. The Bph phosphatase is required for expression of gelE in an Fsr-dependent manner. Additionally, the membrane-anchored protein foldase PrsA is required for GelE activity, but not fsr or gelE gene expression. Disrupting prsA also leads to increased antibiotic sensitivity in biofilms independent of the loss of GelE activity. Together, our results expand the model for gelatinase production in E. faecalis, which has important implications for fundamental studies of GelE function in Enterococcus and also E. faecalis pathogenesis.ImportanceIn Enterococcus faecalis, gelatinase (GelE) is a virulence factor that is also important for biofilm formation and interactions with other microbes as well as the host immune system. The long-standing model for GelE production is that the Fsr quorum sensing system positively regulates expression of gelE. Here, we update that model by identifying two additional factors that contribute to gelatinase production. The biofilm-associated Bph phosphatase regulates the expression of gelE through Fsr, and the peptidyl-prolyl isomerase PrsA is required for production of active GelE through an Fsr-independent mechanism. This provides important insight into how regulatory networks outside of the fsr locus coordinate expression of gelatinase.
Collateral sensitivity as a strategy to suppress resistance emergence: the challenge of diverse evolutionary pathways
Authors: Mandt Rebecca E.K. , Luth Madeline R. , Tye Mark A. , Mazitschek Ralph , Ottilie Sabine , Winzeler Elizabeth A. , Lafuente-Monasterio Maria Jose , Gamo Francisco Javier , Wirth Dyann F. , Lukens Amanda K. 
Published: April 2022

AbstractDrug resistance remains a major obstacle to malaria control and eradication efforts, necessitating the development of novel therapeutic strategies to treat this disease. Drug combinations based on collateral sensitivity, wherein resistance to one drug causes increased sensitivity to the partner drug, have been proposed as an evolutionary strategy to suppress the emergence of resistance in pathogen populations. In this study, we explore collateral sensitivity between compounds targeting the Plasmodium dihydroorotate dehydrogenase (DHODH). We profiled the cross-resistance and collateral sensitivity phenotypes of several DHODH mutant lines to a diverse panel of DHODH inhibitors. We focus on one compound, TCMDC-125334, which was active against all mutant lines tested, including the DHODH C276Y line, which arose in selections with the clinical candidate DSM265. We found that selection of the DHODH C276Y mutant with TCMDC-125334 yielded additional genetic changes in the dhodh locus. These double mutant parasites exhibited decreased sensitivity to TCMDC-125334 and were highly resistant to DSM265. Finally, we tested whether collateral sensitivity could be exploited to suppress the emergence of resistance in the context of combination treatment by exposing wildtype parasites to both DSM265 and TCMDC-125334 simultaneously. This selected for parasites with a DHODH V532A mutation which were cross-resistant to both compounds and were as fit as the wildtype parent in vitro. The emergence of these cross-resistant, evolutionarily fit parasites highlights the mutational flexibility of the DHODH enzyme.
Dissecting the conformational complexity and flipping mechanism of a prokaryotic heme transporter
Authors: Wu Di , Mehdipour Ahmad R , Finke Franziska , Goojani Hojjat G , Groh Roan R , Grund Tamara N , Reichhart Thomas MB , Zimmermann Rita , Welsch Sonja , Bald Dirk , Shepherd Mark , Hummer Gerhard , Safarian Schara 
Published: April 2022

AbstractIron-bound cyclic tetrapyrroles (hemes) are key redox-active cofactors in membrane-integrated oxygen reductases and other bioenergetic enzymes. However, the mechanisms of heme transport and insertion into respiratory chain complexes remain unclear. Here, we used a combination of cellular, biochemical, structural and computational methods to resolve ongoing controversies around the function of the heterodimeric bacterial ABC transporter CydDC. We provide multi-level evidence that CydDC is a heme transporter required for assembly and functional maturation of cytochrome bd, a pharmaceutically relevant drug target. Our systematic single-particle cryo-EM approach combined with atomistic molecular dynamics simulations provides detailed insight into the conformational landscape of CydDC during substrate binding and occlusion. Our simulations reveal that heme binds laterally from the membrane space to the transmembrane region of CydDC, enabled by a highly asymmetrical inward-facing CydDC conformation. During the binding process, heme propionates interact with positively charged residues on the surface and later in the substrate-binding pocket of the transporter, causing the heme orientation to flip 180 degrees. The membrane-accessible heme entry site of CydDC is primarily controlled by the conformational plasticity of CydD transmembrane helix 4, the extended cytoplasmic segment of which also couples heme confinement to a rotational movement of the CydC nucleotide-binding domain. Our cryo-EM data highlight that this signal transduction mechanism is necessary to drive conformational transitions toward occluded and outward-facing states.One Sentence SummaryThe heterodimeric bacterial ABC transporter CydDC is a heme flippase essential for the functional maturation of cytochrome bd.
The structure of the F420-dependent sulfite-detoxifying enzyme from Methanococcales reveals a prototypical sulfite-reductase with assimilatory traits
Authors: Jespersen Marion , Pierik Antonio J. , Wagner Tristan 
Published: April 2022

AbstractThe coenzyme F420-dependent sulfite reductase (Fsr group I) protects hydrogenotrophic methanogens, one of the main contributors in worldwide methane emission, from toxic sulfite. Fsr is a single peptide composed of a F420H2-oxidase and a novel class of sulfite reductase. Both catalytic domains have been proposed to be the ancestors of modern F420-oxido/reductases and dissimilatory/assimilatory sulfite reductases. Here, we describe the X-ray crystal structures of Fsr natively isolated from Methanocaldococcus jannaschii (MjFsr) and Methanothermococcus thermolithotrophicus (MtFsr), respectively refined to 2.30 Å and 1.55 Å resolution. In both organisms, Fsr oligomerizes as a 280-kDa homotetramer, where each siroheme–[4Fe–4S] is catalytically active, in contrast to dissimilatory homologues. The siroheme–[4Fe–4S], embedded in the sulfite reductase domain, is electronically connected to the flavin in the F420H2-oxidase domain by five [4Fe–4S]-clusters. EPR spectroscopy determined the redox potentials of these [4Fe–4S]2+/1+ clusters (−435 to -275 mV), through which electrons flow from FAD to the siroheme–[4Fe–4S]2+/1+ (siroheme, -114 mV; [4Fe–4S] -445 mV). The electron relay is mainly organized by two inserted ferredoxin modules, which stabilize the higher degree of oligomerization. While the F420H2-oxidase part is similar to the β-subunit of F420-reducing hydrogenases, the sulfite reductase domain is structurally analogous to dissimilatory sulfite reductases, whereas its siroheme–[4Fe–4S] cofactor is bound in the same way as in assimilatory ones. Accordingly, the reaction of MtFsr is unidirectional, reducing sulfite or nitrite with F420H2. Our results provide the first structural insights into this unique fusion, a snapshot of a primitive sulfite reductase that turns a poison into an elementary block of Life.
Comprehensive multiomic profiling of somatic mutations in malformations of cortical development
Authors: Chung Changuk , Yang Xiaoxu , Bae Taejeong , Vong Keng Ioi , Mittal Swapnil , Donkels Catharina , Phillips H. Westley , Marsh Ashley P. L. , Breuss Martin W. , Ball Laurel L. , Bernardino Garcia Camila Araújo , George Renee D. , Gu Jing , Xu Mingchu , Barrows Chelsea , James Kiely N. , Stanley Valentina , Nidhiry Anna , Khoury Sami , Howe Gabrielle , Riley Emily , Xu Xin , Copeland Brett , Wang Yifan , Kim Se Hoon , Kang Hoon-Chul , Schulze-Bonhage Andreas , Haas Carola A. , Urbach Horst , Prinz Marco , Gardner Corrine , Gurnett Christina A. , Sattar Shifteh , Nespeca Mark , Gonda David D. , Imai Katsumi , Takahashi Yukitoshi , Chen Robert , Tsai Jin-Wu , Conti Valerio , Guerrini Renzo , Devinsky Orrin , Silva Wilson A. , Machado Helio R. , Mathern Gary W. , Abyzov Alexej , Baldassari Sara , Baulac Stéphanie ,   Focal Cortical Dysplasia Neurogenetics Consortium,   Brain Somatic Mosaicism Network, Gleeson Joseph G. 
Published: April 2022

AbstractMalformations of cortical development (MCD) are neurological conditions displaying focal disruption of cortical architecture and cellular organization arising during embryogenesis, largely from somatic mosaic mutations. Identifying the genetic causes of MCD has been a challenge, as mutations remain at low allelic fractions in brain tissue resected to treat epilepsy. Here, we report a genetic atlas from 317 brain resections, identifying 69 mutated genes through intensive profiling of somatic mutations, combining whole-exome and targeted-amplicon sequencing with functional validation and single-cell sequencing. Genotype-phenotype correlation analysis elucidated specific MCD gene sets associating distinct pathophysiological and clinical phenotypes. The unique spatiotemporal expression patterns identified by comparing single-nucleus transcriptional sequences of mutated genes in control and patient brains implicate critical roles in excitatory neurogenic pools during brain development, and in promoting neuronal hyperexcitability after birth.
Cancer-associated fibroblast-derived ROR2 induces WNT/PCP activation and polarized migration in receiving gastric cancer cells
Authors: Rogers Sally , Zhang Chengting , Anagnostidis Vasilis , Fishel Melissa , Gielen Fabrice , Scholpp Steffen 
Published: April 2022

AbstractBone marrow-derived mesenchymal stem cells and cancer-associated fibroblasts in the tumor-stromal environment have been linked to cancer progression in many studies. These fibroblasts provide signaling factors to the tumor cells that promote proliferation, survival, invasion, and metastasis. One signaling pathway influencing tumor cell behavior is the WNT/Planar Cell Polarity (PCP) signaling in gastric cancer. Here, we show that the gastric tumor cell line, AGS, can respond to the PCP ligand WNT5A, however, express a very low level of the bona-fide WNT/PCP receptor, ROR2. At the same time, we find that CAF display long filopodia and had significantly higher levels of ROR2 than normal gastric fibroblasts. By high-resolution imaging, we observe a direct, cytoneme-mediated transfer of a complex containing ROR2 and WNT5A from CAF to the gastric cancer cells. The amount of ROR2 transferred correlated with JNK signaling in receiving cells, showing a direct requirement for receptor transfer. Co-culture of AGS with CAF expressing a dominant-negative form of ROR2 exhibited reduced actin polarization and migration compared to wild-type CAF. Furthermore, induction of migration via paracrine ROR2 transfer was observed in a zebrafish in vivo model. These unexpected findings demonstrate a fresh role in the direct transfer of a Wnt receptor from a signal-producing cell to a receiving cell and explain the mechanism by which gastric cancer cells expressing low levels of ROR2 can respond to a WNT5A-high tumor microenvironment.