Journal: The Journal of Clinical Investigation

Article Title: Small-molecule modulators of B56-PP2A restore 4E-BP function to suppress eIF4E-dependent translation in cancer cells

doi: 10.1172/JCI176093

Figure Lengend Snippet: ( A ) Cells were treated with vehicle, 20 μM DT-1310, or 20 μM DT-061 and analyzed by immunoblotting. Non-p45, non-p46: rat and human 4E-BP1 that lacks phosphorylation at Thr45/46. Cells were treated for 1 hour (IEC-18), 3 hours (Capan-1, FET), or 5 hours (SNG-M). ( B ) As in A except that cells were treated with 20 μM DT-061 and 80 μM DT-766 alone or in combination. ( C ) Cell lines were pretreated with 120 nM OA or 6 nM Cal-A before addition of DT-061 as indicated for 3 hours and analysis by immunoblotting. ( D ) Cells were transfected with nontargeting siRNA (NT) or siRNA against B56α ( PPP2R5A ) for 48 hours (IEC-18) or 120 hours (with retransfection at 48 hours, FET) before treatment with vehicle or DT-061 and immunoblot analysis. p-64: 4E-BP1 phosphorylated on Ser64 (numbering for rat 4E-BP1). All data are representative of 3 or more independent experiments.

Article Snippet: Dilutions of antibodies for immunoblotting were as follows: 4E-BP1 (Cell Signaling Technology CST-9644, 1:20,000), 4E-BP2 (CST-2845, 1:2,000–1:4,000), 4E-BP3 (Abnova H00008637-M05, 1:500), phospho-64/65 4E-BP1 (CST-9451, 1:500–1:2,000, and Santa Cruz Biotechnology sc-18091-R, 1:500), non-phospho–45/46 4E-BP1 (CST-4923, 1:2,000–1:4,000), GAPDH (CST-5174, 1:60,000), actin (Sigma-Aldrich A2066 and AbClonal AC026, 1:10,000–1:20,000), eIF4E (sc-271480 or sc-9976, 1:2,000–1:4,000), phospho-eIF4E Ser209 (CST-9741, 1:1,000), eIF4G (sc-133155, 1:1,000, or CST-2469, 1:100,000), AKT (CST-9272, 1:3,000), phospho-AKT S473 (CST-4060, 1:20,000), phospho-AKT T308 (CST-2965, 1:1,000–1:8,000), pULK-1 (CST-14202, 1:1,000), total p70S6K (sc-8418, 1:2,000), phospho-p70S6K (AbClonal AP0564, 1:2,000), ERK (CST-9102, 1:2,000), phospho-ERK (CST-9106, 1:2,000), B56α (sc-136045, 1:200), B55α (CST-2290, 1:2,000), ATF4 (Proteintech 10835-1-AP, 1:2,000–1:8,000), TFE3 (CST-14779, 1:2,000), TFEB (CST-4240, 1:1,000), cleaved PARP (CST-5625, 1:1,000), cleaved caspase-3 (CST-9661, 1:500), goat anti-rabbit–HRP (Millipore AP132P, 1:1,000–1:2,000), goat anti-rabbit–HRP (Jackson ImmunoResearch 111-035-144, 1:10,000–1:50,000), and goat anti-mouse–HRP (Bio-Rad 170-6516, 1:3,000).

Techniques: Western Blot, Phospho-proteomics, Transfection

Journal: The Journal of Clinical Investigation

Article Title: Small-molecule modulators of B56-PP2A restore 4E-BP function to suppress eIF4E-dependent translation in cancer cells

doi: 10.1172/JCI176093

Figure Lengend Snippet: SMAPs such as DT-061 and DT-1154 activate a subset of B56-PP2A heterotrimers (B56α-, B56β-, B56ε-PP2A) to dephosphorylate 4E-BP1 and 4E-BP2 at canonical sites. SMAPs/B56-PP2A also dephosphorylate and activate the transcription factors TFE3/TFEB to induce ATF4 transcription. ATF4 accumulation activates transcription of the 4E-BP1 gene, leading to upregulation of 4E-BP1 protein that is also dephosphorylated by B56-PP2A. Active 4E-BP1 prevents formation of the eIF4F translation initiation complex and inhibits eIF4E-dependent translation.

Article Snippet: Dilutions of antibodies for immunoblotting were as follows: 4E-BP1 (Cell Signaling Technology CST-9644, 1:20,000), 4E-BP2 (CST-2845, 1:2,000–1:4,000), 4E-BP3 (Abnova H00008637-M05, 1:500), phospho-64/65 4E-BP1 (CST-9451, 1:500–1:2,000, and Santa Cruz Biotechnology sc-18091-R, 1:500), non-phospho–45/46 4E-BP1 (CST-4923, 1:2,000–1:4,000), GAPDH (CST-5174, 1:60,000), actin (Sigma-Aldrich A2066 and AbClonal AC026, 1:10,000–1:20,000), eIF4E (sc-271480 or sc-9976, 1:2,000–1:4,000), phospho-eIF4E Ser209 (CST-9741, 1:1,000), eIF4G (sc-133155, 1:1,000, or CST-2469, 1:100,000), AKT (CST-9272, 1:3,000), phospho-AKT S473 (CST-4060, 1:20,000), phospho-AKT T308 (CST-2965, 1:1,000–1:8,000), pULK-1 (CST-14202, 1:1,000), total p70S6K (sc-8418, 1:2,000), phospho-p70S6K (AbClonal AP0564, 1:2,000), ERK (CST-9102, 1:2,000), phospho-ERK (CST-9106, 1:2,000), B56α (sc-136045, 1:200), B55α (CST-2290, 1:2,000), ATF4 (Proteintech 10835-1-AP, 1:2,000–1:8,000), TFE3 (CST-14779, 1:2,000), TFEB (CST-4240, 1:1,000), cleaved PARP (CST-5625, 1:1,000), cleaved caspase-3 (CST-9661, 1:500), goat anti-rabbit–HRP (Millipore AP132P, 1:1,000–1:2,000), goat anti-rabbit–HRP (Jackson ImmunoResearch 111-035-144, 1:10,000–1:50,000), and goat anti-mouse–HRP (Bio-Rad 170-6516, 1:3,000).

Techniques:

Journal: Emerging Microbes & Infections

Article Title: To be or not to be phosphorylated: understanding the role of Ebola virus nucleoprotein in the dynamic interplay with the transcriptional activator VP30 and the host phosphatase PP2A-B56

doi: 10.1080/22221751.2024.2447612

Figure Lengend Snippet: Transcription activation requires a close spatial and orientational interplay between NP, PP2A-B56α, and VP30. (A) EBOV NP mutants carrying mutations at the PP2A-B56α (orange) and VP30 (green) binding interfaces that were utilized for the following experiments. (B) Transcription and replication competent virus-like particle (trVLP) assay with a tetracistronic minigenome, reporter gene activity in producer cells (p0). 72 hours post transfection (hpt), luciferase activity was measured reflecting EBOV-specific transcription. NPwt set to 100%. Mean of individual experiments, ns: not statistically significant. (C) Reporter gene activity in infected target cells (p1). Generated trVLPs from (B) supernatants were collected and concentrated via ultracentrifugation to infect naïve p1 cells. Luciferase activity was measured 72 hpt reflecting primary transcription. NPwt set to 100%. Mean of individual experiments. (D) Expression of NP and VP40 in p0 cell lysates and incorporation into trVLPs analysed by WB analysis. Samples generated as described in panels B and C. (E–F) Co-immunoprecipitation of NP mutants with B56α (E) or VP30 (F) in HEK293 cells using anti-flag antibody covered magnetic beads. Flag-tagged NP is precipitated together with potential binding partners that are subsequently detected through WB. On the right side, quantification from individual experiments. NPwt set to 100%. (G) VP30 phosphorylation in the presence of NP upon recombinant expression in HeLa cells. VP30AA was included as positive control for dephosphorylated VP30 (lane 11 ). On the right side, quantification from individual experiments. NPwt set to 100%.

Article Snippet: We could recently determine the underlying mechanism of how EBOV and also Marburg virus VP30 are dephosphorylated by PP2A: the regulatory subunit B56α of PP2A as well as its substrate VP30 directly bind to NP, that thereby acts as a scaffold protein bringing both proteins in close contact, a mechanism that is conserved among filoviruses [ , ].

Techniques: Activation Assay, Binding Assay, Virus, Activity Assay, Transfection, Luciferase, Infection, Generated, Expressing, Immunoprecipitation, Magnetic Beads, Phospho-proteomics, Recombinant, Positive Control

Journal: Emerging Microbes & Infections

Article Title: To be or not to be phosphorylated: understanding the role of Ebola virus nucleoprotein in the dynamic interplay with the transcriptional activator VP30 and the host phosphatase PP2A-B56

doi: 10.1080/22221751.2024.2447612

Figure Lengend Snippet: Rescue and characterization of recEBOV-NP mutants with an increased distance between the binding sites of PP2A-B56α and VP30. (A) Successfully rescued recEBOV as indicated. Bottom, scheme of NP with the naturally emerged compensatory mutation NP-T603I in the recEBOV-NP58aa context. (B) Growth kinetics of the in panel A introduced recEBOVs. VeroE6 cells were infected with the indicated MOI and supernatant was collected at 0-, 1-, 2-, 3-, 4-, and 7-dpi. Viral titres were determined by TCID 50 /ml and a mean from three independent experiments is shown. (C) Viral inclusion body formation in HuH7 cells infected with the respective viruses (MOI 0.3) at 24 hpi. NP and VP40 were stained with specific antibodies, and nuclei are stained with DAPI. (D) Protein expression in lysates and supernatants at 24 hpi from the samples generated in (C). (E) Viral protein incorporation in equal amounts of infectious virus particles (PFU). 2 × 10 5 PFU were concentrated by ultracentrifugation for each of the respected recEBOVs and analysed by WB. (F) Viral genome copies in equal amounts of infectious virus particles (PFU). Mean of 5 individual experiments is shown. C t values (as 45– C t ) are shown based on EBOV-specific RT-qPCR. (G) Ratio of incorporated viral proteins in recEBOV purified from HuH7 cells determined by MS/MS. Mean and standard deviation determined from four independent experiments. For individual measurements, see supplement S4. (H) Cryo-electron microscopy images of recEBOVs purified from HuH7 cells.

Article Snippet: We could recently determine the underlying mechanism of how EBOV and also Marburg virus VP30 are dephosphorylated by PP2A: the regulatory subunit B56α of PP2A as well as its substrate VP30 directly bind to NP, that thereby acts as a scaffold protein bringing both proteins in close contact, a mechanism that is conserved among filoviruses [ , ].

Techniques: Binding Assay, Mutagenesis, Infection, Staining, Expressing, Generated, Virus, Quantitative RT-PCR, Purification, Tandem Mass Spectroscopy, Standard Deviation, Cryo-Electron Microscopy

Journal: Emerging Microbes & Infections

Article Title: To be or not to be phosphorylated: understanding the role of Ebola virus nucleoprotein in the dynamic interplay with the transcriptional activator VP30 and the host phosphatase PP2A-B56

doi: 10.1080/22221751.2024.2447612

Figure Lengend Snippet: Model of NP and VP30 phosphorylation contributing to an efficient EBOV life cycle. NP phosphorylation state differs between virions and in cells. EBOV NP is phosphorylated at position T603 in virions, upon infection of target cells; NP-T603 requires dephosphorylation to support efficient viral transcription and replication. At a later time point during the viral replication cycle, NP-T603 is re-phosphorylated and finally packaged into newly formed virions. NP serves as a key player in the tight interplay of VP30-dependent transcription initiation by facilitating VP30 dephosphosphorylation through PP2A-B56α.

Article Snippet: We could recently determine the underlying mechanism of how EBOV and also Marburg virus VP30 are dephosphorylated by PP2A: the regulatory subunit B56α of PP2A as well as its substrate VP30 directly bind to NP, that thereby acts as a scaffold protein bringing both proteins in close contact, a mechanism that is conserved among filoviruses [ , ].

Techniques: Phospho-proteomics, Infection, De-Phosphorylation Assay