Journal: Human molecular genetics

Article Title: A humanized yeast model reveals dominant-negative properties of neuropathy-associated alanyl-tRNA synthetase mutations.

doi: 10.1093/hmg/ddad054

Figure Lengend Snippet: Figure 1. G102R and R329H AARS1 are loss-of-function alleles that repress yeast cell growth in the presence of wild-type AARS1. (A) Yeast harboring an endogenous doxycycline-repressible ALA1 locus were transformed with a p413 vector with no insert and a pAG425 vector to express either wild-type or mutant human AARS1. Cultures were plated undiluted or diluted on media lacking histidine and leucine, and containing galactose/raffinose and doxycycline. (B) Similar experiment as shown in panel A; here, yeast were first transformed with a p413 vector expressing wild-type human AARS1. For both panels, the vectors present in each experiment are indicated across the top, the dilution of the spotted yeast cultured is indicated on the left side of the image, and the media conditions are indicated across the bottom of the image (his = histidine; leu = leucine; gal = galactose; raf = raffinose; dox = doxycycline). Representative images are shown from thirteen (for G102R) or sixteen (for R329H) biological replicates. A cartoon on the bottom left illustrates the experimental conditions for all samples.

Article Snippet: For yeast protein and HEK293T co-immunoprecipitation experiments, the AARS1 antibody (Bethyl Laboratories A303-473A) was used at 1:1000 dilution.

Techniques: Transformation Assay, Plasmid Preparation, Mutagenesis, Expressing, Cell Culture

Journal: Human molecular genetics

Article Title: A humanized yeast model reveals dominant-negative properties of neuropathy-associated alanyl-tRNA synthetase mutations.

doi: 10.1093/hmg/ddad054

Figure Lengend Snippet: Figure 2. G102R and R329H AARS1 dimerize with wild-type AARS1. (A) HEK293T cells were transfected with vectors to co-express wild-type and mutant human AARS1, and a western blot was performed to detect the resulting proteins along with endogenous loading controls. The image is representative of three independent replicates. A cartoon along the top illustrates the constructs employed in the experiments, and the presence or absence of each construct is indicated across the top of the gel image. Protein molecular weights are indicated in kilodaltons (kDa) along the left side of the image and antibodies are indicated along the right side. (B) After immunoprecipitation with an anti-6xHis antibody, a western blot was performed to detect co-immunoprecipitated proteins. A representative image from five (for R329H) or three (for G102R) independent replicates are shown. This image is annotated as in panel A. (C) After immunoprecipitation with an anti-FLAG antibody, a western blot was performed to detect co-immunoprecipitated proteins. A representative image from two independent replicates is shown. This image is annotated as in panels A and B. (D) After treating patient and control samples with a protein cross-linking agent, a western blot was performed to detect endogenous AARS1 protein. The image is representative of four independent technical replicates. Samples and conditions are indicated across the top of the image, protein molecular weights are indicated in kilodaltons (kDa) along the left side, and the antibody employed is indicated on the right side. DSS = disuccinimidyl suberate. (E) The percentage of dimeric AARS1 protein signal in the total AARS1 protein signal (D) was quantified with ImageJ. The mean of four technical replicates is shown, with error bars representing one standard deviation. A one-way ANOVA with Tukey’s multiple comparisons tests [F(2,9) = 0.3602, P = 0.7071] was performed to determine if there was a statistically significant difference between R329H/+ cells and either of the two controls.

Article Snippet: For yeast protein and HEK293T co-immunoprecipitation experiments, the AARS1 antibody (Bethyl Laboratories A303-473A) was used at 1:1000 dilution.

Techniques: Transfection, Mutagenesis, Western Blot, Construct, Immunoprecipitation, Control, Standard Deviation

Journal: Human molecular genetics

Article Title: A humanized yeast model reveals dominant-negative properties of neuropathy-associated alanyl-tRNA synthetase mutations.

doi: 10.1093/hmg/ddad054

Figure Lengend Snippet: Figure 3. Engineering dimer-reducing AARS1 variants. (A) A cartoon generated from PyMOL illustrates the crystal structure of the AARS1 C-terminal dimerization domain (left side). One subunit from the dimer is shown in green, and the other in purple. Amino-acid residues that contact the opposite subunit are shown in dark green or dark purple. The residues targeted in this assay are shown in pink and labeled in the inset. On the right side of this panel is a second cartoon that illustrates the AARS1 C-terminal dimerization domain with the Q855∗mutation. The dashed circles indicate the globular domain that is ablated by the premature stop codon. (B) Yeast harboring a doxycycline-repressible endogenous ALA1 locus were transformed with a pAG425 vector to express either wild-type or mutant human AARS1 (i.e. one of the engineered mutations affecting the residues highlighted in panel A). Cultures were plated undiluted or diluted on media lacking leucine, and containing galactose/raffinose and doxycycline. A representative image of four biological replicates is shown. The dilution of the spotted yeast cultured is indicated on the left and the media conditions are indicated across the bottom (leu = leucine; gal = galactose; raf = raffinose; dox = doxycycline). (C) Yeast protein lysates were subjected to western blot analysis to detect the human AARS1 proteins expressed from wild-type and mutant expression constructs, which are indicated across the top. Yeast was grown in galactose and raffinose media lacking leucine, with no doxycycline. A representative image of three biological replicates is shown.

Article Snippet: For yeast protein and HEK293T co-immunoprecipitation experiments, the AARS1 antibody (Bethyl Laboratories A303-473A) was used at 1:1000 dilution.

Techniques: Generated, Labeling, Transformation Assay, Plasmid Preparation, Mutagenesis, Cell Culture, Western Blot, Expressing, Construct

Journal: Human molecular genetics

Article Title: A humanized yeast model reveals dominant-negative properties of neuropathy-associated alanyl-tRNA synthetase mutations.

doi: 10.1093/hmg/ddad054

Figure Lengend Snippet: Figure 4. Q855∗AARS1 impairs dimerization with wild-type AARS1. (A) HEK293T cells were transfected with vectors to co-express wild-type or Q855∗

Article Snippet: For yeast protein and HEK293T co-immunoprecipitation experiments, the AARS1 antibody (Bethyl Laboratories A303-473A) was used at 1:1000 dilution.

Techniques: Transfection

Journal: Human molecular genetics

Article Title: A humanized yeast model reveals dominant-negative properties of neuropathy-associated alanyl-tRNA synthetase mutations.

doi: 10.1093/hmg/ddad054

Figure Lengend Snippet: Figure 5. Reducing dimerization of G102R and R329H with wild-type AARS1 rescues yeast growth. (A) Yeast with the doxycycline-repressible endogenous ALA1 locus was transformed with an empty p413 vector and a pAG425 vector expressing wild-type or mutant human AARS1. Cultures were plated undiluted or diluted on media lacking histidine and leucine, and containing galactose/raffinose and doxycycline. (B) Similar to strains shown in panel A, except that yeast was transformed with p413 expressing wild-type human AARS1. For both panels, the vectors present in each experiment are indicated across the top, the dilution of the spotted yeast cultured is indicated on the left, and the media conditions are indicated across the bottom (his = histidine; leu = leucine; gal = galactose; raf = raffinose; dox = doxycycline). (C) Yeast spot intensity was quantified using ImageJ. Bars represent the mean and one standard deviation. Thirteen biological replicates were assessed for G757∗and wild-type AARS1, eight for R329H and R329H + Q855∗, and seven for G102R and G102R + Q855∗. The indicated fold-change between the G102R strain and the G102R + Q855∗strain, and the fold-change between the R329H strain and the R329H + Q855∗strain, were both calculated using the mean of each sample. To compare yeast growth to the strain expressing both wild-type and G757∗AARS1, a one-way ANOVA with Dunnett’s multiple comparisons test was performed [F(5,50) = 19.90, P < 0.0001].

Article Snippet: For yeast protein and HEK293T co-immunoprecipitation experiments, the AARS1 antibody (Bethyl Laboratories A303-473A) was used at 1:1000 dilution.

Techniques: Transformation Assay, Plasmid Preparation, Expressing, Mutagenesis, Cell Culture, Standard Deviation

Journal: Human molecular genetics

Article Title: A humanized yeast model reveals dominant-negative properties of neuropathy-associated alanyl-tRNA synthetase mutations.

doi: 10.1093/hmg/ddad054

Figure Lengend Snippet: Figure 6. R329C, R329S and R326W AARS1 are loss-of-function alleles that dominantly repress yeast cell growth. (A) Yeast with the doxycycline- repressible endogenous ALA1 locus was transformed with a p413 vector with no insert, and a pAG425 vector expressing wild-type or mutant AARS1. Yeast cultures were spotted undiluted or diluted on media lacking histidine and leucine, and containing galactose/raffinose and doxycycline. (B) A similar experiment to that described in panel A, except that yeast expresses wild-type human AARS1 from p413. For both panels, the vectors present in each experiment are indicated across the top, the dilution of the spotted yeast cultured is indicated on the left, and the media conditions are shown across the bottom (his = histidine; leu = leucine; gal = galactose; raf = raffinose; dox = doxycycline). Images are representative of three replicates.

Article Snippet: For yeast protein and HEK293T co-immunoprecipitation experiments, the AARS1 antibody (Bethyl Laboratories A303-473A) was used at 1:1000 dilution.

Techniques: Transformation Assay, Plasmid Preparation, Expressing, Mutagenesis, Cell Culture

Journal: Human molecular genetics

Article Title: A humanized yeast model reveals dominant-negative properties of neuropathy-associated alanyl-tRNA synthetase mutations.

doi: 10.1093/hmg/ddad054

Figure Lengend Snippet: Figure 7. Reducing dimerization of R329C, R329S or R326W with wild-type AARS1 rescues yeast growth. (A) Yeast with the doxycycline-repressible ALA1 locus was transformed with an empty p413 vector and a pAG425 vector expressing either wild-type or mutant AARS1. Yeast was spotted undiluted or diluted on media lacking histidine and leucine, and containing galactose/raffinose and doxycycline. (B) An experiment similar to that described in panel A, except that yeast expresses wild-type human AARS1 from the p413 vector. (C) Yeast spot intensity was quantified using ImageJ analysis; bars represent the mean and one standard deviation. At least three biological replicates were assessed for all variants. The indicated fold change between the strains expressing R329C, R329S or R326W, and their counterpart with Q855∗in cis, was calculated using the mean intensity of each condition. To compare yeast growth to that of the strain expressing both wild-type and G757∗AARS1, a one-way ANOVA with Dunnett’s multiple comparisons test was performed [F(7,27) = 14.72, P < 0.0001].

Article Snippet: For yeast protein and HEK293T co-immunoprecipitation experiments, the AARS1 antibody (Bethyl Laboratories A303-473A) was used at 1:1000 dilution.

Techniques: Transformation Assay, Plasmid Preparation, Expressing, Mutagenesis, Standard Deviation

Journal: Translational Neurodegeneration

Article Title: Ecto-GPR37: a potential biomarker for Parkinson’s disease

doi: 10.1186/s40035-021-00232-7

Figure Lengend Snippet: GPR37 protein density and mRNA levels in post-mortem brain tissues from neurological controls (NCs) and Parkinson’s disease (PD) subjects. a Immunoblots showing the expression of GPR37 in the SN of NC and PD subjects (Braak PD stage 4, Braak PD 5 and Braak PD 6). Extracts from the human post-mortem SN were analysed by SDS-PAGE and immunoblotted using rabbit anti-GPR37-C and rabbit anti-α-actinin antibodies. The right dashed outlined area (*) is shown with enhanced contrast and corresponds to lane 1 (NC). The different GPR37 forms included precursor (70 kDa), full-length mature (93 kDa) and GPR37 N-terminus-cleaved forms (76 kDa, 52 kDa, 47 kDa, 43 kDa and 39–40 kDa). b Relative quantification of GPR37 protein density in the SN. The immunoblots corresponding to the precursor (Pre) and cleaved GPR37 forms from the NC ( n = 5), Braak PD stage 4 ( n = 5), Braak PD stage 5 ( n = 5) and Braak PD stage 6 ( n = 5) subjects were quantified by densitometric scanning. Values were normalized to α-actinin in each lane to correct for protein loading. Results are expressed as the percentage (mean ± SEM) of the NC. * P < 0.05, ** P < 0.01 vs NC, one-way ANOVA followed by Dunnett’s post-hoc test. c RT-qPCR assessment of GPR37 mRNA expression in post-mortem SN from NCs ( n = 10), Braak PD stage 3/4 ( n = 7) and Braak PD stage 5/6 ( n = 17) subjects. Mean values of the housekeeping genes ( GUS-B , XPNPEP1 , AARS and HPRT ) were used to normalize samples. Results are expressed as fold-change (mean ± SEM) of the NCs. ** P < 0.01 vs NC, one-way ANOVA followed by Dunnett’s post-hoc test

Article Snippet: Parallel amplification reactions were carried out using 20× TaqMan Gene Expression Assays and 2× TaqMan Universal PCR Master Mix (Applied Biosystems). β-Glucuronidase ( GUS-β; Ref: Hs00939627_m1), X-prolyl aminopeptidase (aminopeptidase P) 1 ( XPNPEP1 , Ref: Hs00958026_m1), alanyl-transfer RNA synthase ( AARS , Ref: Hs00609836_m1) and hypoxanthine-guanine phosphoribosyltransferase ( HPRT , ref: Hs02800695_m1) were used as housekeeping genes for normalization of GPR37 (Ref: Hs00173744_m1) expression.

Techniques: Western Blot, Expressing, SDS Page, Quantitative Proteomics, Quantitative RT-PCR

Journal: Translational Neurodegeneration

Article Title: Ecto-GPR37: a potential biomarker for Parkinson’s disease

doi: 10.1186/s40035-021-00232-7

Figure Lengend Snippet: GPR37 mRNA expression and ecto-GPR37 levels in AD. GPR37 mRNA expression in post-mortem frontal cortex ( a ) and entorhinal cortex ( b ) from NCs (panel a : n = 17; b : n = 5), Braak AD stage 3/4 ( a : n = 9; b : n = 4) and Braak AD stage 5/6 ( a : n = 20; b : n = 5) subjects was assessed by RT-qPCR. Mean values of the housekeeping genes ( GUS-B , XPNPEP1 , AARS and HPRT ) were used to normalize samples. Results are expressed as fold-change (mean ± SEM) of the NCs. c Detection of ecto-GPR37 in the CSF from NC ( n = 22) and AD ( n = 23) participants recruited at the University Medical School of Göttingen. d ROC curve analysis of CSF ecto-GPR37

Article Snippet: Parallel amplification reactions were carried out using 20× TaqMan Gene Expression Assays and 2× TaqMan Universal PCR Master Mix (Applied Biosystems). β-Glucuronidase ( GUS-β; Ref: Hs00939627_m1), X-prolyl aminopeptidase (aminopeptidase P) 1 ( XPNPEP1 , Ref: Hs00958026_m1), alanyl-transfer RNA synthase ( AARS , Ref: Hs00609836_m1) and hypoxanthine-guanine phosphoribosyltransferase ( HPRT , ref: Hs02800695_m1) were used as housekeeping genes for normalization of GPR37 (Ref: Hs00173744_m1) expression.

Techniques: Expressing, Quantitative RT-PCR