Identification of novel inner membrane complex and apical annuli proteins of the malaria parasite Plasmodium falciparum

The inner membrane complex (IMC) is a defining feature of apicomplexan parasites, which confers stability and shape to the cell, functions as a scaffolding compartment during the formation of daughter cells and plays an important role in motility and invasion during different life cycle stages of these single-celled organisms. To explore the IMC proteome of the malaria parasite Plasmodium falciparum we applied a proximity-dependent biotin identification (BioID)-based proteomics approach, using the established IMC marker protein Photosensitized INA-Labelled protein 1 (PhIL1) as bait in asexual blood-stage parasites. Subsequent mass spectrometry-based peptide identification revealed enrichment of 12 known IMC proteins and several uncharacterized candidate proteins. We validated nine of these previously uncharacterized proteins by endogenous GFP-tagging. Six of these represent new IMC proteins, while three proteins have a distinct apical localization that most likely represents structures described as apical annuli in Toxoplasma gondii. Additionally, various Kelch13 interacting candidates were identified, suggesting an association of the Kelch13 compartment and the IMC in schizont and merozoite stages. This work extends the number of validated IMC proteins in the malaria parasite and reveals for the first time the existence of apical annuli proteins in P. falciparum. Additionally, it provides evidence for a spatial association between the Kelch13 compartment and the IMC in late blood-stage parasites

Retinal regions shape human and murine Müller cell proteome profile and functionality

The human macula is a highly specialized retinal region with pit‐like morphology and rich in cones. How Müller cells, the principal glial cell type in the retina, are adapted to this environment is still poorly understood. We compared proteomic data from cone‐ and rod‐rich retinae from human and mice and identified different expression profiles of cone‐ and rod‐associated Müller cells that converged on pathways representing extracellular matrix and cell adhesion. In particular, epiplakin (EPPK1), which is thought to play a role in intermediate filament organization, was highly expressed in macular Müller cells. Furthermore, EPPK1 knockout in a human Müller cell‐derived cell line led to a decrease in traction forces as well as to changes in cell size, shape, and filopodia characteristics. We here identified EPPK1 as a central molecular player in the region‐specific architecture of the human retina, which likely enables specific functions under the immense mechanical loads in vivo

Joint Observation of the Galactic Center with MAGIC and CTA-LST-1

MAGIC is a system of two Imaging Atmospheric Cherenkov Telescopes (IACTs), designed to detect very-high-energy gamma rays, and is operating in stereoscopic mode since 2009 at the Observatorio del Roque de Los Muchachos in La Palma, Spain. In 2018, the prototype IACT of the Large-Sized Telescope (LST-1) for the Cherenkov Telescope Array, a next-generation ground-based gamma-ray observatory, was inaugurated at the same site, at a distance of approximately 100 meters from the MAGIC telescopes. Using joint observations between MAGIC and LST-1, we developed a dedicated analysis pipeline and established the threefold telescope system via software, achieving the highest sensitivity in the northern hemisphere. Based on this enhanced performance, MAGIC and LST-1 have been jointly and regularly observing the Galactic Center, a region of paramount importance and complexity for IACTs. In particular, the gamma-ray emission from the dynamical center of the Milky Way is under debate. Although previous measurements suggested that a supermassive black hole Sagittarius A* plays a primary role, its radiation mechanism remains unclear, mainly due to limited angular resolution and sensitivity. The enhanced sensitivity in our novel approach is thus expected to provide new insights into the question. We here present the current status of the data analysis for the Galactic Center joint MAGIC and LST-1 observations

MAGIC and H.E.S.S. detect VHE gamma rays from the blazar OT081 for the first time: a deep multiwavelength study

https://pos.sissa.it/395/815/pdfPublished versio

The effect of antimalarial drugs on intracellular pH in «Plasmodium falciparum»

Malaria is one of the most severe public health problems worldwide and a leading cause of death and disease in developing countries. The alarming spread of resistance to antimalarials accentuates the need for the better biochemical and physiological understanding of the interaction of malaria parasites with these drugs in order to develop novel therapeutic strategies that either inhibit or circumvent resistance mechanisms. The digestive vacuole (DV) of Plasmodium falciparum is the site of action of many antimalarials and plays a pivotal role in drug resistance. Its membrane is thought to contain pumps and transporters that are implicated in the maintenance of an acidic pH in the DV lumen (pHDV) and the transport of antimalarials. The low pHDV is important for the functions of this compartment, including hemoglobin degradation and heme detoxification. pH alterations in response to quinoline-containing antimalarials (QCAs) have not yet been quantitatively determined and the mechanisms of action of these drugs are still unclear.The QCAs chloroquine (CQ), quinine (QN) and mefloquine (MQ) are weak bases, increase pHDV in drug-sensitive parasites and may thus negatively affect heme detoxification pathways, causing the accumulation of toxic byproducts and ultimately the death of the parasite. In contrast, drug-resistant parasites are thought to avoid changes in pHDV via an increased efflux of the antimalarial from this organelle. The P. falciparum chloroquine resistance transporter (PfCRT) has been suggested to mediate proton-coupled efflux of CQ and QN from the DV into the cytosol of the parasite, reducing the accumulation of these drugs at their site of action. Furthermore, specific inhibition of the vacuolar-type H+-ATPase (V-ATPase) that acidifies the DV lumen may lead to an increase in pHDV. To test these hypotheses, single live parasites expressing pHluorin, a ratiometric pH-sensitive green fluorescent protein, were imaged under physiological conditions at 37°C using confocal fluorescence microscopy. At concentrations of 500 nM, CQ, QN and MQ significantly decreased the cytosolic pH by 0.2 – 0.3 units and increased pHDV by 0.5 – 0.6 units within seconds in both sensitive (3D7) and resistant parasites (Dd2). In Dd2, the PfCRT inhibitor verapamil (VP) circumvented the decrease in cytosolic pH caused by CQ and QN, while it did not modify the pH response to CQ or QN in 3D7 nor to MQ in either of the two strains. Interestingly, VP is known to sensitize Dd2 to both CQ and QN. Inhibiting the V-ATPase in P. falciparum caused a profound disruption of intracellular pH regulation: ConA almost completely abolished and BafA1 reduced the transvacuolar pH gradient. The pH effects were reversible within 15 min and parasite morphology was affected after 6 h but not within 2 h of exposure to the inhibitors.Our quantitative data indicate that vacuolar alkalinization is probably not a determinant of QCA resistance and are consistent with the hypothesis that proton-coupled efflux of CQ and QN from the DV is mediated by PfCRTCQR and sensitive to VP in drug-resistant parasites. In contrast, MQ is probably not transported by PfCRT. Our results suggest that BafA1 and ConA1 do not bind covalently to the V-ATPase and that a disruption of the transvacuolar pH gradient can by tolerated by P. falciparum for at least 2 hours, whereas the viability of the parasite is affected after 6 h.Le paludisme représente un des problèmes de santé publique les plus sérieux dans le monde, et constitue une cause majeure de mortalité et de maladie dans les pays en voie de développement. L'inquiétante progression de la résistance aux antipaludéens accentue le besoin pressant d'approfondir nos connaissances sur les mécanismes d'interaction biochimique et physiologique entre les antipaludéens et le parasite pour le développement de stratégies thérapeutiques innovantes.La vacuole digestive (DV) du Plasmodium falciparum est le site d'action de nombreux antipaludéens et joue un rôle pivot dans la résistance au traitement. Sa membrane semble contenir des pompes et des transporteurs servant, entre autre, au maintien d'un pH acide dans le lumen de la vacuole digestive (pHDV), ainsi qu'au transport des antipaludéens. Un pHDV faible est important pour les fonctions de ce compartiment, comprenant la dégradation de l'hémoglobine et la détoxification de l'hème. Les altérations du pH en réponse aux antipaludéens contenant de la quinoline (QCAs) n'ont pas encore été analysées quantitativement et les mécanismes d'action de ces médicaments demeurent toutefois obscurs.Les QCAs, comme la chloroquine (CQ), la quinine (QN) et la mefloquine (MQ), sont des bases faibles, augmentent le pHDV des parasites sensibles aux médicaments et de ce fait, pourraient affecter négativement les voies de détoxification de l'hème, causant ainsi l'accumulation des produits toxiques et finalement la mort du parasite. A l'opposé, il est possible que les parasites résistants aux médicaments puissent éviter les variations du pHDV par un efflux augmenté des antipaludéens depuis cet organite. Il a été suggéré que le PfCRT (P. falciparum chloroquine resistance transporter) intervenait dans l'efflux proton-couplé de CQ et QN depuis la vacuole digestive jusqu'au cytosol du parasite, diminuant l'accumulation de ces médicaments à leur site d'action. En outre, l'inhibition spécifique de la H+-ATPase du type vacuolaire (V-ATPase), qui acidifie le lumen de la vacuole digestive, pourrait entraîner une élévation du pHDV. Des parasites vivants et exprimant la pHluorin ratiométrique, une protéine verte fluorescente sensible au pH, ont été examinés au par microscopie confocale à fluorescence à 37°C sous des conditions physiologiques. À des concentrations de 500 nM, la CQ, la QN et la MQ ont abaissé le pH cytosolique de 0.2 – 0.3 unités et augmenté le pHDV de 0.5 – 0.6 unités de façon significative en quelques secondes dans des parasites sensibles (3D7) et résistants (Dd2). Le traitement de Dd2 avec l'inhibiteur du PfCRT vérapamil (VP) a éliminé la baisse du pH cytosolique normalement entrainé par la CQ et la QN. VP n'a toutefois pas modifié la réponse du pH à la CQ ou la QN dans le 3D7, ni à la MQ dans aucune des deux souches. Il est intéressant de noter que le VP est reconnu pour sensibiliser Dd2 à la CQ et à la QN. L'inhibition de la V-ATPase dans le P. falciparum a entrainé un profond dysfonctionnement de la régulation du pH intracellulaire. Le gradient transvacuolaire du pH a été presque complètement aboli par ConA et réduit par BafA1. Les effets sur le pH ont été réversibles après 15 minutes et la morphologie du parasite a été affectée après 6 heures d'incubation, mais pas dans les 2 heures suivant l'administration du médicament.Nos données quantitatives indiquent que l'alcalinisation vacuolaire n'est probablement pas un facteur déterminant la résistance aux QCAs et sont compatibles avec l'hypothèse selon laquelle l'efflux proton-couplé de la CQ et de la QN de la vacuole digestive est induite par le transporteur PfCRTCQR et est sensible au VP dans les parasites résistants aux médicaments. Au contraire, la MQ n'est probablement pas transporté par le PfCRT. Nos résultats portent à croire qu'une perturbation du gradient du pH transvacuolaire est tolérée par P. falciparum pendant au moins 2 heures, tandis que la viabilité du parasite est affectée après 6 heures d'incubation

Obesity-Induced TNFα and IL-6 Signaling: The Missing Link between Obesity and Inflammation—Driven Liver and Colorectal Cancers

Obesity promotes the development of numerous cancers, such as liver and colorectal cancers, which is at least partly due to obesity-induced, chronic, low-grade inflammation. In particular, the recruitment and activation of immune cell subsets in the white adipose tissue systemically increase proinflammatory cytokines, such as tumor necrosis factor α (TNFα) and interleukin-6 (IL-6). These proinflammatory cytokines not only impair insulin action in metabolic tissues, but also favor cancer development. Here, we review the current state of knowledge on how obesity affects inflammatory TNFα and IL-6 signaling in hepatocellular carcinoma and colorectal cancers

Biochemical and cellular characterisation of the Plasmodium falciparum M1 alanyl aminopeptidase (PfM1AAP) and M17 leucyl aminopeptidase (PfM17LAP)

AbstractThe Plasmodium falciparum M1 alanyl aminopeptidase and M17 leucyl aminopeptidase, PfM1AAP and PfM17LAP, are potential targets for novel anti-malarial drug development. Inhibitors of these aminopeptidases have been shown to kill malaria parasites in culture and reduce parasite growth in murine models. The two enzymes may function in the terminal stages of haemoglobin digestion, providing free amino acids for protein synthesis by the rapidly growing intra-erythrocytic parasites. Here we have performed a comparative cellular and biochemical characterisation of the two enzymes. Cell fractionation and immunolocalisation studies reveal that both enzymes are associated with the soluble cytosolic fraction of the parasite, with no evidence that they are present within other compartments, such as the digestive vacuole (DV). Enzyme kinetic studies show that the optimal pH of both enzymes is in the neutral range (pH 7.0–8.0), although PfM1AAP also possesses some activity (&lt; 20%) at the lower pH range of 5.0–5.5. The data supports the proposal that PfM1AAP and PfM17LAP function in the cytoplasm of the parasite, likely in the degradation of haemoglobin-derived peptides generated in the DV and transported to the cytosol.</jats:p

Coronin 2A (CRN5) expression is associated with colorectal adenoma-adenocarcinoma sequence and oncogenic signalling

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Coronin 2A (CRN5) expression is associated with colorectal adenoma-adenocarcinoma sequence and oncogenic signalling

Background: Coronin proteins are known as regulators of actin-based cellular processes, and some of them are associated with the malignant progression of human cancer. Here, we show that expression of coronin 2A is up-regulated in human colon carcinoma. Methods: This study included 26 human colon tumour specimens and 9 normal controls. Expression and localisation of coronin 2A was studied by immunohistochemistry, immunofluorescence imaging, cell fractionation, and immunoblotting. Functional roles of coronin 2A were analysed by over-expression and knock-down of the protein. Protein interactions were studied by co-immunoprecipitation and pull-down experiments, mass spectrometry analyses, and in vitro kinase and methylation assays. Results: Histopathological investigation revealed that the expression of coronin 2A in colon tumour cells is up-regulated during the adenoma-adenocarcinoma progression. At the subcellular level, coronin 2A localised to multiple compartments, i.e. F-actin stress fibres, the front of lamellipodia, focal adhesions, and the nuclei. Over-expression of coronin 2A led to a reduction of F-actin stress fibres and elevated cell migration velocity. We identified two novel direct coronin 2A interaction partners. The interaction of coronin 2A with MAPK14 (mitogen activated protein kinase 14 or MAP kinase p38a) led to phosphorylation of coronin 2A and also to activation of the MAPK14 pathway. Moreover, coronin 2A interacted with PRMT5 (protein arginine N-methyltransferase 5), which modulates the sensitivity of tumour cells to TRAIL-induced cell death. Conclusions: We show that increased expression of coronin 2A is associated with the malignant phenotype of human colon carcinoma. Moreover, we linked coronin 2A to MAPK14 and PRMT5 signalling pathways involved in tumour progression