bluuebunny/crossref_metadata_2025_split huggingface dataset:
Evaluation of PRKACA as a Therapeutic Target for Fibrolamellar Carcinoma
Authors: Schalm Stefanie S. , O’Hearn Erin , Wilson Kevin , LaBranche Timothy P. , Silva Grace , Zhang Zhuo , DiPietro Lucian , Bifulco Neil , Woessner Richard , Stransky Nicolas , Sappal Darshan , Campbell Robert , Lobbardi Riadh , Palmer Michael , Kim Joseph , Ye Chaoyang , Dorsch Marion , Lengauer Christoph , Guzi Timothy , Kadambi Vivek , Garner Andrew , Hoeflich Klaus P. 
Published: February 2022

AbstractBackground & AimsFibrolamellar carcinoma (FLC) is a rare, difficult-to-treat liver cancer primarily affecting pediatric and adolescent patients, and for which precision medicine approaches have historically not been possible. The DNAJB1-PRKACA gene fusion was identified as a driver of FLC pathogenesis. We aimed to assess whether FLC tumors maintain dependency on this gene fusion and determine if PRKACA is a viable therapeutic target.MethodsFLC patient-derived xenograft (PDX) shRNA cell lines were implanted subcutaneously into female NOD-SCID mice and tumors were allowed to develop prior to randomization to doxycycline (to induce knockdown) or control groups. Tumor development was assessed every 2 days. To assess the effect of treatment with novel selective PRKACA small molecule kinase inhibitors, BLU0588 and BLU2864, FLC PDX tumor cells were implanted subcutaneously into NOD-SCID mice and tumors allowed to develop. Mice were randomized to treatment (BLU0588 and BLU2864, orally, once daily) or control groups and tumor size determined as above.ResultsKnockdown of DNAJB1-PRKACA reversed a FLC-specific gene signature and reduced PDX tumor growth in mice compared to the control group. Furthermore, FLC PDX tumor growth was significantly reduced with BLU0588 and BLU2864 treatment versus control (P = 0.003 and P = 0.0005, respectively).ConclusionsWe demonstrated, using an inducible knockdown and small molecule approaches, that FLC PDX tumors were dependent upon DNAJB1-PRKACA fusion activity. In addition, this study serves as a proof-of-concept that PRKACA is a viable therapeutic target for FLC and warrants further investigation.
Orally active mGluR2/3 metabotropic antagonist pro-drug mimics the beneficial effects of physical exercise on neurogenesis, behavior, and exercise-related molecular pathways in an Alzheimer’s disease mouse model
Authors: Perez-Garcia Georgina , Bicak Mesude , Buros Jacqueline , Haure-Mirande Jean-Vianney , Perez Gissel M. , Otero-Pagan Alena , Gama Sosa Miguel A. , De Gasperi Rita , Sano Mary , Gage Fred H. , Barlow Carrolee , Dudley Joel T. , Glicksberg Benjamin S. , Wang Yanzhuang , Readhead Benjamin , Ehrlich Michelle E. , Elder Gregory A. , Gandy Sam 
Published: February 2022

AbstractModulation of physical activity represents an important intervention that may delay, slow, or prevent mild cognitive impairment (MCI) or dementia due to Alzheimer’s disease (AD). One mechanism proposed to underlie the beneficial effect of physical exercise involves the apparent stimulation of adult hippocampal neurogenesis (AHN). BCI-838 is a pro-drug whose active metabolite BCI-632 is an antagonist at the group II metabotropic glutamate receptor (mGluR2/3). We previously demonstrated that administration of BCI-838 to a mouse model of cerebrovascular accumulation of oligomeric AβE22Q (APPE693Q= “Dutch APP”) reduced learning behavior impairment and anxiety, both of which are associated with the phenotype of the Dutch APP mice. Here we show that (i) administration of BCI-838, physical exercise, or a combination of BCI-838 and physical exercise enhanced AHN in a four-month old mouse model of AD amyloid pathology (APPKM670/671NL/ PSEN1Δexon9 = APP/PS1), (ii) administration of BCI-838 alone or associated with physical exercise led to stimulation of AHN and improvement in both spatial and recognition memory, (iii) significantly, the hippocampal dentate gyrus transcriptome of APP/PS1 mice following BCI-838 treatment up-regulated brain-derived neurotrophic factor (BDNF), PIK3C2A of the PI3K-MTOR pathway, and metabotropic glutamate receptors, and down-regulated EIF5A of ketamine-modulating mTOR activity, and finally (iv) qPCR findings validate a significantly strong association between increased BDNF levels and BCI-838 treatment. Our study points to BCI-838 as a safe and orally active compound capable of mimicking the beneficial effect of exercise on AHN, learning behavior, and anxiety in a mouse model of AD neuropathology.
Three-dimensional analysis and in vivo imaging for sperm release and transport in the murine seminiferous tubule
Authors: Kanazawa Yuta , Omotehara Takuya , Nakata Hiroki , Hirashima Tsuyoshi , Itoh Masahiro 
Published: February 2022

AbstractIntroductionSpermatozoa released from Sertoli cells must be transported to the epididymis. However, the contribution of the peristaltic motion in the seminiferous tubule to sperm release and transport remains unclear. We, therefore, investigated luminal flow and movements in the seminiferous tubules by three-dimensional analysis and in vivo imaging.Materials and MethodsSerial testicular sections were cut in 5-μm-thick and 50-μm-interval and stained by PAS-hematoxylin. After the three-dimensional reconstruction of the seminiferous tubules, the localization of the flowing spermatozoa and stages observed in the sections were recorded in each reconstructed tubule. The luminal movements in the seminiferous tubule were observed by in vivo imaging using a fluorescent-reporter mouse and two-photon excitation microscopy system.ResultsFlowing spermatozoa were mainly scattered in the lumina at stage VII/VIII, and clustered spermatozoa were also found in some regions. The clustered spermatozoa were observed at zero to two regions in each seminiferous tubule. Flowing spermatozoa were also found in the opposite direction to the rete testis. The flagellum direction of the spermatozoa attached to the seminiferous epithelium was reversed within a few seconds to a few tens of seconds when observed by in vivo imaging. The epithelium at the inner curve of the seminiferous tubule moved more actively and attached fewer spermatozoa compared to that at the outer curve.DiscussionThis study revealed the presence of repeatedly reversed luminal flow in the seminiferous tubule. Such movements are suggested to help the sperm release from the Sertoli cells and the following aggregation of the released spermatozoa.
Mechanistic insights into intramembrane proteolysis by E. coli site-2 protease homolog RseP
Authors: Imaizumi Yuki , Takanuki Kazunori , Miyake Takuya , Takemoto Mizuki , Hirata Kunio , Hirose Mika , Oi Rika , Kobayashi Tatsuya , Miyoshi Kenichi , Aruga Rie , Yokoyama Tatsuhiko , Katagiri Shizuka , Matsuura Hiroaki , Iwasaki Kenji , Kato Takayuki , Kaneko Mika K. , Kato Yukinari , Tajiri Michiko , Akashi Satoko , Nureki Osamu , Hizukuri Yohei , Akiyama Yoshinori , Nogi Terukazu 
Published: February 2022

AbstractSite-2 proteases are a conserved family of intramembrane proteases that cleave transmembrane substrates to regulate signal transduction and maintain proteostasis. Here, we elucidated crystal structures of inhibitor-bound forms of bacterial site-2 proteases including E. coli RseP. Our observations are consistent with a rearrangement of the RseP domains surrounding the active center to expose the substrate-binding site where a conserved electrostatic linkage between the transmembrane and membrane-associated domains mediates the conformational changes, suggesting that RseP has a gating mechanism to regulate substrate entry. Mutational analysis also supports that the substrate transmembrane helix is unwound by strand addition to the intramembrane β sheet and is clamped at the active center for efficient cleavage. Furthermore, this substrate accommodation mechanism appears to be common across distinct intramembrane proteases.
Domain expansion and functional diversification in vertebrate reproductive proteins
Authors: Rivera Alberto M. , Wilburn Damien B. , Swanson Willie J. 
Published: February 2022

AbstractThe rapid evolution of fertilization proteins can result in remarkable diversity in their structure and function. Many of the proteins in vertebrate egg coats contain copies (1-6) of the ZP-N domain. These ZP-N domains can facilitate multiple reproduction functions, including species-specific sperm recognition. We integrated phylogenetics and machine learning to investigate how ZP-N domains diversified in structure and function. The most C-terminal ZP-N domain of each paralog is associated with another domain type (ZP-C) which together form a “ZP module.” All modular ZP-N domains were phylogenetically distinct from non-modular or free ZP-N domains. Machine learning-based classification identified 8 residues that form a stabilizing network in modular ZP-N domains that is absent in free domains. Positive selection was identified in some free ZP-N domains. Our findings suggest that purifying selection has conserved an essential structural core in modular ZP-N domains, while free N-terminal domains have been able to experience functionally diversify.
Exploring the possible role of hybridization in the evolution of photosynthetic pathways in Flaveria (Asteraceae), the prime model of C4 photosynthesis evolution
Authors: Morales-Briones Diego F. , Kadereit Gudrun 
Published: February 2022

AbstractFlaveria (Asteraceae) is the prime model for the study of C4 photosynthesis evolution and seems to support a stepwise acquisition of the pathway through C3-C4 intermediate phenotypes, still existing in Flaveria today. Molecular phylogenies of Flaveria based on concatenated data matrices are currently used to reconstruct the complex sequence of trait shifts during C4 evolution. To assess the possible role of hybridization in C4 evolution in Flaveria, we re-analyzed transcriptome data of 17 Flaveria species to infer the extent of gene tree discordance and possible reticulation events. We found massive gene tree discordance as well as reticulation along the backbone and within clades containing C3-C4 intermediate and C4-like species. An early hybridization event between two C3 species might have triggered C4 evolution in the genus. The clade containing all C4 species plus the C4-like species F. vaginata and F. palmeri is robust but of hybrid origin involving F. angustifolia and F. sonorensis (both C3-C4 intermediate) as parental lineages. Hybridization seems to be a driver of C4 evolution in Flaveria and likely promoted the fast acquisition of C4 traits. This new insight can be used in further exploring C4 evolution and can inform C4 bioengineering efforts.
Selective inhibition of protein secretion by abrogating receptor-coat interactions during ER export
Authors: Gomez-Navarro Natalia , Maldutyte Julija , Poljak Kristina , Peak-Chew Sew-Yeu , Orme Jonathon , Bisnett Brittany J. , Lamb Caitlin H. , Boyce Michael , Gianni Davide , Miller Elizabeth A. 
Published: February 2022

AbstractProtein secretion is an essential cellular process that permits cell growth, movement and communication. Traffic of proteins within the eukaryotic secretory pathway is mediated by transport intermediates that bud from one compartment, populated with appropriate cargo proteins, and fuse with a downstream compartment to deliver their contents. Here, we explore the possibility that protein secretion can be selectively inhibited by perturbing protein-protein interactions that drive capture into transport vesicles. Human PCSK9 is a major determinant of cholesterol metabolism, whose secretion is mediated by a specific cargo adaptor of the ER export machinery, SEC24A. We map a series of protein-protein interactions between PCSK9, its ER export receptor, SURF4, and SEC24A, that mediate secretion of PCSK9. We show that the interaction between SURF4 and SEC24A can be inhibited by 4-PBA, a small molecule that occludes a cargo-binding domain of SEC24. This inhibition reduces secretion of PCSK9 and additional SURF4 clients that we identify by mass spectrometry, leaving other secreted cargoes unaffected. We propose that selective small molecule inhibition of cargo recognition by SEC24 is a potential therapeutic intervention for atherosclerosis and other diseases that are modulated by secreted proteins.
G protein-coupled estrogen receptor activation by Bisphenol-A disrupts protection from apoptosis conferred by estrogen receptors ERα and ERβ in pancreatic beta cells
Authors: Babiloni-Chust Ignacio , dos Santos Reinaldo S. , Medina-Gali Regla M. , Perez-Serna Atenea A. , Encinar José-Antonio , Martinez-Pinna Juan , Gustafsson Jan-Ake , Marroqui Laura , Nadal Angel 
Published: February 2022

Abstract17β-estradiol protects pancreatic β-cells from apoptosis via the estrogen receptors ERα, ERβ and GPER. Conversely, the endocrine disruptor Bisphenol-A (BPA), which exerts multiple effects in this cell type via the same estrogen receptors, increased basal apoptosis. The molecular initiated events that trigger these opposite actions have yet to be identified. We demonstrated that combined genetic downregulation and pharmacological blockade of each estrogen receptor increased apoptosis to a different extent. The increase in apoptosis induced by BPA was diminished by the pharmacological blockade or the genetic silencing of GPER, and it was partially reproduced by the GPER agonist G1. BPA and G1-induced apoptosis were abolished upon pharmacological inhibition, silencing of ERα and ERβ, or in dispersed islet cells from ERβ knockout (BERKO) mice. Yet, the ERα and ERβ agonists, PPT and DPN, respectively, had no effect on beta cell viability. To exert their biological actions, ERα and ERβ form homodimers and heterodimers. Molecular dynamic simulations together with proximity ligand assay and coimmunoprecipitation experiments indicated that the interaction of BPA with ERα and ERβ as well as the GPER activation by G1 decreased ERαβ heterodimers. We propose that ERαβ heterodimers play an antiapoptotic role in beta cells and that BPA- and G1-induced decrease in ERαβ heterodimers leads to beta cell apoptosis. Unveiling how different estrogenic chemicals affect the crosstalk among estrogen receptors should help to identify diabetogenic endocrine disruptors.HighlightsPharmacological blockade and gene silencing of estrogen receptors ERα, ERβ and GPER indicate that they are antiapoptotic in basal conditions.GPER activation by G1 and BPA triggered apoptosis via a crosstalk with ERα and ERβ.BPA interaction with ERα and ERβ as well as GPER activation decreased ERαβ heterodimers, which was associated to increased apoptosis.This pathway represents a novel molecular initiating event underlying the pro-apoptotic effect of BPAThe EndoC-βH1 cell line may be a valid model of human β-cells for identifying diabetogenic pollutants.
A subset of gut leukocytes have telomerase-dependent “hyper-long” telomeres and require telomerase for function in zebrafish
Authors: Ellis Pam S. , Martins Raquel R. , Thompson Emily J. , Farhat Asma , Renshaw Stephen A. , Henriques Catarina M. 
Published: February 2022

ABSTRACTTelomerase, the enzyme capable of elongating telomeres, is usually restricted in human somatic cells, which contributes to progressive telomere shortening with cell-division and ageing. Using the zebrafish model, we show that subsets gut immune cells have telomerase-dependent “hyper-long” telomeres, which we identified as being predominantly macrophages and dendritics (mpeg1.1+ and cd45+mhcII+). Notably, mpeg+ macrophages have much longer telomeres in the gut than in their haematopoietic tissue of origin, suggesting that there is a gut-specific modulation of telomerase in these cells.Moreover, we show that a subset of gut mpeg+ cells express telomerase (tert) in young WT zebrafish, but that the relative proportion of these cells decreases with ageing. Importantly, this is accompanied by telomere shortening and DNA damage responses with ageing and a telomerase-dependent decrease in expression of autophagy and immune activation markers. Finally, these telomerase-dependent molecular alterations are accompanied by impaired phagocytosis of E.coli and increased gut permeability in vivo. Together, our data show that limiting levels of telomerase lead to changes in gut immunity and gut permeability, which, together, are likely contributors to local and systemic tissue degeneration and increased susceptibility to infection with ageing.GRAPHICAL ABSTRACTOur data show that limiting levels of telomerase contribute to alterations in gut immunity, namely increased apoptosis, decreased autophagy and immune activation of mpeg+ macrophages in the gut. This is accompanied by a decreased ability to clear pathogens and increased gut permeability. Together, these are likely contributors to local and systemic tissue degeneration and susceptibility to infection with ageing.
Lipid-mediated nuclear moonlighting of cytosolic glyceraldehyde-3-phosphate dehydrogenase in plant heat response
Authors: Kim Sang-Chul , Yao Shuaibing , Zhang Qun , Wang Xuemin 
Published: February 2022

AbstractCytosolic glyceraldehyde-3-phosphate dehydrogenase (GAPC) is a glycolytic enzyme, but it undergoes stress-induced nuclear translocation for moonlighting and regulating gene expression. To elucidate how the cytosolic enzyme moves into the nuclei under stress, we show that the plasma membrane-associated phospholipase Dδ(PLDδ) and its product phosphatidic acid (PA) promote heat-induced nuclear translocation of GAPC. The GAPC nuclear accumulation and Arabidopsis seedling tolerance to heat stress were reduced in pldδ, which was restored by genetic complementation with intact PLDδ, but not with catalytically inactive enzyme. GAPC overexpression enhanced the seedling thermotolerance and the expression of heat-inducible genes, but this was not observed when GAPC was overexpressed in the pldδ background. The GAPC nuclear accumulation and seedling thermotolerance were also decreased by application with a vesicle trafficking inhibitor brefeldin A (BFA) or zinc that inhibited the PA-GAPC interaction. Heat stress elevated PA levels in nuclei from wild-type, but not from pldδ and BFA-treated plants. Lipid labeling and fluorescence resonance energy transfer analyses demonstrated heat-induced nuclear co-localization of PA and GAPC, which was impaired by BFA or zinc treatment. Taken together, our data suggest that PLDδ-produced PA mediates nuclear translocation of GAPC via lipid-protein interaction and vesicle trafficking for plants to cope with heat.One sentence summaryThe lipid mediator phosphatidic acid produced by a plasma membrane-associated phospholipase D mediates the nuclear moonlighting of cytosolic glyceraldehyde-3-phosphate dehydrogenase under heat.
Active site center redesign increases protein stability preserving catalysis
Authors: Romero-Romero Maria Luisa , Garcia-Seisdedos Hector , Ibarra-Molero Beatriz 
Published: February 2022

ABSTRACTThe stabilization of natural proteins is a long-standing desired goal in protein engineering. Optimizing the hydrophobicity of the protein core often results in extensive stability enhancements. However, the presence of totally or partially buried catalytic charged residues, essential for protein function, has limited the applicability of this strategy. Here, focusing on the thioredoxin, we aimed to augment protein stability by removing buried charged residues in the active site without loss of catalytic activity. To this end, we performed a charged-to-hydrophobic substitution of a buried and functional group, resulting in a significant stability increase yet abolishing catalytic activity. Then, to simulate the catalytic role of the buried ionizable group, we designed a combinatorial library of variants targeting a set of seven surface residues adjacent to the active site. Notably, more than 50% of the library variants restored, to some extent, the catalytic activity. The combination of experimental study of 2% of the library with the prediction of the whole mutational space by Partial Least-squares regression revealed that a single point mutation at the protein surface is sufficient to fully restore the catalytic activity without thermostability cost. As a result, we engineered one of the highest thermal stability reported for a protein with a natural occurring fold (138 °C). Further, our hyperstable variant preserves the catalytic activity both in vitro and in vivo.SIGNIFICANCEThe major driving force of protein folding is the hydrophobic effect, and increasing the protein core hydrophobicity essentially increases protein stability. Active sites often contain buried ionizable groups, which can be essential for function but dramatically reduce protein stability. Thus, increasing the protein core hydrophobicity cannot be applied to enzyme active sites without a functional cost. We propose a method to enhance protein stability by overcoming this obstacle. We show that catalytic properties of buried charges can be mimicked with surface mutations, thus paving the way to unlock the optimization of the hydrophobic core to stabilize enzymes.
Modifications of lipoic arm by reactive nitrogen species regulate α-ketoacid dehydrogenases
Authors: Seim Gretchen L. , Fang Zixiang , Pagliarini David J. , Fan Jing 
Published: February 2022

AbstractMitochondrial α-ketoacid dehydrogenases, including the pyruvate dehydrogenase complex (PDHC) and the oxoglutarate dehydrogenase complex (OGDC), are a family of multi-subunit enzyme complexes that use a lipoic arm to transfer an acyl group to coenzyme A (CoA). The regulation of α-ketoacid dehydrogenases plays crucial roles in mitochondrial metabolism and cellular energy homeostasis. We previously found that PDHC and OGDC become profoundly inhibited in macrophages upon classical activation, causing substantial remodeling of the TCA cycle. This inhibition was driven by the loss of the catalytically active lipoic moiety; however, the molecular mechanism causing this loss was not clear. Here we show that reactive nitrogen species (RNS), which are produced by activated macrophages, can cause a series of thiol-modifications to the lipoic arm that inactivate PDHC and OGDC. CoA-SNO, the non-enzymatic product between RNS and the E2 subunit’s natural substrate CoA, plays a key role in efficiently delivering RNS mediated modifications onto the lipoic arm. This work reveals a new biochemical mechanism capable of substantially regulating mitochondrial α-ketoacid dehydrogenases, which has potential relevance for a range of physiological and pathological conditions.