Endothelial tip cells are essential for VEGF-induced angiogenesis, but underlying mechanisms are elusive. The Ena/VASP protein family, consisting of EVL, VASP, and Mena, plays a pivotal role in axon guidance. Given that axonal growth cones and endothelial tip cells share many common features, from the morphological to the molecular level, we investigated the role of Ena/VASP proteins in angiogenesis. EVL and VASP, but not Mena, are expressed in endothelial cells of the postnatal mouse retina. Global deletion of EVL (but not VASP) compromises the radial sprouting of the vascular plexus in mice. Similarly, endothelial-specific EVL deletion compromises the radial sprouting of the vascular plexus and reduces the endothelial tip cell density and filopodia formation. Gene sets involved in blood vessel development and angiogenesis are down-regulated in EVL-deficient P5-retinal endothelial cells. Consistently, EVL deletion impairs VEGF-induced endothelial cell proliferation and sprouting, and reduces the internalization and phosphorylation of VEGF receptor 2 and its downstream signaling via the MAPK/ERK pathway. Together, we show that endothelial EVL regulates sprouting angiogenesis via VEGF receptor-2 internalization and signaling.

Technical bias can cause lack of reproducibility. While harder to identify than other bias, it can cause consistent systemic errors in experimental data and analysis.

Esophageal squamous cell carcinoma (ESCC) is the most common subtype of esophageal cancer worldwide. The most commonly mutated gene in ESCC is TP53. Using a combinatorial genetic and carcinogenic approach, we generate a novel mouse model of ESCC expressing either mutant or null p53 and show that mutant p53 exhibits enhanced tumorigenic properties and displays a distinct genomic profile. Through RNA-seq analysis, we identify several endocytic recycling genes, including Rab Coupling Protein (Rab11-FIP1), which are significantly downregulated in mutant p53 tumor cells. In 3-dimensional (3D) organoid models, genetic knockdown of Rab11-FIP1 results in increased organoid size. Loss of Rab11-FIP1 increases tumor cell invasion in part through mutant p53 but also in an independent manner. Furthermore, loss of Rab11-FIP1 in human ESCC cell lines decreases E-cadherin expression and increases mesenchymal lineage-specific markers, suggesting induction of epithelial-mesenchymal transition (EMT). Rab11-FIP1 regulates EMT through direct inhibition of Zeb1, a key EMT transcriptional factor. Our novel findings reveal that Rab11-FIP1 regulates organoid formation, tumor cell invasion, and EMT.

Phosphorylation is one of the most dynamic and widespread post-translational modifications regulating virtually every aspect of eukaryotic cell biology. Here, we assemble a dataset from 75 independent phosphoproteomic experiments performed in our laboratory using Saccharomyces cerevisiae. We report 30,902 phosphosites identified from cells cultured in a range of DNA damage conditions and/or arrested in distinct cell cycle stages. To generate a comprehensive resource for the budding yeast community, we aggregate our dataset with the Saccharomyces Genome Database and another recently published study, resulting in over 46,000 budding yeast phosphosites. With the goal of enhancing the identification of functional phosphorylation events, we perform computational positioning of phosphorylation sites on available 3D protein structures and systematically identify events predicted to regulate protein complex architecture. Results reveal hundreds of phosphorylation sites mapping to or near protein interaction interfaces, many of which result in steric or electrostatic "clashes" predicted to disrupt the interaction. With the advancement of Cryo-EM and the increasing number of available structures, our approach should help drive the functional and spatial exploration of the phosphoproteome.

Genome architecture and chromatin dynamics govern the fate and identify of a cell. Recent advances in mapping chromatin landscapes offer valuable tools for the acquisition of accurate information regarding chromatin dynamics. Here we discuss recent findings linking chromatin dynamics to cell fate control. Specifically, chromatin undergoes a binary off/on switch during iPSC reprogramming, closing and opening loci occupied by somatic and pluripotency transcription factors, respectively. This logic of a binary off/on switch may also be operational in cell fate control during normal development and implies that further approaches could potentially be developed to direct cell fate changes both in vitro and in vivo.

Bactericidal antibiotics are powerful agents due to their ability to convert essential bacterial functions into lethal processes. However, many important bacterial pathogens are remarkably tolerant against bactericidal antibiotics due to inducible damage repair responses. The cell wall damage response two-component system VxrAB of the gastrointestinal pathogen Vibrio cholerae promotes high-level β-lactam tolerance and controls a gene network encoding highly diverse functions, including negative control over multiple iron uptake systems. How this system contributes to tolerance is poorly understood. Here, we show that β-lactam antibiotics cause an increase in intracellular free iron levels and collateral oxidative damage, which is exacerbated in the ∆vxrAB mutant. Mutating major iron uptake systems dramatically increases ∆vxrAB tolerance to β-lactams. We propose that VxrAB reduces antibiotic-induced toxic iron and concomitant metabolic perturbations by downregulating iron uptake transporters and show that iron sequestration enhances tolerance against β-lactam therapy in a mouse model of cholera infection. Our results suggest that a microorganism's ability to counteract diverse antibiotic-induced stresses promotes high-level antibiotic tolerance and highlights the complex secondary responses elicited by antibiotics.

Differentiation of naïve peripheral B cells into terminally differentiated plasma cells is characterized by epigenetic alterations, yet the epigenetic mechanisms that control B-cell fate remain unclear. Here, we identified a role for the histone H3K79 methyltransferase DOT1L in controlling B-cell differentiation. Mouse B cells lacking Dot1L failed to establish germinal centers (GC) and normal humoral immune responses in vivo. In vitro, activated B cells in which Dot1L was deleted showed aberrant differentiation and prematurely acquired plasma cell characteristics. Similar results were obtained when DOT1L was chemically inhibited in mature B cells in vitro. Mechanistically, combined epigenomics and transcriptomics analysis revealed that DOT1L promotes expression of a pro-proliferative, pro-GC program. In addition, DOT1L indirectly supports the repression of an anti-proliferative plasma cell differentiation program by maintaining the repression of Polycomb Repressor Complex 2 (PRC2) targets. Our findings show that DOT1L is a key modulator of the core transcriptional and epigenetic landscape in B cells, establishing an epigenetic barrier that warrants B-cell naivety and GC B-cell differentiation.

Retinoblastoma protein (RB) encoded by Rb1 is a prominent inducer of cell cycle arrest (CCA). The hormone progesterone (P4 ) promotes CCA in the uterine epithelium and previous studies indicated that P4 activates RB by reducing the phosphorylated, inactive form of RB. Here, we show that embryo implantation is impaired in uterine-specific Rb1 knockout mice. We observe persistent cell proliferation of the Rb1-deficient uterine epithelium until embryo attachment, loss of epithelial necroptosis, and trophoblast phagocytosis, which correlates with subsequent embryo invasion failure, indicating that Rb1-induced CCA and necroptosis of uterine epithelium are involved in embryo invasion. Pre-implantation P4 supplementation is sufficient to restore these defects and embryo invasion. In Rb1-deficient uterine epithelial cells, TNFα-primed necroptosis is impaired, which is rescued by the treatment with a CCA inducer thymidine or P4 through the upregulation of TNF receptor type 2. TNFα is expressed in the luminal epithelium and the embryo at the embryo attachment site. These results provide evidence that uterine Rb1-induced CCA is involved in TNFα-primed epithelial necroptosis at the implantation site for successful embryo invasion.

Proteins of the ADF/cofilin family play a central role in the disassembly of actin filaments, and their activity must be tightly regulated in cells. Recently, the oxidation of actin filaments by the enzyme MICAL1 was found to amplify the severing action of cofilin through unclear mechanisms. Using single filament experiments in vitro, we found that actin filament oxidation by MICAL1 increases, by several orders of magnitude, both cofilin binding and severing rates, explaining the dramatic synergy between oxidation and cofilin for filament disassembly. Remarkably, we found that actin oxidation bypasses the need for cofilin activation by dephosphorylation. Indeed, non-activated, phosphomimetic S3D-cofilin binds and severs oxidized actin filaments rapidly, in conditions where non-oxidized filaments are unaffected. Finally, tropomyosin Tpm1.8 loses its ability to protect filaments from cofilin severing activity when actin is oxidized by MICAL1. Together, our results show that MICAL1-induced oxidation of actin filaments suppresses their physiological protection from the action of cofilin. We propose that, in cells, direct post-translational modification of actin filaments by oxidation is a way to trigger their disassembly.

Lysine succinylation (Ksucc) is an evolutionarily conserved and widespread post-translational modification. Histone acetyltransferase 1 (HAT1) is a type B histone acetyltransferase, regulating the acetylation of both histone and non-histone proteins. However, the role of HAT1 in succinylation modulation remains unclear. Here, we employ a quantitative proteomics approach to study succinylation in HepG2 cancer cells and find that HAT1 modulates lysine succinylation on various proteins including histones and non-histones. HAT1 succinylates histone H3 on K122, contributing to epigenetic regulation and gene expression in cancer cells. Moreover, HAT1 catalyzes the succinylation of PGAM1 on K99, resulting in its increased enzymatic activity and the stimulation of glycolytic flux in cancer cells. Clinically, HAT1 is significantly elevated in liver cancer, pancreatic cancer, and cholangiocarcinoma tissues. Functionally, HAT1 succinyltransferase activity and the succinylation of PGAM1 by HAT1 play critical roles in promoting tumor progression in vitro and in vivo. Thus, we conclude that HAT1 is a succinyltransferase for histones and non-histones in tumorigenesis.

Dynamic control of ubiquitination by deubiquitinating enzymes is essential for almost all biological processes. Ubiquitin-specific peptidase 22 (USP22) is part of the SAGA complex and catalyzes the removal of mono-ubiquitination from histones H2A and H2B, thereby regulating gene transcription. However, novel roles for USP22 have emerged recently, such as tumor development and cell death. Apart from apoptosis, the relevance of USP22 in other programmed cell death pathways still remains unclear. Here, we describe a novel role for USP22 in controlling necroptotic cell death in human tumor cell lines. Loss of USP22 expression significantly delays TNFα/Smac mimetic/zVAD.fmk (TBZ)-induced necroptosis, without affecting TNFα-mediated NF-κB activation or extrinsic apoptosis. Ubiquitin remnant profiling identified receptor-interacting protein kinase 3 (RIPK3) lysines 42, 351, and 518 as novel, USP22-regulated ubiquitination sites during necroptosis. Importantly, mutation of RIPK3 K518 reduced necroptosis-associated RIPK3 ubiquitination and amplified necrosome formation and necroptotic cell death. In conclusion, we identify a novel role of USP22 in necroptosis and further elucidate the relevance of RIPK3 ubiquitination as crucial regulator of necroptotic cell death.

Energy storage and growth are coordinated in response to nutrient status of animals. How nutrient-regulated signaling pathways control these processes in vivo remains insufficiently understood. Here, we establish an atypical MAP kinase, ERK7, as an inhibitor of adiposity and growth in Drosophila. ERK7 mutant larvae display elevated triacylglycerol (TAG) stores and accelerated growth rate, while overexpressed ERK7 is sufficient to inhibit lipid storage and growth. ERK7 expression is elevated upon fasting and ERK7 mutant larvae display impaired survival during nutrient deprivation. ERK7 acts in the fat body, the insect counterpart of liver and adipose tissue, where it controls the subcellular localization of chromatin-binding protein PWP1, a growth-promoting downstream effector of mTOR. PWP1 maintains the expression of sugarbabe, encoding a lipogenic Gli-similar family transcription factor. Both PWP1 and Sugarbabe are necessary for the increased growth and adiposity phenotypes of ERK7 loss-of-function animals. In conclusion, ERK7 is an anti-anabolic kinase that inhibits lipid storage and growth while promoting survival on fasting conditions.

Metabolic reprogramming of non-cancer cells residing in a tumor microenvironment, as a result of the adaptations to cancer-derived metabolic and non-metabolic factors, is an emerging aspect of cancer-host interaction. We show that in normal and cancer-associated fibroblasts, breast cancer-secreted extracellular vesicles suppress mTOR signaling upon amino acid stimulation to globally reduce mRNA translation. This is through delivery of cancer-derived miR-105 and miR-204, which target RAGC, a component of Rag GTPases that regulate mTORC1 signaling. Following amino acid starvation and subsequent re-feeding, 13 C-arginine labeling of de novo synthesized proteins shows selective translation of proteins that cluster to specific cellular functional pathways. The repertoire of these newly synthesized proteins is altered in fibroblasts treated with cancer-derived extracellular vesicles, in addition to the overall suppressed protein synthesis. In human breast tumors, RAGC protein levels are inversely correlated with miR-105 in the stroma. Our results suggest that through educating fibroblasts to reduce and re-prioritize mRNA translation, cancer cells rewire the metabolic fluxes of amino acid pool and dynamically regulate stroma-produced proteins during periodic nutrient fluctuations.

One major factor that contributes to the virulence of Pseudomonas aeruginosa is its ability to reside and replicate unchallenged inside airway epithelial cells. The mechanism by which P. aeruginosa escapes destruction by intracellular host defense mechanisms, such as autophagy, is not known. Here, we show that the type III secretion system effector protein ExoS facilitates P. aeruginosa survival in airway epithelial cells by inhibiting autophagy in host cells. Autophagy inhibition is independent of mTOR activity, as the latter is also inhibited by ExoS, albeit by a different mechanism. Deficiency of the critical autophagy gene Atg7 in airway epithelial cells, both in vitro and in mouse models, greatly enhances the survival of ExoS-deficient P. aeruginosa but does not affect the survival of ExoS-containing bacteria. The inhibitory effect of ExoS on autophagy and mTOR depends on the activity of its ADP-ribosyltransferase domain. Inhibition of mTOR is caused by ExoS-mediated ADP ribosylation of RAS, whereas autophagy inhibition is due to the suppression of autophagic Vps34 kinase activity.

Comment on EMBO Rep. 2021 Feb 3;22(2):e50785.

Comment in EMBO Rep. 2021 Feb 3;22(2):e52180.

Advanced paternal age can have deleterious effects on various traits in the next generation. Here, we establish a paternal-aging model in mice to understand the molecular mechanisms of transgenerational epigenetics. Whole-genome target DNA methylome analyses of sperm from aged mice reveal more hypo-methylated genomic regions enriched in REST/NRSF binding motifs. Gene set enrichment analyses also reveal the upregulation of REST/NRSF target genes in the forebrain of embryos from aged fathers. Offspring derived from young mice administrated with a DNA de-methylation drug phenocopy the abnormal vocal communication of pups derived from aged fathers. In conclusion, hypo-methylation of sperm DNA can be a key molecular feature modulating neurodevelopmental programs in offspring by causing fluctuations in the expression of REST/NRSF target genes.

Although iron is required for cell proliferation, iron-dependent programmed cell death serves as a critical barrier to tumor growth and metastasis. Emerging evidence suggests that iron-mediated lipid oxidation also facilitates immune eradication of cancer. However, the regulatory mechanisms of iron metabolism in cancer remain unclear. Here we identify OTUD1 as the deubiquitinase of iron-responsive element-binding protein 2 (IREB2), selectively reduced in colorectal cancer. Clinically, downregulation of OTUD1 is highly correlated with poor outcome of cancer. Mechanistically, OTUD1 promotes transferrin receptor protein 1 (TFRC)-mediated iron transportation through deubiquitinating and stabilizing IREB2, leading to increased ROS generation and ferroptosis. Moreover, the presence of OTUD1 promotes the release of damage-associated molecular patterns (DAMPs), which in turn recruits the leukocytes and strengthens host immune response. Reciprocally, depletion of OTUD1 limits tumor-reactive T-cell accumulation and exacerbates colon cancer progression. Our data demonstrate that OTUD1 plays a stimulatory role in iron transportation and highlight the importance of OTUD1-IREB2-TFRC signaling axis in host antitumor immunity.

Comment in EMBO Rep. 2021 Apr 7;22(4):e52614.

Cell signalling governs cellular behaviour and is therefore subject to tight spatiotemporal regulation. Signalling output is modulated by specialized cell membranes and vesicles which contain unique combinations of lipids and proteins. The phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2 ), an important component of the plasma membrane as well as other subcellular membranes, is involved in multiple processes, including signalling. However, which enzymes control the turnover of non-plasma membrane PI(4,5)P2 , and their impact on cell signalling and function at the organismal level are unknown. Here, we identify Paladin as a vascular PI(4,5)P2 phosphatase regulating VEGFR2 endosomal signalling and angiogenesis. Paladin is localized to endosomal and Golgi compartments and interacts with vascular endothelial growth factor receptor 2 (VEGFR2) in vitro and in vivo. Loss of Paladin results in increased internalization of VEGFR2, over-activation of extracellular regulated kinase 1/2, and hypersprouting of endothelial cells in the developing retina of mice. These findings suggest that inhibition of Paladin, or other endosomal PI(4,5)P2 phosphatases, could be exploited to modulate VEGFR2 signalling and angiogenesis, when direct and full inhibition of the receptor is undesirable.

The rumen microbiome - a remarkable example of obligatory symbiosis with high ecological and social relevance.

Despite its limited resources, Portugal has gained a prominent position in research on malaria. Several historical and personal factors have contributed to this achievement.

Sleep homeostasis is crucial for sleep regulation. The role of epigenetic regulation in sleep homeostasis is unestablished. Previous studies showed that octopamine is important for sleep homeostasis. However, the regulatory mechanism of octopamine reception in sleep is unknown. In this study, we identify an epigenetic regulatory cascade (Stuxnet-Polycomb-Octβ2R) that modulates the octopamine receptor in Drosophila. We demonstrate that stuxnet positively regulates Octβ2R through repression of Polycomb in the ellipsoid body of the adult fly brain and that Octβ2R is one of the major receptors mediating octopamine function in sleep homeostasis. In response to octopamine, Octβ2R transcription is inhibited as a result of stuxnet downregulation. This feedback through the Stuxnet-Polycomb-Octβ2R cascade is crucial for sleep homeostasis regulation. This study demonstrates a Stuxnet-Polycomb-Octβ2R-mediated epigenetic regulatory mechanism for octopamine reception, thus providing an example of epigenetic regulation of sleep homeostasis.

Centrosomes, composed of two centrioles and pericentriolar material, organize mitotic spindles during cell division and template cilia during interphase. The first few divisions during mouse development occur without centrioles, which form around embryonic day (E) 3. However, disruption of centriole biogenesis in Sas-4 null mice leads to embryonic arrest around E9. Centriole loss in Sas-4-/- embryos causes prolonged mitosis and p53-dependent cell death. Studies in vitro discovered a similar USP28-, 53BP1-, and p53-dependent mitotic surveillance pathway that leads to cell cycle arrest. In this study, we show that an analogous pathway is conserved in vivo where 53BP1 and USP28 are upstream of p53 in Sas-4-/- embryos. The data indicate that the pathway is established around E7 of development, four days after the centrioles appear. Our data suggest that the newly formed centrioles gradually mature to participate in mitosis and cilia formation around the beginning of gastrulation, coinciding with the activation of mitotic surveillance pathway upon centriole loss.

Mutations in the nuclear trypsin-like serine protease FAM111A cause Kenny-Caffey syndrome (KCS2) with hypoparathyroidism and skeletal dysplasia or perinatally lethal osteocraniostenosis (OCS). In addition, FAM111A was identified as a restriction factor for certain host range mutants of the SV40 polyomavirus and VACV orthopoxvirus. However, because FAM111A function is poorly characterized, its roles in restricting viral replication and the etiology of KCS2 and OCS remain undefined. We find that FAM111A KCS2 and OCS patient mutants are hyperactive and cytotoxic, inducing apoptosis-like phenotypes such as disruption of nuclear structure and pore distribution, in a protease-dependent manner. Moreover, wild-type FAM111A activity causes similar nuclear phenotypes, including the loss of nuclear barrier function, when SV40 host range mutants attempt to replicate in restrictive cells. Interestingly, pan-caspase inhibitors do not block these FAM111A-induced phenotypes, implying it acts independently or upstream of caspases. In this regard, we identify nucleoporins and the associated GANP transcription/replication factor as FAM111A interactors and candidate targets. Overall, we reveal a potentially unifying mechanism through which deregulated FAM111A activity restricts viral replication and causes KCS2 and OCS.