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Tropospheric ozone, one of the most phytotoxic air pollutants, may specially impose in long-lived forest trees substantial reduction in productivity and biomass. European beech saplings grown in lysimeter around areas were used to monitor proteomic changes upon elevated ozone concentrations following four vegetation periods of exposure. A proteome study based on highly sensitive two-dimensional fluorescence difference gel electrophoresis (2-D DIGE) was performed to identify protein changes in European beech, the most important deciduous tree in Central Europe. Main emphasis was on identifying differentially expressed proteins after long-time period of ozone exposure under natural conditions rather than short-term responses or reactions under controlled conditions. Our results clearly demonstrate a response of European beech saplings to long-term ozone fumigation at the protein level. We indicate changes in the protein abundance of 142 protein spots; among them 59 were increased and 83 decreased following three years of elevated ozone exposure. As the first step, 40 proteins were identified by a homology driven mass spectrometric approach. Some of the identified proteins have been previously described in the context of short-term ozone responses in plants, indicating, at least for certain cellular functions, the congruence of plant reactions following short- and long-term ozone exposure. Under elevated ozone exposure, abundance of proteins related to the Calvin cycle and photosynthetic electron transport chain were decreased whereas the abundance of proteins regarding the carbon metabolism/catabolism were increased.

Deciphering breast cancer treatment resistance remains hindered by the lack of models that can successfully capture the four-dimensional dynamics of the tumor microenvironment. Here, we show that microextrusion bioprinting can reproducibly generate distinct cancer and stromal compartments integrating cells relevant to human pathology. Our findings unveil the functional maturation of this millimeter-sized model, showcasing the development of a hypoxic cancer core and an increased surface proliferation. Maturation was also driven by the presence of cancer-associated fibroblasts (CAF) that induced elevated microvascular-like structures complexity. Such modulation was concomitant to extracellular matrix remodeling, with high levels of collagen and matricellular proteins deposition by CAF, simultaneously increasing tumor stiffness and recapitulating breast cancer fibrotic development. Importantly, our bioprinted model faithfully reproduced response to treatment, further modulated by CAF. Notably, CAF played a protective role for cancer cells against radiotherapy, facilitating increased paracrine communications. This model holds promise as a platform to decipher interactions within the microenvironment and evaluate stroma-targeted drugs in a context relevant to human pathology., Competing Interests: All authors declare that there is no conflict of interest., (© 2024 The Authors.)

Immune checkpoint inhibitors have produced encouraging results in cancer patients. However, the majority of ß-catenin-mutated tumors have been described as lacking immune infiltrates and resistant to immunotherapy. The mechanisms by which oncogenic ß-catenin affects immune surveillance remain unclear. Herein, we highlighted the involvement of ß-catenin in the regulation of the exosomal pathway and, by extension, in immune/cancer cell communication in hepatocellular carcinoma (HCC). We showed that mutated ß-catenin represses expression of SDC4 and RAB27A , two main actors in exosome biogenesis, in both liver cancer cell lines and HCC patient samples. Using nanoparticle tracking analysis and live-cell imaging, we further demonstrated that activated ß-catenin represses exosome release. Then, we demonstrated in 3D spheroid models that activation of β-catenin promotes a decrease in immune cell infiltration through a defect in exosome secretion. Taken together, our results provide the first evidence that oncogenic ß-catenin plays a key role in exosome biogenesis. Our study gives new insight into the impact of ß-catenin mutations on tumor microenvironment remodeling, which could lead to the development of new strategies to enhance immunotherapeutic response., Competing Interests: CD, JV, AB, IM, AR, JD, MP, PB, DP, ND, MV, BB, CB, VM No competing interests declared, (© 2024, Dantzer et al.)

Bioprinting applications in the clinical field generate great interest, but developing suitable biomaterial inks for medical settings is a challenge. Placental tissues offer a promising solution due to their abundance, stability, and status as medical waste. They contain basement membrane components, have a clinical history, and support angiogenesis. This study formulates bioinks from two placental tissues, amnion (AM) and chorion (CHO), and compares their unique extracellular matrix (ECM) and growth factor compositions. Rheological properties of the bioinks are evaluated for bioprinting and maturation of human endothelial cells. Both AM and Cho-derived bioinks sustained human endothelial cell viability, proliferation, and maturation, promoting optimal vasculogenesis. These bioinks derived from human sources have significant potential for tissue engineering applications, particularly in supporting vasculogenesis. This research contributes to the advancement of tissue engineering and regenerative medicine, bringing everyone closer to clinically viable bioprinting solutions using placental tissues as valuable biomaterials., (© 2023 The Authors. Advanced Healthcare Materials published by Wiley-VCH GmbH.)

Entosis is a process that leads to the formation of cell-in-cell structures commonly found in cancers. Here, we identified entosis in hepatocellular carcinoma and the loss of Rnd3 (also known as RhoE) as an efficient inducer of this mechanism. We characterized the different stages and the molecular regulators of entosis induced after Rnd3 silencing. We demonstrated that this process depends on the RhoA/ROCK pathway, but not on E-cadherin. The proteomic profiling of entotic cells allowed us to identify LAMP1 as a protein upregulated by Rnd3 silencing and implicated not only in the degradation final stage of entosis, but also in the full mechanism. Moreover, we found a positive correlation between the presence of entotic cells and the metastatic potential of tumors in human patient samples. Altogether, these data suggest the involvement of entosis in liver tumor progression and highlight a new perspective for entosis analysis in medicine research as a novel therapeutic target., (© 2024. The Author(s).)

Male infertility is common and complex, presenting a wide range of heterogeneous phenotypes. Although about 50% of cases are estimated to have a genetic component, the underlying cause often remains undetermined. Here, from whole-exome sequencing on samples from 168 infertile men with asthenoteratozoospermia due to severe sperm flagellum, we identified homozygous ZMYND12 variants in four unrelated patients. In sperm cells from these individuals, immunofluorescence revealed altered localization of DNAH1, DNALI1, WDR66, and TTC29. Axonemal localization of ZMYND12 ortholog TbTAX-1 was confirmed using the Trypanosoma brucei model. RNAi knock-down of TbTAX-1 dramatically affected flagellar motility, with a phenotype similar to the sperm from men bearing homozygous ZMYND12 variants. Co-immunoprecipitation and ultrastructure expansion microscopy in T. brucei revealed TbTAX-1 to form a complex with TTC29. Comparative proteomics with samples from Trypanosoma and Ttc29 KO mice identified a third member of this complex: DNAH1. The data presented revealed that ZMYND12 is part of the same axonemal complex as TTC29 and DNAH1, which is critical for flagellum function and assembly in humans, and Trypanosoma . ZMYND12 is thus a new asthenoteratozoospermia-associated gene, bi-allelic variants of which cause severe flagellum malformations and primary male infertility., Competing Interests: DD, GM, CB, JB, PL, MT, JD, DR, CL, EL, ZW, JE, AA, AD, SH, RZ, SM, LH, XJ, YS, CL, NT, AS, MB, VS, CC, ZK, CA, PR, AT, MB, CC No competing interests declared, (© 2023, Dacheux, Martinez et al.)

Microglia are specialized brain-resident macrophages that arise from primitive macrophages colonizing the embryonic brain 1 . Microglia contribute to multiple aspects of brain development, but their precise roles in the early human brain remain poorly understood owing to limited access to relevant tissues 2-6 . The generation of brain organoids from human induced pluripotent stem cells recapitulates some key features of human embryonic brain development 7-10 . However, current approaches do not incorporate microglia or address their role in organoid maturation 11-21 . Here we generated microglia-sufficient brain organoids by coculturing brain organoids with primitive-like macrophages generated from the same human induced pluripotent stem cells (iMac) 22 . In organoid cocultures, iMac differentiated into cells with microglia-like phenotypes and functions (iMicro) and modulated neuronal progenitor cell (NPC) differentiation, limiting NPC proliferation and promoting axonogenesis. Mechanistically, iMicro contained high levels of PLIN2 + lipid droplets that exported cholesterol and its esters, which were taken up by NPCs in the organoids. We also detected PLIN2 + lipid droplet-loaded microglia in mouse and human embryonic brains. Overall, our approach substantially advances current human brain organoid approaches by incorporating microglial cells, as illustrated by the discovery of a key pathway of lipid-mediated crosstalk between microglia and NPCs that leads to improved neurogenesis., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

Background & Aims: Hepatocellular adenomas (HCAs) are rare, benign, liver tumours classified at the clinicopathological, genetic, and proteomic levels. The β-catenin-activated (b-HCA) subtypes harbour several mutation types in the β-catenin gene ( CTNNB1 ) associated with different risks of malignant transformation or bleeding. Glutamine synthetase is a surrogate marker of β-catenin pathway activation associated with the risk of malignant transformation. Recently, we revealed an overexpression of glutamine synthetase in the rims of exon 3 S45-mutated b-HCA and exon 7/8-mutated b-HCA compared with the rest of the tumour. A difference in vascularisation was found in this rim shown by diffuse CD34 staining only at the tumour centre. Here, we aimed to characterise this tumour heterogeneity to better understand its physiopathological involvement., Methods: Using mass spectrometry imaging, genetic, and proteomic analyses combined with laser capture microdissection, we compared the tumour centre with the tumour rim and with adjacent non-tumoural tissue., Results: The tumour rim harboured the same mutation as the tumour centre, meaning both parts belong to the same tumour. Mass spectrometry imaging showed different spectral profiles between the rim and the tumour centre. Proteomic profiling revealed the significant differential expression of 40 proteins at the rim compared with the tumour centre. The majority of these proteins were associated with metabolism, with an expression profile comparable with a normal perivenous hepatocyte expression profile., Conclusions: The difference in phenotype between the tumour centres and tumour rims of exon 3 S45-mutated b-HCA and exon 7/8-mutated b-HCA does not depend on CTNNB1 mutational status. In a context of sinusoidal arterial pathology, tumour heterogeneity at the rim harbours perivenous characteristics and could be caused by a functional peripheral venous drainage., Impact and Implications: Tumour heterogeneity was revealed in β-catenin-mutated hepatocellular adenomas (b-HCAs) via the differential expression of glutamine synthase at tumour rims. The combination of several spatial approaches (mass spectrometry imaging, genetic, and proteomic analyses) after laser capture microdissection allowed identification of a potential role for peripheral venous drainage underlying this difference. Through this study, we were able to illustrate that beyond a mutational context, many factors can downstream regulate gene expression and contribute to different clinicopathological phenotypes. We believe that the combinations of spatial analyses that we used could be inspiring for all researchers wanting to access heterogeneity information of liver tumours., Competing Interests: The authors declare that they have no conflicts of interest. Please refer to the accompanying ICMJE disclosure forms for further details., (© 2023 The Authors.)

Bronchi of chronic obstructive pulmonary disease (COPD) are the site of extensive cell infiltration, allowing persistent contact between resident cells and immune cells. Tissue fibrocytes interaction with CD8 + T cells and its consequences were investigated using a combination of in situ , in vitro experiments and mathematical modeling. We show that fibrocytes and CD8 + T cells are found in the vicinity of distal airways and that potential interactions are more frequent in tissues from COPD patients compared to those of control subjects. Increased proximity and clusterization between CD8 + T cells and fibrocytes are associated with altered lung function. Tissular CD8 + T cells from COPD patients promote fibrocyte chemotaxis via the CXCL8-CXCR1/2 axis. Live imaging shows that CD8 + T cells establish short-term interactions with fibrocytes, that trigger CD8 + T cell proliferation in a CD54- and CD86-dependent manner, pro-inflammatory cytokines production, CD8 + T cell cytotoxic activity against bronchial epithelial cells and fibrocyte immunomodulatory properties. We defined a computational model describing these intercellular interactions and calibrated the parameters based on our experimental measurements. We show the model's ability to reproduce histological ex vivo characteristics, and observe an important contribution of fibrocyte-mediated CD8 + T cell proliferation in COPD development. Using the model to test therapeutic scenarios, we predict a recovery time of several years, and the failure of targeting chemotaxis or interacting processes. Altogether, our study reveals that local interactions between fibrocytes and CD8 + T cells could jeopardize the balance between protective immunity and chronic inflammation in the bronchi of COPD patients., Competing Interests: EE, EM, JS, PH, PE, TT, JD, AL, AS, HB, MT, RH, RM, FL, PV, CC No competing interests declared, MZ MZ reports personal fees from AstraZeneca, Boehringer Ingelheim, Novartis, Chiesi, GlaxoSmithKline and non-financial support Lilly outside the submitted work, PG POG has a patent (EP 3050574: Use of plerixafor for treating and/or preventing acute exacerbations of chronic obstructive pulmonary disease) granted. POG reports grants, personal fees and non-financial support from AstraZeneca, personal fees and non-financial support from Chiesi, personal fees and non-financial support from GlaxoSmithKline, personal fees and non-financial support from Novartis, personal fees and non-financial support from Sanofi, outside the submitted work, PB PB has a patent (EP N3050574: Use of plerixafor for treating and/or preventing acute exacerbations of chronic obstructive pulmonary disease) granted. PB reports grants from AstraZeneca, Glaxo-Smith-Kline, Novartis, Chiesi, which support COBRA during the conduct of the study; grants and personal fees from AstraZeneca, BoehringerIngelheim, Novartis, personal fees and non-financial support from Chiesi, Sanofi, Menarini, outside the submitted work, ID ID has a patent (EP 3050574: Use of plerixafor for treating and/or preventing acute exacerbations of chronic obstructive pulmonary disease) granted, (© 2023, Eyraud et al.)

In mammals, about 99% of mitochondrial proteins are synthesized in the cytosol as precursors that are subsequently imported into the organelle. The mitochondrial health and functions rely on an accurate quality control of these imported proteins. Here, we show that the E3 ubiquitin ligase F box/leucine-rich-repeat protein 6 (FBXL6) regulates the quality of cytosolically translated mitochondrial proteins. Indeed, we found that FBXL6 substrates are newly synthesized mitochondrial ribosomal proteins. This E3 binds to chaperones involved in the folding and trafficking of newly synthesized peptide and to ribosomal-associated quality control proteins. Deletion of these interacting partners is sufficient to hamper interactions between FBXL6 and its substrate. Furthermore, we show that cells lacking FBXL6 fail to degrade specifically mistranslated mitochondrial ribosomal proteins. Finally, showing the role of FBXL6-dependent mechanism, FBXL6-knockout (KO) cells display mitochondrial ribosomal protein aggregations, altered mitochondrial metabolism, and inhibited cell cycle in oxidative conditions., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Fibrillin-1 is an extracellular matrix protein that assembles into microfibrils which provide critical functions in large blood vessels and other tissues. Mutations in the fibrillin-1 gene are associated with cardiovascular, ocular, and skeletal abnormalities in Marfan syndrome. Here, we reveal that fibrillin-1 is critical for angiogenesis which is compromised by a typical Marfan mutation. In the mouse retina vascularization model, fibrillin-1 is present in the extracellular matrix at the angiogenic front where it colocalizes with microfibril-associated glycoprotein-1, MAGP1. In Fbn1 C1041G/+ mice, a model of Marfan syndrome, MAGP1 deposition is reduced, endothelial sprouting is decreased, and tip cell identity is impaired. Cell culture experiments confirmed that fibrillin-1 deficiency alters vascular endothelial growth factor-A/Notch and Smad signaling which regulate the acquisition of endothelial tip cell/stalk cell phenotypes, and we showed that modulation of MAGP1 expression impacts these pathways. Supplying the growing vasculature of Fbn1 C1041G/+ mice with a recombinant C-terminal fragment of fibrillin-1 corrects all defects. Mass spectrometry analyses showed that the fibrillin-1 fragment alters the expression of various proteins including ADAMTS1, a tip cell metalloprotease and matrix-modifying enzyme. Our data establish that fibrillin-1 is a dynamic signaling platform in the regulation of cell specification and matrix remodeling at the angiogenic front and that mutant fibrillin-1-induced defects can be rescued pharmacologically using a C-terminal fragment of the protein. These findings, identify fibrillin-1, MAGP1, and ADAMTS1 in the regulation of endothelial sprouting, and contribute to our understanding of how angiogenesis is regulated. This knowledge may have critical implications for people with Marfan syndrome.

Fungal secretomes are known to contain a multitude of components involved in nutrition, cell growth or biotic interactions. Recently, extra-cellular vesicles have been identified in a few fungal species. Here, we used a multidisciplinary approach to identify and characterize extracellular vesicles produced by the plant necrotroph Botrytis cinerea . Transmission electron microscopy of infectious hyphae and hyphae grown in vitro revealed extracellular vesicles of various sizes and densities. Electron tomography showed the co-existence of ovoid and tubular vesicles and pointed to their release via the fusion of multi-vesicular bodies with the cell plasma membrane. The isolation of these vesicles and exploration of their protein content using mass spectrometry led to the identification of soluble and membrane proteins involved in transport, metabolism, cell wall synthesis and remodeling, proteostasis, oxidoreduction and traffic. Confocal microscopy highlighted the capacity of fluorescently labeled vesicles to target cells of B. cinerea , cells of the fungus Fusarium graminearum , and onion epidermal cells but not yeast cells. In addition, a specific positive effect of these vesicles on the growth of B. cinerea was quantified. Altogether, this study broadens our view on the secretion capacity of B. cinerea and its cell-to-cell communication.

Candida lusitaniae is an emerging opportunistic pathogenic yeast capable of shifting from yeast to pseudohyphae form, and it is one of the few Candida species with the ability to reproduce sexually. In this study, we showed that a dpp3 Δ mutant, inactivated for a putative pyrophosphatase, is impaired in cell separation, pseudohyphal growth and mating. The defective phenotypes were not restored after the reconstruction of a wild-type DPP3 locus, reinforcing the hypothesis of the presence of an additional mutation that we suspected in our previous study. Genetic crosses and genome sequencing identified an additional mutation in MED15 , encoding a subunit of the mediator complex that functions as a general transcriptional co-activator in Eukaryotes. We confirmed that inactivation of MED15 was responsible for the defective phenotypes by rescuing the dpp3 Δ mutant with a wild-type copy of MED15 and constructing a med15 Δ knockout mutant that mimics the phenotypes of dpp3 Δ in vitro. Proteomic analyses revealed the biological processes under the control of Med15 and involved in hyphal growth, cell separation and mating. This is the first description of the functions of MED15 in the regulation of hyphal growth, cell separation and mating, and the pathways involved in C. lusitaniae .

The Toxoplasma gondii tachyzoite is a singled-cell obligate intracellular parasite responsible for the acute phase of toxoplasmosis. This polarized cell exhibits an apical complex, a hallmark of the phylum Apicomplexa, essential for motility, invasion, and egress from the host cell. Located on the opposite end of the cell is the basal complex, an elaborated cytoskeletal structure that also plays critical roles in the lytic cycle of the parasite, being involved in motility, cell division, constriction and cytokinesis, as well as intravacuolar cell-cell communication. Nevertheless, only a few proteins of this structure have been described and functionally assessed. In this study, we used spatial proteomics to identify new basal complex components (BCC), and in situ imaging, including ultrastructure expansion microscopy, to position them. We thus confirmed the localization of nine BCCs out of the 12 selected candidates and assigned them to different sub-compartments of the basal complex, including two new domains located above the basal ring and below the posterior cup. Their functional investigation revealed that none of these BCCs are essential for parasite growth in vitro . However, one BCC is critical for constricting of the basal complex, likely through direct interaction with the class VI myosin heavy chain J (MyoJ), and for gliding motility. Four other BCCs, including a phosphatase and a guanylate-binding protein, are involved in the formation and/or maintenance of the intravacuolar parasite connection, which is required for the rosette organization and synchronicity of cell division., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Roumégous, Abou Hammoud, Fuster, Dupuy, Blancard, Salin, Robinson, Renesto, Tardieux and Frénal.)

Signaling from ciliary microdomains controls developmental processes in metazoans. Trypanosome transmission requires development and migration in the tsetse vector alimentary tract. Flagellar cAMP signaling has been linked to parasite social motility (SoMo) in vitro, yet uncovering control of directed migration in fly organs is challenging. Here we show that the composition of an adenylate cyclase (AC) complex in the flagellar tip microdomain is essential for tsetse salivary gland (SG) colonization and SoMo. Cyclic AMP response protein 3 (CARP3) binds and regulates multiple AC isoforms. CARP3 tip localization depends on the cytoskeletal protein FLAM8. Re-localization of CARP3 away from the tip microdomain is sufficient to abolish SoMo and fly SG colonization. Since intrinsic development is normal in carp3 and flam8 knock-out parasites, AC complex-mediated tip signaling specifically controls parasite migration and thereby transmission. Participation of several developmentally regulated receptor-type AC isoforms may indicate the complexity of the in vivo signals perceived., (© 2022. The Author(s).)

The fungal cell wall occupies a central place in the interaction between fungi and their environment. This study focuses on the role of the putative polysaccharide synthase Cps1 in the physiology, development and virulence of the grey mold-causing agent Botrytis cinerea . Deletion of the Bccps1 gene does not affect the germination of the conidia (asexual spores) or the early mycelial development, but it perturbs hyphal expansion after 24 h, revealing a two-phase hyphal development that has not been reported so far. It causes a severe reduction of mycelial growth in a solid medium and modifies hyphal aggregation into pellets in liquid cultures. It strongly impairs plant penetration, plant colonization and the formation of sclerotia (survival structures). Loss of the BcCps1 protein associates with a decrease in glucans and glycoproteins in the fungus cell wall and the up-accumulation of 132 proteins in the mutant's exoproteome, among which are fungal cell wall enzymes. This is accompanied by an increased fragility of the mutant mycelium, an increased sensitivity to some environmental stresses and a reduced adhesion to plant surface. Taken together, the results support a significant role of Cps1 in the cell wall biology of B. cinerea .

Combined therapy with anti-BRAF plus anti-MEK is currently used as first-line treatment of patients with metastatic melanomas harboring the somatic BRAF V600E mutation. However, the main issue with targeted therapy is the acquisition of tumor cell resistance. In a majority of resistant melanoma cells, the resistant process consists in epithelial-to-mesenchymal transition (EMT). This process called phenotype switching makes melanoma cells more invasive. Its signature is characterized by MITF low, AXL high, and actin cytoskeleton reorganization through RhoA activation. In parallel of this phenotype switching phase, the resistant cells exhibit an anarchic cell proliferation due to hyper-activation of the MAP kinase pathway. We show that a majority of human melanoma overexpress discoidin domain receptor 2 (DDR2) after treatment. The same result was found in resistant cell lines presenting phenotype switching compared to the corresponding sensitive cell lines. We demonstrate that DDR2 inhibition induces a decrease in AXL expression and reduces stress fiber formation in resistant melanoma cell lines. In this phenotype switching context, we report that DDR2 control cell and tumor proliferation through the MAP kinase pathway in resistant cells in vitro and in vivo. Therefore, inhibition of DDR2 could be a new and promising strategy for countering this resistance mechanism., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Rhinovirus (RV) infection of the bronchial epithelium is implicated in the vast majority of severe asthma exacerbations. Interestingly, the susceptibility of bronchial epithelium to RV infection is increased in persons with asthma. Bronchial smooth muscle (BSM) remodeling is an important feature of severe asthma pathophysiology, and its reduction using bronchial thermoplasty has been associated with a significant decrease in the exacerbation rate. We hypothesized that asthmatic BSM can play a role in RV infection of the bronchial epithelium. Using an original co-culture model between bronchial epithelium and BSM cells, we show that asthmatic BSM cells increase RV replication in bronchial epithelium following RV infection. These findings are related to the increased production of CCL20 by asthmatic BSM cells. Moreover, we demonstrate an original downregulation of the activity of the epithelial protein kinase RNA-activated (PKR) antiviral pathway. Finally, we identify a direct bottom-up effect of asthmatic BSM cells on bronchial epithelium susceptibility to RV infection., Competing Interests: Declaration of interests, (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: Diffuse Midline Glioma, H3K27M-mutant (DMG) is a rare, highly aggressive pediatric tumor affecting the brainstem, and is one of the deadliest cancers. Currently available treatment options such as chemotherapy and radiotherapy do only modestly prolong survival. In this pathology, H3K27 mutations deregulate Polycomb Repressive Complex 2 (PRC2), including enzymatic activity of EZH2, which is therefore under investigation as a therapeutic target., Methods: We used a chemical EZH2 inhibitor, GSK126, small interfering RNAs, and a CRISPR/Cas9 knockout approaches in a series of DMG tumor cell lines to investigate metabolic treatment responses by proteomic analysis. A combination strategy was elaborated and studied in primary and established DMG cells, spheroid 3D cultures, and in vivo in a chick chorio-allantoic membrane DMG assay and an orthotopic intracranial DMG mouse model., Results: GSK126 shows significant ( P < .05-.001) inhibitory effects in in vitro cell proliferation assays and induces apoptosis. Chemical targeting of EZH2 induced expression of proteins implicated in cholesterol metabolism. Low-dose GSK126 treatment together with statins revealed strong growth inhibition in combinatorial treatments, but not in single treatments, both in DMG cells in vitro , in DMG spheroid cultures, and in chick and mouse in vivo models ( P < .05). All statistical tests were two-sided., Conclusions: Our results reveal an unexpected GSK126-inducible sensitivity to cholesterol biosynthesis inhibitors in highly aggressive pediatric glioma that warrants further evaluation as treatment strategy. This combinatorial therapy should have few side effects because of the low doses used to achieve significant anti-tumor activity., (© The Author(s) 2022. Published by Oxford University Press, the Society for Neuro-Oncology and the European Association of Neuro-Oncology.)

Antibody-mediated rejection (ABMR) is the leading cause of allograft failure in kidney transplantation. Its histological hallmark is represented by lesions of glomerulitis i.e., inflammatory cells within glomeruli. Current therapies for ABMR fail to prevent chronic allograft damage i.e., transplant glomerulopathy, leading to allograft loss. We used laser microdissection of glomeruli from formalin-fixed allograft biopsies combined with mass spectrometry-based proteomics to describe the proteome modification of 11 active and 10 chronic active ABMR cases compared to 8 stable graft controls. Of 1335 detected proteins, 77 were deregulated in glomerulitis compared to stable grafts, particularly involved in cellular stress mediated by interferons type I and II, leukocyte activation and microcirculation remodeling. Three proteins extracted from this protein profile, TYMP, WARS1 and GBP1, showed a consistent overexpression by immunohistochemistry in glomerular endothelial cells that may represent relevant markers of endothelial stress during active ABMR. In transplant glomerulopathy, 137 proteins were deregulated, which favor a complement-mediated mechanism, wound healing processes through coagulation activation and ultimately a remodeling of the glomerular extracellular matrix, as observed by light microscopy. This study brings novel information on glomerular proteomics of ABMR in kidney transplantation, and highlights potential targets of diagnostic and therapeutic interest.

Calcium ions (Ca 2+ ) play important and diverse roles in the regulation of autophagy, cell death and differentiation. Here, we investigated the impact of Ca 2+ in regulating acute promyelocytic leukemia (APL) cell fate in response to the anti-cancer agent all-trans retinoic acid (ATRA). We observed that ATRA promotes calcium entry through store-operated calcium (SOC) channels into acute promyelocytic leukemia (APL) cells. This response is associated with changes in the expression profiles of ORAI1 and STIM1, two proteins involved in SOC channels activation, as well as with a significant upregulation of several key proteins associated to calcium signaling. Moreover, ATRA treatment of APL cells led to a significant activation of calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK2) and its downstream effector AMP-activated protein kinase (AMPK), linking Ca 2+ signaling to autophagy. Pharmacological inhibition of SOC channels and CAMKK2 enhanced ATRA-induced cell differentiation and death. Altogether, our results unravel an ATRA-elicited signaling pathway that involves SOC channels/CAMKK2 activation, induction of autophagy, inhibition of cellular differentiation and suppression of cell death. We suggest that SOC channels and CAMKK2 may constitute novel drug targets for potentiating the anti-cancer effect of ATRA in APL patients.

Background: Bronchial smooth muscle (BSM) remodelling in asthma is related to an increased mitochondrial biogenesis and enhanced BSM cell proliferation in asthma. Since mitochondria produce the highest levels of cellular energy and fatty acid β-oxidation is the most powerful way to produce ATP, we hypothesised that, in asthmatic BSM cells, energetic metabolism is shifted towards the β-oxidation of fatty acids., Objectives: We aimed to characterise BSM cell metabolism in asthma both in vitro and ex vivo to identify a novel target for reducing BSM cell proliferation., Methods: 21 asthmatic and 31 non-asthmatic patients were enrolled. We used metabolomic and proteomic approaches to study BSM cells. Oxidative stress, ATP synthesis, fatty acid endocytosis, metabolite production, metabolic capabilities, mitochondrial networks, cell proliferation and apoptosis were assessed on BSM cells. Fatty acid content was assessed in vivo using matrix-assisted laser desorption/ionisation spectrometry imaging., Results: Asthmatic BSM cells were characterised by an increased rate of mitochondrial respiration with a stimulated ATP production and mitochondrial β-oxidation. Fatty acid consumption was increased in asthmatic BSM both in vitro and ex vivo . Proteome remodelling of asthmatic BSM occurred via two canonical mitochondrial pathways. The levels of carnitine palmitoyl transferase (CPT)2 and low-density lipoprotein (LDL) receptor, which internalise fatty acids through mitochondrial and cell membranes, respectively, were both increased in asthmatic BSM cells. Blocking CPT2 or LDL receptor drastically and specifically reduced asthmatic BSM cell proliferation., Conclusion: This study demonstrates a metabolic switch towards mitochondrial β-oxidation in asthmatic BSM and identifies fatty acid metabolism as a new key target to reduce BSM remodelling in asthma., Competing Interests: Conflict of interest: P. Esteves reports other (postdoctoral salary) from Fondation pour la Recherche Médicale, grants from Fondation Bordeaux université (FGLMR/AVAD), during the conduct of the study. Conflict of interest: L. Blanc reports other (salary) from Fondation pour la Recherche Médicale, during the conduct of the study. Conflict of interest: A. Celle has nothing to disclose. Conflict of interest: I. Dupin has a delivered patent (EP 15152886 “New compositions and methods of treating and/or preventing chronic obstructive pulmonary disease”), and a submitted patent (EP 20173595.8 “New compositions and methods of treating COVID-19 disease”), all outside the submitted work. Conflict of interest: E. Maurat has nothing to disclose. Conflict of interest: N. Amoedo has nothing to disclose. Conflict of interest: G. Cardouat has nothing to disclose. Conflict of interest: O. Ousova has nothing to disclose. Conflict of interest: L. Gales has nothing to disclose. Conflict of interest: F. Bellvert has nothing to disclose. Conflict of interest: H. Begueret has nothing to disclose. Conflict of interest: M. Thumerel has nothing to disclose. Conflict of interest: J-W. Dupuy has nothing to disclose. Conflict of interest: N. Desbenoit has nothing to disclose. Conflict of interest: R. Marthan has nothing to disclose. Conflict of interest: P-O. Girodet reports grants, personal fees and non-financial support from AstraZeneca, personal fees and non-financial support from Chiesi, GlaxoSmithKline, Novartis and Sanofi, outside the submitted work. Conflict of interest: R. Rossignol has nothing to disclose. Conflict of interest: P. Berger reports grants from Fondation pour la Recherche Médicale, during the conduct of the study; grants and personal fees from Novartis, grants, personal fees and non-financial support from Boehringer Ingelheim, personal fees and non-financial support from Chiesi, AstraZeneca and Sanofi, non-financial support from Menarinni and TEVA, outside the submitted work; and has a patent EP 15152886.6 “New compositions and methods of treating and/or preventing chronic obstructive pulmonary disease” issued, a patent 22605-FR “Geometric characterization of airways using MRI” pending, and a patent EP 20173595.8 “New compositions and methods of treating COVID-19 disease” pending. Conflict of interest: T. Trian reports grants from Agence Nationale pour la Recherche, during the conduct of the study., (Copyright ©The authors 2021. For reproduction rights and permissions contact permissions@ersnet.org.)

Background: LRP-1 is a multifunctional scavenger receptor belonging to the LDLR family. Due to its capacity to control pericellular levels of various growth factors and proteases, LRP-1 plays a crucial role in membrane proteome dynamics, which appears decisive for tumor progression., Methods: LRP-1 involvement in a TNBC model was assessed using an RNA interference strategy in MDA-MB-231 cells. In vivo, tumorigenic and angiogenic effects of LRP-1-repressed cells were evaluated using an orthotopic xenograft model and two angiogenic assays (Matrigel ® plugs, CAM). DCE-MRI, FMT, and IHC were used to complete a tumor longitudinal follow-up and obtain morphological and functional vascular information. In vitro, HUVECs' angiogenic potential was evaluated using a tumor secretome, subjected to a proteomic analysis to highlight LRP-1-dependant signaling pathways., Results: LRP-1 repression in MDA-MB-231 tumors led to a 60% growth delay because of, inter alia, morphological and functional vascular differences, confirmed by angiogenic models. In vitro, the LRP-1-repressed cells secretome restrained HUVECs' angiogenic capabilities. A proteomics analysis revealed that LRP-1 supports tumor growth and angiogenesis by regulating TGF-β signaling and plasminogen/plasmin system., Conclusions: LRP-1, by its wide spectrum of interactions, emerges as an important matricellular player in the control of cancer-signaling events such as angiogenesis, by supporting tumor vascular morphology and functionality.

Background and Aims: Through an exploratory proteomic approach based on typical hepatocellular adenomas (HCAs), we previously identified a diagnostic biomarker for a distinctive subtype of HCA with high risk of bleeding, already validated on a multicenter cohort. We hypothesized that the whole protein expression deregulation profile could deliver much more informative data for tumor characterization. Therefore, we pursued our analysis with the characterization of HCA proteomic profiles, evaluating their correspondence with the established genotype/phenotype classification and assessing whether they could provide added diagnosis and prognosis values., Approach and Results: From a collection of 260 cases, we selected 52 typical cases of all different subgroups on which we built a reference HCA proteomics database. Combining laser microdissection and mass-spectrometry-based proteomic analysis, we compared the relative protein abundances between tumoral (T) and nontumoral (NT) liver tissues from each patient and we defined a specific proteomic profile of each of the HCA subgroups. Next, we built a matching algorithm comparing the proteomic profile extracted from a patient with our reference HCA database. Proteomic profiles allowed HCA classification and made diagnosis possible, even for complex cases with immunohistological or genomic analysis that did not lead to a formal conclusion. Despite a well-established pathomolecular classification, clinical practices have not substantially changed and the HCA management link to the assessment of the malignant transformation risk remains delicate for many surgeons. That is why we also identified and validated a proteomic profile that would directly evaluate malignant transformation risk regardless of HCA subtype., Conclusions: This work proposes a proteomic-based machine learning tool, operational on fixed biopsies, that can improve diagnosis and prognosis and therefore patient management for HCAs., (© 2021 by the American Association for the Study of Liver Diseases.)

Glycosomes are peroxisome-related organelles of trypanosomatid parasites containing metabolic pathways, such as glycolysis and biosynthesis of sugar nucleotides, usually present in the cytosol of other eukaryotes. UDP-glucose pyrophosphorylase (UGP), the enzyme responsible for the synthesis of the sugar nucleotide UDP-glucose, is localized in the cytosol and glycosomes of the bloodstream and procyclic trypanosomes, despite the absence of any known peroxisome-targeting signal (PTS1 and PTS2). The questions that we address here are (i) is the unusual glycosomal biosynthetic pathway of sugar nucleotides functional and (ii) how is the PTS-free UGP imported into glycosomes? We showed that UGP is imported into glycosomes by piggybacking on the glycosomal PTS1-containing phosphoenolpyruvate carboxykinase (PEPCK) and identified the domains involved in the UGP/PEPCK interaction. Proximity ligation assays revealed that this interaction occurs in 3 to 10% of glycosomes, suggesting that these correspond to organelles competent for protein import. We also showed that UGP is essential for the growth of trypanosomes and that both the glycosomal and cytosolic metabolic pathways involving UGP are functional, since the lethality of the knockdown UGP mutant cell line ( RNAi UGP, where RNAi indicates RNA interference) was rescued by expressing a recoded UGP (rUGP) in the organelle ( RNAi UGP/ EXP rUGP-GPDH, where GPDH is glycerol-3-phosphate dehydrogenase). Our conclusion was supported by targeted metabolomic analyses (ion chromatography-high-resolution mass spectrometry [IC-HRMS]) showing that UDP-glucose is no longer detectable in the RNAi UGP mutant, while it is still produced in cells expressing UGP exclusively in the cytosol (PEPCK null mutant) or glycosomes ( RNAi UGP/ EXP rUGP-GPDH). Trypanosomatids are the only known organisms to have selected functional peroxisomal (glycosomal) sugar nucleotide biosynthetic pathways in addition to the canonical cytosolic ones. IMPORTANCE Unusual compartmentalization of metabolic pathways within organelles is one of the most enigmatic features of trypanosomatids. These unicellular eukaryotes are the only organisms that sequestered glycolysis inside peroxisomes (glycosomes), although the selective advantage of this compartmentalization is still not clear. Trypanosomatids are also unique for the glycosomal localization of enzymes of the sugar nucleotide biosynthetic pathways, which are also present in the cytosol. Here, we showed that the cytosolic and glycosomal pathways are functional. As in all other eukaryotes, the cytosolic pathways feed glycosylation reactions; however, the role of the duplicated glycosomal pathways is currently unknown. We also showed that one of these enzymes (UGP) is imported into glycosomes by piggybacking on another glycosomal enzyme (PEPCK); they are not functionally related. The UGP/PEPCK association is unique since all piggybacking examples reported to date involve functionally related interacting partners, which broadens the possible combinations of carrier-cargo proteins being imported as hetero-oligomers.

Fungi are the most prevalent plant pathogens, causing annually important damages. To infect and colonize their hosts, they secrete effectors including hydrolytic enzymes able to kill and macerate plant tissues. These secreted proteins are transported from the Endoplasmic Reticulum and the Golgi apparatus to the extracellular space through intracellular vesicles. In pathogenic fungi, intracellular vesicles were described but their biogenesis and their role in virulence remain unclear. In this study, we report the essential role of clathrin heavy chain (CHC) in the pathogenicity of Botrytis cinerea , the agent of gray mold disease. To investigate the importance of this protein involved in coat vesicles formation in eukaryotic cells, a T-DNA insertional mutant reduced in the expression of the CHC-encoding gene, and a mutant expressing a dominant-negative form of CHC were studied. Both mutants were strongly affected in pathogenicity. Characterization of the mutants revealed altered infection cushions and an important defect in protein secretion. This study demonstrates the essential role of clathrin in the infectious process of a plant pathogenic fungus and more particularly its role in virulence factors delivery., Competing Interests: M-PL was employed by the company Bayer SAS. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Souibgui, Bruel, Choquer, de Vallée, Dieryckx, Dupuy, Latorse, Rascle and Poussereau.)

Previous studies have shown that Reptin is overexpressed in hepatocellular carcinoma and that it is necessary for in vitro proliferation and cell survival. However, its pathophysiological role in vivo remains unknown. We aimed to study the role of Reptin in hepatocyte proliferation after regeneration using a liver Reptin knock-out model (Reptin LKO ). Interestingly, hepatocyte proliferation is strongly impaired in Reptin LKO mice 36 h after partial hepatectomy, associated with a decrease of cyclin-A expression and mTORC1 and MAPK signalling, leading to an impaired liver regeneration. Moreover, in the Reptin LKO model, we have observed a progressive loss of Reptin invalidation associated with an atypical liver regeneration. Hypertrophic and proliferative hepatocytes gradually replace Reptin KO hypotrophic hepatocytes. To conclude, our results show that Reptin is required for hepatocyte proliferation in vivo and liver regeneration and that it plays a crucial role in hepatocyte survival and liver homeostasis., (© 2021 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background & Aims: A single point mutation in the Z-variant of alpha 1-antitrypsin (Z-AAT) alone can lead to both a protein folding and trafficking defect, preventing its exit from the endoplasmic reticulum (ER), and the formation of aggregates that are retained as inclusions within the ER of hepatocytes. These defects result in a systemic AAT deficiency (AATD) that causes lung disease, whereas the ER-retained aggregates can induce severe liver injury in patients with ZZ-AATD. Unfortunately, therapeutic approaches are still limited and liver transplantation represents the only curative treatment option . To overcome this limitation, a better understanding of the molecular basis of ER aggregate formation could provide new strategies for therapeutic intervention., Methods: Our functional and omics approaches here based on human hepatocytes from patients with ZZ-AATD have enabled the identification and characterisation of the role of the protein disulfide isomerase (PDI) A4/ERP72 in features of AATD-mediated liver disease., Results: We report that 4 members of the PDI family (PDIA4, PDIA3, P4HB, and TXNDC5) are specifically upregulated in ZZ-AATD liver samples from adult patients. Furthermore, we show that only PDIA4 knockdown or alteration of its activity by cysteamine treatment can promote Z-AAT secretion and lead to a marked decrease in Z aggregates. Finally, detailed analysis of the Z-AAT interactome shows that PDIA4 silencing provides a more conducive environment for folding of the Z mutant, accompanied by reduction of Z-AAT-mediated oxidative stress, a feature of AATD-mediated liver disease., Conclusions: PDIA4 is involved in AATD-mediated liver disease and thus represents a therapeutic target for inhibition by drugs such as cysteamine. PDI inhibition therefore represents a potential therapeutic approach for treatment of AATD., Lay Summary: Protein disulfide isomerase (PDI) family members, and particularly PDIA4, are upregulated and involved in alpha 1-antitrypsin deficiency (AATD)-mediated liver disease in adults. PDI inhibition upon cysteamine treatment leads to improvements in features of AATD and hence represents a therapeutic approach for treatment of AATD-mediated liver disease., Competing Interests: The authors have no potential conflicts (financial, professional, or personal) relevant to the manuscript. Please refer to the accompanying ICMJE disclosure forms for further details., (© 2021 The Authors.)

The necrotrophic plant-pathogen fungus Botrytis cinerea produces multicellular appressoria dedicated to plant penetration, named infection cushions (IC). A microarray analysis was performed to identify genes upregulated in mature IC. The expression data were validated by RT-qPCR analysis performed in vitro and in planta, proteomic analysis of the IC secretome and biochemical assays. 1231 upregulated genes and 79 up-accumulated proteins were identified. The data support the secretion of effectors by IC: phytotoxins, ROS, proteases, cutinases, plant cell wall-degrading enzymes and plant cell death-inducing proteins. Parallel upregulation of sugar transport and sugar catabolism-encoding genes would indicate a role of IC in nutrition. The data also reveal a substantial remodelling of the IC cell wall and suggest a role for melanin and chitosan in IC function. Lastly, mutagenesis of two upregulated genes in IC identified secreted fasciclin-like proteins as actors in the pathogenesis of B. cinerea. These results support the role of IC in plant penetration and also introduce other unexpected functions for this fungal organ, in colonization, necrotrophy and nutrition of the pathogen., (© 2021 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Gram-negative bacteria export a large variety of antimicrobial compounds by forming two-membrane spanning tripartite multidrug efflux systems composed of an inner membrane transporter, an outer membrane channel and a periplasmic adaptor protein. Here we present the co-expression, purification and first electron microscopy insights of the Escherichia coli EmrAB-TolC tripartite Major Facilitator Superfamily (MSF) efflux system as a whole complex stabilized by Amphipol polymer. The structure reveals a 33 nm long complex delineated by the Amphipol belt at both extremities. Comparison of projection structures of EmrAB-TolC and AcrAB-TolC indicates that the outer membrane protein TolC linked to the periplasmic adaptor EmrA protein form an extended periplasmic canal. The overall length of EmrAB-TolC complex is similar to that of AcrAB-TolC with a probable tip-to-tip interaction between EmrA and TolC unveiling how the adaptor protein connects TolC and EmrB embedded in the inner membrane., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The genome of trypanosomatids rearranges by using repeated sequences as platforms for amplification or deletion of genomic segments. These stochastic recombination events have a direct impact on gene dosage and foster the selection of adaptive traits in response to environmental pressure. We provide here such an example by showing that the phosphoenolpyruvate carboxykinase (PEPCK) gene knockout (Δpepck) leads to the selection of a deletion event between two tandemly arranged fumarate reductase (FRDg and FRDm2) genes to produce a chimeric FRDg-m2 gene in the Δpepck∗ cell line. FRDg is expressed in peroxisome-related organelles, named glycosomes, expression of FRDm2 has not been detected to date, and FRDg-m2 is nonfunctional and cytosolic. Re-expression of FRDg significantly impaired growth of the Δpepck∗ cells, but FRD enzyme activity was not required for this negative effect. Instead, glycosomal localization as well as the covalent flavinylation motif of FRD is required to confer growth retardation and intracellular accumulation of reactive oxygen species (ROS). The data suggest that FRDg, similar to Escherichia coli FRD, can generate ROS in a flavin-dependent process by transfer of electrons from NADH to molecular oxygen instead of fumarate when the latter is unavailable, as in the Δpepck background. Hence, growth retardation is interpreted as a consequence of increased production of ROS, and rearrangement of the FRD locus liberates Δpepck∗ cells from this obstacle. Interestingly, intracellular production of ROS has been shown to be required to complete the parasitic cycle in the insect vector, suggesting that FRDg may play a role in this process., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Diffuse midline glioma (DMG) is a pediatric malignancy with poor prognosis. Most children die less than one year after diagnosis. Recently, mutations in histone H3 have been identified and are believed to be oncogenic drivers. Targeting this epigenetic abnormality using histone deacetylase (HDAC) inhibitors such as panobinostat (PS) is therefore a novel therapeutic option currently evaluated in clinical trials., Methods: BH3 profiling revealed engagement in an irreversible apoptotic process of glioma cells exposed to PS confirmed by annexin-V/propidium iodide staining. Using proteomic analysis of 3 DMG cell lines, we identified 2 proteins deregulated after PS treatment. We investigated biological effects of their downregulation by silencing RNA but also combinatory effects with PS treatment in vitro and in vivo using a chick embryo DMG model. Electron microscopy was used to validate protein localization., Results: Scaffolding proteins EBP50 and IRSp53 were upregulated by PS treatment. Reduction of these proteins in DMG cell lines leads to blockade of proliferation and migration, invasion, and an increase of apoptosis. EBP50 was found to be expressed in cytoplasm and nucleus in DMG cells, confirming known oncogenic locations of the protein. Treatment of glioma cells with PS together with genetic or chemical inhibition of EBP50 leads to more effective reduction of cell growth in vitro and in vivo., Conclusion: Our data reveal a specific relation between HDAC inhibitors and scaffolding protein deregulation which might have a potential for therapeutic intervention for cancer treatment., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Until recently, 10% of hepatocellular adenomas (HCAs) remained unclassified (UHCA). Among the UHCAs, the sonic hedgehog HCA (shHCA) was defined by focal deletions that fuse the promoter of Inhibin beta E chain with GLI1. Prostaglandin D2 synthase was proposed as immunomarker. In parallel, our previous work using proteomic analysis showed that most UHCAs constitute a homogeneous subtype associated with overexpression of argininosuccinate synthase (ASS1). To clarify the use of ASS1 in the HCA classification and avoid misinterpretations of the immunohistochemical staining, the aims of this work were to study (1) the link between shHCA and ASS1 overexpression and (2) the clinical relevance of ASS1 overexpression for diagnosis. Molecular, proteomic, and immunohistochemical analyses were performed in UHCA cases of the Bordeaux series. The clinico-pathological features, including ASS1 immunohistochemical labeling, were analyzed on a large international series of 67 cases. ASS1 overexpression and the shHCA subgroup were superimposed in 15 cases studied by molecular analysis, establishing ASS1 overexpression as a hallmark of shHCA. Moreover, the ASS1 immunomarker was better than prostaglandin D2 synthase and only found positive in 7 of 22 shHCAs. Of the 67 UHCA cases, 58 (85.3%) overexpressed ASS1, four cases were ASS1 negative, and in five cases ASS1 was noncontributory. Proteomic analysis performed in the case of doubtful interpretation of ASS1 overexpression, especially on biopsies, can be a support to interpret such cases. ASS1 overexpression is a specific hallmark of shHCA known to be at high risk of bleeding. Therefore, ASS1 is an additional tool for HCA classification and clinical diagnosis., (© 2020 The Authors. Hepatology Communications published by Wiley Periodicals, Inc., on behalf of the American Association for the Study of Liver Diseases.)

Background: Therapeutic outcomes using the multikinase inhibitors, sorafenib and regorafenib, remain unsatisfactory for patients with advanced hepatocellular carcinoma (HCC). Thus, new drug modalities are needed. We recently reported the remarkable capacity of miR-4510 to impede the growth of HCC and hepatoblastoma through Glypican-3 (GPC3) targeting and Wnt pathway inactivation., Methods: To identify new targets of miR-4510, we used a label-free proteomic approach and reported down-regulation of RAF proto-oncogene serine/threonine-protein kinase (RAF1) by miR-4510. Because the tumourigenic role of RAF1 in HCC is controversial, we further studied RAF1:miR-4510 interactions using cellular, molecular as well as functional approaches and a chicken chorioallantoic membrane (CAM) xenograft model., Results: We found an increase in RAF1 protein in 59.3% of HCC patients and a specific up-regulation of its transcript in proliferative tumours. We showed that miR-4510 inactivates the RAS/RAF/MEK/ERK pathway and reduces the expression of downstream targets (ie c-Fos proto-oncogene [FOS]) through RAF1 direct targeting. At a cellular level, miR-4510 inhibited HCC cell proliferation and migration and induced senescence in part by lowering RAF1 messenger RNA (mRNA) and protein expression. Finally, we confirmed the pro-tumoural function of RAF1 protein in HCC cells and its ability to sustain HCC tumour progression in vitro and in vivo., Conclusions: In this work, we confirm that RAF1 acts as an oncogene in HCC and further demonstrate that miR-4510 acts as a strong tumour suppressor in the liver by targeting many proto-oncogenes, including GPC3 and RAF1, and subsequently controlling key biological and signalling pathways among which Wnt and RAS/RAF/MEK/ERK signals., (© 2019 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

The basic understanding of the biological effects of eukaryotic translation initiation factors (EIFs) remains incomplete, notably for their roles independent of protein translation. Different EIFs exhibit nuclear localization and DNA-related functions have been proposed, but the understanding of EIFs novel functions beyond protein translation lacks of integrative analyses between the genomic and the proteomic levels. Here, the noncanonical function of EIF3F was studied in human lung adenocarcinoma by combining methods that revealed both the protein-protein and the protein-DNA interactions of this factor. We discovered that EIF3F promotes cell metastasis in vivo. The underpinning molecular mechanisms involved the regulation of a cluster of 34 metastasis-promoting genes including Snail2, as revealed by proteomics combined with immuno-affinity purification of EIF3F and ChIP-seq/Q-PCR analyses. The interaction between EIF3F and signal transducer and activator of transcription 3 (STAT3) controlled the EIF3F-mediated increase in Snail2 expression and cellular invasion, which were specifically abrogated using the STAT3 inhibitor Nifuroxazide or knockdown approaches. Furthermore, EIF3F overexpression reprogrammed energy metabolism through the activation of AMP-activated protein kinase and the stimulation of oxidative phosphorylation. Our findings demonstrate the role of EIF3F in the molecular control of cell migration, invasion, bioenergetics, and metastasis. The discovery of a role for EIF3F-STAT3 interaction in the genetic control of cell migration and metastasis in human lung adenocarcinoma could lead to the development of diagnosis and therapeutic strategies.

The gray mold fungus Botrytis cinerea is a necrotrophic pathogen able to infect hundreds of host plants, including high-value crops such as grapevine, strawberry and tomato. In order to decipher its infectious strategy, a library of 2,144 mutants was generated by random insertional mutagenesis using Agrobacterium tumefaciens- mediated transformation (ATMT). Twelve mutants exhibiting total loss of virulence toward different host plants were chosen for detailed analyses. Their molecular characterization revealed a single T-DNA insertion in different loci. Using a proteomics approach, the secretome of four of these strains was compared to that of the parental strain and a common profile of reduced lytic enzymes was recorded. Significant variations in this profile, notably deficiencies in the secretion of proteases and hemicellulases, were observed and validated by biochemical tests. They were also a hallmark of the remaining eight non-pathogenic strains, suggesting the importance of these secreted proteins in the infection process. In the twelve non-pathogenic mutants, the differentiation of infection cushions was also impaired, suggesting a link between the penetration structures and the secretion of proteins involved in the virulence of the pathogen., (Copyright © 2019 de Vallée, Bally, Bruel, Chandat, Choquer, Dieryckx, Dupuy, Kaiser, Latorse, Loisel, Mey, Morgant, Rascle, Schumacher, Simon, Souibgui, Viaud, Villalba and Poussereau.)

Background: Glioblastomas are heterogeneous tumors composed of a necrotic and tumor core and an invasive periphery., Methods: Here, we performed a proteomics analysis of laser-capture micro-dissected glioblastoma core and invasive areas of patient-derived xenografts., Results: Bioinformatics analysis identified enriched proteins in central and invasive tumor areas. Novel markers of invasion were identified, the genes proteolipid protein 1 (PLP1) and Dynamin-1 (DNM1), which were subsequently validated in tumors and by functional assays., Conclusions: In summary, our results identify new networks and molecules that may play an important role in glioblastoma development and may constitute potential novel therapeutic targets., (© The Author(s) 2019. Published by Oxford University Press, the Society for Neuro-Oncology and the European Association of Neuro-Oncology.)

G-quadruplex ligands exert their antiproliferative effects through telomere-dependent and telomere-independent mechanisms, but the inter-relationships among autophagy, cell growth arrest and cell death induced by these ligands remain largely unexplored. Here, we demonstrate that the G-quadruplex ligand 20A causes growth arrest of cancer cells in culture and in a HeLa cell xenografted mouse model. This response is associated with the induction of senescence and apoptosis. Transcriptomic analysis of 20A treated cells reveals a significant functional enrichment of biological pathways related to growth arrest, DNA damage response and the lysosomal pathway. 20A elicits global DNA damage but not telomeric damage and activates the ATM and autophagy pathways. Loss of ATM following 20A treatment inhibits both autophagy and senescence and sensitizes cells to death. Moreover, disruption of autophagy by deletion of two essential autophagy genes ATG5 and ATG7 leads to failure of CHK1 activation by 20A and subsequently increased cell death. Our results, therefore, identify the activation of ATM by 20A as a critical player in the balance between senescence and apoptosis and autophagy as one of the key mediators of such regulation. Thus, targeting the ATM/autophagy pathway might be a promising strategy to achieve the maximal anticancer effect of this compound., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Protein kinase A (PKA), the main effector of cAMP in eukaryotes, is a paradigm for the mechanisms of ligand-dependent and allosteric regulation in signalling. Here we report the orthologous but cAMP-independent PKA of the protozoan Trypanosoma and identify 7-deaza-nucleosides as potent activators (EC 50  ≥ 6.5 nM) and high affinity ligands (K D  ≥ 8 nM). A co-crystal structure of trypanosome PKA with 7-cyano-7-deazainosine and molecular docking show how substitution of key amino acids in both CNB domains of the regulatory subunit and its unique C-terminal αD helix account for this ligand swap between trypanosome PKA and canonical cAMP-dependent PKAs. We propose nucleoside-related endogenous activators of Trypanosoma brucei PKA (TbPKA). The existence of eukaryotic CNB domains not associated with binding of cyclic nucleotides suggests that orphan CNB domains in other eukaryotes may bind undiscovered signalling molecules. Phosphoproteome analysis validates 7-cyano-7-deazainosine as powerful cell-permeable inducer to explore cAMP-independent PKA signalling in medically important neglected pathogens.

The bloodstream forms of Trypanosoma brucei (BSF), the parasite protist causing sleeping sickness, primarily proliferate in the blood of their mammalian hosts. The skin and adipose tissues were recently identified as additional major sites for parasite development. Glucose was the only carbon source known to be used by bloodstream trypanosomes to feed their central carbon metabolism, however, the metabolic behaviour of extravascular tissue-adapted parasites has not been addressed yet. Since the production of glycerol is an important primary function of adipocytes, we have adapted BSF trypanosomes to a glucose-depleted but glycerol-rich culture medium (CMM&#95;Glyc/GlcNAc) and compared their metabolism and proteome to those of parasites grown in standard glucose-rich conditions (CMM&#95;Glc). BSF were shown to consume 2-folds more oxygen per consumed carbon unit in CMM&#95;Glyc/GlcNAc and were 11.5-times more sensitive to SHAM, a specific inhibitor of the plant-like alternative oxidase (TAO), which is the only mitochondrial terminal oxidase expressed in BSF. This is consistent with (i) the absolute requirement of the mitochondrial respiratory activity to convert glycerol into dihydroxyacetone phosphate, as deduced from the updated metabolic scheme and (ii) with the 1.8-fold increase of the TAO expression level compared to the presence of glucose. Proton NMR analysis of excreted end products from glycerol and glucose metabolism showed that these two carbon sources are metabolised through the same pathways, although the contributions of the acetate and succinate branches are more important in the presence of glycerol than glucose (10.2% versus 3.4% of the excreted end products, respectively). In addition, metabolomic analyses by mass spectrometry showed that, in the absence of glucose, 13C-labelled glycerol was incorporated into hexose phosphates through gluconeogenesis. As expected, RNAi-mediated down-regulation of glycerol kinase expression abolished glycerol metabolism and was lethal for BSF grown in CMM&#95;Glyc/GlcNAc. Interestingly, BSF have adapted their metabolism to grow in CMM&#95;Glyc/GlcNAc by concomitantly increasing their rate of glycerol consumption and decreasing that of glucose. However, the glycerol kinase activity was 7.8-fold lower in CMM&#95;Glyc/GlcNAc, as confirmed by both western blotting and proteomic analyses. This suggests that the huge excess in glycerol kinase that is not absolutely required for glycerol metabolism, might be used for another yet undetermined non-essential function in glucose rich-conditions. Altogether, these data demonstrate that BSF trypanosomes are well-adapted to glycerol-rich conditions that could be encountered by the parasite in extravascular niches, such as the skin and adipose tissues., Competing Interests: The authors have declared that no competing interests exist.

The phytopathogenic fungus Botrytis cinerea is able to infect a wide variety of plants and plant tissues with differing chemical compositions. During its interaction with the host, this pathogen modulates its ambient pH by secreting acids or ammonia. In this work, we examined the Pal/Pac pathway, the fungal ambient pH-responsive signalling circuit, and investigated the role of the PacC transcription factor. Characterization of the BcpacC deletion mutant revealed an alteration of both fungal growth and virulence depending on the pH of the culture medium or of the host tissue. The pathogenicity of the mutant was altered on plants exhibiting a neutral pH and not on plants with acidic tissues. The capacity of the mutant to acidify its environment and, more particularly, to produce oxalic acid was affected, as was production of reactive oxygen species. Finally, proteomic profiling of the mutant secretome revealed significant changes in plant cell wall polysaccharides proteins and lipid degradation and oxidoreduction, highlighting the importance of BcPacC in the necrotrophic lifestyle of B. cinerea., (© 2018 Society for Applied Microbiology and John Wiley & Sons Ltd.)

The ubiquitin proteasome system (UPS) regulates many cellular functions by degrading key proteins. Notably, the role of UPS in regulating mitochondrial metabolic functions is unclear. Here, we show that ubiquitination occurs in different mitochondrial compartments, including the inner mitochondrial membrane, and that turnover of several metabolic proteins is UPS dependent. We specifically detailed mitochondrial ubiquitination and subsequent UPS-dependent degradation of succinate dehydrogenase subunit A (SDHA), which occurred when SDHA was minimally involved in mitochondrial energy metabolism. We demonstrate that SDHA ubiquitination occurs inside the organelle. In addition, we show that the specific inhibition of SDHA degradation by UPS promotes SDHA-dependent oxygen consumption and increases ATP, malate, and citrate levels. These findings suggest that the mitochondrial metabolic machinery is also regulated by the UPS., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Invadosomes are F-actin-based structures involved in extracellular matrix degradation, cell invasion, and metastasis formation. Analyzing their proteome is crucial to decipher their molecular composition, to understand their mechanisms, and to find specific elements to target them. However, the specific analysis of invadosomes is challenging, because it is difficult to maintain their integrity during isolation. In addition, classical purification methods often suffer from contaminations, which may impair data validation. To ensure the specific identification of invadosome components, we here develop a method that combines laser microdissection and mass spectrometry, enabling the analysis of subcellular structures in their native state based on low amounts of input material. Using this combinatorial method, we show that invadosomes contain specific components of the translational machinery, in addition to known marker proteins. Moreover, functional validation reveals that protein translation activity is an inherent property of invadosomes, which is required to maintain invadosome structure and activity.

RNA polymerase (Pol) III transcribes small untranslated RNAs that are essential for cellular homeostasis and growth. Its activity is regulated by inactivation of tumor suppressor proteins and overexpression of the oncogene c-MYC, but the concerted action of these tumor-promoting factors on Pol III transcription has not yet been assessed. In order to comprehensively analyse the regulation of Pol III transcription during tumorigenesis we employ a model system that relies on the expression of five genetic elements to achieve cellular transformation. Expression of these elements in six distinct transformation intermediate cell lines leads to the inactivation of TP53, RB1, and protein phosphatase 2A, as well as the activation of RAS and the protection of telomeres by TERT, thereby conducting to full tumoral transformation of IMR90 fibroblasts. Transformation is accompanied by moderately enhanced levels of a subset of Pol III-transcribed RNAs (7SK; MRP; H1). In addition, mRNA and/or protein levels of several Pol III subunits and transcription factors are upregulated, including increased protein levels of TFIIIB and TFIIIC subunits, of SNAPC1 and of Pol III subunits. Strikingly, the expression of POLR3G and of SNAPC1 is strongly enhanced during transformation in this cellular transformation model. Collectively, our data indicate that increased expression of several components of the Pol III transcription system accompanied by a 2-fold increase in steady state levels of a subset of Pol III RNAs is sufficient for sustaining tumor formation.

The quantification of monoclonal antibodies (mAbs) such as bevacizumab, a recombinant humanized immunoglobulin G1 (hIgG1), in biological fluids, is an essential prerequisite to any pharmacokinetic preclinical and clinical study. To date, reference techniques used to quantify mAbs rely on enzyme-linked immunosorbent assay (ELISA) lacking specificity. Furthermore, the commercially available ELISA kit to quantify bevacizumab in human plasma only assesses the free fraction of the drug. However, the conditions of storage and analysis of plasma samples could alter the physiological equilibrium between the free, bound and partially bound forms of bevacizumab and this could result in over- or underestimation of drug concentration. We developed a new assay for absolute quantification of total fraction of bevacizumab by liquid chromatography tandem mass spectrometry (LC-MS/MS) basing identification and quantification of bevacizumab on two specific peptides. In this report we compare our assay with two internal standard (IS) calibration approaches: one using a different human mAb (Trastuzumab) and the other using a stable isotope labeled specific peptide. After enrichment by affinity chromatography on protein A and concentration by ultrafiltration, human plasma samples were proteolyzed by trypsin. Linearity was established from 12.5 to 500μg/mL with an interday accuracy ranging from 101.7 to 110.6% and precision from 7.0% to 9.9%. This study demonstrates the importance of the choice of the IS in quantifying bevacizumab in human plasma and highlights the difficulty of reaching a reliable proteolysis with a sufficient recovery. We developed a reliable and cost-effective LC-MS/MS method to quantify total plasmatic fraction of bevacizumab in human plasma. Through our development we proposed a generic methodology easily transposable to quantify all IgG1 subclass very useful for clinical pharmacokinetics studies., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Hepatocellular adenomas (HCAs) are rare benign tumors divided into three main subgroups defined by pathomolecular features, HNF1A (H-HCA), mutated β-catenin (b-HCA), and inflammatory (IHCA). In the case of unclassified HCAs (UHCAs), which are currently identified by default, a high risk of bleeding remains a clinical issue. The objective of this study was to explore UHCA proteome with the aim to identify specific biomarkers. Following dissection of the tumoral (T) and nontumoral (NT) tissue on formalin-fixed, paraffin-embedded HCA tissue sections using laser capture methodology, we performed mass spectrometry analysis to compare T and NT protein expression levels in H-HCA, IHCA, b-HCA, UHCA, and focal nodular hyperplasia. Using this methodology, we searched for proteins which are specifically deregulated in UHCA. We demonstrate that proteomic profiles allow for discriminating known HCA subtypes through identification of classical biomarkers in each HCA subgroup. We observed specific up-regulation of the arginine synthesis pathway associated with overexpression of argininosuccinate synthase (ASS1) and arginosuccinate lyase in UHCA. ASS1 immunohistochemistry identified all the UHCA, of which 64.7% presented clinical bleeding manifestations. Interestingly, we demonstrated that the significance of ASS1 was not restricted to UHCA, but also encompassed certain hemorrhagic cases in other HCA subtypes, particularly IHCA., Conclusion: ASS1 + HCA combined with a typical hematoxylin and eosin stain aspect defined a new HCA subgroup at a high risk of bleeding. (Hepatology 2017;66:2016-2028)., (© 2017 by the American Association for the Study of Liver Diseases.)

Background: Aedes albopictus is a vector of arboviruses that cause severe diseases in humans such as Chikungunya, Dengue and Zika fevers. The vector competence of Ae. albopictus varies depending on the mosquito population involved and the virus transmitted. Wolbachia infection status in believed to be among key elements that determine viral transmission efficiency. Little is known about the cellular functions mobilized in Ae. albopictus during co-infection by Wolbachia and a given arbovirus. To decipher this tripartite interaction at the molecular level, we performed a proteome analysis in Ae. albopictus C6/36 cells mono-infected by Wolbachia wAlbB strain or Chikungunya virus (CHIKV), and bi-infected., Results: We first confirmed significant inhibition of CHIKV by Wolbachia. Using two-dimensional gel electrophoresis followed by nano liquid chromatography coupled with tandem mass spectrometry, we identified 600 unique differentially expressed proteins mostly related to glycolysis, translation and protein metabolism. Wolbachia infection had greater impact on cellular functions than CHIKV infection, inducing either up or down-regulation of proteins associated with metabolic processes such as glycolysis and ATP metabolism, or structural glycoproteins and capsid proteins in the case of bi-infection with CHIKV. CHIKV infection inhibited expression of proteins linked with the processes of transcription, translation, lipid storage and miRNA pathways., Conclusions: The results of our proteome profiling have provided new insights into the molecular pathways involved in tripartite Ae. albopictus-Wolbachia-CHIKV interaction and may help defining targets for the better implementation of Wolbachia-based strategies for disease transmission control.

Background: Insertion of retroviral genome DNA occurs in the chromatin of the host cell. This step is modulated by chromatin structure as nucleosomes compaction was shown to prevent HIV-1 integration and chromatin remodeling has been reported to affect integration efficiency. LEDGF/p75-mediated targeting of the integration complex toward RNA polymerase II (polII) transcribed regions ensures optimal access to dynamic regions that are suitable for integration. Consequently, we have investigated the involvement of polII-associated factors in the regulation of HIV-1 integration., Results: Using a pull down approach coupled with mass spectrometry, we have selected the FACT (FAcilitates Chromatin Transcription) complex as a new potential cofactor of HIV-1 integration. FACT is a histone chaperone complex associated with the polII transcription machinery and recently shown to bind LEDGF/p75. We report here that a tripartite complex can be formed between HIV-1 integrase, LEDGF/p75 and FACT in vitro and in cells. Biochemical analyzes show that FACT-dependent nucleosome disassembly promotes HIV-1 integration into chromatinized templates, and generates highly favored nucleosomal structures in vitro. This effect was found to be amplified by LEDGF/p75. Promotion of this FACT-mediated chromatin remodeling in cells both increases chromatin accessibility and stimulates HIV-1 infectivity and integration., Conclusions: Altogether, our data indicate that FACT regulates HIV-1 integration by inducing local nucleosomes dissociation that modulates the functional association between the incoming intasome and the targeted nucleosome.