Journal: Genes and immunity

Article Title: Genetic variations in GPSM3 associated with protection from rheumatoid arthritis affect its transcript abundance

doi: 10.1038/gene.2016.3

Figure Lengend Snippet: GPSM3 transcript abundance in whole blood from individuals homozygous for the minor allele of the rs204989/rs204991 haploblock (the “m/m” genotype) is significantly lower than in whole blood from individuals homozygous for the major allele of the haploblock (the “M/M” genotype). ( A ) Schematic representation of identified polymorphisms with respect to the predicted transcription start site of the human GPSM3 gene. ( B ) Scatter plot representation of GPSM3 mRNA abundance, measured in whole blood by qRT-PCR, as normalized to the average level measured in M/M individuals. Individual results for homozygous major allele samples (M/M; n = 53) are depicted by solid circles, and homozygous minor allele samples (m/m; n=11) are depicted by open circles. An average decrease (Δμ) in GPSM3 transcript abundance of 24.1% (t = 3.80, df = 62, p = 0.0003) was observed in homozygous minor allele samples. Error bars represent S.E.M. ( C ) Descriptive statistics of the three cohorts of volunteers genotyped in this study: Minor allele frequencies were 23% for rs204989 and rs204991 in the rheumatoid arthritis samples versus 18% in the disease-free controls (Fisher's exact test; p = 0.4839). Risk/minor allele frequency for rs2812378 was 30.0% in the rheumatoid arthris samples versus 25.0% in the disease-free controls (p = 0.5267). Risk/minor allele frequency for the HLA gene region biallelic SNP rs6457620 was 35.0% in the rheumatoid arthritis samples and 45.0% in the disease-free matched controls (p = 0.1938).

Article Snippet: SNP genotypes were determined using the Type-it Fast Probe PCR Kit (Qiagen), TaqMan SNP genotyping probes, and primers for rs204989 ( GPSM3 ), rs204991 ( GPSM3 ), rs2812378 ( CCL21 ), and rs6457620 ( HLA gene region) (Life Technologies; cat. #C_3293828_20, C_2412452_20, C_16113556_10, and C_29315329_10, respectively) using the manufacturer's suggested protocols.

Techniques: Quantitative RT-PCR

Journal: Genes and immunity

Article Title: Genetic variations in GPSM3 associated with protection from rheumatoid arthritis affect its transcript abundance

doi: 10.1038/gene.2016.3

Figure Lengend Snippet: Linkage of the GPSM3 SNP haploblock and the known RA risk allele in the HLA gene region, rs6457620 [C>G], is more pronounced in RA samples than controls, but this linkage does not result in a rs6457620 genotype-dependent effect on GPSM3 transcript abundance in whole blood. ( A ) Descriptive statistics of all 200 volunteers, including the RA (n = 50), disease-free matched control (n = 50), and young healthy control volunteers recruited for the current study. These data are stratified by GPSM3 SNP haplotype, showing that the rs6457620 [G] risk allele frequency amongst homozygous ancestral GPSM3 haploblock individuals (M/M) is 41.3%, heterozygous individuals (M/m) is 56.3%, and homozygous minor GPSM3 haploblock (m/m) is 60.7%. These data are significantly different from chance distribution (Fisher-Freeman-Halton exact test; p = 0.0066). ( B ) Analyses of GPSM3 SNP haploblock linkage within RA samples stratified by rs6457620 genotype, again exhibiting statistically significant difference from chance distribution (Fisher-Freeman-Halton exact test; p = 0.0123). ( C ) Within the disease-free matched control samples, GPSM3 SNP minor allele (m) frequency, as stratified by rs6457620 genotype, is not significantly different from chance distribution as determined by Fisher-Freeman-Halton exact test (p = 0.2739). ( D ) Graphical representation of minor allele frequencies (MAFs; of European [‘eu’] population), physical distance (in basepairs), and linkage disequilibrium (LD) value (as quantified by heatmap) between the three linked SNPs of the GPSM3 SNP haploblock (rs204989, rs204990, rs204991) and the HLA gene region SNP rs6547620, as obtained using the NIEHS SNPinfo webserver ( http://snpinfo.niehs.nih.gov/ ) from NCBI dbSNP data (ref. ). ( E ) Despite modest linkage between GPSM3 SNP minor alleles (m) and the rs6457620 risk allele (G), there is no effect of the genotype at HLA gene region SNP rs6457620 on GPSM3 transcript abundance. Significance determined by one-way ANOVA with Bonferroni post-hoc .

Article Snippet: SNP genotypes were determined using the Type-it Fast Probe PCR Kit (Qiagen), TaqMan SNP genotyping probes, and primers for rs204989 ( GPSM3 ), rs204991 ( GPSM3 ), rs2812378 ( CCL21 ), and rs6457620 ( HLA gene region) (Life Technologies; cat. #C_3293828_20, C_2412452_20, C_16113556_10, and C_29315329_10, respectively) using the manufacturer's suggested protocols.

Techniques: Control

Journal: Genes and immunity

Article Title: Genetic variations in GPSM3 associated with protection from rheumatoid arthritis affect its transcript abundance

doi: 10.1038/gene.2016.3

Figure Lengend Snippet: Functional consequences of SNPs rs204989, rs204990, rs204991, and rs3096688 on GPSM3 promoter activity. ( A ) Functional results upon subcloning a 3.5 kb-region immediately 5′ to the GPSM3 transcription start site from M/M and m/m genotype volunteers, leading to wild type (pGL3-M) and “minor”/polymorphic (pGL3-m) promoter-driven expression of firefly luciferase in HEK293T cells. Luciferase activity is reported as a ratio of GPSM3 promoter-dependent firefly luciferase activity (FLuc) to control Renilla luciferase activity (pRL-TK control vector) and normalized to the average level measured from the wild type pGL3-M vector (set to 1.00; dotted line). Introduction of independent minor alleles (denoted “+###”) into the wild type pGL3-M vector is seen to differentially affect resultant firefly luciferase activity. ( B ) Restoration of the major allele at rs204989 in the pGL3-m vector ( i.e. , “pGL-m-989”) restores wild type GPSM3 promoter activity. All data are compiled from three independent experiments. Error measure is S.E.M. Significance was determined using one-way ANOVA after controlling for multiple comparisons with Dunnett's test using pGL3-M as the defined control.

Article Snippet: SNP genotypes were determined using the Type-it Fast Probe PCR Kit (Qiagen), TaqMan SNP genotyping probes, and primers for rs204989 ( GPSM3 ), rs204991 ( GPSM3 ), rs2812378 ( CCL21 ), and rs6457620 ( HLA gene region) (Life Technologies; cat. #C_3293828_20, C_2412452_20, C_16113556_10, and C_29315329_10, respectively) using the manufacturer's suggested protocols.

Techniques: Functional Assay, Activity Assay, Subcloning, Expressing, Luciferase, Control, Plasmid Preparation

Journal: Genes and immunity

Article Title: Genetic variations in GPSM3 associated with protection from rheumatoid arthritis affect its transcript abundance

doi: 10.1038/gene.2016.3

Figure Lengend Snippet: Putative transcription factor binding sites within the human GPSM3 promoter potentially disrupted by the minor allele of SNP rs204989 (arrows), as predicted by the JASPAR database: ( A ) Fos/Jun (AP1) heterodimer; (B) C/EBP-β.

Article Snippet: SNP genotypes were determined using the Type-it Fast Probe PCR Kit (Qiagen), TaqMan SNP genotyping probes, and primers for rs204989 ( GPSM3 ), rs204991 ( GPSM3 ), rs2812378 ( CCL21 ), and rs6457620 ( HLA gene region) (Life Technologies; cat. #C_3293828_20, C_2412452_20, C_16113556_10, and C_29315329_10, respectively) using the manufacturer's suggested protocols.

Techniques: Binding Assay

Journal: Genes and immunity

Article Title: Genetic variations in GPSM3 associated with protection from rheumatoid arthritis affect its transcript abundance

doi: 10.1038/gene.2016.3

Figure Lengend Snippet: Lentiviral-mediated shRNA knockdown of endogenous GPSM3 expression in the human monocytic THP-1 cell line disrupts migration toward MCP-1 ( A ) Western blot showing whole lysate (40 μg) immunoreactivity with mouse monoclonal anti-GPSM3 antibody 35.5.1 (ref. ) and with anti-β-actin as a loading control. ( B ) Real-time Transwell migration analysis of indicated calcein-AM stained THP-1 cell lines as shown by change in the ratio of MCP-1-induced to vehicle-induced relative fluorescence units (RFU) over a 30 minute timeframe. ( C ) qRT-PCR data from indicated stable knockdown or scrambled control THP-1 cell line total RNA preparations. There was no significant difference in CCR2 mRNA abundance in the THP-1 cell lines as determined by one-way ANOVA (F = 2.40; p = 0.119). ( D ) Regression analysis-derived rate of THP-1 cell line transmigration over initial migratory period; differences between each cell line was calculated by one-way ANOVA. ( E ) Regression analysis-derived maximum THP-1 cell transmigration over 30 minute timecourese; differences between each cell line were calculated by one-way ANOVA. All data are reported as means with error bars representing S.E.M.

Article Snippet: SNP genotypes were determined using the Type-it Fast Probe PCR Kit (Qiagen), TaqMan SNP genotyping probes, and primers for rs204989 ( GPSM3 ), rs204991 ( GPSM3 ), rs2812378 ( CCL21 ), and rs6457620 ( HLA gene region) (Life Technologies; cat. #C_3293828_20, C_2412452_20, C_16113556_10, and C_29315329_10, respectively) using the manufacturer's suggested protocols.

Techniques: shRNA, Knockdown, Expressing, Migration, Western Blot, Control, Staining, Fluorescence, Quantitative RT-PCR, Derivative Assay, Transmigration Assay

Journal: Frontiers in Molecular Neuroscience

Article Title: Light-Induced Thiol Oxidation of Recoverin Affects Rhodopsin Desensitization

doi: 10.3389/fnmol.2018.00474

Figure Lengend Snippet: Identification of multimeric oxidized forms of recoverin in the retina of albino rats and pigmented rabbits exposed to different doses of visual light illumination. Western blotting under non-reducing or reducing conditions of recoverin fractions extracted from the rat retinas illuminated in vivo for 14 h with metal halide lamp (2,500 lx, ‘exp’) (A) , and rabbit retinas illuminated in vivo for 3 h with halogen lamp following scheme 1 (2,200 lx, ‘exp S1’) or scheme 2 (30,000 lx, ‘exp S2’) (B) . The recoverin fractions obtained from the dark-adapted retinas were used as a control (‘contr’). ‘M,’ ‘M2,’ and ‘A’ denote monomeric, dimeric, and multimeric/aggregated forms of recoverin, respectively. Volumes of the loaded recoverin samples were chosen to ensure nearly equivalent bands of the monomeric protein. The numbers in left-hand columns indicate the molecular masses of protein markers in kDa. (C) The weight fractions of monomeric and dimeric forms of rabbit recoverin estimated from the Western blotting data from at least three independent in vivo experiments.

Article Snippet: The resulting protein samples were adjusted to pH 6.2, concentrated and analyzed by non-reducing or reducing Western blotting using affinity-purified rabbit polyclonal anti-recoverin antibodies ( ) or mouse monoclonal anti-recoverin antibodies (Santa Cruz Biotechnology), for rat and rabbit retinal extracts, respectively.

Techniques: Western Blot, In Vivo, Control

Journal: Frontiers in Molecular Neuroscience

Article Title: Light-Induced Thiol Oxidation of Recoverin Affects Rhodopsin Desensitization

doi: 10.3389/fnmol.2018.00474

Figure Lengend Snippet: Identification of monomeric oxidized forms of recoverin in the retina of pigmented rabbits exposed to different doses of visual light illumination. MALDI-TOF/TOF mass spectra of monomeric recoverin ([MH] + molecular ions of full-length proteins) extracted from the rabbit retinas illuminated in vivo for 3 h with halogen lamp following scheme 1 (2,200 lx) (A) or scheme 2 (30,000 lx) (B) .

Article Snippet: The resulting protein samples were adjusted to pH 6.2, concentrated and analyzed by non-reducing or reducing Western blotting using affinity-purified rabbit polyclonal anti-recoverin antibodies ( ) or mouse monoclonal anti-recoverin antibodies (Santa Cruz Biotechnology), for rat and rabbit retinal extracts, respectively.

Techniques: In Vivo

Journal: Frontiers in Molecular Neuroscience

Article Title: Light-Induced Thiol Oxidation of Recoverin Affects Rhodopsin Desensitization

doi: 10.3389/fnmol.2018.00474

Figure Lengend Snippet: The parameters of Ca 2+ -binding, thermal denaturation and membrane association of the recoverin variants.

Article Snippet: The resulting protein samples were adjusted to pH 6.2, concentrated and analyzed by non-reducing or reducing Western blotting using affinity-purified rabbit polyclonal anti-recoverin antibodies ( ) or mouse monoclonal anti-recoverin antibodies (Santa Cruz Biotechnology), for rat and rabbit retinal extracts, respectively.

Techniques: Binding Assay, Membrane

Journal: Frontiers in Molecular Neuroscience

Article Title: Light-Induced Thiol Oxidation of Recoverin Affects Rhodopsin Desensitization

doi: 10.3389/fnmol.2018.00474

Figure Lengend Snippet: Structural properties of recoverin forms. (A,B) Far-UV CD spectra of Ca 2+ -free (A) or Ca 2+ -loaded (B) RmRec (4 μM, solid curves), OmRec (4 μM, dotted curves) and dRec (2 μM, thick solid curves) at 20°C, pH 8.2. (C) Thermal dependencies of fluorescence spectrum maximum position for Ca 2+ -free (open symbols) and Ca 2+ -loaded (filled symbols) RmRec (14 μM, squares), OmRec (14 μM, circles) and dRec (7 μM, triangles) samples at pH 7.3. (D) The binding of bis-ANS (1 μM) to Ca 2+ -free (dashed curves) or Ca 2+ -loaded (solid curves) RmRec (6 μM, medium curves), C39D mutant (6 μM, thin curves) and dRec (3 μM, thick curves) monitored by fluorescence emission spectrum of the dye at 20°C, pH 7.3.

Article Snippet: The resulting protein samples were adjusted to pH 6.2, concentrated and analyzed by non-reducing or reducing Western blotting using affinity-purified rabbit polyclonal anti-recoverin antibodies ( ) or mouse monoclonal anti-recoverin antibodies (Santa Cruz Biotechnology), for rat and rabbit retinal extracts, respectively.

Techniques: Circular Dichroism, Fluorescence, Binding Assay, Mutagenesis

Journal: Frontiers in Molecular Neuroscience

Article Title: Light-Induced Thiol Oxidation of Recoverin Affects Rhodopsin Desensitization

doi: 10.3389/fnmol.2018.00474

Figure Lengend Snippet: The secondary structure fractions for the recoverin forms estimated from the far-UV CD data shown in Figures 6A,B using CDPro software package ( Sreerama et al., 2000 ).

Article Snippet: The resulting protein samples were adjusted to pH 6.2, concentrated and analyzed by non-reducing or reducing Western blotting using affinity-purified rabbit polyclonal anti-recoverin antibodies ( ) or mouse monoclonal anti-recoverin antibodies (Santa Cruz Biotechnology), for rat and rabbit retinal extracts, respectively.

Techniques: Software

Journal: Frontiers in Molecular Neuroscience

Article Title: Light-Induced Thiol Oxidation of Recoverin Affects Rhodopsin Desensitization

doi: 10.3389/fnmol.2018.00474

Figure Lengend Snippet: Functional properties of the recoverin forms. (A) The binding of 30 μM RmRec (open circles) and 15 μM dRec (open squares) to photoreceptor membranes. Recoverin was mixed with urea-washed photoreceptor membranes in the presence of 0.11–500 μM [Ca 2+ ] free at 37°C, pH 8.0 and the membranes were separated by ultracentrifugation. The fractions of membrane-bound protein evaluated by SDS-PAGE were plotted versus [Ca 2+ ] free and the plots were fitted to the 4-parameter Hill equation. Solid and dashed curves represent best fits for RmRec and dRec, respectively. The inset: fractions of the Ca 2+ -free and Ca 2+ -saturated protein bound to the membranes. (B) Inhibition of GRK1 by RmRec or C39D mutant (40 μM), or dRec (20 μM). Rhodopsin phosphorylation by GRK1 in the presence of [γ - 32 P]ATP was monitored at high [Ca 2+ ] free (200 μM Ca 2+ , filled bars) or low [Ca 2+ ] free (0.01 μM, open bars) by phosphorimaging radioautography.

Article Snippet: The resulting protein samples were adjusted to pH 6.2, concentrated and analyzed by non-reducing or reducing Western blotting using affinity-purified rabbit polyclonal anti-recoverin antibodies ( ) or mouse monoclonal anti-recoverin antibodies (Santa Cruz Biotechnology), for rat and rabbit retinal extracts, respectively.

Techniques: Functional Assay, Binding Assay, Membrane, SDS Page, Inhibition, Mutagenesis, Phospho-proteomics

Journal: Frontiers in Molecular Neuroscience

Article Title: Light-Induced Thiol Oxidation of Recoverin Affects Rhodopsin Desensitization

doi: 10.3389/fnmol.2018.00474

Figure Lengend Snippet: The affinity of Ca 2+ -bound recoverin variants to N-terminal domain of GRK1 (N-GRK1) and its fragment corresponding to the residues M1-S25 (1-25GRK1), estimated using SPR spectroscopy according to the heterogeneous ligand model (1).

Article Snippet: The resulting protein samples were adjusted to pH 6.2, concentrated and analyzed by non-reducing or reducing Western blotting using affinity-purified rabbit polyclonal anti-recoverin antibodies ( ) or mouse monoclonal anti-recoverin antibodies (Santa Cruz Biotechnology), for rat and rabbit retinal extracts, respectively.

Techniques: Spectroscopy

Journal: Frontiers in Molecular Neuroscience

Article Title: Light-Induced Thiol Oxidation of Recoverin Affects Rhodopsin Desensitization

doi: 10.3389/fnmol.2018.00474

Figure Lengend Snippet: Position of C39 in three-dimensional structure of recoverin bound to membrane and GRK1. (A) Topology of recoverin on membrane surface built based on NMR structures of myristoylated Ca 2+ -bound protein [PDB entry 1JSA ( ; )]. C39 (orange), calcium ions (yellow), myristoyl residue (green) and the basic residues (K5, K11, K37, R43, and K84) in close contact with the membrane are indicated. (B) The structure of recoverin complex with GRK1 [PDB entry 2I94 ]. N-terminal amphipathic helix of GRK1 (magenta), calcium ions (yellow) and C39 (orange) are indicated. The images were created using PyMol Molecular Graphics System v.1.4.1 (Schrödinger, LLC).

Article Snippet: The resulting protein samples were adjusted to pH 6.2, concentrated and analyzed by non-reducing or reducing Western blotting using affinity-purified rabbit polyclonal anti-recoverin antibodies ( ) or mouse monoclonal anti-recoverin antibodies (Santa Cruz Biotechnology), for rat and rabbit retinal extracts, respectively.

Techniques: Membrane, Residue

Journal: Frontiers in Molecular Neuroscience

Article Title: Light-Induced Thiol Oxidation of Recoverin Affects Rhodopsin Desensitization

doi: 10.3389/fnmol.2018.00474

Figure Lengend Snippet: Hypothetical scheme describing potential roles of disulfide dimer and thiol-oxidized monomer of recoverin in the mechanisms of photoreceptor apoptosis induced by visual light. The designations are as follows: Arr, arrestin; dArr, disulfide dimer of arrestin; GRK1, G-protein coupled kinase-1; Gt, transducin; PDE6, rod cGMP-specific 3’,5’-cyclic phosphodiesterase; Rho, rhodopsin; p Rho, phosphorylated rhodopsin; OmRec, monomeric recoverin with C39 converted into sulfinic acid; dRec, disulfide dimer of recoverin; JNK3, c-Jun N-terminal kinase 3; c-Jun/c-Fos (AP-1), activator protein 1 – heterodimeric transcription factor; nNOS, neuronal nitric oxide synthase 1; GC, guanylate cyclase; MDM2, mouse double minute 2 homolog – E3 ubiquitin-protein ligase; UPR, unfolded protein response; CHOP, C/EBP homologous protein – pro-apoptotic transcription factor; PERK, protein kinase R (PKR)-like endoplasmic reticulum kinase – translation initiation factor 2-alpha kinase 3; p eLF2α, phosphorylated translation initiation factor 2α; ATF4, activating transcription factor 4; Bcl2, B-cell lymphoma 2 – apoptosis regulator protein. For details, refer to section “Discussion.”

Article Snippet: The resulting protein samples were adjusted to pH 6.2, concentrated and analyzed by non-reducing or reducing Western blotting using affinity-purified rabbit polyclonal anti-recoverin antibodies ( ) or mouse monoclonal anti-recoverin antibodies (Santa Cruz Biotechnology), for rat and rabbit retinal extracts, respectively.

Techniques: Ubiquitin Proteomics