isopentenyl pyrophosphate  (Echelon Biosciences)


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    Echelon Biosciences isopentenyl pyrophosphate
    Isopentenyl Pyrophosphate, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    isopentenyl diphosphate ipp  (Echelon Biosciences)


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    Echelon Biosciences isopentenyl diphosphate ipp
    Isopentenyl Diphosphate Ipp, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    isopentenyl diphosphate ipp  (Echelon Biosciences)


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    Echelon Biosciences isopentenyl diphosphate ipp
    Isopentenyl Diphosphate Ipp, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ipp  (Echelon Biosciences)


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    Echelon Biosciences ipp
    Ipp, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ipp  (Echelon Biosciences)


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    Echelon Biosciences ipp
    Growth under temperature stress requires <t>IPP</t> synthesis and protein farnesylation. (A) Prenylphosphate substrates for protein prenylation are derived from the nonmevalonate MEP pathway. MEP pathway products, IPP and dimethylallyl pyrophosphate (DMAPP), serve as precursors to FPP used by FTase and GGPP used by GGTase in protein prenylation. FSM treatment inhibits production of IPP and DMAPP. Farnesyltransferase <t>inhibitors</t> <t>(FTI</t> or BMS) inhibit protein farnesylation, while geranylgeranyltransferase inhibitors (GGTI) prevent protein geranylgeranylation. (B) Parasites were treated with FSM (5 μM), farnesyltransferase inhibitors (FTI [10 μM] and BMS [200 nM]), or geranylgeranyltransferase inhibitor GGTI (2 μM) for 24 h prior to a 6-h heat (40°C) or cold (25°C) shock. (C and D) FSM-treated parasite growth is significantly reduced after heat shock (C) and cold shock (D). (E) Inhibition of farnesylation by treating parasites with FTI or BMS significantly reduced growth after temperature stress. Growth in GGTI-treated parasites is unchanged after heat or cold shock. (C to E) n = 6; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001. (C and D) 2-way ANOVA, P values adjusted for multiple comparisons using Sidak’s multiple-comparison test. (E) Within each treatment group, the normalized control was compared to temperature shock sample by unpaired t test with Welch’s correction. Abbreviations: ctrl, control, hs, heat shock, cs, cold shock.
    Ipp, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Protein Prenylation and Hsp40 in Thermotolerance of Plasmodium falciparum Malaria Parasites"

    Article Title: Protein Prenylation and Hsp40 in Thermotolerance of Plasmodium falciparum Malaria Parasites

    Journal: mBio

    doi: 10.1128/mBio.00760-21

    Growth under temperature stress requires IPP synthesis and protein farnesylation. (A) Prenylphosphate substrates for protein prenylation are derived from the nonmevalonate MEP pathway. MEP pathway products, IPP and dimethylallyl pyrophosphate (DMAPP), serve as precursors to FPP used by FTase and GGPP used by GGTase in protein prenylation. FSM treatment inhibits production of IPP and DMAPP. Farnesyltransferase inhibitors (FTI or BMS) inhibit protein farnesylation, while geranylgeranyltransferase inhibitors (GGTI) prevent protein geranylgeranylation. (B) Parasites were treated with FSM (5 μM), farnesyltransferase inhibitors (FTI [10 μM] and BMS [200 nM]), or geranylgeranyltransferase inhibitor GGTI (2 μM) for 24 h prior to a 6-h heat (40°C) or cold (25°C) shock. (C and D) FSM-treated parasite growth is significantly reduced after heat shock (C) and cold shock (D). (E) Inhibition of farnesylation by treating parasites with FTI or BMS significantly reduced growth after temperature stress. Growth in GGTI-treated parasites is unchanged after heat or cold shock. (C to E) n = 6; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001. (C and D) 2-way ANOVA, P values adjusted for multiple comparisons using Sidak’s multiple-comparison test. (E) Within each treatment group, the normalized control was compared to temperature shock sample by unpaired t test with Welch’s correction. Abbreviations: ctrl, control, hs, heat shock, cs, cold shock.
    Figure Legend Snippet: Growth under temperature stress requires IPP synthesis and protein farnesylation. (A) Prenylphosphate substrates for protein prenylation are derived from the nonmevalonate MEP pathway. MEP pathway products, IPP and dimethylallyl pyrophosphate (DMAPP), serve as precursors to FPP used by FTase and GGPP used by GGTase in protein prenylation. FSM treatment inhibits production of IPP and DMAPP. Farnesyltransferase inhibitors (FTI or BMS) inhibit protein farnesylation, while geranylgeranyltransferase inhibitors (GGTI) prevent protein geranylgeranylation. (B) Parasites were treated with FSM (5 μM), farnesyltransferase inhibitors (FTI [10 μM] and BMS [200 nM]), or geranylgeranyltransferase inhibitor GGTI (2 μM) for 24 h prior to a 6-h heat (40°C) or cold (25°C) shock. (C and D) FSM-treated parasite growth is significantly reduced after heat shock (C) and cold shock (D). (E) Inhibition of farnesylation by treating parasites with FTI or BMS significantly reduced growth after temperature stress. Growth in GGTI-treated parasites is unchanged after heat or cold shock. (C to E) n = 6; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001. (C and D) 2-way ANOVA, P values adjusted for multiple comparisons using Sidak’s multiple-comparison test. (E) Within each treatment group, the normalized control was compared to temperature shock sample by unpaired t test with Welch’s correction. Abbreviations: ctrl, control, hs, heat shock, cs, cold shock.

    Techniques Used: Derivative Assay, Inhibition

    Inhibition of either IPP synthesis or protein farnesylation results in reduced membrane association of HSP40. (A and B) Representative anti-HSP40 immunoblots of control- and FSM (20 μM)-treated (A) or FTI (10 μM)-treated (B) P. falciparum total lysate and membrane fractions. (C and D) Quantification of several immunoblots adjusted to loading control. HSP40 is significantly reduced in the membrane fraction after inhibition of IPP synthesis (C) and inhibition of farnesylation (D). Anti-HAD1 and anti-Exp-2, loading controls for total lysate and membrane fractions, respectively. **, P ≤ 0.01; ***, P ≤ 0.001 unpaired t test with Welch’s correction. (E and F) HSP40 membrane association is reduced after FTI treatment. Apparent membrane-associated HSP40 (10 nm gold particles) is reduced after inhibition of farnesylation. The number of membrane-associated HSP40 per micrograph is quantified for control and treated parasites (F). A single control cohort was quantified. A decrease in the number of membrane-associated HSP40 particles is observed. *, P ≤ 0.05, unpaired t test with Welch’s correction. Scale, 500 nm.
    Figure Legend Snippet: Inhibition of either IPP synthesis or protein farnesylation results in reduced membrane association of HSP40. (A and B) Representative anti-HSP40 immunoblots of control- and FSM (20 μM)-treated (A) or FTI (10 μM)-treated (B) P. falciparum total lysate and membrane fractions. (C and D) Quantification of several immunoblots adjusted to loading control. HSP40 is significantly reduced in the membrane fraction after inhibition of IPP synthesis (C) and inhibition of farnesylation (D). Anti-HAD1 and anti-Exp-2, loading controls for total lysate and membrane fractions, respectively. **, P ≤ 0.01; ***, P ≤ 0.001 unpaired t test with Welch’s correction. (E and F) HSP40 membrane association is reduced after FTI treatment. Apparent membrane-associated HSP40 (10 nm gold particles) is reduced after inhibition of farnesylation. The number of membrane-associated HSP40 per micrograph is quantified for control and treated parasites (F). A single control cohort was quantified. A decrease in the number of membrane-associated HSP40 particles is observed. *, P ≤ 0.05, unpaired t test with Welch’s correction. Scale, 500 nm.

    Techniques Used: Inhibition, Western Blot

    Both IPP synthesis and protein farnesylation influence HSP40 protein-protein interactions. Candidate protein interactors were determined by mass spectrometry after IP of parasite lysate with anti-HSP40. Results for FSM (5 μM)- and FTI (10 μM)-treated parasites are compared to untreated controls ( n = 3). UniProt and GeneIDs are provided in Table S2. Heat map of normalized log 2 -transformed data was generated using NG-CHM Heat Map Builder. Gene Ontology (GO) annotations are indicated by colored bars.
    Figure Legend Snippet: Both IPP synthesis and protein farnesylation influence HSP40 protein-protein interactions. Candidate protein interactors were determined by mass spectrometry after IP of parasite lysate with anti-HSP40. Results for FSM (5 μM)- and FTI (10 μM)-treated parasites are compared to untreated controls ( n = 3). UniProt and GeneIDs are provided in Table S2. Heat map of normalized log 2 -transformed data was generated using NG-CHM Heat Map Builder. Gene Ontology (GO) annotations are indicated by colored bars.

    Techniques Used: Mass Spectrometry, Transformation Assay, Generated

    Localization of GAPDH, but not its glycolytic function, is IPP- or farnesylation-dependent. (A to C) Inhibition of IPP synthesis and farnesylation reduced membrane association of GAPDH. (A) Representative anti-GAPDH immunoblots of control-, FSM (20 μM)-, and FTI (10 μM)-treated P. falciparum . (B and C) Quantification of several immunoblots adjusted with loading control. Anti-Hsp70 (1:5,000) and anti-PM-V (1:500) were used as loading controls for total lysate and membrane fractions, respectively. n = 5 to 6; ****, P ≤ 0.0001, unpaired t test with Welch’s correction.
    Figure Legend Snippet: Localization of GAPDH, but not its glycolytic function, is IPP- or farnesylation-dependent. (A to C) Inhibition of IPP synthesis and farnesylation reduced membrane association of GAPDH. (A) Representative anti-GAPDH immunoblots of control-, FSM (20 μM)-, and FTI (10 μM)-treated P. falciparum . (B and C) Quantification of several immunoblots adjusted with loading control. Anti-Hsp70 (1:5,000) and anti-PM-V (1:500) were used as loading controls for total lysate and membrane fractions, respectively. n = 5 to 6; ****, P ≤ 0.0001, unpaired t test with Welch’s correction.

    Techniques Used: Inhibition, Western Blot

    Glycolytic and pentose phosphate pathway metabolite levels remain constant under IPP- and farnesylation-deficient conditions. Levels of glycolytic and pentose phosphate pathway intermediates were measured by liquid chromatography with tandem mass spectrometry and normalized based on parasitemia of each individual sample to give concentration per cell. No significant changes are observed after treatment with FSM (5 μM) or FTI (10 μM). n = 3, unpaired t test with Welch’s correction.
    Figure Legend Snippet: Glycolytic and pentose phosphate pathway metabolite levels remain constant under IPP- and farnesylation-deficient conditions. Levels of glycolytic and pentose phosphate pathway intermediates were measured by liquid chromatography with tandem mass spectrometry and normalized based on parasitemia of each individual sample to give concentration per cell. No significant changes are observed after treatment with FSM (5 μM) or FTI (10 μM). n = 3, unpaired t test with Welch’s correction.

    Techniques Used: Liquid Chromatography, Mass Spectrometry, Concentration Assay

    isopentenyl pyrophosphate  (Echelon Biosciences)


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    Echelon Biosciences isopentenyl pyrophosphate
    Isopentenyl Pyrophosphate, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ipp  (Echelon Biosciences)


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    Echelon Biosciences ipp
    Alpaca B30.2 domain binds PAg similarly to human B30.2 domain. ITC binding isotherm traces of alpaca B30.2 domain with <t>either</t> <t>HMBPP</t> or <t>IPP</t> show interaction between the protein and PAgs. The binding measurements were all performed using 100 μM protein in the cell and 2 mM ligand in the syringe. The interaction with HMBPP is shown on the Left, whereas the interaction with IPP is shown on the Right. The buffer controls are shown in open circle (HMBPP) or open triangle (IPP); the trace for protein interaction is in filled circle (HMBPP) or filled triangle (IPP). The binding curves in red shown in both plots were fit assuming one-site binding. The reported molar ratio is the ratio of PAg:B30.2 domain.
    Ipp, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Alpaca ( Vicugna pacos ), the first nonprimate species with a phosphoantigen-reactive Vγ9Vδ2 T cell subset"

    Article Title: Alpaca ( Vicugna pacos ), the first nonprimate species with a phosphoantigen-reactive Vγ9Vδ2 T cell subset

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.1909474117

    Alpaca B30.2 domain binds PAg similarly to human B30.2 domain. ITC binding isotherm traces of alpaca B30.2 domain with either HMBPP or IPP show interaction between the protein and PAgs. The binding measurements were all performed using 100 μM protein in the cell and 2 mM ligand in the syringe. The interaction with HMBPP is shown on the Left, whereas the interaction with IPP is shown on the Right. The buffer controls are shown in open circle (HMBPP) or open triangle (IPP); the trace for protein interaction is in filled circle (HMBPP) or filled triangle (IPP). The binding curves in red shown in both plots were fit assuming one-site binding. The reported molar ratio is the ratio of PAg:B30.2 domain.
    Figure Legend Snippet: Alpaca B30.2 domain binds PAg similarly to human B30.2 domain. ITC binding isotherm traces of alpaca B30.2 domain with either HMBPP or IPP show interaction between the protein and PAgs. The binding measurements were all performed using 100 μM protein in the cell and 2 mM ligand in the syringe. The interaction with HMBPP is shown on the Left, whereas the interaction with IPP is shown on the Right. The buffer controls are shown in open circle (HMBPP) or open triangle (IPP); the trace for protein interaction is in filled circle (HMBPP) or filled triangle (IPP). The binding curves in red shown in both plots were fit assuming one-site binding. The reported molar ratio is the ratio of PAg:B30.2 domain.

    Techniques Used: Binding Assay

    isopentenyl diphosphate ipp  (Echelon Biosciences)


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    Echelon Biosciences isopentenyl diphosphate ipp
    Isopentenyl Diphosphate Ipp, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Echelon Biosciences ipp
    Analysis of catalytic activity of Trypanosoma cruzi farnesyl pyrophosphate synthase (tcFPPS). (a) Scheme of coupled enzymatic assay for analysis of tcFPPS activity. tcFPPS reaction: Synthesis of farnesyl pyrophosphate (FPP) and pyrophosphate (PPi) from geranyl <t>pyrophosphate</t> <t>(GPP)</t> and isopentenyl diphosphate <t>(IPP).</t> Pyruvate phosphate dikinase (PPDK) reaction: Conversion of PPi into ATP using AMP, and with the associated conversion of phosphoenol pyruvate (PEP) into pyruvate (Pyr) and phosphate (PO 4 2− ). Luciferase reaction: ATP drives the conversion of luciferin into oxyluciferin using O 2 and Mg 2+ . This is followed by the spontaneous reaction that generates light. AMP and pyrophosphate are recycled into the PPDK reaction. (b) Enzymatic activity monitored continuously as the increase in luminescence (relative light unit, RLU) as a function of time (in seconds) in a standard assay mixture consisting of 10 nM tcFPPS, 0.32 U PPDK, luciferase/luciferin reaction kit containing Mg 2+ , 0.5 mM PEP, 1 μM GPP and 2.5 μM IPP, 0.4 mM AMP in HEPES‐EDTA at pH 7.0. (c) Activity of tcFPPS immobilized to a full sensor chip surface on the bench. Reagents were pipetted directly to the chip surface. Samples were extracted every 10 s, and the luminescence was measured according to the standard procedure in microplates. Activities were determined directly after immobilization (top, circles), after a 2‐min incubation with 40 μM risendronate (middle, squares), and after a 2‐min incubation with guanidinium‐HCl (bottom, triangles)
    Ipp, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Establishing Trypanosoma cruzi farnesyl pyrophosphate synthase as a viable target for biosensor driven fragment‐based lead discovery"

    Article Title: Establishing Trypanosoma cruzi farnesyl pyrophosphate synthase as a viable target for biosensor driven fragment‐based lead discovery

    Journal: Protein Science : A Publication of the Protein Society

    doi: 10.1002/pro.3834

    Analysis of catalytic activity of Trypanosoma cruzi farnesyl pyrophosphate synthase (tcFPPS). (a) Scheme of coupled enzymatic assay for analysis of tcFPPS activity. tcFPPS reaction: Synthesis of farnesyl pyrophosphate (FPP) and pyrophosphate (PPi) from geranyl pyrophosphate (GPP) and isopentenyl diphosphate (IPP). Pyruvate phosphate dikinase (PPDK) reaction: Conversion of PPi into ATP using AMP, and with the associated conversion of phosphoenol pyruvate (PEP) into pyruvate (Pyr) and phosphate (PO 4 2− ). Luciferase reaction: ATP drives the conversion of luciferin into oxyluciferin using O 2 and Mg 2+ . This is followed by the spontaneous reaction that generates light. AMP and pyrophosphate are recycled into the PPDK reaction. (b) Enzymatic activity monitored continuously as the increase in luminescence (relative light unit, RLU) as a function of time (in seconds) in a standard assay mixture consisting of 10 nM tcFPPS, 0.32 U PPDK, luciferase/luciferin reaction kit containing Mg 2+ , 0.5 mM PEP, 1 μM GPP and 2.5 μM IPP, 0.4 mM AMP in HEPES‐EDTA at pH 7.0. (c) Activity of tcFPPS immobilized to a full sensor chip surface on the bench. Reagents were pipetted directly to the chip surface. Samples were extracted every 10 s, and the luminescence was measured according to the standard procedure in microplates. Activities were determined directly after immobilization (top, circles), after a 2‐min incubation with 40 μM risendronate (middle, squares), and after a 2‐min incubation with guanidinium‐HCl (bottom, triangles)
    Figure Legend Snippet: Analysis of catalytic activity of Trypanosoma cruzi farnesyl pyrophosphate synthase (tcFPPS). (a) Scheme of coupled enzymatic assay for analysis of tcFPPS activity. tcFPPS reaction: Synthesis of farnesyl pyrophosphate (FPP) and pyrophosphate (PPi) from geranyl pyrophosphate (GPP) and isopentenyl diphosphate (IPP). Pyruvate phosphate dikinase (PPDK) reaction: Conversion of PPi into ATP using AMP, and with the associated conversion of phosphoenol pyruvate (PEP) into pyruvate (Pyr) and phosphate (PO 4 2− ). Luciferase reaction: ATP drives the conversion of luciferin into oxyluciferin using O 2 and Mg 2+ . This is followed by the spontaneous reaction that generates light. AMP and pyrophosphate are recycled into the PPDK reaction. (b) Enzymatic activity monitored continuously as the increase in luminescence (relative light unit, RLU) as a function of time (in seconds) in a standard assay mixture consisting of 10 nM tcFPPS, 0.32 U PPDK, luciferase/luciferin reaction kit containing Mg 2+ , 0.5 mM PEP, 1 μM GPP and 2.5 μM IPP, 0.4 mM AMP in HEPES‐EDTA at pH 7.0. (c) Activity of tcFPPS immobilized to a full sensor chip surface on the bench. Reagents were pipetted directly to the chip surface. Samples were extracted every 10 s, and the luminescence was measured according to the standard procedure in microplates. Activities were determined directly after immobilization (top, circles), after a 2‐min incubation with 40 μM risendronate (middle, squares), and after a 2‐min incubation with guanidinium‐HCl (bottom, triangles)

    Techniques Used: Activity Assay, Enzymatic Assay, Luciferase, Incubation

    Effect of variations in enzyme and substrate concentrations on catalytic rate of coupled FPPS reaction
    Figure Legend Snippet: Effect of variations in enzyme and substrate concentrations on catalytic rate of coupled FPPS reaction

    Techniques Used:

    ipp  (Echelon Biosciences)


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    Echelon Biosciences ipp
    Growth under temperature stress requires <t>IPP</t> synthesis and protein farnesylation. (A) Prenylphosphate substrates for protein prenylation are derived from the non-mevalonate MEP pathway. MEP pathway products, IPP and dimethylallyl pyrophosphate (DMAPP) serve as precursors to FPP used by FTase and GGPP used by GGTase in protein prenylation. FSM treatment inhibits production of IPP and DMAPP. Farnesyltransferase <t>inhibitors</t> <t>(FTI</t> or BMS) inhibit protein farnesylation, while geranylgeranyltransferase inhibitors (GGTI) prevent protein geranylgeranylation. (B) Parasites were treated with FSM (5 μM), farnesyltransferase inhibitors [FTI (10μM) and BMS (200nM)] or geranylgeranyltransferase inhibitor GGTI (2μM) for 24 hours prior to a 6 hour heat (40°C) or cold (25°C) shock. (C,D) FSM-treated parasite growth is significantly reduced after heat shock (C) and cold shock (D). (E) Inhibition of farnesylation by treating parasites with FTI or BMS significantly reduced growth after temperature stress. Growth in GGTI-treated parasites is unchanged after heat or cold shock. (C-E) n = 6; ** p≤0.01; *** p≤0.001; **** p≤0.0001, (C,D) 2-way ANOVA, p-values adjusted for multiple comparisons using Sidak’s multiple comparison test, (E) within each treatment group the normalized control was compared to temperature shock sample by unpaired t-test with Welch’s correction. Abbreviations: control (ctrl), heat shock (hs), cold shock (cs).
    Ipp, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Protein prenylation and Hsp40 in thermotolerance of Plasmodium falciparum malaria parasites"

    Article Title: Protein prenylation and Hsp40 in thermotolerance of Plasmodium falciparum malaria parasites

    Journal: bioRxiv

    doi: 10.1101/842468

    Growth under temperature stress requires IPP synthesis and protein farnesylation. (A) Prenylphosphate substrates for protein prenylation are derived from the non-mevalonate MEP pathway. MEP pathway products, IPP and dimethylallyl pyrophosphate (DMAPP) serve as precursors to FPP used by FTase and GGPP used by GGTase in protein prenylation. FSM treatment inhibits production of IPP and DMAPP. Farnesyltransferase inhibitors (FTI or BMS) inhibit protein farnesylation, while geranylgeranyltransferase inhibitors (GGTI) prevent protein geranylgeranylation. (B) Parasites were treated with FSM (5 μM), farnesyltransferase inhibitors [FTI (10μM) and BMS (200nM)] or geranylgeranyltransferase inhibitor GGTI (2μM) for 24 hours prior to a 6 hour heat (40°C) or cold (25°C) shock. (C,D) FSM-treated parasite growth is significantly reduced after heat shock (C) and cold shock (D). (E) Inhibition of farnesylation by treating parasites with FTI or BMS significantly reduced growth after temperature stress. Growth in GGTI-treated parasites is unchanged after heat or cold shock. (C-E) n = 6; ** p≤0.01; *** p≤0.001; **** p≤0.0001, (C,D) 2-way ANOVA, p-values adjusted for multiple comparisons using Sidak’s multiple comparison test, (E) within each treatment group the normalized control was compared to temperature shock sample by unpaired t-test with Welch’s correction. Abbreviations: control (ctrl), heat shock (hs), cold shock (cs).
    Figure Legend Snippet: Growth under temperature stress requires IPP synthesis and protein farnesylation. (A) Prenylphosphate substrates for protein prenylation are derived from the non-mevalonate MEP pathway. MEP pathway products, IPP and dimethylallyl pyrophosphate (DMAPP) serve as precursors to FPP used by FTase and GGPP used by GGTase in protein prenylation. FSM treatment inhibits production of IPP and DMAPP. Farnesyltransferase inhibitors (FTI or BMS) inhibit protein farnesylation, while geranylgeranyltransferase inhibitors (GGTI) prevent protein geranylgeranylation. (B) Parasites were treated with FSM (5 μM), farnesyltransferase inhibitors [FTI (10μM) and BMS (200nM)] or geranylgeranyltransferase inhibitor GGTI (2μM) for 24 hours prior to a 6 hour heat (40°C) or cold (25°C) shock. (C,D) FSM-treated parasite growth is significantly reduced after heat shock (C) and cold shock (D). (E) Inhibition of farnesylation by treating parasites with FTI or BMS significantly reduced growth after temperature stress. Growth in GGTI-treated parasites is unchanged after heat or cold shock. (C-E) n = 6; ** p≤0.01; *** p≤0.001; **** p≤0.0001, (C,D) 2-way ANOVA, p-values adjusted for multiple comparisons using Sidak’s multiple comparison test, (E) within each treatment group the normalized control was compared to temperature shock sample by unpaired t-test with Welch’s correction. Abbreviations: control (ctrl), heat shock (hs), cold shock (cs).

    Techniques Used: Derivative Assay, Inhibition

    Inhibition of either IPP synthesis or protein farnesylation results in reduced membrane-association of HSP40. (A,B) Representative anti-HSP40 immunoblots of control and FSM (20μM) treated (A) or FTI (10μM) treated (B) P. falciparum total lysate and membrane fractions. (C,D) Quantification of several immunoblots adjusted to loading control. HSP40 is significantly reduced in the membrane fraction after inhibition of IPP synthesis (C) and inhibition of farnesylation (D). Anti-HAD1 and Anti-Exp-2, loading controls for total lysate and membrane fractions, respectively. ** p≤0.01, *** p≤0.001 unpaired t-test with Welch’s correction. (E-H) HSP40 membrane association is reduced after FSM and FTI treatment. Apparent membrane-associated HSP40 (10nm gold particles, arrowheads) is reduced after inhibition of IPP synthesis and farnesylation. The number of membrane-associated HSP40 per micrograph is quantified for control and treated parasites (G,H). A single control cohort was quantified. A decrease in the number of membrane-associated HSP40 particles is observed. * p≤0.05, unpaired t-test with Welch’s correction. Scale, 500 nm.
    Figure Legend Snippet: Inhibition of either IPP synthesis or protein farnesylation results in reduced membrane-association of HSP40. (A,B) Representative anti-HSP40 immunoblots of control and FSM (20μM) treated (A) or FTI (10μM) treated (B) P. falciparum total lysate and membrane fractions. (C,D) Quantification of several immunoblots adjusted to loading control. HSP40 is significantly reduced in the membrane fraction after inhibition of IPP synthesis (C) and inhibition of farnesylation (D). Anti-HAD1 and Anti-Exp-2, loading controls for total lysate and membrane fractions, respectively. ** p≤0.01, *** p≤0.001 unpaired t-test with Welch’s correction. (E-H) HSP40 membrane association is reduced after FSM and FTI treatment. Apparent membrane-associated HSP40 (10nm gold particles, arrowheads) is reduced after inhibition of IPP synthesis and farnesylation. The number of membrane-associated HSP40 per micrograph is quantified for control and treated parasites (G,H). A single control cohort was quantified. A decrease in the number of membrane-associated HSP40 particles is observed. * p≤0.05, unpaired t-test with Welch’s correction. Scale, 500 nm.

    Techniques Used: Inhibition, Western Blot

    (A) Gene ontology analysis reveals three significant HSP40-associated biological processes in asexual P. falciparum. All significant associations are lost in FSM-treated parasites. (B,C) The interactions of HSP40 with Tubulin α chain [Tub α; Q6ZLZ9], Tubulin chain [Tub β; Q7KQL5], Actin-1 [Act1; Q8I4X0], and GAPDH [Q8IKK7] are significantly reduced after inhibition of IPP synthesis and/or farnesylation. Interestingly, interactions with HSP70 [Q8IB24] are significantly increased after farnesylation inhibition, but not after inhibition of IPP synthesis. Protein-protein interactions were determined by mass spectrometry after IP of parasite lysate with anti-HSP40. Results for FSM (5μM)- and FTI (10μM)-treated parasites are compared to untreated controls. n = 3; multiple unpaired t-tests.
    Figure Legend Snippet: (A) Gene ontology analysis reveals three significant HSP40-associated biological processes in asexual P. falciparum. All significant associations are lost in FSM-treated parasites. (B,C) The interactions of HSP40 with Tubulin α chain [Tub α; Q6ZLZ9], Tubulin chain [Tub β; Q7KQL5], Actin-1 [Act1; Q8I4X0], and GAPDH [Q8IKK7] are significantly reduced after inhibition of IPP synthesis and/or farnesylation. Interestingly, interactions with HSP70 [Q8IB24] are significantly increased after farnesylation inhibition, but not after inhibition of IPP synthesis. Protein-protein interactions were determined by mass spectrometry after IP of parasite lysate with anti-HSP40. Results for FSM (5μM)- and FTI (10μM)-treated parasites are compared to untreated controls. n = 3; multiple unpaired t-tests.

    Techniques Used: Inhibition, Mass Spectrometry

    (A-C) Inhibition of IPP synthesis and farnesylation reduced membrane association of GAPDH. (A) Representative anti-GAPDH immunoblots of control, FSM (20μM) and FTI (10μM) treated P. falciparum . (B,C) Quantification of several immunoblots adjusted with loading control. Anti-Hsp70 (1:5,000) and anti-PM-V (1:500) were used as loading controls for total lysate and membrane fractions, respectively. n = 5-6; **** p≤0.0001, unpaired t-test with Welch’s correction. (D) GAPDH catalyzes the conversion of glyceraldehyde 3-phosphate (GAPD) to 1,3-bisphosphoglycerate, which is further reduced to 2/3-phosphoglycerate (2/3-PGA). (E,F) Levels of GAPD and 2/3-PGA were measured by liquid chromatography with tandem mass spectrometry and normalized based on parasitemia of each individual sample to give concentration per cell. The amount of GAPD and 2/3-PGA is unchanged after treatment with FSM (5μM) or FTI (10μM). n=3, unpaired t-test with Welch’s correction.
    Figure Legend Snippet: (A-C) Inhibition of IPP synthesis and farnesylation reduced membrane association of GAPDH. (A) Representative anti-GAPDH immunoblots of control, FSM (20μM) and FTI (10μM) treated P. falciparum . (B,C) Quantification of several immunoblots adjusted with loading control. Anti-Hsp70 (1:5,000) and anti-PM-V (1:500) were used as loading controls for total lysate and membrane fractions, respectively. n = 5-6; **** p≤0.0001, unpaired t-test with Welch’s correction. (D) GAPDH catalyzes the conversion of glyceraldehyde 3-phosphate (GAPD) to 1,3-bisphosphoglycerate, which is further reduced to 2/3-phosphoglycerate (2/3-PGA). (E,F) Levels of GAPD and 2/3-PGA were measured by liquid chromatography with tandem mass spectrometry and normalized based on parasitemia of each individual sample to give concentration per cell. The amount of GAPD and 2/3-PGA is unchanged after treatment with FSM (5μM) or FTI (10μM). n=3, unpaired t-test with Welch’s correction.

    Techniques Used: Inhibition, Western Blot, Liquid Chromatography, Mass Spectrometry, Concentration Assay

    Glycolytic and pentose phosphate pathway metabolite levels remain constant under IPP and farnesylation-deficient conditions. Levels of glycolytic and pentose phosphate pathway intermediates were measured by liquid chromatography with tandem mass spectrometry and normalized based on parasitemia of each individual sample to give concentration per cell. No significant changes are observed after treatment with FSM (5µM) or FTI (10µM). n=3, unpaired t-test with Welch’s correction.
    Figure Legend Snippet: Glycolytic and pentose phosphate pathway metabolite levels remain constant under IPP and farnesylation-deficient conditions. Levels of glycolytic and pentose phosphate pathway intermediates were measured by liquid chromatography with tandem mass spectrometry and normalized based on parasitemia of each individual sample to give concentration per cell. No significant changes are observed after treatment with FSM (5µM) or FTI (10µM). n=3, unpaired t-test with Welch’s correction.

    Techniques Used: Liquid Chromatography, Mass Spectrometry, Concentration Assay

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