The 麻豆社区 麻豆社区 Publications database contains details of all publications resulting from our research groups and  Pre-prints by 麻豆社区 authors can be viewed on the 麻豆社区's . We believe that free and open access to the outputs of publicly鈥恌unded research offers significant social and economic benefits, as well as aiding the development of new research. We are working to provide Open Access to as many publications as possible and these can be identified below by the padlock icon. Where this hasn't been possible, subscriptions may be required to view the full text.

Open Access
Young KA, Mulholland KE, Wojdyla K, Casanova SA, Lai T, Fearnley GW, Antrobus R, Barton PR, Anderson KR, Biggins L, Andrews SR, Mahler-Araujo B, Sharpe HJ Signalling,Bioinformatics

PTPRK is a receptor tyrosine phosphatase linked to the regulation of growth factor signalling and tumour suppression. It is stabilized at the plasma membrane by trans homophilic interactions upon cell-cell contact. It regulates cell-cell adhesion, but is also reported to regulate numerous cancer-associated signalling pathways. However, its signalling mechanism remains to be determined. Here, we find that PTPRK regulates cell adhesion signalling, suppresses invasion and promotes collective, directed migration in colorectal cancer cells. In vivo, PTPRK supports recovery from inflammation-induced colitis. In addition, we confirm that PTPRK functions as a tumour suppressor in the mouse colon and in colorectal cancer xenografts. PTPRK regulates growth factor and adhesion signalling, and suppresses epithelial to mesenchymal transition (EMT). Contrary to the prevailing notion that PTPRK directly dephosphorylates EGFR, we find that PTPRK regulation of both EGFR and EMT is independent of its catalytic function. This suggests that additional adaptor and scaffold functions are important features of PTPRK signalling.

+view abstract Journal of cell science, PMID: 38904097

Open Access
Kreisinger J, Dooley J, Singh K, 膶铆啪kov谩 D, Schmiedov谩 L, Bendov谩 B, Liston A, Moudra A Immunology

Microbiome research has gained much attention in recent years as the importance of gut microbiota in regulating host health becomes increasingly evident. However, the impact of radiation on the microbiota in the murine bone marrow transplantation model is still poorly understood. In this paper, we present key findings from our study on how radiation, followed by bone marrow transplantation with or without T cell depletion, impacts the microbiota in the ileum and caecum. Our findings show that radiation has different effects on the microbiota of the two intestinal regions, with the caecum showing increased interindividual variation, suggesting an impaired ability of the host to regulate microbial symbionts, consistent with the Anna Karenina principle. Additionally, we observed changes in the ileum composition, including an increase in bacterial taxa that are important modulators of host health, such as and . In contrast, radiation in the caecum was associated with an increased abundance of several common commensal taxa in the gut, including and . Finally, we found that high doses of radiation had more substantial effects on the caecal microbiota of the T-cell-depleted group than that of the non-T-cell-depleted group. Overall, our results contribute to a better understanding of the complex relationship between radiation and the gut microbiota in the context of bone marrow transplantation and highlight the importance of considering different intestinal regions when studying microbiome responses to environmental stressors.

+view abstract Frontiers in microbiology, PMID: 38903788

Open Access
Moli猫re A, Park JYC, Goyala A, Vayndorf EM, Zhang B, Hsiung KC, Jung Y, Kwon S, Statzer C, Meyer D, Nguyen R, Chadwick J, Thompson MA, Schumacher B, Lee SV, Essmann CL, MacArthur MR, Kaeberlein M, David D, Gems D, Ewald CY Signalling

Little is known about the possibility of reversing age-related biological changes when they have already occurred. To explore this, we have characterized the effects of reducing insulin/IGF-1 signaling (IIS) during old age. Reduction of IIS throughout life slows age-related decline in diverse species, most strikingly in the nematode Caenorhabditis elegans. Here we show that even at advanced ages, auxin-induced degradation of DAF-2 in single tissues, including neurons and the intestine, is still able to markedly increase C. elegans lifespan. We describe how reversibility varies among senescent changes. While senescent pathologies that develop in mid-life were not reversed, there was a rejuvenation of the proteostasis network, manifesting as a restoration of the capacity to eliminate otherwise intractable protein aggregates that accumulate with age. Moreover, resistance to several stressors was restored. These results support several new conclusions. (1) Loss of resilience is not solely a consequence of pathologies that develop in earlier life. (2) Restoration of proteostasis and resilience by inhibiting IIS is a plausible cause of the increase in lifespan. And (3), most interestingly, some aspects of the age-related transition from resilience to frailty can be reversed to a certain extent. This raises the possibility that the effect of IIS and related pathways on resilience and frailty during aging in higher animals might possess some degree of reversibility.

+view abstract GeroScience, PMID: 38900346

Open Access
Burton OT, Bricard O, Tareen S, Gergelits V, Andrews S, Biggins L, Roca CP, Whyte C, Junius S, Brajic A, Pasciuto E, Ali M, Lemaitre P, Schlenner SM, Ishigame H, Brown BD, Dooley J, Liston A Immunology,Bioinformatics

The tissues are the site of many important immunological reactions, yet how the immune system is controlled at these sites remains opaque. Recent studies have identified Foxp3 regulatory T (Treg) cells in non-lymphoid tissues with unique characteristics compared with lymphoid Treg cells. However, tissue Treg cells have not been considered holistically across tissues. Here, we performed a systematic analysis of the Treg cell population residing in non-lymphoid organs throughout the body, revealing shared phenotypes, transient residency, and common molecular dependencies. Tissue Treg cells from different non-lymphoid organs shared T聽cell receptor (TCR) sequences, with functional capacity to drive multi-tissue Treg cell entry and were tissue-agnostic on tissue homing. Together, these results demonstrate that the tissue-resident Treg cell pool in most non-lymphoid organs, other than the gut, is largely constituted by broadly self-reactive Treg cells, characterized by transient multi-tissue migration. This work suggests common regulatory mechanisms may allow pan-tissue Treg cells to safeguard homeostasis across the body.

+view abstract Immunity, PMID: 38897202

Open Access
Corujo-Simon E, Bates LE, Yanagida A, Jones K, Clark S, von Meyenn F, Reik W, Nichols J Epigenetics

To implant in the uterus, mammalian embryos form blastocysts comprising trophectoderm (TE) surrounding an inner cell mass (ICM), confined to the polar region by the expanding blastocoel. The mode of implantation varies between species. Murine embryos maintain a single layered TE until they implant in the characteristic thick deciduum, whereas human blastocysts attach via polar TE directly to the uterine wall. Using immunofluorescence (IF) of rapidly isolated ICMs, blockade of RNA and protein synthesis in whole embryos, or 3D visualization of immunostained embryos, we provide evidence of multi-layering in human polar TE before implantation. This may be required for rapid uterine invasion to secure the developing human embryo and initiate formation of the placenta. Using sequential fluorescent labeling, we demonstrate that the majority of inner TE in human blastocysts arises from existing outer cells, with no evidence of conversion from the ICM in the context of the intact embryo.

+view abstract Developmental cell, PMID: 38889726

脕lvarez-S谩nchez A, Grinat J, Doria-Borrell P, Mellado-L贸pez M, Pedrera-Alc贸cer 脡, Malenchini M, Meseguer S, Hemberger M, P茅rez-Garc铆a V Epigenetics

The glycosylphosphatidylinositol (GPI) biosynthetic pathway in the endoplasmic reticulum (ER) is crucial for generating GPI-anchored proteins (GPI-APs), which are translocated to the cell surface and play a vital role in cell signaling and adhesion. This study focuses on two integral components of the GPI pathway, the PIGL and PIGF proteins, and their significance in trophoblast biology. We show that GPI pathway mutations impact on placental development impairing the differentiation of the syncytiotrophoblast (SynT), and especially the SynT-II layer, which is essential for the establishment of the definitive nutrient exchange area within the placental labyrinth. CRISPR/Cas9 knockout of Pigl and Pigf in mouse trophoblast stem cells (mTSCs) confirms the role of these GPI enzymes in syncytiotrophoblast differentiation. Mechanistically, impaired GPI-AP generation induces an excessive unfolded protein response (UPR) in the ER in mTSCs growing in stem cell conditions, akin to what is observed in human preeclampsia.聽Upon differentiation, the impairment of the GPI pathway hinders the induction of WNT signaling for early SynT-II development.聽Remarkably, the transcriptomic profile of Pigl- and Pigf-deficient cells separates human patient placental samples into preeclampsia and control groups, suggesting an involvement of Pigl and Pigf in establishing a preeclamptic gene signature. Our study unveils the pivotal role of GPI biosynthesis in early placentation and uncovers a new preeclampsia gene expression profile associated with mutations in the GPI biosynthesis pathway, providing novel molecular insights into placental development with implications for enhanced patient stratification and timely interventions.

+view abstract Cellular and molecular life sciences : CMLS, PMID: 38819479

Open Access
Valsakumar D, Voigt P Epigenetics

Nucleosomes constitute the fundamental building blocks of chromatin. They are comprised of DNA wrapped around a histone octamer formed of two copies each of the four core histones H2A, H2B, H3, and H4. Nucleosomal histones undergo a plethora of posttranslational modifications that regulate gene expression and other chromatin-templated processes by altering chromatin structure or by recruiting effector proteins. Given their symmetric arrangement, the sister histones within a nucleosome have commonly been considered to be equivalent and to carry the same modifications. However, it is now clear that nucleosomes can exhibit asymmetry, combining differentially modified sister histones or different variants of the same histone within a single nucleosome. Enabled by the development of novel tools that allow generating asymmetrically modified nucleosomes, recent biochemical and cell-based studies have begun to shed light on the origins and functional consequences of nucleosomal asymmetry. These studies indicate that nucleosomal asymmetry represents a novel regulatory mechanism in the establishment and functional readout of chromatin states. Asymmetry expands the combinatorial space available for setting up complex sets of histone marks at individual nucleosomes, regulating multivalent interactions with histone modifiers and readers. The resulting functional consequences of asymmetry regulate transcription, poising of developmental gene expression by bivalent chromatin, and the mechanisms by which oncohistones deregulate chromatin states in cancer. Here, we review recent progress and current challenges in uncovering the mechanisms and biological functions of nucleosomal asymmetry.

+view abstract Biochemical Society transactions, PMID: 38778762

Open Access
Kruger RE, Frum T, Brumm AS, Hickey SL, Niakan KK, Aziz F, Shammami MA, Roberts JG, Ralston A Epigenetics

Bone Morphogenic Protein (BMP) signaling plays an essential and highly conserved role in embryo axial patterning in animal species. However, in mammalian embryos, which develop inside the mother, early development includes a preimplantation stage, which does not occur in externally developing embryos. During preimplantation, the epiblast is segregated from extraembryonic lineages that enable implantation and development in utero. Yet, the requirement for BMP signaling in is imprecisely defined in mouse early embryos. Here, we show that, in contrast to prior reports, BMP signaling (SMAD1/5/9 phosphorylation) is not detectable until implantation when it is detected in the primitive endoderm - an extraembryonic lineage. Moreover, preimplantation development appears normal following deletion of maternal and zygotic Smad4, an essential effector of canonical BMP signaling. In fact, mice lacking maternal Smad4 are viable. Finally, we uncover a new requirement for zygotic Smad4 in epiblast scaling and cavitation immediately after implantation, via a mechanism involving FGFR/ERK attenuation. Altogether, our results demonstrate no role for BMP4/SMAD4 in the first lineage decisions during mouse development. Rather, multi-pathway signaling among embryonic and extraembryonic cell types drives epiblast morphogenesis post-implantation.

+view abstract Development (Cambridge, England), PMID: 38752427

Open Access
Shah P, Hill R, Dion C, Clark SJ, Abakir A, Willems J, Arends MJ, Garaycoechea JI, Leitch HG, Reik W, Crossan GP Epigenetics

Mutations in DNA damage response (DDR) factors are associated with human infertility, which affects up to 15% of the population. The DDR is required during germ cell development and meiosis. One pathway implicated in human fertility is DNA translesion synthesis (TLS), which allows replication impediments to be bypassed. We find that TLS is essential for pre-meiotic germ cell development in the embryo. Loss of the central TLS component, REV1, significantly inhibits the induction of human PGC-like cells (hPGCLCs). This is recapitulated in mice, where deficiencies in TLS initiation (Rev1 or Pcna) or extension (Rev7鈥) result in a鈥>鈥150-fold reduction in the number of primordial germ cells (PGCs) and complete sterility. In contrast, the absence of TLS does not impact the growth, function, or homeostasis of somatic tissues. Surprisingly, we find a complete failure in both activation of the germ cell transcriptional program and in DNA demethylation, a critical step in germline epigenetic reprogramming. Our findings show that for normal fertility, DNA repair is required not only for meiotic recombination but for progression through the earliest stages of germ cell development in mammals.

+view abstract Nature communications, PMID: 38702312

Open Access
Azzi C, Rayon T Signalling, Epigenetics

Temporal control is central to deploy and coordinate genetic programs during development. At present, there is limited understanding of the molecular mechanisms that govern the duration and speed of developmental processes. Timing mechanisms may run in parallel and/or interact with each other to integrate temporal signals throughout the organism. In this piece, we consider findings on the extrinsic control of developmental tempo and discuss the intrinsic roles of cell cycle, metabolic rates, protein turnover, and post-transcriptional mechanisms in the regulation of tempo during neural development.

+view abstract Current opinion in genetics & development, PMID: 38648722

Open Access
Abnizova I, Stapel C, Boekhorst RT, Lee JTH, Hemberg M Epigenetics

Regulation of transcription is central to the emergence of new cell types during development, and it often involves activation of genes via proximal and distal regulatory regions. The activity of regulatory elements is determined by transcription factors (TFs) and epigenetic marks, but despite extensive mapping of such patterns, the extraction of regulatory principles remains challenging.

+view abstract BMC biology, PMID: 38600550

Open Access
Adamowski M, Sharma Y, Molcan T, Wo艂odko K, Kelsey G, Galv茫o AM Epigenetics

Obesity is associated with increased ovarian inflammation and the establishment of leptin resistance. We presently investigated the role of impaired leptin signalling on transcriptional regulation in granulosa cells (GCs) collected from genetically obese mice. Furthermore, we characterised the association between ovarian leptin signalling, the activation of the NOD-like receptor protein 3 (NLRP3) inflammasome and macrophage infiltration in obese mice. After phenotype characterisation, ovaries were collected from distinct group of animals for protein and mRNA expression analysis: (i) mice subjected to a diet-induced obesity (DIO) protocol, where one group was fed a high-fat diet (HFD) and another a standard chow diet (CD) for durations of 4 or 16聽weeks; (ii) mice genetically deficient in the long isoform of the leptin receptor (ObRb; db/db); (iii) mice genetically deficient in leptin (ob/ob); and (iv) mice rendered pharmacologically hyperleptinemic (LEPT). Next, GCs from antral follicles isolated from db/db and ob/ob mice were subjected to transcriptome analysis. Transcriptional analysis revealed opposing profiles in genes associated with steroidogenesis and prostaglandin action between the genetic models, despite the similarities in body weight. Furthermore, we observed no changes in the mRNA and protein levels of NLRP3 inflammasome components in the ovaries of db/db mice or in markers of M1 and M2 macrophage infiltration. This contrasted with the downregulation of NLRP3 inflammasome components and M1 markers in ob/ob and 16-wk HFD-fed mice. We concluded that leptin signalling regulates NLRP3 inflammasome activation and the expression of M1 markers in the ovaries of obese mice in an ObRb-dependent and ObRb-independent manner. Furthermore, we found no changes in the expression of leptin signalling and NLRP3 inflammasome genes in GCs from db/db and ob/ob mice, which was associated with no effects on macrophage infiltration genes, despite the dysregulation of genes associated with steroidogenesis in homozygous obese db/db. Our results suggest that: (i) the crosstalk between leptin signalling, NLRP3 inflammasome and macrophage infiltration takes place in ovarian components other than the GC compartment; and (ii) transcriptional changes in GCs from homozygous obese ob/ob mice suggest structural rearrangement and organisation, whereas in db/db mice the impairment in steroidogenesis and secretory activity.

+view abstract Scientific reports, PMID: 38580672

Cheetham M, Davies D, Hall C, Petersen CC, Schulte R, Walker R Flow Cytometry

Cell sorting is a technique commonly used in academic and biotechnology laboratories in order to separate out cells or particles of interest from heterogeneous populations. Cell sorters use the same principles as flow cytometry analyzers, but instead of cell populations passing to the waste of the instrument, they can be collected for further studies including DNA sequencing as well as other genomic, in vitro and in vivo experiments. This chapter aims to give an overview of cell sorting, the different types of cell sorters, details on how a cell sorter works, as well as protocols that are useful when embarking on a journey with cell sorting.

+view abstract Methods in molecular biology (Clifton, N.J.), PMID: 38526785

Superti-Furga G, Agostinho M, Bury J, Cook S, Durinx C, Ender A, van Luenen H, Lund AH, Medema RH, Mi膮czy艅ska M, Nickel D, Pelicci PG, Puisieux A, Ripatti S, Sander M, Schubeler D, Serrano L, Sommer T, Sonne-Hansen K, Toman膷谩k P, Vives J, Vontas J, Bettencourt-Dias M Signalling, Epigenetics, Immunology

The diverse range of organizations contributing to the global research ecosystem is believed to enhance the overall quality and resilience of its output. Mid-sized autonomous research institutes, distinct from universities, play a crucial role in this landscape. They often lead the way in new research fields and experimental methods, including those in social and organizational domains, which are vital for driving innovation. The EU-LIFE alliance was established with the goal of fostering excellence by developing and disseminating best practices among European biomedical research institutes. As directors of the 15 EU-LIFE institutes, we have spent a decade comparing and refining our processes. Now, we are eager to share the insights we've gained. To this end, we have crafted this Charter, outlining 10 principles we deem essential for research institutes to flourish and achieve ground-breaking discoveries. These principles, detailed in the Charter, encompass excellence, independence, training, internationality and inclusivity, mission focus, technological advancement, administrative innovation, cooperation, societal impact, and public engagement. Our aim is to inspire the establishment of new institutes that adhere to these principles and to raise awareness about their significance. We are convinced that they should be viewed a crucial component of any national and international innovation strategies.

+view abstract FEBS letters, PMID: 38514456

Panova V, Richard AC Immunology

Upon lymphocyte stimulation, accumulation of intracellular NAD(H) reflects the strength of antigen receptor signals and controls the rate of cell cycle entry and proliferation (see related Research Article by Turner .).

+view abstract Science immunology, PMID: 38489351

Giaccari C, Cecere F, Argenziano L, Pagano A, Galvao A, Acampora D, Rossi G, Hay Mele B, Acurzio B, Coonrod S, Cubellis MV, Cerrato F, Andrews S, Cecconi S, Kelsey G, Riccio A Epigenetics

Maternal inactivation of genes encoding components of the subcortical maternal complex (SCMC) and its associated member, PADI6, generally results in early embryo lethality. In humans, SCMC gene variants were found in the healthy mothers of children affected by multilocus imprinting disturbances (MLID). However, how the SCMC controls the DNA methylation required to regulate imprinting remains poorly defined. We generated a mouse line carrying a missense variant that was identified in a family with Beckwith-Wiedemann syndrome and MLID. If homozygous in female mice, this variant resulted in interruption of embryo development at the two-cell stage. Single-cell multiomic analyses demonstrated defective maturation of mutant oocytes and incomplete DNA demethylation, down-regulation of zygotic genome activation (ZGA) genes, up-regulation of maternal decay genes, and developmental delay in two-cell embryos developing from mutant oocytes but little effect on genomic imprinting. Western blotting and immunofluorescence analyses showed reduced levels of UHRF1 in oocytes and abnormal localization of DNMT1 and UHRF1 in both oocytes and zygotes. Treatment with 5-azacytidine reverted DNA hypermethylation but did not rescue the developmental arrest of mutant embryos. Taken together, this study demonstrates that PADI6 controls both nuclear and cytoplasmic oocyte processes that are necessary for preimplantation epigenetic reprogramming and ZGA.

+view abstract Genes & development, PMID: 38453481

Chen X, Tsvetkov AS, Shen HM, Isidoro C, Ktistakis NT, Linkermann A, Koopman WJH, Simon HU, Galluzzi L, Luo S, Xu D, Gu W, Peulen O, Cai Q, Rubinsztein DC, Chi JT, Zhang DD, Li C, Toyokuni S, Liu J, Roh JL, Dai E, Juhasz G, Liu W, Zhang J, Yang M, Liu J, Zhu LQ, Zou W, Piacentini M, Ding WX, Yue Z, Xie Y, Petersen M, Gewirtz DA, Mandell MA, Chu CT, Sinha D, Eftekharpour E, Zhivotovsky B, Besteiro S, Gabrilovich DI, Kim DH, Kagan VE, Bayir H, Chen GC, Ayton S, L眉nemann JD, Komatsu M, Krautwald S, Loos B, Baehrecke EH, Wang J, Lane JD, Sadoshima J, Yang WS, Gao M, M眉nz C, Thumm M, Kampmann M, Yu D, Lipinski MM, Jones JW, Jiang X, Zeh HJ, Kang R, Klionsky DJ, Kroemer G, Tang D Signalling

Macroautophagy/autophagy is a complex degradation process with a dual role in cell death that is influenced by the cell types that are involved and the stressors they are exposed to. Ferroptosis is an iron-dependent oxidative form of cell death characterized by unrestricted lipid peroxidation in the context of heterogeneous and plastic mechanisms. Recent studies have shed light on the involvement of specific types of autophagy (e.g. ferritinophagy, lipophagy, and clockophagy) in initiating or executing ferroptotic cell death through the selective degradation of anti-injury proteins or organelles. Conversely, other forms of selective autophagy (e.g. reticulophagy and lysophagy) enhance the cellular defense against ferroptotic damage. Dysregulated autophagy-dependent ferroptosis has implications for a diverse range of pathological conditions. This review aims to present an updated definition of autophagy-dependent ferroptosis, discuss influential substrates and receptors, outline experimental methods, and propose guidelines for interpreting the results.

+view abstract Autophagy, PMID: 38442890

Open Access
Nakanoh S, Sham K, Ghimire S, Mohorianu I, Rayon T, Vallier L Signalling, Epigenetics

Mechanisms specifying amniotic ectoderm and surface ectoderm are unresolved in humans due to their close similarities in expression patterns and signal requirements. This lack of knowledge hinders the development of protocols to accurately model human embryogenesis. Here, we developed a human pluripotent stem cell model to investigate the divergence between amniotic and surface ectoderms. In the established culture system, cells differentiated into functional amnioblast-like cells. Single-cell RNA sequencing analyses of amnioblast differentiation revealed an intermediate cell state with enhanced surface ectoderm gene expression. Furthermore, when the differentiation started at the confluent condition, cells retained the expression profile of surface ectoderm. Collectively, we propose that human amniotic ectoderm and surface ectoderm are specified along a common nonneural ectoderm trajectory based on cell density. Our culture system also generated extraembryonic mesoderm-like cells from the primed pluripotent state. Together, this study provides an integrative understanding of the human nonneural ectoderm development and a model for embryonic and extraembryonic human development around gastrulation.

+view abstract Science advances, PMID: 38427729

Briffa A, Menon G, Movilla Miangolarra A, Howard M Epigenetics

Understanding the mechanistic basis of epigenetic memory has proven to be a difficult task due to the underlying complexity of the systems involved in its establishment and maintenance. Here, we review the role of computational modeling in helping to unlock this complexity, allowing the dissection of intricate feedback dynamics. We focus on three forms of epigenetic memory encoded in gene regulatory networks, DNA methylation, and histone modifications and discuss the important advantages offered by plant systems in their dissection. We summarize the main modeling approaches involved and highlight the principal conceptual advances that the modeling has enabled through iterative cycles of predictive modeling and experiments. Lastly, we discuss remaining gaps in our understanding and how intertwined theory and experimental approaches might help in their resolution. Expected final online publication date for the , Volume 75 is May 2024. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

+view abstract Annual review of plant biology, PMID: 38424070

Weatherdon L, Stuart K, Cassidy MA, de la G谩ndara AM, Okkenhaug H, Muellener M, Mckenzie G, Cook SJ, Gilley R Signalling,Imaging

The RAS-regulated RAF-MEK1/2-ERK1/2 signalling pathway is activated in cancer due to mutations in RAS proteins (especially KRAS), BRAF, CRAF, MEK1 and MEK2. Whilst inhibitors of KRASG12C (lung adenocarcinoma) and BRAF and MEK1/2 (melanoma and colorectal cancer) are clinically approved, acquired resistance remains a problem. Consequently, the search for new inhibitors (especially of RAS proteins), new inhibitor modalities and regulators of this pathway, which may be new drug targets, continues and increasingly involves cell-based screens with small molecules or genetic screens such as RNAi, CRISPR or Protein Interference. Here we describe cell lines that exhibit doxycycline-dependent expression KRASG12V or BRAFV600E and harbour a stably integrated EGR1:EmGFP reporter gene that can be detected by flow cytometry, high-content microscopy or immunoblotting. KRASG12V or BRAFV600E-driven EmGFP expression is inhibited by MEK1/2 or ERK1/2 inhibitors (MEKi and ERKi). BRAFi inhibit BRAFV600E-driven EmGFP expression but enhance the response to KRASG12V, recapitulating paradoxical activation of wild type RAF proteins. In addition to small molecules, expression of iDab6, encoding a RAS-specific antibody fragment inhibited KRASG12V- but not BRAFV600E-driven EmGFP expression. Finally, substitution of EmGFP for a bacterial nitroreductase gene allowed KRASG12V or BRAFV600E to drive cell death in the presence of a pro-drug, which may allow selection of pathway inhibitors that promote survival. These cell lines should prove useful for cell-based screens to identify new regulators of KRAS- or BRAF-dependent ERK1/2 signalling (drug target discovery) as well as screening or triaging 'hits' from drug discovery screens.

+view abstract The Biochemical journal, PMID: 38381045

Rebak AS, Hendriks IA, Elsborg JD, Buch-Larsen SC, Nielsen CH, Terslev L, Kirsch R, Damgaard D, Doncheva NT, Lennartsson C, Ryk忙r M, Jensen LJ, Christophorou MA, Nielsen ML Epigenetics

Despite the importance of citrullination in physiology and disease, global identification of citrullinated proteins, and the precise targeted sites, has remained challenging. Here we employed quantitative-mass-spectrometry-based proteomics to generate a comprehensive atlas of citrullination sites within the HL60 leukemia cell line following differentiation into neutrophil-like cells. We identified 14,056 citrullination sites within 4,008 proteins and quantified their regulation upon inhibition of the citrullinating enzyme PADI4. With this resource, we provide quantitative and site-specific information on thousands of PADI4 substrates, including signature histone marks and transcriptional regulators. Additionally, using peptide microarrays, we demonstrate the potential clinical relevance of certain identified sites, through distinct reactivities of antibodies contained in synovial fluid from anti-CCP-positive and anti-CCP-negative people with rheumatoid arthritis. Collectively, we describe the human citrullinome at a systems-wide level, provide a resource for understanding citrullination at the mechanistic level and link the identified targeted sites to rheumatoid arthritis.

+view abstract Nature structural & molecular biology, PMID: 38321148

Park AY, Leney-Greene M, Lynberg M, Gabrielski JQ, Xu X, Schwarz B, Zheng L, Balasubramaniyam A, Ham H, Chao B, Zhang Y, Matthews HF, Cui J, Yao Y, Kubo S, Chanchu JM, Morawski AR, Cook SA, Jiang P, Ravell JC, Cheng YH, George A, Faruqi A, Pagalilauan AM, Bergerson JRE, Ganesan S, Chauvin SD, Aluri J, Edwards-Hicks J, Bohrnsen E, Tippett C, Omar H, Xu L, Butcher GW, Pascall J, Karakoc-Aydiner E, Kiykim A, Maecker H, Tezcan 陌, Esenboga S, Heredia RJ, Akata D, Tekin S, Kara A, Kuloglu Z, Unal E, Kendirli T, Dogu F, Karabiber E, Atkinson TP, Cochet C, Filhol O, Bosio CM, Davis MM, Lifton RP, Pearce EL, Daumke O, Aytekin C, 艦ahin GE, Aksu A脺, Uzel G, Koneti Rao V, Sari S, Boztug K, Cagdas D, Haskologlu S, Ikinciogullari A, Schwefel D, Vilarinho S, Baris S, Ozen A, Su HC, Lenardo MJ Immunology

Preserving cells in a functional, non-senescent state is a major goal for extending human healthspans. Model organisms reveal that longevity and senescence are genetically controlled, but how genes control longevity in different mammalian tissues is unknown. Here, we report a new human genetic disease that causes cell senescence, liver and immune dysfunction, and early mortality that results from deficiency of GIMAP5, an evolutionarily conserved GTPase selectively expressed in lymphocytes and endothelial cells. We show that GIMAP5 restricts the pathological accumulation of long-chain ceramides (CERs), thereby regulating longevity. GIMAP5 controls CER abundance by interacting with protein kinase CK2 (CK2), attenuating its ability to activate CER synthases. Inhibition of CK2 and CER synthase rescues GIMAP5-deficient T cells by preventing CER overaccumulation and cell deterioration. Thus, GIMAP5 controls longevity assurance pathways crucial for immune function and healthspan in mammals.

+view abstract Nature immunology, PMID: 38172257

Open Access
Sunshine HL, Cicchetto AC, Kaczor-Urbanowicz KE, Ma F, Pi D, Symons C, Turner M, Shukla V, Christofk HR, Vallim TA, Iruela-Arispe ML Immunology

Vascular morphogenesis requires a delicate gradient of Notch signaling controlled, in part, by the distribution of ligands (Dll4 and Jagged1). How Jagged1 (JAG1) expression is compartmentalized in the vascular plexus remains unclear. Here, we show that Jag1 mRNA is a direct target of zinc-finger protein 36 (ZFP36), an RNA-binding protein involved in mRNA decay that we find robustly induced by vascular endothelial growth factor (VEGF). Endothelial cells lacking ZFP36 display high levels of JAG1 and increase angiogenic sprouting in聽vitro. Furthermore, mice lacking Zfp36 in endothelial cells display mispatterned and increased levels of JAG1 in the developing retinal vascular plexus. Abnormal levels of JAG1 at the sprouting front alters NOTCH1 signaling, increasing the number of tip cells, a phenotype that is rescued by imposing haploinsufficiency of Jag1. Our findings reveal an important feedforward loop whereby VEGF stimulates ZFP36, consequently suppressing Jag1 to enable adequate levels of Notch signaling during sprouting angiogenesis.

+view abstract Cell reports, PMID: 38157296

Wang Y, Wakelam MJO, Bankaitis VA, McDermott MI Signalling

Phospholipase D (PLD) hydrolyses phosphatidylcholine (PtdCho) to produce free choline and the critically important lipid signaling molecule phosphatidic acid (PtdOH). Since the initial discovery of PLD activities in plants and bacteria, PLDs have been identified in a diverse range of organisms spanning the taxa. While widespread interest in these proteins grew following the discovery of mammalian isoforms, research into the PLDs of non-mammalian organisms has revealed a fascinating array of functions ranging from roles in microbial pathogenesis, to the stress responses of plants and the developmental patterning of flies. Furthermore, studies in non-mammalian model systems have aided our understanding of the entire PLD superfamily, with translational relevance to human biology and health. Increasingly, the promise for utilization of non-mammalian PLDs in biotechnology is also being recognized, with widespread potential applications ranging from roles in lipid synthesis, to their exploitation for agricultural and pharmaceutical applications.

+view abstract Advances in biological regulation, PMID: 38081756