Journal: Science Advances

Article Title: Phosphorylation of shiftless is important for inhibiting the programmed −1 ribosomal frameshift

doi: 10.1126/sciadv.adw7471

Figure Lengend Snippet: ( A ) Schematic diagram of three AirID constructs used for PDB. ( B ) Working flow of the proximity biotinylation for identifying kinases responsible for SFL phosphorylation. WB, Western blot. ( C ) The expression plasmids (A) were transfected into HEK293T cells, and 0.5 μM biotins were supplemented in the cell culture to promote biotinylation. The cells were harvested and lysed 16 hours posttransfection and analyzed by Western blot. Input: proteins before SA magnetic bead enrichment. Output: proteins after SA magnetic bead enrichment. Representative of three independent experiments. ( D ) LC-MS/MS analysis of SA magnetic bead–enriched samples from three independent biological replicates. Gray dots were proteins detected in both the 10- and 35-nm groups; black dot was SFL, and red dots were Ser/Thr kinases with expression levels at least fourfold higher than the AirID group (both 10 and 35 nm), among which six kinases (indicated with blue fonts) were selected for further analysis. FC, fold change. ( E ) Left: The purple and blue circles represent Ser/Thr kinases with expression levels up-regulated at least fourfold compared to the AirID group in the 10- and 35-nm subgroups. The green circle includes 58 kinases predicted by the PhosphositePlus software. Right: Six Ser/Thr kinases were selected because they represented the consensus of three cycles. ( F ) Phos-Tag PAGE analysis of in vitro kinase assays. Six selected kinases were tested for the ability to phosphorylate SFL. Phosphorylation species are marked by red numbers. Representative of two independent experiments. ( G ) Identification of phosphorylation sites in SFL from the marked bands in (F) by LC-MS/MS. ( H ) Expression of HIV-1 proteins in HEK293T cells with knockdown of EEF2K , NEK9 , or PBK genes in the presence or absence of SFL. Representative of two independent experiments.

Article Snippet: The kinases tested include EEF2K (MCE, HY-P701676), MAP3K1 (MERCK, SRP5047), MAP3K7 (Sino Biological, M15-13G), NEK9 (Sino Biological, N11-11G), PBK (Sino Biological, T14-10G), and TTK (MERCK, SRP5095).

Techniques: Construct, Phospho-proteomics, Western Blot, Expressing, Transfection, Cell Culture, Liquid Chromatography with Mass Spectroscopy, Software, In Vitro, Knockdown

Journal: Acta Neuropathologica Communications

Article Title: Activity of translation regulator eukaryotic elongation factor-2 kinase is increased in Parkinson disease brain and its inhibition reduces alpha synuclein toxicity

doi: 10.1186/s40478-018-0554-9

Figure Lengend Snippet: eEF2K expression and activity in PD brain. a Quantitation of p-eEF2 (T56) IHC (3,3′-Diaminobenzidine-DAB staining) in postmortem hippocampus- Hip (CA1 and CA2 fields) and midbrain- MB (SN-substantia nigra, and PAG-peri-aqueductal gray matter) sections from 3 control and 6 PD cases (Additional file : Table S1; counts from at least 6 high power fields from each control or PD section; Mann–Whitney test, * p < 0.05, *** p < 0.005; error bars indicate Mean ± S.D.). b - d eEF2K mRNA expression in control and PD striatum ( b ), medial substantia nigra ( b ) and dorsal nucleus of vagus nerve ( d ). The following publicly available transcriptomic profile datasets were analyzed on the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO) platform: Striatum ( b )- dataset GEO accession # GSE28894, Illumina human Ref-8 v2.0 expression beadchip platform, probe ID ILMN_1789171, controls n = 15 and PD n = 15; Medial substantia nigra ( c )- dataset GEO accession # GSE8397, Affymetrix Human Genome U133B Array, probe ID 225546_at, controls n = 8 and PD n = 15; Dorsal nucleus of vagus ( d )- dataset GEO accession # GSE43490, Agilent-014850 Whole Human Genome Microarray, probe ID A_24_P716162, controls n = 6 and PD n = 7. (Mann–Whitney test, * p < 0.05; error bars in 3b-d indicate Mean ± S.D.). e Relative eEF2K mRNA expression in human iPSCs derived cultured midbrain control (WT, n = 3) or A53T ( n = 2) mutation carrying organoids (T-test, * p < 0.05; error bars indicate Mean ± S.D.)

Article Snippet: Additional reagents and biochemical assays employed during these studies include: pool of small interference RNAs (SiRNAs) targeting mouse eEF2K (Santa Cruz, #sc-39,012), Cell Titer Glo ATP measurement kit (Promega, #G7570), Lactate dehydrogenase (LDH) fluorometric assay (Novus Biologicals, #NBP2–54851), Seahorse Mito stress test kit (Agilent, #103015–100), 2′,7′-dichlorodihydrofluorescein diacetate (DCFDA) fluorescent ROS reagent (ThermoFisher, #D399), and MitoTracker Green fluorescent reagent for mitochondrial mass (ThermoFisher, #M7514).

Techniques: Expressing, Activity Assay, Quantitation Assay, Staining, Control, MANN-WHITNEY, Gene Expression, Microarray, Derivative Assay, Cell Culture, Mutagenesis

Journal: Acta Neuropathologica Communications

Article Title: Activity of translation regulator eukaryotic elongation factor-2 kinase is increased in Parkinson disease brain and its inhibition reduces alpha synuclein toxicity

doi: 10.1186/s40478-018-0554-9

Figure Lengend Snippet: Brain eEF2K expression and activity in transgenic M83 +/+ PD mice. a eEF2K mRNA levels in whole brain homogenates from transgenic M83 +/+ PD mice intramuscularly (IM) injected bilaterally with phosphate buffered saline (PBS, n = 10) or pre-formed fibrillar (PFF, n = 13) mouse wild type AS. (Mann–Whitney test, *** p < 0.005; error bars indicate Mean ± S.D.). b - c Western blot analysis of p-eEF2 (T56) and p-ASyn (S129) in whole brain homogenates from transgenic M83 +/+ PD mice intramuscularly (IM) injected bilaterally with phosphate buffered saline (PBS) or pre-formed fibrillar (PFF) mouse wild type AS ( b ), and corresponding densitometry analysis ( c ) ( n = 7/group; Mann–Whitney test, * p < 0.05, *** p < 0.005; error bars indicate Mean ± S.D.)

Article Snippet: Additional reagents and biochemical assays employed during these studies include: pool of small interference RNAs (SiRNAs) targeting mouse eEF2K (Santa Cruz, #sc-39,012), Cell Titer Glo ATP measurement kit (Promega, #G7570), Lactate dehydrogenase (LDH) fluorometric assay (Novus Biologicals, #NBP2–54851), Seahorse Mito stress test kit (Agilent, #103015–100), 2′,7′-dichlorodihydrofluorescein diacetate (DCFDA) fluorescent ROS reagent (ThermoFisher, #D399), and MitoTracker Green fluorescent reagent for mitochondrial mass (ThermoFisher, #M7514).

Techniques: Expressing, Activity Assay, Transgenic Assay, Injection, Saline, MANN-WHITNEY, Western Blot

Journal: Acta Neuropathologica Communications

Article Title: Activity of translation regulator eukaryotic elongation factor-2 kinase is increased in Parkinson disease brain and its inhibition reduces alpha synuclein toxicity

doi: 10.1186/s40478-018-0554-9

Figure Lengend Snippet: Effects of eEF2K inhibition on human AS cytotoxicity in differentiated N2A cells. a - b Western blot analysis of p-eEF2 (T56) levels in N2A cells subsequent to transient overexpression of human wild type or mutant A53T AS, with or without siRNA mediated eEF2K knockdown ( a ), and corresponding densitometry analysis ( b ) ( n = 6–9/group from three independent experiments; One-way ANOVA post-hoc Bonferroni test, * p < 0.05, *** p < 0.005; error bars indicate Mean ± S.E.M). c Measurements of cytotoxicity by lactate dehydrogenase-LDH release in the culture medium ( c ) and FACS analysis of propidium iodide-PI staining ( d ) in N2A cells subsequent to transient overexpression of human wild type or mutant A53T AS, with or without siRNA mediated eEF2K knockdown ( n = 9–12/group from three independent experiments; One-way ANOVA post-hoc Bonferroni test, * p < 0.05, ** p < 0.01, *** p < 0.005, NS = not significant; error bars indicate Mean ± S.D.)

Article Snippet: Additional reagents and biochemical assays employed during these studies include: pool of small interference RNAs (SiRNAs) targeting mouse eEF2K (Santa Cruz, #sc-39,012), Cell Titer Glo ATP measurement kit (Promega, #G7570), Lactate dehydrogenase (LDH) fluorometric assay (Novus Biologicals, #NBP2–54851), Seahorse Mito stress test kit (Agilent, #103015–100), 2′,7′-dichlorodihydrofluorescein diacetate (DCFDA) fluorescent ROS reagent (ThermoFisher, #D399), and MitoTracker Green fluorescent reagent for mitochondrial mass (ThermoFisher, #M7514).

Techniques: Inhibition, Western Blot, Over Expression, Mutagenesis, Knockdown, Staining

Journal: Acta Neuropathologica Communications

Article Title: Activity of translation regulator eukaryotic elongation factor-2 kinase is increased in Parkinson disease brain and its inhibition reduces alpha synuclein toxicity

doi: 10.1186/s40478-018-0554-9

Figure Lengend Snippet: Effects of eEF2K inhibition on mitochondrial dysfunction and oxidative stress induced by human AS in differentiated N2A cells. a - b Measurements of basal oxygen consumption rate-OCR ( b ) and ATP levels ( c ) in N2A cells subsequent to transient overexpression of human wild type or mutant A53T AS, with or without siRNA mediated eEF2K knockdown ( n = 9–12/group from three independent experiments; Unpaired T-test, * p < 0.05, ** p < 0.01, *** p < 0.005; error bars indicate Mean ± S.D.). c Flow cytometry analysis of reactive oxygen species (ROS), measured by DCFDA staining, in N2A cells subsequent to transient overexpression of human wild type or mutant A53T AS, with or without siRNA mediated eEF2K knockdown ( n = 9/group from three independent experiments; Unpaired T-test, * p < 0.05, ** p < 0.01, *** p < 0.005; error bars indicate Mean ± S.D.)

Article Snippet: Additional reagents and biochemical assays employed during these studies include: pool of small interference RNAs (SiRNAs) targeting mouse eEF2K (Santa Cruz, #sc-39,012), Cell Titer Glo ATP measurement kit (Promega, #G7570), Lactate dehydrogenase (LDH) fluorometric assay (Novus Biologicals, #NBP2–54851), Seahorse Mito stress test kit (Agilent, #103015–100), 2′,7′-dichlorodihydrofluorescein diacetate (DCFDA) fluorescent ROS reagent (ThermoFisher, #D399), and MitoTracker Green fluorescent reagent for mitochondrial mass (ThermoFisher, #M7514).

Techniques: Inhibition, Over Expression, Mutagenesis, Knockdown, Flow Cytometry, Staining

Journal: Biochemical Journal

Article Title: eEF2K enhances expression of PD-L1 by promoting the translation of its mRNA

doi: 10.1042/bcj20200697

Figure Lengend Snippet: Figure 1. eEF2K upregulates PD-L1 protein expression in PC3 and A549 cells.

Article Snippet: Lentiviral shRNA infection and NK-92 killing assays Lentiviral non-targeting control (sh-NC) shRNA or shRNA targeting eEF2K (sh-eEF2K; sc-39011-V) were purchased from Santa Cruz Biotechnology.

Techniques: Expressing

Journal: Biochemical Journal

Article Title: eEF2K enhances expression of PD-L1 by promoting the translation of its mRNA

doi: 10.1042/bcj20200697

Figure Lengend Snippet: Figure 2. Ablation of eEF2K shifts PD-L1 mRNAs from polysomal to non/subpolysomal fractions in PC3 cells.

Article Snippet: Lentiviral shRNA infection and NK-92 killing assays Lentiviral non-targeting control (sh-NC) shRNA or shRNA targeting eEF2K (sh-eEF2K; sc-39011-V) were purchased from Santa Cruz Biotechnology.

Techniques:

Journal: Biochemical Journal

Article Title: eEF2K enhances expression of PD-L1 by promoting the translation of its mRNA

doi: 10.1042/bcj20200697

Figure Lengend Snippet: Figure 3. PD-L1 mRNAs are more rapidly degraded in eEF2K-null PC3 cells. (A) Cells were cultured in in growth medium (control), or medium without FBS (no FBS), or medium without glucose (no

Article Snippet: Lentiviral shRNA infection and NK-92 killing assays Lentiviral non-targeting control (sh-NC) shRNA or shRNA targeting eEF2K (sh-eEF2K; sc-39011-V) were purchased from Santa Cruz Biotechnology.

Techniques: Cell Culture, Control

Journal: Biochemical Journal

Article Title: eEF2K enhances expression of PD-L1 by promoting the translation of its mRNA

doi: 10.1042/bcj20200697

Figure Lengend Snippet: Figure 4. PD-L1 translation is up-regulated by eEF2K through a bypass of uORF-mediated translational repression in

Article Snippet: Lentiviral shRNA infection and NK-92 killing assays Lentiviral non-targeting control (sh-NC) shRNA or shRNA targeting eEF2K (sh-eEF2K; sc-39011-V) were purchased from Santa Cruz Biotechnology.

Techniques:

Journal: Biochemical Journal

Article Title: eEF2K enhances expression of PD-L1 by promoting the translation of its mRNA

doi: 10.1042/bcj20200697

Figure Lengend Snippet: Figure 5. eEF2K-null PC3 cells are more susceptible to immune killing.

Article Snippet: Lentiviral shRNA infection and NK-92 killing assays Lentiviral non-targeting control (sh-NC) shRNA or shRNA targeting eEF2K (sh-eEF2K; sc-39011-V) were purchased from Santa Cruz Biotechnology.

Techniques:

Journal: Biochemical Journal

Article Title: eEF2K enhances expression of PD-L1 by promoting the translation of its mRNA

doi: 10.1042/bcj20200697

Figure Lengend Snippet: Figure 6. High levels of EEF2K positively correlate with enhanced CD274 gene expression and poor survival rates in

Article Snippet: Lentiviral shRNA infection and NK-92 killing assays Lentiviral non-targeting control (sh-NC) shRNA or shRNA targeting eEF2K (sh-eEF2K; sc-39011-V) were purchased from Santa Cruz Biotechnology.

Techniques: Gene Expression

Journal: International Journal of Molecular Sciences

Article Title: Targeting Protein Translation in Melanoma by Inhibiting EEF-2 Kinase Regulates Cholesterol Metabolism though SREBP2 to Inhibit Tumour Development

doi: 10.3390/ijms23073481

Figure Lengend Snippet: Knockdown of EEF2K inhibited melanoma cell proliferation. Data from the TCGA database suggest poorer survival with EEF2K overexpression ( A ) in melanoma patients. n = 115 for melanoma patients with low EEF2K; n = 105 for melanoma patients with high EEF2K. p = 0.2194. The data are available through the UCSC Xena Cancer Browser. Western blot showing knockdown of EEF2K with siRNA #1 in UACC 903, 1205 Lu and C8161Cl9 cell lines ( B ). Alpha-enolase served as the control for protein loading. Western blot showing knockdown of EEF2K with siRNAs #2, 3, 4 in UACC 903 ( C ). siRNA knockdown of EEF2K (siEEF2K) significantly reduced the growth of 1205 Lu ( D ) cells after 72 h in an MTS metabolism assay. Additionally, siRNA #1 dose dependently reduced the growth of 1205 Lu ( E ) and UACC 903 cells ( F ). siRNA to BRAF served as a positive control while scrambled (scr) siRNA served as the negative control. Significance was compared to scrambled knockdown by one-way ANOVA followed by Dunnett’s as post-hoc analysis. Experiments were replicated for n = 3 and representative graphs are shown.

Article Snippet: Blots were probed with antibodies according to each supplier’s recommendations: antibodies to EEF2K, p-EEF2K, alpha-enolase, LDL-R, HMGCR, SREBP2 and secondary antibodies conjugated with horseradish peroxidase from Santa Cruz Biotechnology.

Techniques: Knockdown, Over Expression, Western Blot, Control, Positive Control, Negative Control

Journal: International Journal of Molecular Sciences

Article Title: Targeting Protein Translation in Melanoma by Inhibiting EEF-2 Kinase Regulates Cholesterol Metabolism though SREBP2 to Inhibit Tumour Development

doi: 10.3390/ijms23073481

Figure Lengend Snippet: EEF2K modulated cholesterol metabolism in cancer cells. Genetic knockdown of EEF2K using siRNA (siEEF2K) reduced the cholesterol levels in 1205 Lu ( A ), UACC 903 ( B ) melanoma cell lines compared to scrambled siRNA knockdown. The cholesterol levels were normalized to the total protein content in cells. Significance was compared to scrambled siRNA knockdown (siScr) by t -test analysis ( n = 3). Knockdown of EEF2K led to a dose dependent reduction of major cholesterol biomarkers LDLR, HMGCR and SREBP2 as measured by protein levels. Alpha-enolase served as a protein loading control ( C ). The cholesterol levels were rescued by the addition of LDL or mevalonic acid (Mev) to the media of both UACC 903 ( D ) and 1205 Lu cells ( E ). Significance was compared to scrambled knockdown (siScr) by one-way ANOVA followed by Dunnett’s as post-hoc analysis. Similarly, addition of LDL and mevalonic acid to the cell growth media rescued the proliferation of both UACC 903 ( F ) and 1205 Lu ( G ) cells. Significance was compared to scrambled knockdown (siScr) by two-way ANOVA followed by Dunnett’s as post-hoc analysis ( n = 3).

Article Snippet: Blots were probed with antibodies according to each supplier’s recommendations: antibodies to EEF2K, p-EEF2K, alpha-enolase, LDL-R, HMGCR, SREBP2 and secondary antibodies conjugated with horseradish peroxidase from Santa Cruz Biotechnology.

Techniques: Knockdown, Control

Journal: International Journal of Molecular Sciences

Article Title: Targeting Protein Translation in Melanoma by Inhibiting EEF-2 Kinase Regulates Cholesterol Metabolism though SREBP2 to Inhibit Tumour Development

doi: 10.3390/ijms23073481

Figure Lengend Snippet: Pharmacological knockdown of EEF2K reduced cholesterol levels in melanoma. The pharmacological inhibitor of EEF2K, NH125 ( A ) significantly reduced the proliferation of melanoma cells after 72 h using an MTS assay ( B ) and decreased EEF2K phosphorylation ( C ) compared to total EEF2K levels. Alpha-enolase served as a protein loading control. Experiments were replicated three times. 5 µM NH125 reduced the cholesterol levels in UACC 903 ( D ) and 1205 Lu ( E ) cells compared to control DMSO. Significance was measured by one-way ANOVA followed by Dunnett’s as the post-hoc analysis. Lovastatin treatment served as a positive control for the inhibition of cholesterol synthesis ( n = 3). Mechanistically, NH125 dose dependently lowered the LDLR, HMGCR, SREBP2 and SREBP1 levels in UACC 903 cells to reduce cholesterol levels ( F ).

Article Snippet: Blots were probed with antibodies according to each supplier’s recommendations: antibodies to EEF2K, p-EEF2K, alpha-enolase, LDL-R, HMGCR, SREBP2 and secondary antibodies conjugated with horseradish peroxidase from Santa Cruz Biotechnology.

Techniques: Knockdown, MTS Assay, Phospho-proteomics, Control, Positive Control, Inhibition

Journal: International Journal of Molecular Sciences

Article Title: Targeting Protein Translation in Melanoma by Inhibiting EEF-2 Kinase Regulates Cholesterol Metabolism though SREBP2 to Inhibit Tumour Development

doi: 10.3390/ijms23073481

Figure Lengend Snippet: Targeting EEF2K by siRNA and NanoNH125 inhibited melanoma tumour growth through reduction of cholesterol levels. The siRNA-mediated knockdown of EEF2K reduced melanoma tumour development ( A ). The EEF2K siRNA knocked down UACC 903 cells were injected subcutaneously into nude mice and tumour growth kinetics were measured and compared to scrambled siRNA controls ( n = 8). The significance at each time point was compared to scrambled siRNA (siScr) by two-way ANOVA followed by Dunnett’s as post-hoc analysis. BRAF knockdown served as a positive control (A). Western blots confirming the required knockdown of EEF2K for 8 days while alpha-tubulin served as loading control ( B ). NH125 inhibited tumour growth of UACC 903 xenografts compared to DMSO control by 34% when dosed at 0.5 mg/kg ( n = 8) ( C ). The significance at each time point was compared to DMSO controls by two-way ANOVA followed by Dunnett’s as the post-hoc analysis. Higher doses of NH125 could not be tested because of toxicity. NanoNH125 significantly inhibited tumour growth of UACC 903 ( D ) and 1205 Lu ( E ) xenografts by 65% and 72%, respectively, compared to empty liposome vehicle control at 2 mg/kg following 28 days of treatment ( n = 8). The significance at each time point was compared to DMSO controls by two-way ANOVA followed by Dunnett’s as the post-hoc analysis. NanoNH125 did not significantly affect animal body weight ( D , E -insets) compared to empty liposome vehicle control. The NH125 treatment reduced cholesterol levels in tumours normalized by weight to tumours treated with empty liposome controls ( F ). To determine whether dietary supplementation with LDL can reverse the tumour inhibition mediated by siRNA-mediated EEF2K downregulation, 2 to 6 mg of LDL were orally administered to mice daily for three days prior to tumour cell implantation and continued daily for the duration of the experiment. Daily dietary supplementation of LDL slightly reversed the siEEF2K-mediated tumour inhibition . The increase in tumour growth with LDL supplementation was predicted to be subtle as these types of studies in animals are notoriously difficult to achieve. Since the reversal of the siEEF2K effect on tumour development was subtle, it may be due to insufficient pre-treatment or an inappropriate delivery route to achieve the optimal cholesterol levels needed for a full reversal.

Article Snippet: Blots were probed with antibodies according to each supplier’s recommendations: antibodies to EEF2K, p-EEF2K, alpha-enolase, LDL-R, HMGCR, SREBP2 and secondary antibodies conjugated with horseradish peroxidase from Santa Cruz Biotechnology.

Techniques: Knockdown, Injection, Positive Control, Western Blot, Control, Inhibition

Journal: International Journal of Molecular Sciences

Article Title: Targeting Protein Translation in Melanoma by Inhibiting EEF-2 Kinase Regulates Cholesterol Metabolism though SREBP2 to Inhibit Tumour Development

doi: 10.3390/ijms23073481

Figure Lengend Snippet: Schematics of mechanisms by which targeting EEF2K with siRNA or NH125 inhibit melanoma cell survival. Active EEF2K mediates protein translation through EEF2 and increases the levels of SREBP2, LDLR and HMGCR to increase cellular cholesterol and promote cell growth in cancer cells. Targeting EEF2K by siRNA and NH125 decreases the expressions of SREBP2, LDLR and HMGCR, which results in reduced cellular cholesterol and increased cell death.

Article Snippet: Blots were probed with antibodies according to each supplier’s recommendations: antibodies to EEF2K, p-EEF2K, alpha-enolase, LDL-R, HMGCR, SREBP2 and secondary antibodies conjugated with horseradish peroxidase from Santa Cruz Biotechnology.

Techniques: