eef1akmt4  (Proteintech)

Journal: Genes & Diseases

Article Title: EEF1AKMT4-eEF1A2 synergistically facilitates the progression of GBC by promoting ribosomal protein output

doi: 10.1016/j.gendis.2025.101619

Figure Lengend Snippet: EEF1AKMT4 trimethylates eEF1A2 at K36 site in GBC. (A) Spatial distribution of the five main methylation sites in the protein structure of eEF1A2 combined with GDP (PDB: 6ra9). Most of the methylation sites were located in the nucleotide-binding domain of eEF1A2. (B) Relative mRNA expression of five methylases, EEF1AKMT4, METTL13, N6AMT2, METTL21B, and METTL10, which are responsible for methylation of K36, K55, K79, K165, and K318 sites, respectively, was quantified by qRT-PCR in 10 cases of gallbladder cancer tissues and their patient-paired normal tissues. (C) Coomassie blue staining of anti-eEF1A2 co-immunoprecipitation in GBCSD, SGC996, and HEK293T cell lines. Target bands are indicated by a black box and were cut and subjected to LC-MS/MS to analyze the methylation status. (D) Methylation status at the K36, K55, K79, K165, and K318 sites of eEF1A2 in GBCSD, SGC996, and HEK293T cell lines were analyzed. (E) Representative tandem mass spectra identifying in vitro tri-methylated (upper) and non-methylated (lower) eEF1A2K36. m/z for b and y ions observed in the spectra are indicated in red and blue, respectively. (F) Histogram showing the methylation changes in K36, K55, K79, K165, and K318 before (upper) and after (lower) knockdown of EEF1AKMT4 in GBCSD cells. (G) Selected ion chromatograms for non-, mono-, di-, and trimethyl eEF1AK36 peptides from GluC digestion of endogenous eEF1A2 immunoprecipitated from whole-cell lysates of GBCSD, indicating the methylation status shift after EEF1AKMT4 knockdown. (H) Western blot analysis of the knockdown efficiency of EEF1AKMT4 in GBCSD and the status of eEF1A2 and eEF1A2K36me3. (I) Expression of eEF1AKMT4 and eEF1A2 K36me3 levels in GBC tumor tissues and paired normal tissues were examined by Western blotting and analyzed using ImageJ. Statistical significance between groups was assessed using Student's t-test. ns, not significant; ∗, p < 0.05; ∗∗, p < 0.01.

Article Snippet: pcDNA3.1 plasmids encoding wide eEF1A2 and EEF1AKMT4 were purchased from Biosun Company. eEF1A2 K36R and EEF1AKMT4 D88A mutations were constructed through PCR-mediated site-directed mutagenesis using a QuickChange Site-Directed Mutagenesis Kit (Agilent Stratagene, CA, USA).

Techniques: Methylation, Binding Assay, Expressing, Quantitative RT-PCR, Staining, Immunoprecipitation, Liquid Chromatography with Mass Spectroscopy, In Vitro, Knockdown, Western Blot

Journal: Genes & Diseases

Article Title: EEF1AKMT4-eEF1A2 synergistically facilitates the progression of GBC by promoting ribosomal protein output

doi: 10.1016/j.gendis.2025.101619

Figure Lengend Snippet: Knockdown of EEF1AKMT4 inhibits the malignant phenotype of GBC while its overexpression is not tumor-promoting. (A–B) Expression levels of EEF1AKMT4 in HEK293T, RBE, NOZ, GBCSD, SGC996, and OCUG-1 were detected using qRT-PCR (A) and Western blotting (B). (C) EEF1AKMT4 was knocked down or overexpressed in the GBCSD and SGC996 cell lines, and validated by Western blotting. (D) CCK-8 assays were performed to identify alterations in cell proliferation after EEF1AKMT4 knockdown in GBCSD and SGC996 cells. (E) Clone formation assay was performed in EEF1AKMT4 knockdown and vector control cells in GBCSD and SGC996 cells. (F) Wound healing assays were performed to investigate the effect of EEF1AKMT4 knockdown on the migration ability of the GBCSD and SGC996 cell lines. (G) Transwell assays with matrigel were performed to evaluate the invasive ability of GBCSD and SGC996 cells after EEF1AKMT4 knockdown. (H) CCK-8 assays were performed to identify alterations in cell proliferation after EEF1AKMT4 overexpression in GBCSD and SGC996 cells. ( I ) Transwell assays with matrigel were performed to evaluate the invasive ability of GBCSD and SGC996 cells after EEF1AKMT4 overexpression. Statistical significance between subgroups was assessed using the Student's t-test. ns, not significant; ∗∗∗, p < 0.001.

Article Snippet: pcDNA3.1 plasmids encoding wide eEF1A2 and EEF1AKMT4 were purchased from Biosun Company. eEF1A2 K36R and EEF1AKMT4 D88A mutations were constructed through PCR-mediated site-directed mutagenesis using a QuickChange Site-Directed Mutagenesis Kit (Agilent Stratagene, CA, USA).

Techniques: Knockdown, Over Expression, Expressing, Quantitative RT-PCR, Western Blot, CCK-8 Assay, Tube Formation Assay, Plasmid Preparation, Control, Migration

Journal: Genes & Diseases

Article Title: EEF1AKMT4-eEF1A2 synergistically facilitates the progression of GBC by promoting ribosomal protein output

doi: 10.1016/j.gendis.2025.101619

Figure Lengend Snippet: K36 site trimethylation is essential for the tumor-promoting effect of eEF1A2. (A) Western blot analysis of the expression of EEF1AKMT4, eEF1A2 K36me3, and eEF1A2 in eEF1A2-knockdown GBCSD SGC996 cells complemented with vector, eEF1A2 WT, or eEF1A2 K36R. (B) CCK-8 assays in eEF1A2-knockdown GBCSD SGC996 cells complemented with vector, eEF1A2 WT, or eEF1A2 K36R. (C) Clone formation assays of cells treated with scrambled shRNA, or eEF1A2-knockdowned GBCSD SGC996 cells complemented with vector, eEF1A2 WT or eEF1A2 K36R. (D) Wound healing assays of cells treated with scrambled shRNA, or eEF1A2-knockdowned GBCSD SGC996 cells complemented with vector, eEF1A2 WT or eEF1A2 K36R. (E) Transwell assays with matrigel of cells treated with scrambled shRNA, or eEF1A2-knockdowned GBCSD SGC996 cells complemented with the vector, eEF1A2 WT, or eEF1A2 K36R. (F) Western blot analysis of the expression of EEF1AKMT4, eEF1A2 K36me3, and eEF1A2 in EEF1AKMT4-knockdown GBCSD cells complemented with vector, EEF1AKMT4 WT, or EEF1AKMT4 D88A. (G) CCK-8 assays in EEF1AKMT4-knockdown GBCSD SGC996 cells complemented with vector, EEF1AKMT4 WT or EEF1AKMT4 D88A. (H) Clone formation assays of cells treated with scrambled shRNA, or EEF1AKMT4-knockdowned GBCSD SGC996 cells complemented with vector, EEF1AKMT4 WT or EEF1AKMT4 D88A. (I) Wound healing assays of cells treated with scrambled shRNA, or EEF1AKMT4-knockdowned GBCSD SGC996 cells complemented with vector, EEF1AKMT4 WT or EEF1AKMT4 D88A. (J) Transwell assays with matrigel of cells treated with scrambled shRNA, or EEF1AKMT4-knockdowned GBCSD SGC996 cells complemented with the vector, EEF1AKMT4 WT, or EEF1AKMT4 D88A. (K) In vivo subcutaneous xenografts were established. (L–M) Tumor weight (L) and volume (M) of the subcutaneous xenografts were measured. (N) Comparison of popliteal lymph node size. (O) Comparison of the volume of metastasized popliteal lymph nodes. (P) Representative images of a nude mouse model of popliteal LN metastasis. HE staining of the lymph nodes was performed. Scale bar: 100 μm ns, not significant; ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001.

Article Snippet: pcDNA3.1 plasmids encoding wide eEF1A2 and EEF1AKMT4 were purchased from Biosun Company. eEF1A2 K36R and EEF1AKMT4 D88A mutations were constructed through PCR-mediated site-directed mutagenesis using a QuickChange Site-Directed Mutagenesis Kit (Agilent Stratagene, CA, USA).

Techniques: Western Blot, Expressing, Knockdown, Plasmid Preparation, CCK-8 Assay, shRNA, In Vivo, Comparison, Staining

Journal: Genes & Diseases

Article Title: EEF1AKMT4-eEF1A2 synergistically facilitates the progression of GBC by promoting ribosomal protein output

doi: 10.1016/j.gendis.2025.101619

Figure Lengend Snippet: eEF1A2 K36 trimethylation affects protein output by affecting its GTP enzyme activity. (A) FLAG-tagged eEF1A2 WT and eEF1A2 K36R were overexpressed in HEK293T cells. Flag-co-IP and subsequent LC-MS/MS were performed to identify the differences in binding proteins between eEF1A2 WT and K36R mutations. The binding proteins of eEF1A2 WT (left) and eEF1A2 K36R (right) in HEK293T cells were analyzed by KEGG enrichment analysis. (B) Heatmap showing the differentially expressed genes after eEF1A2 knockdown in GBCSD cell lines. (C) The differentially expressed genes after eEF1A2 knockdown were analyzed by KEGG enrichment analysis. (D) Western blot analysis of the expression of ERK1/2 and AKT in EEF1AKMT4-knockdown or eEF1A2-knockdown GBCSD cells. (E) qPCR analysis of the mRNA expression of ERK1/2 and AKT in EEF1AKMT4-knockdown or eEF1A2-knockdown GBCSD cells. (F) SUnSET assays were performed under the indicated conditions to analyze the effect of eEF1A2 K36 methylation status on the protein synthesis rate in GBCSD (left) and SGC996(right) cell lines. These results revealed reduced protein production in eEF1A2 K36me0 cells. (G) Purification of eEF1A2 ± K36me3 protein with anti-eEF1A2 co-immunoprecipitation in EEF1AKMT wt and knockdown cells. Top panel: Western blot validation of EEF1AKMT4; middle panels: Western blot analysis with the indicated antibodies against eEF1A2 purified from 293 T cells; bottom panel: Coomassie stain of purified eEF1A2 protein. (H) In vitro GTP hydrolysis by trimethylated or unmethylated eEF1A2. eEF1A2 ± K36me3 purified was incubated with increasing amounts of GTP at 37 °C for 3 h. Kinetic parameters were obtained by fitting the Michaelis–Menten equation to plot the velocity of phosphate formation against GTP concentration. (I) K36me3 increases the catalytic efficiency of GTP hydrolysis by eEF1A2. The Michaelis–Menten kinetic parameters of eEF1A2 ± K36me3 are shown.

Article Snippet: pcDNA3.1 plasmids encoding wide eEF1A2 and EEF1AKMT4 were purchased from Biosun Company. eEF1A2 K36R and EEF1AKMT4 D88A mutations were constructed through PCR-mediated site-directed mutagenesis using a QuickChange Site-Directed Mutagenesis Kit (Agilent Stratagene, CA, USA).

Techniques: Activity Assay, Co-Immunoprecipitation Assay, Liquid Chromatography with Mass Spectroscopy, Binding Assay, Knockdown, Western Blot, Expressing, Methylation, Purification, Immunoprecipitation, Biomarker Discovery, Staining, In Vitro, Incubation, Concentration Assay

Journal: Genes & Diseases

Article Title: EEF1AKMT4-eEF1A2 synergistically facilitates the progression of GBC by promoting ribosomal protein output

doi: 10.1016/j.gendis.2025.101619

Figure Lengend Snippet: A schematic model shows the effects of EEF1AKMT4 and eEF1A2 on regulating ribosomal protein synthesis. EEF1AKMT4 trimethylates eEF1A2 at K36 and fuels its GTPase activity. Elevated eEF1A2 enzyme activity promotes the protein output of several oncogenic growth signals including AKT and ERK. Overall, upregulated eEF1A2 expression together with the fuel of EEF1AKMT4 promotes the progression of GBC and LN metastasis.

Article Snippet: pcDNA3.1 plasmids encoding wide eEF1A2 and EEF1AKMT4 were purchased from Biosun Company. eEF1A2 K36R and EEF1AKMT4 D88A mutations were constructed through PCR-mediated site-directed mutagenesis using a QuickChange Site-Directed Mutagenesis Kit (Agilent Stratagene, CA, USA).

Techniques: Activity Assay, Expressing

Journal: Genes & Diseases

Article Title: EEF1AKMT4-eEF1A2 synergistically facilitates the progression of GBC by promoting ribosomal protein output

doi: 10.1016/j.gendis.2025.101619

Figure Lengend Snippet: EEF1AKMT4 trimethylates eEF1A2 at K36 site in GBC. (A) Spatial distribution of the five main methylation sites in the protein structure of eEF1A2 combined with GDP (PDB: 6ra9). Most of the methylation sites were located in the nucleotide-binding domain of eEF1A2. (B) Relative mRNA expression of five methylases, EEF1AKMT4, METTL13, N6AMT2, METTL21B, and METTL10, which are responsible for methylation of K36, K55, K79, K165, and K318 sites, respectively, was quantified by qRT-PCR in 10 cases of gallbladder cancer tissues and their patient-paired normal tissues. (C) Coomassie blue staining of anti-eEF1A2 co-immunoprecipitation in GBCSD, SGC996, and HEK293T cell lines. Target bands are indicated by a black box and were cut and subjected to LC-MS/MS to analyze the methylation status. (D) Methylation status at the K36, K55, K79, K165, and K318 sites of eEF1A2 in GBCSD, SGC996, and HEK293T cell lines were analyzed. (E) Representative tandem mass spectra identifying in vitro tri-methylated (upper) and non-methylated (lower) eEF1A2K36. m/z for b and y ions observed in the spectra are indicated in red and blue, respectively. (F) Histogram showing the methylation changes in K36, K55, K79, K165, and K318 before (upper) and after (lower) knockdown of EEF1AKMT4 in GBCSD cells. (G) Selected ion chromatograms for non-, mono-, di-, and trimethyl eEF1AK36 peptides from GluC digestion of endogenous eEF1A2 immunoprecipitated from whole-cell lysates of GBCSD, indicating the methylation status shift after EEF1AKMT4 knockdown. (H) Western blot analysis of the knockdown efficiency of EEF1AKMT4 in GBCSD and the status of eEF1A2 and eEF1A2K36me3. (I) Expression of eEF1AKMT4 and eEF1A2 K36me3 levels in GBC tumor tissues and paired normal tissues were examined by Western blotting and analyzed using ImageJ. Statistical significance between groups was assessed using Student's t-test. ns, not significant; ∗, p < 0.05; ∗∗, p < 0.01.

Article Snippet: EEF1AKMT4 , Proteintech , Cat No. 15418-1-AP.

Techniques: Methylation, Binding Assay, Expressing, Quantitative RT-PCR, Staining, Immunoprecipitation, Liquid Chromatography with Mass Spectroscopy, In Vitro, Knockdown, Western Blot

Journal: Genes & Diseases

Article Title: EEF1AKMT4-eEF1A2 synergistically facilitates the progression of GBC by promoting ribosomal protein output

doi: 10.1016/j.gendis.2025.101619

Figure Lengend Snippet: Knockdown of EEF1AKMT4 inhibits the malignant phenotype of GBC while its overexpression is not tumor-promoting. (A–B) Expression levels of EEF1AKMT4 in HEK293T, RBE, NOZ, GBCSD, SGC996, and OCUG-1 were detected using qRT-PCR (A) and Western blotting (B). (C) EEF1AKMT4 was knocked down or overexpressed in the GBCSD and SGC996 cell lines, and validated by Western blotting. (D) CCK-8 assays were performed to identify alterations in cell proliferation after EEF1AKMT4 knockdown in GBCSD and SGC996 cells. (E) Clone formation assay was performed in EEF1AKMT4 knockdown and vector control cells in GBCSD and SGC996 cells. (F) Wound healing assays were performed to investigate the effect of EEF1AKMT4 knockdown on the migration ability of the GBCSD and SGC996 cell lines. (G) Transwell assays with matrigel were performed to evaluate the invasive ability of GBCSD and SGC996 cells after EEF1AKMT4 knockdown. (H) CCK-8 assays were performed to identify alterations in cell proliferation after EEF1AKMT4 overexpression in GBCSD and SGC996 cells. ( I ) Transwell assays with matrigel were performed to evaluate the invasive ability of GBCSD and SGC996 cells after EEF1AKMT4 overexpression. Statistical significance between subgroups was assessed using the Student's t-test. ns, not significant; ∗∗∗, p < 0.001.

Article Snippet: EEF1AKMT4 , Proteintech , Cat No. 15418-1-AP.

Techniques: Knockdown, Over Expression, Expressing, Quantitative RT-PCR, Western Blot, CCK-8 Assay, Tube Formation Assay, Plasmid Preparation, Control, Migration

Journal: Genes & Diseases

Article Title: EEF1AKMT4-eEF1A2 synergistically facilitates the progression of GBC by promoting ribosomal protein output

doi: 10.1016/j.gendis.2025.101619

Figure Lengend Snippet: K36 site trimethylation is essential for the tumor-promoting effect of eEF1A2. (A) Western blot analysis of the expression of EEF1AKMT4, eEF1A2 K36me3, and eEF1A2 in eEF1A2-knockdown GBCSD SGC996 cells complemented with vector, eEF1A2 WT, or eEF1A2 K36R. (B) CCK-8 assays in eEF1A2-knockdown GBCSD SGC996 cells complemented with vector, eEF1A2 WT, or eEF1A2 K36R. (C) Clone formation assays of cells treated with scrambled shRNA, or eEF1A2-knockdowned GBCSD SGC996 cells complemented with vector, eEF1A2 WT or eEF1A2 K36R. (D) Wound healing assays of cells treated with scrambled shRNA, or eEF1A2-knockdowned GBCSD SGC996 cells complemented with vector, eEF1A2 WT or eEF1A2 K36R. (E) Transwell assays with matrigel of cells treated with scrambled shRNA, or eEF1A2-knockdowned GBCSD SGC996 cells complemented with the vector, eEF1A2 WT, or eEF1A2 K36R. (F) Western blot analysis of the expression of EEF1AKMT4, eEF1A2 K36me3, and eEF1A2 in EEF1AKMT4-knockdown GBCSD cells complemented with vector, EEF1AKMT4 WT, or EEF1AKMT4 D88A. (G) CCK-8 assays in EEF1AKMT4-knockdown GBCSD SGC996 cells complemented with vector, EEF1AKMT4 WT or EEF1AKMT4 D88A. (H) Clone formation assays of cells treated with scrambled shRNA, or EEF1AKMT4-knockdowned GBCSD SGC996 cells complemented with vector, EEF1AKMT4 WT or EEF1AKMT4 D88A. (I) Wound healing assays of cells treated with scrambled shRNA, or EEF1AKMT4-knockdowned GBCSD SGC996 cells complemented with vector, EEF1AKMT4 WT or EEF1AKMT4 D88A. (J) Transwell assays with matrigel of cells treated with scrambled shRNA, or EEF1AKMT4-knockdowned GBCSD SGC996 cells complemented with the vector, EEF1AKMT4 WT, or EEF1AKMT4 D88A. (K) In vivo subcutaneous xenografts were established. (L–M) Tumor weight (L) and volume (M) of the subcutaneous xenografts were measured. (N) Comparison of popliteal lymph node size. (O) Comparison of the volume of metastasized popliteal lymph nodes. (P) Representative images of a nude mouse model of popliteal LN metastasis. HE staining of the lymph nodes was performed. Scale bar: 100 μm ns, not significant; ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001.

Article Snippet: EEF1AKMT4 , Proteintech , Cat No. 15418-1-AP.

Techniques: Western Blot, Expressing, Knockdown, Plasmid Preparation, CCK-8 Assay, shRNA, In Vivo, Comparison, Staining

Journal: Genes & Diseases

Article Title: EEF1AKMT4-eEF1A2 synergistically facilitates the progression of GBC by promoting ribosomal protein output

doi: 10.1016/j.gendis.2025.101619

Figure Lengend Snippet: eEF1A2 K36 trimethylation affects protein output by affecting its GTP enzyme activity. (A) FLAG-tagged eEF1A2 WT and eEF1A2 K36R were overexpressed in HEK293T cells. Flag-co-IP and subsequent LC-MS/MS were performed to identify the differences in binding proteins between eEF1A2 WT and K36R mutations. The binding proteins of eEF1A2 WT (left) and eEF1A2 K36R (right) in HEK293T cells were analyzed by KEGG enrichment analysis. (B) Heatmap showing the differentially expressed genes after eEF1A2 knockdown in GBCSD cell lines. (C) The differentially expressed genes after eEF1A2 knockdown were analyzed by KEGG enrichment analysis. (D) Western blot analysis of the expression of ERK1/2 and AKT in EEF1AKMT4-knockdown or eEF1A2-knockdown GBCSD cells. (E) qPCR analysis of the mRNA expression of ERK1/2 and AKT in EEF1AKMT4-knockdown or eEF1A2-knockdown GBCSD cells. (F) SUnSET assays were performed under the indicated conditions to analyze the effect of eEF1A2 K36 methylation status on the protein synthesis rate in GBCSD (left) and SGC996(right) cell lines. These results revealed reduced protein production in eEF1A2 K36me0 cells. (G) Purification of eEF1A2 ± K36me3 protein with anti-eEF1A2 co-immunoprecipitation in EEF1AKMT wt and knockdown cells. Top panel: Western blot validation of EEF1AKMT4; middle panels: Western blot analysis with the indicated antibodies against eEF1A2 purified from 293 T cells; bottom panel: Coomassie stain of purified eEF1A2 protein. (H) In vitro GTP hydrolysis by trimethylated or unmethylated eEF1A2. eEF1A2 ± K36me3 purified was incubated with increasing amounts of GTP at 37 °C for 3 h. Kinetic parameters were obtained by fitting the Michaelis–Menten equation to plot the velocity of phosphate formation against GTP concentration. (I) K36me3 increases the catalytic efficiency of GTP hydrolysis by eEF1A2. The Michaelis–Menten kinetic parameters of eEF1A2 ± K36me3 are shown.

Article Snippet: EEF1AKMT4 , Proteintech , Cat No. 15418-1-AP.

Techniques: Activity Assay, Co-Immunoprecipitation Assay, Liquid Chromatography with Mass Spectroscopy, Binding Assay, Knockdown, Western Blot, Expressing, Methylation, Purification, Immunoprecipitation, Biomarker Discovery, Staining, In Vitro, Incubation, Concentration Assay

Journal: Genes & Diseases

Article Title: EEF1AKMT4-eEF1A2 synergistically facilitates the progression of GBC by promoting ribosomal protein output

doi: 10.1016/j.gendis.2025.101619

Figure Lengend Snippet: A schematic model shows the effects of EEF1AKMT4 and eEF1A2 on regulating ribosomal protein synthesis. EEF1AKMT4 trimethylates eEF1A2 at K36 and fuels its GTPase activity. Elevated eEF1A2 enzyme activity promotes the protein output of several oncogenic growth signals including AKT and ERK. Overall, upregulated eEF1A2 expression together with the fuel of EEF1AKMT4 promotes the progression of GBC and LN metastasis.

Article Snippet: EEF1AKMT4 , Proteintech , Cat No. 15418-1-AP.

Techniques: Activity Assay, Expressing

Journal: Nucleic Acids Research

Article Title: Methylation of human eukaryotic elongation factor alpha (eEF1A) by a member of a novel protein lysine methyltransferase family modulates mRNA translation

doi: 10.1093/nar/gkx432

Figure Lengend Snippet: Gene structure and targeting strategy for the endothelin converting enzyme 2 ( ECE2 ) /EEF1AKMT4 locus. ( A ) Topology diagram of the 7BS MTase fold. α-helices and β-strands are depicted as grey boxes (denoted Z and A–E) and black arrows (denoted 1–7), respectively, and annotated according to previously established nomenclature . ( B ) Organization and annotation of the human ECE2 locus. Top, current annotation of the ECE2 locus. Regions in the genomic DNA corresponding to annotated exons of ECE2 (located on the forward stand) and CAMK2N2 (located on the reverse strand) are indicated in blue and green, respectively (based on the annotated gene structure according to Ensembl (assembly GRCh37)). Middle, tracks representing spliced expressed sequence tags (ESTs) exported from the UCSC genome browser. Bottom, alternative gene model supported by the EST data. ( C ) Gene targeting strategy and disruption of the EEF1AKMT4 locus by CRISPR/Cas9 technology. Top, schematic representation of the EEF1AKMT4 gene. Exons and introns are represented by boxes (gray, coding region; white, untranslated regions) and lines, respectively. An arrow indicates the region targeted by CRISPR. Bottom, DNA and protein sequence of targeted region of EEF1AKMT4 locus in HAP-1 wild-type (WT) and EEF1AKMT4 knockout (KO) cells. The conserved motif Post I is indicated by a rectangle and the last residue of the motif, D88, is shown in red. ( D ) Structural support for an involvement of D88 in coordinating AdoMet binding. The figure is based on a previously published structure (PDB ID: 2PXX) of eEF1A-KMT4 in complex with S -adenosylhomocysteine (AdoHcy; the unmethylated counterpart of AdoMet) . AdoHcy and D88 are represented as sticks and the second β-strand (β2) is represented as a cartoon. Possible hydrogen bonds between the carboxyl group of D88 and the ribose moiety of AdoHcy are shown (dashed lines). The figure was generated using the PyMOL Molecular Graphics System, Version 1.3 (Schrodinger, LLC).

Article Snippet: HAP-1 EEF1AKMT4 gene KO cells were generated using the CRISPR/Cas9 system as a custom project performed by Horizon Genomics (formerly Haplogen).

Techniques: Sequencing, CRISPR, Knock-Out, Binding Assay, Generated

Journal: Nucleic Acids Research

Article Title: Methylation of human eukaryotic elongation factor alpha (eEF1A) by a member of a novel protein lysine methyltransferase family modulates mRNA translation

doi: 10.1093/nar/gkx432

Figure Lengend Snippet: Identification of eEF1A proteins as candidate substrates for eEF1A-KMT4. ( A ) eEF1A-KMT4-mediated methylation in protein extracts. Protein extracts from HAP-1 wild-type (WT) or EEF1AKMT4 KO cells were incubated with [ 3 H]-AdoMet and with recombinant eEF1A-KMT4 as indicated. Top panel, Ponceau S-stained membrane of sodium dodecyl sulphate (SDS-PAGE)-separated methylation reactions. Bottom, visualization of methylation by fluorography of membrane in upper panel. ( B ) Mutation of D88 abrogates the enzymatic activity of eEF1A-KMT4. Protein extract from HAP-1 EEF1AKMT4 KO cells incubated with [ 3 H]-AdoMet in the presence of either recombinant WT eEF1A-KMT4 or the corresponding D88A mutant protein. Reactions were analyzed as in (A). ( C ) Outline of chromatography based enrichment strategy for eEF1A-KMT4 substrates. In vitro methylated protein extract was loaded onto an anion exchange (Q) column and, in turn, the flow-through (FTQ) was loaded onto a cation exchange (S) column. Proteins were eluted from both columns with a step gradient of increasing NaCl. ( D ) Enrichment of the ∼50 kDa eEF1A-KMT4 substrate. Proteins were eluted from ion exchange columns with a step gradient ramped from 150 mM (0.15) to 1 M (1.0) NaCl according to the scheme depicted in (C). Collected fractions were analyzed by fluorography as in (A). ( E ) SDS-PAGE analysis of the main substrate-enriched fraction (0.3S). The fraction containing the bulk of methylated substrate in (D) eluted at 300 mM NaCl from the S column (0.3S). The most prominent proteins in the major ∼50 kDaband were identified as eEF1A1 and eEF1A2 by mass spectrometry (MS) (See Table ).

Article Snippet: HAP-1 EEF1AKMT4 gene KO cells were generated using the CRISPR/Cas9 system as a custom project performed by Horizon Genomics (formerly Haplogen).

Techniques: Methylation, Incubation, Recombinant, Staining, SDS Page, Mutagenesis, Activity Assay, Chromatography, In Vitro, Mass Spectrometry

Journal: Nucleic Acids Research

Article Title: Methylation of human eukaryotic elongation factor alpha (eEF1A) by a member of a novel protein lysine methyltransferase family modulates mRNA translation

doi: 10.1093/nar/gkx432

Figure Lengend Snippet: eEF1A-KMT4-mediated methylation of K36 in eEF1A in vivo . ( A ) EEF1AKMT4 KO abrogates eEF1A-K36 methylation in human cells. Ion chromatograms representing peptides of the different methylation states of K36 in eEF1A proteins from HAP-1 WT and EEF1AKMT4 knock out (KO) cells are shown. The retention time for analyzed peptides is indicated (arrow) and their relative abundances are indicated (obtained by integration of the area of the relevant peaks; expressed as the percentage of the sum of all peaks). Tandem mass spectra supporting identity of analyzed peptides are shown in Supplementary Figure S2. ( B ) eEF1A1 and eEF1A2 are both methylated at K36 in vivo . Chromatograms represent the methylation status of K36 in TAP-tagged eEF1A that was affinity-purified from HEK293-derived cells ectopically expressing TAP-tagged eEF1A1 or eEF1A2. ( C ) Methylation status of eEF1A-K36 in mammalian organs. Chromatograms representing the methylation status of K36 in eEF1A obtained from rat brain, liver, intestine and kidney are shown.

Article Snippet: HAP-1 EEF1AKMT4 gene KO cells were generated using the CRISPR/Cas9 system as a custom project performed by Horizon Genomics (formerly Haplogen).

Techniques: Methylation, In Vivo, Knock-Out, Affinity Purification, Derivative Assay, Expressing

Journal: Nucleic Acids Research

Article Title: Methylation of human eukaryotic elongation factor alpha (eEF1A) by a member of a novel protein lysine methyltransferase family modulates mRNA translation

doi: 10.1093/nar/gkx432

Figure Lengend Snippet: The effect of EEF1AKMT4 KO on the translational landscape. ( A ) Differential expression plot showing the log 2 -fold changes on the mRNA and ribosome footprint level in EEF1AKMT4 KO relative to WT cells. Indicated are: not significantly changed genes (DEseq2 padj >0.05) (gray), genes differentially expressed at the mRNA level only (green), genes with differential ribosome footprinting only (blue) and genes that were changed at both the mRNA and ribosome footprinting levels (red). ( B ) GO analysis (Cellular Process) for genes significantly upregulated on the footprint level only. n = 322, numbers in brackets indicate genes matching this category. ( C) GO analysis (Cellular Process) for genes significantly downregulated on the footprint level only. n = 501, numbers in brackets indicate genes matching this category. ( D ) Ribosome occupancy in EEF1AKMT4 KO cells compared with WT cells. A-site occupancy and +1 occupancy are normalized to untranslated downstream codons (mean ± SD, n = 3). Symbol size indicates the relative frequency of codons.

Article Snippet: HAP-1 EEF1AKMT4 gene KO cells were generated using the CRISPR/Cas9 system as a custom project performed by Horizon Genomics (formerly Haplogen).

Techniques: Expressing, Footprinting