Dengue Virus Replication Is Associated with Catecholamine Biosynthesis and Metabolism in Hepatocytes

Previously, the association between the catecholamine biosynthetic enzyme L-Dopa decarboxylase (DDC) and Dengue virus (DV) replication was demonstrated in liver cells and was found to be mediated at least by the interaction between DDC and phosphoinositide 3-kinase (PI3K). Here, we show that biogenic amines production and uptake impede DV replication in hepatocytes and monocytes, while the virus reduces catecholamine biosynthesis, metabolism, and transport. To examine how catecholamine biosynthesis/metabolism influences DV, first, we verified the role of DDC by altering DDC expression. DDC silencing enhanced virus replication, but not translation, attenuated the negative effect of DDC substrates on the virus and reduced the infection related cell death. Then, the role of the downstream steps of the catecholamine biosynthesis/metabolism was analyzed by chemical inhibition of the respective enzymes, application of their substrates and/or their products; moreover, reserpine, the inhibitor of the vesicular monoamine transporter 2 (VMAT2), was used to examine the role of uptake/storage of catecholamines on DV. Apart from the role of each enzyme/transporter, these studies revealed that the dopamine uptake, and not the dopamine-signaling, is responsible for the negative effect on DV. Accordingly, all treatments expected to enhance the accumulation of catecholamines in the cell cytosol suppressed DV replication. This was verified by the use of chemical inducers of catecholamine biosynthesis. Last, the cellular redox alterations due to catecholamine oxidation were not related with the inhibition of DV replication. In turn, DV apart from its negative impact on DDC, inhibits tyrosine hydroxylase, dopamine beta-hydroxylase, monoamine oxidase, and VMAT2 expression

<i>Pb</i>S2P shows partial co-localisation with the cis-Golgi marker ERD2.

<p>(A) Double labelling IFA of <i>P</i>. <i>berghei</i> schizont cultures using α-HA (3F10) for detection of <i>Pb</i>S2P (red) and α-ERD2 as a Golgi marker (green) showing partial, or in some cases complete, co-localisation. Nuclei are stained with Hoechst (blue). Scale bar 5 μM.</p

<i>Plasmodium</i> M50 proteases.

<p>(A) Conserved catalytic motifs (HExxH and NxxPxxxxDG- highlighted red in grey boxes) from a multiple sequence alignment of S2P orthologues from <i>Plasmodium</i> species and related apicomplexan parasites. (B) 3D homology model of <i>Pb</i>S2P (right panel—PbANKA_1404100) using the open conformation of <i>Methanocaldococcus jannaschii</i> S2P (left panel—PDB id: 3B4R) as a template and Phyre2 as program. The first transmembrane domain is labelled in orange, the second to fourth in blue, and the fifth and sixth in lime green, respectively. The catalytic zinc atom is depicted in red and the catalytic residues are shown surrounding the zinc atom as blue sticks. The orientation within the lipid membrane is also indicated. (C) Magnification of the active site in the <i>Pb</i>S2P homology model, illustrating the structural conservation of the catalytic residues. Strictly conserved residues are shown as sticks and are labelled in black for <i>Pb</i>S2P and blue for the respective homologous amino acid residues in <i>M</i>. <i>jannaschii</i> S2P.</p

<i>s2p(-)</i> parasites show no defects in sexual development nor sporogony.

<p>(A) Exflagellation assay showing formation of exflagellation centres. Mean values (±SD) from three independent experiments are shown. Differences were non-significant (Mann-Whitney test). (B) Ookinete conversion rates of WT and <i>s2p(-)</i> parasites after staining with an antibody against the surface antigen P28 and enumeration of ookinetes, zygotes and macrogametes. Shown are mean values (±SD) from three independent experiments. Differences were non-significant (2way-ANOVA). (C) Immunofluorescence analysis of <i>Anopheles gambiae</i> epithelia sheets infected with WT or <i>s2p(-)</i> parasites. Both strains induce an epithelial response as shown by the SRPN6 antibody (red). Ookinetes are stained with an antibody against surface protein P28 (green) and nuclei are stained with TO-PRO 3 (blue). Scale bar 10 μM. (D) Oocyst numbers of <i>s2p(-)</i> strain compared to the parental WT line after standard membrane feeding assay of <i>An</i>. <i>gambiae</i> mosquitoes from two independent experiments. Black bars show mean values (±SEM). Differences were non-significant (Mann-Whitney test).</p

Expression and localisation of <i>Pb</i>S2P.

<p>(A) Relative expression levels of <i>PbS2P</i> as determined by qRT-PCR from cDNAs of schizonts (schz), sporozoites (spz), 24h liver stages (LS24) and 48h liver stages (LS48). Transcript levels were normalised to <i>PbHSP70</i> and <i>GFP</i>. (B) Western blot analysis of <i>Pb</i>S2P-HA whole protein extract from purified schizonts of transgenic <i>PbS2P-HA</i> parasites using an α-HA antibody. <i>Pb</i>S2P-HA migrates at 35kDa. (C) Immunofluorescence analysis (IFA) of <i>Pb</i>S2P-HA merozoites, ookinete, oocyst, and salivary gland sporozoites using α-HA (3F10) for detection of <i>Pb</i>S2P (red) and Hoechst stain for the nucleus (blue). For delineation of parasites the following antibodies (green) were used: α-HSP70, schizonts/merozoites; α-MTIP, ookinete and sporozoite; α-PbCap380, oocyst. Prominent localisation of <i>Pb</i>S2P in proximity to the nucleus is present in all invasive stages. Star, apical end of ookinete. Scale bar 5 μM.</p

<i>s2p(-)</i> parasites show delayed transmission and growth, resulting in reduced virulence.

<p>(A) Kaplan-Meier analysis of time to patency after inoculation of 10,000 WT or <i>s2p(-)</i> salivary gland sporozoites into C57BL/6 mice (WT n = 9, <i>s2p(-)</i> n = 13). **, <i>P</i> < 0.01 (Log rank [Mantel-Cox] test). (B) Blood stage growth curve of the same mice showing the difference in growth rate of WT <i>vs</i>. <i>s2p(-)</i> parasites, alongside blood stage development of mice infected through bite back from infected <i>A</i>. <i>stephensi</i> mosquitoes (Wt bb <i>vs</i>. <i>s2p(-)</i> bb). In all cases mean values (± SD) are shown. **<i>P</i> < 0.01, ***<i>P</i> < 0.001 (Multiple t-test comparison). Significance is shown as follows: Black stars: Sporozoite injection experiment, Grey stars: Bite back experiment. (C) Kaplan-Meier curve of mice developing experimental cerebral malaria (ECM) over time after injection of 10,000 WT or <i>s2p(-)</i> salivary gland sporozoites (WT n = 9, <i>s2p(-)</i> n = 13). ***, <i>P</i> < 0.001 (Log rank [Mantel-Cox] test). (D, E) Parasitaemia levels of C57BL/6 mice after infection with (D) 10,000 (<i>n</i> = 3 each) or (E) 1,000 (<i>n</i> = 5 each) WT or <i>s2p(-)</i> iRBCs, respectively, as determined by Giemsa stained blood smears. <i>s2p(-)</i> parasites exhibit slower growth rates compared to the WT line. Mean values (± SD) are shown *<i>P</i> < 0.05 **<i>P</i> < 0.01, ***<i>P</i> < 0.001 (Multiple t-test comparison). (F) Kaplan-Meier curve of time to ECM development after patency. A one day delay in ECM symptoms was observed in mice infected with 10,000 <i>s2p(-)</i> iRBCs (<i>P</i> = 0.11 (Log rank [Mantel-Cox] test). Mice infected with 1,000 WT iRBC developed ECM symptoms at day 8 (4/5) and 9 (1/5), while 2/5 <i>s2p(-)</i> infected mice developed ECM at day 11 and 3/5 remained free of ECM symptoms. **<i>P</i> < 0.01 (Log rank [Mantel-Cox] test).</p

Kinome-wide synthetic lethal screen identifies PANK4 as a modulator of temozolomide resistance in glioblastoma

Temozolomide (TMZ) represents the cornerstone of therapy for glioblastoma (GBM). However, acquisition of resistance limits its therapeutic potential. The human kinome is an undisputable source of druggable targets, still, current knowledge remains confined to a limited fraction of it, with a multitude of under-investigated proteins yet to be characterized. Here, following a kinome-wide RNAi screen, pantothenate kinase 4 (PANK4) isuncovered as a modulator of TMZ resistance in GBM. Validation of PANK4 across various TMZ-resistant GBM cell models, patient-derived GBM cell lines, tissue samples, as well as in vivo studies, corroborates the potential translational significance of these findings. Moreover, PANK4 expression is induced during TMZ treatment, and its expression is associated with a worse clinical outcome. Furthermore, a Tandem Mass Tag (TMT)-based quantitative proteomic approach, reveals that PANK4 abrogation leads to a significant downregulation of a host of proteins with central roles in cellular detoxification and cellular response to oxidative stress. More specifically, as cells undergo genotoxic stress during TMZ exposure, PANK4 depletion represents a crucial event that can lead to accumulation of intracellular reactive oxygen species (ROS) and subsequent cell death. Collectively, a previously unreported role for PANK4 in mediating therapeutic resistance to TMZ in GBM is unveiled.</p

Kinome-wide synthetic lethal screen identifies PANK4 as a modulator of temozolomide resistance in glioblastoma

Temozolomide (TMZ) represents the cornerstone of therapy for glioblastoma (GBM). However, acquisition of resistance limits its therapeutic potential. The human kinome is an undisputable source of druggable targets, still, current knowledge remains confined to a limited fraction of it, with a multitude of under-investigated proteins yet to be characterized. Here, following a kinome-wide RNAi screen, pantothenate kinase 4 (PANK4) isuncovered as a modulator of TMZ resistance in GBM. Validation of PANK4 across various TMZ-resistant GBM cell models, patient-derived GBM cell lines, tissue samples, as well as in vivo studies, corroborates the potential translational significance of these findings. Moreover, PANK4 expression is induced during TMZ treatment, and its expression is associated with a worse clinical outcome. Furthermore, a Tandem Mass Tag (TMT)-based quantitative proteomic approach, reveals that PANK4 abrogation leads to a significant downregulation of a host of proteins with central roles in cellular detoxification and cellular response to oxidative stress. More specifically, as cells undergo genotoxic stress during TMZ exposure, PANK4 depletion represents a crucial event that can lead to accumulation of intracellular reactive oxygen species (ROS) and subsequent cell death. Collectively, a previously unreported role for PANK4 in mediating therapeutic resistance to TMZ in GBM is unveiled.</p

Kinome‐Wide Synthetic Lethal Screen Identifies PANK4 as a Modulator of Temozolomide Resistance in Glioblastoma

Temozolomide (TMZ) represents the cornerstone of therapy for glioblastoma (GBM). However, acquisition of resistance limits its therapeutic potential. The human kinome is an undisputable source of druggable targets, still, current knowledge remains confined to a limited fraction of it, with a multitude of under‐investigated proteins yet to be characterized. Here, following a kinome‐wide RNAi screen, pantothenate kinase 4 (PANK4) isuncovered as a modulator of TMZ resistance in GBM. Validation of PANK4 across various TMZ‐resistant GBM cell models, patient‐derived GBM cell lines, tissue samples, as well as in vivo studies, corroborates the potential translational significance of these findings. Moreover, PANK4 expression is induced during TMZ treatment, and its expression is associated with a worse clinical outcome. Furthermore, a Tandem Mass Tag (TMT)‐based quantitative proteomic approach, reveals that PANK4 abrogation leads to a significant downregulation of a host of proteins with central roles in cellular detoxification and cellular response to oxidative stress. More specifically, as cells undergo genotoxic stress during TMZ exposure, PANK4 depletion represents a crucial event that can lead to accumulation of intracellular reactive oxygen species (ROS) and subsequent cell death. Collectively, a previously unreported role for PANK4 in mediating therapeutic resistance to TMZ in GBM is unveiled.</p

Targeted Deletion of a Plasmodium Site-2 Protease Impairs Life Cycle Progression in the Mammalian Host

Site-2 proteases (S2P) belong to the M50 family of metalloproteases, which typically perform essential roles by mediating activation of membrane-bound transcription factors through regulated intramembrane proteolysis (RIP). Protease-dependent liberation of dormant transcription factors triggers diverse cellular responses, such as sterol regulation, Notch signalling and the unfolded protein response. Plasmodium parasites rely on regulated proteolysis for controlling essential pathways throughout the life cycle. In this study we examine the Plasmodium-encoded S2P in a murine malaria model and show that it is expressed in all stages of Plasmodium development. Localisation studies by endogenous gene tagging revealed that in all invasive stages the protein is in close proximity to the nucleus. Ablation of PbS2P by reverse genetics leads to reduced growth rates during liver and blood infection and, hence, virulence attenuation. Strikingly, absence of PbS2P was compatible with parasite life cycle progression in the mosquito and mammalian hosts under physiological conditions, suggesting redundant or dispensable roles in vivo