Journal: Frontiers in Cell and Developmental Biology

Article Title: Loss of the Essential Autophagy Regulators FIP200 or Atg5 Leads to Distinct Effects on Focal Adhesion Composition and Organization

doi: 10.3389/fcell.2020.00733

Figure Lengend Snippet: Specific loss of FIP200 results in elevated phospo-ERK1/2 across the time course of fibronectin-induced cell spreading. (A) MCF10A wild-type and FIP200 KO cells were kept in suspension in EGF depleted growth media for 1 hour prior to plating on 10 μg/ml fibronectin for the indicated times. Time zero indicates cells in suspension. Western blot analysis was performed on lysates harvested at the indicated times. Immunoblotting was performed against the indicated proteins. (B) Quantitation of Ser473 Akt phosphorylation western blot integrated density values normalized to actin from wild-type and FIP200 KO cells. (C) Fold change in Akt Ser473 or ERK1 Thr202 and ERK2 Tyr204 phosphorylation from zero time point following normalization to actin from wild-type and FIP200 KO cells. (D) MCF10A wild-type and Atg5 KO cells were kept in suspension in EGF depleted growth media for 1 hour prior to plating on 10 μg/ml fibronectin for the indicated times. Time zero indicates cells in suspension. Western blot analysis was performed on lysates harvested at the indicated times. Immunoblotting was performed against the indicated proteins. (E) Quantitation of Ser473 Akt phosphorylation western blot integrated density values normalized to actin from wild-type and Atg5 KO cells. (F) Fold change in Akt Ser473 or ERK1 Thr202 and ERK2 Tyr204 phosphorylation from zero time point following normalization to actin from wild-type and Atg5 KO cells. Results represent at least 3 independent experiments and error bars are the SEM. * p < 0.05.

Article Snippet: The antibodies used and their dilutions for western blot (WB) and immunofluorescence microscopy (IF) were as follows: FIP200/RB1CC1 rabbit polyclonal (Proteintech; 17250-1-Ab) WB 1:1000, IF 1:200; LC3B/MAP1LC3B rabbit polyclonal (Novus Biologicals; NB100-2220) WB 1:2000, IF 1:200; p62/SQSTM1 rabbit polyclonal (ProSci; 5449) WB 1:2000, IF 1:300; actin mouse monoclonal (612656) WB 1:3000, p-Akt ser473 rabbit monoclonal (560378) WB 1:800, fibronectin mouse monoclonal (610077) WB 1:2000, IF1:200; E-cadherin mouse monoclonal (610181) WB 1:4000, paxillin mouse monoclonal (610619) WB 1:2000, and FAK pY397 (61172) WB 1:1000, IF 1:200 were purchased from BD Transduction Laboratories; phospho-p44/42 MAPK (Erk1/2) (137F5) rabbit monoclonal (Cell Signalling Technology) WB 1:1000; FAK mouse polyclonal (BioLegend; 603801) WB 1:1000; vinculin mouse monoclonal (EMD Millipore; MAB3574) WB 1:2000, IF 1:200; Atg5 rabbit polyclonal (Cell Signalling; 2630S) WB 1:3000; GAPDH rabbit polyclonal (ProSci; 3781) WB 1:1000.

Techniques: Western Blot, Quantitation Assay

Journal: Nature ecology & evolution

Article Title: Birth of a pathway for sulfur metabolism in early amniote evolution

doi: 10.1038/s41559-020-1232-4

Figure Lengend Snippet: a , Venn diagram of the in silico comparison of Gallus gallus and Homo sapiens PLPomes summarizing the numbers of shared and unique genes. Accession numbers of the proteins identified as cysteine lyase (CL), cystathionine beta-synthase (CBS), cysteic acid decarboxylase (CAD) are indicated. b , Reactions catalyzed by CL: synthesis of cysteate (1) and synthesis of lanthionine (2). c , Reactions catalyzed by CBS: synthesis of cystathionine (3) and conversion of serine into cysteine via addition of hydrogen sulfide (4). d , Conservation of the catalytic lysine (K119) and non conservative substitutions of active site residues of Gallus gallus CBS-like (XP_015156382). Numeration is according to the human CBS sequence. e, Gg CL and Gg CBS domain composition; the dashed line indicates gene truncation for recombinant protein expression in Gg CL (K396) and Gg CBS (S427). f , Time-resolved 1 H NMR spectra of cysteine (5 mM) in the presence of Na 2 SO 3 (7 mM) and Gg CL (1 μM), showing complete conversion into cysteic acid. ( g ) Time-resolved 1 H NMR spectra of serine (5 mM) in the presence of Na 2 S (30 mM) and Gg CBS (4 μM), showing complete conversion into cysteine. h , Gg CAD domain composition. ( i ) Time-resolved 1 H NMR spectra of cysteic acid (5 mM) in the presence of Gg CAD (1 μM), showing complete conversion into taurine. j , Example of Gg CAD kinetics in the presence of cysteic acid (CA) or cysteine sulfinic acid (CSA); the black curve is the fitting of the experimental points with the integrated Michaelis-Menten equation with K M = 6.95 ± 3.23 mM, k cat = 10.54 ± 3.46 s −1 . k , Specific activities of Gallus , Homo , and Danio CSAD orthologs with CA or CSA substrates. Data are means ± SDV of three independent experiments.

Article Snippet: To acknowledge its substrate specificity, we propose to name CA decarboxylase (CAD) the enzyme encoded by the Gallus gene annotated as CSAD based on its orthology .

Techniques: In Silico, Comparison, Sequencing, Recombinant, Expressing

Journal: Nature ecology & evolution

Article Title: Birth of a pathway for sulfur metabolism in early amniote evolution

doi: 10.1038/s41559-020-1232-4

Figure Lengend Snippet: a , Multiple alignment of CSAD orthologs from (1) non-sauropsids and (2) sauropsids. Conserved differences between groups are shaded in green. Residues that recognize PLP (yellow) or line the active site cavity (white) or are within 5 Å from the active site cavity (blue) in the human holo CSAD structure (PDB code 2JIS) are indicated by filled circles; positions of site-directed mutants are indicated by red arrows. b , Specific activities of wild-type (WT), single (Q467V, T470A), and double (Q467V/T470A) Gg CAD mutants with CA and CSA substrates. c , 1 H NMR spectra showing decarboxylation activity of wild-type (WT), single (Q467V, T470A) and double (Q467V-T470A) mutants in the presence of cysteic acid (right) and cysteine sulfinic acid (left) after 5’ of reaction stopped with 1M HCl.

Article Snippet: To acknowledge its substrate specificity, we propose to name CA decarboxylase (CAD) the enzyme encoded by the Gallus gene annotated as CSAD based on its orthology .

Techniques: Activity Assay

Journal: Nature ecology & evolution

Article Title: Birth of a pathway for sulfur metabolism in early amniote evolution

doi: 10.1038/s41559-020-1232-4

Figure Lengend Snippet: The identified pathway for sulfonated amino acids biosynthesis is shown in comparison with the known pathway for taurine biosynthesis. The dashed line shows recycle of hydrogen sulfide into cysteine catalyzed by CBS. Cysteine and sulfite sulfur atoms are denoted in different colors to highlight the different sources of sulfur. Enzymes are indicated by EC numbers (if any) and protein abbreviations as follows: cystathionine beta-synthase (CBS), cysteine lyase (CL), cysteic acid decarboxylase (CAD), cysteine dioxygenase (CDO), cysteine sulfinic acid decarboxylase (CSAD), flavin-containing monooxygenase 1 (FMO1).

Article Snippet: To acknowledge its substrate specificity, we propose to name CA decarboxylase (CAD) the enzyme encoded by the Gallus gene annotated as CSAD based on its orthology .

Techniques: Comparison

Journal: Nature ecology & evolution

Article Title: Birth of a pathway for sulfur metabolism in early amniote evolution

doi: 10.1038/s41559-020-1232-4

Figure Lengend Snippet: a , Venn diagram of the in silico comparison of Gallus gallus and Homo sapiens PLPomes summarizing the numbers of shared and unique genes. Accession numbers of the proteins identified as cysteine lyase (CL), cystathionine beta-synthase (CBS), cysteic acid decarboxylase (CAD) are indicated. b , Reactions catalyzed by CL: synthesis of cysteate (1) and synthesis of lanthionine (2). c , Reactions catalyzed by CBS: synthesis of cystathionine (3) and conversion of serine into cysteine via addition of hydrogen sulfide (4). d , Conservation of the catalytic lysine (K119) and non conservative substitutions of active site residues of Gallus gallus CBS-like (XP_015156382). Numeration is according to the human CBS sequence. e, Gg CL and Gg CBS domain composition; the dashed line indicates gene truncation for recombinant protein expression in Gg CL (K396) and Gg CBS (S427). f , Time-resolved 1 H NMR spectra of cysteine (5 mM) in the presence of Na 2 SO 3 (7 mM) and Gg CL (1 μM), showing complete conversion into cysteic acid. ( g ) Time-resolved 1 H NMR spectra of serine (5 mM) in the presence of Na 2 S (30 mM) and Gg CBS (4 μM), showing complete conversion into cysteine. h , Gg CAD domain composition. ( i ) Time-resolved 1 H NMR spectra of cysteic acid (5 mM) in the presence of Gg CAD (1 μM), showing complete conversion into taurine. j , Example of Gg CAD kinetics in the presence of cysteic acid (CA) or cysteine sulfinic acid (CSA); the black curve is the fitting of the experimental points with the integrated Michaelis-Menten equation with K M = 6.95 ± 3.23 mM, k cat = 10.54 ± 3.46 s −1 . k , Specific activities of Gallus , Homo , and Danio CSAD orthologs with CA or CSA substrates. Data are means ± SDV of three independent experiments.

Article Snippet: However, Gallus has a bona fide ortholog (XP_025001259; see and ) of mammalian cysteine sulfinic acid decarboxylase (CSAD), a protein reportedly able to catalyze CA decarboxylation, albeit with lower efficiency with respect to its CSA substrate .

Techniques: In Silico, Comparison, Sequencing, Recombinant, Expressing

Journal: Nature ecology & evolution

Article Title: Birth of a pathway for sulfur metabolism in early amniote evolution

doi: 10.1038/s41559-020-1232-4

Figure Lengend Snippet: a , Multiple alignment of CSAD orthologs from (1) non-sauropsids and (2) sauropsids. Conserved differences between groups are shaded in green. Residues that recognize PLP (yellow) or line the active site cavity (white) or are within 5 Å from the active site cavity (blue) in the human holo CSAD structure (PDB code 2JIS) are indicated by filled circles; positions of site-directed mutants are indicated by red arrows. b , Specific activities of wild-type (WT), single (Q467V, T470A), and double (Q467V/T470A) Gg CAD mutants with CA and CSA substrates. c , 1 H NMR spectra showing decarboxylation activity of wild-type (WT), single (Q467V, T470A) and double (Q467V-T470A) mutants in the presence of cysteic acid (right) and cysteine sulfinic acid (left) after 5’ of reaction stopped with 1M HCl.

Article Snippet: However, Gallus has a bona fide ortholog (XP_025001259; see and ) of mammalian cysteine sulfinic acid decarboxylase (CSAD), a protein reportedly able to catalyze CA decarboxylation, albeit with lower efficiency with respect to its CSA substrate .

Techniques: Activity Assay

Journal: Nature ecology & evolution

Article Title: Birth of a pathway for sulfur metabolism in early amniote evolution

doi: 10.1038/s41559-020-1232-4

Figure Lengend Snippet: a-d , In situ hybridization analysis of CL expression in chick embryos at Hamburger-Hamilton developmental stages 4, 10, 18, and 24, sorted from left to right. e-h , In situ hybridization analysis of CBS expression at HH stages 4, 10, 18, and 24, sortexd from left to right. i-l , In situ hybridization analysis of CSAD expression at HH stages 4, 10, 18, and 24, sorted from left to right. Scale bars: 1mm ( a-c, e-g, i-k ) 5mm ( d, h, i ). m , NCBI Sequence-viewer representation of the genomic region on Gallus gallus chromosome 1 (annotation release 104) encompassing the CBS and CL genes. Gene exon structure is represented by green segments. Blue bars represent RNA-seq exon coverage (log2 scaled) for aggregate, kidney (SAMEA2201372), liver (SAMEA2201470), and duodenum (SAMN03376186) datasets. n , NCBI Sequence-viewer representation of the genomic region on Gallus gallus chromosome 33 encompassing the CSAD gene; tracks are as in panel m.

Article Snippet: However, Gallus has a bona fide ortholog (XP_025001259; see and ) of mammalian cysteine sulfinic acid decarboxylase (CSAD), a protein reportedly able to catalyze CA decarboxylation, albeit with lower efficiency with respect to its CSA substrate .

Techniques: In Situ Hybridization, Expressing, Sequencing, RNA Sequencing

Journal: Nature ecology & evolution

Article Title: Birth of a pathway for sulfur metabolism in early amniote evolution

doi: 10.1038/s41559-020-1232-4

Figure Lengend Snippet: The identified pathway for sulfonated amino acids biosynthesis is shown in comparison with the known pathway for taurine biosynthesis. The dashed line shows recycle of hydrogen sulfide into cysteine catalyzed by CBS. Cysteine and sulfite sulfur atoms are denoted in different colors to highlight the different sources of sulfur. Enzymes are indicated by EC numbers (if any) and protein abbreviations as follows: cystathionine beta-synthase (CBS), cysteine lyase (CL), cysteic acid decarboxylase (CAD), cysteine dioxygenase (CDO), cysteine sulfinic acid decarboxylase (CSAD), flavin-containing monooxygenase 1 (FMO1).

Article Snippet: However, Gallus has a bona fide ortholog (XP_025001259; see and ) of mammalian cysteine sulfinic acid decarboxylase (CSAD), a protein reportedly able to catalyze CA decarboxylation, albeit with lower efficiency with respect to its CSA substrate .

Techniques: Comparison

Journal: Gene Expression

Article Title: HNF4α Regulates CSAD to Couple Hepatic Taurine Production to Bile Acid Synthesis in Mice

doi: 10.3727/105221618X15277685544442

Figure Lengend Snippet: Hepatocyte nuclear factor 4α (HNF4α) binds mouse CSAD promoter to induce its gene transcription. (A–C) C57BL/6J male mice (n = 5) were injected with Ad-Null or Ad-shHNF4α at a dose of 1 × 109 pfu/mouse. After 7 days, mice were fasted overnight, and tissues were collected. Liver mRNA and protein were measured. The mRNA results are expressed as mean ± SEM. *Statistical significance versus Ad-Null. Densitometry was performed using ImageJ software and normalized to loading controls. (D) Luciferase reporter constructs (0.2 μg), β-gal expression construct (0.05 μg), and 0.1 μg of pcDNA3.0 or HNF4α expression plasmid were cotransfected into AML12 cells. Luciferase and β-gal activities were measured 48 h later. Controls (pcDNA3.0) for different reporter constructs were normalized to “1” for comparison. *Statistical significance versus pcDNA3.0. The indicated promoter length is relative to the transcriptional start site of the Csad gene as “+1”. The “ATG” start codon of the Csad gene starts at +169 bp. (E) Chromatin immunoprecipitation (ChIP) assay detection of HNF4α occupancy to the proximal promoter region of the Csad chromatin in mouse livers. The ChIP assay real-time PCR primer pairs amplify the −57/+60 Csad chromatin region. (F) Wild-type (WT) and HNF4α mutant CSAD-(−913/+60)-luc constructs (0.2 μg), β-gal expression construct (0.05 μg), and 0.1 μg of pcDNA3.0 or HNF4α expression plasmid were cotransfected into AML12 cells. Luciferase and β-gal activities were measured 48 h later. The putative HNF4α binding site and mutant sequences are shown below the bar graph. *Statistical significance versus pcDNA3.0. #Statistical significance versus WT-Luc. (G) EMSA detection of HNF4α binding to the WT but not the mutant HNF4α binding site in the CSAD promoter DNA probe. Mut, CSAD probe with mutations introduced into the HNF4α binding site as shown in (C). All reporter assay results are shown as mean ± SD of triplicate assays.

Article Snippet: Luciferase Reporter Assay Mouse CSAD promoter fragments were generated by PCR and cloned into PGL3-basic vector (Promega, Madison, WI, USA).

Techniques: Injection, Software, Luciferase, Construct, Expressing, Plasmid Preparation, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Mutagenesis, Binding Assay, Reporter Assay

Journal: Gene Expression

Article Title: HNF4α Regulates CSAD to Couple Hepatic Taurine Production to Bile Acid Synthesis in Mice

doi: 10.3727/105221618X15277685544442

Figure Lengend Snippet: Small heterodimer partner (SHP) represses HNF4α transactivation of the mouse CSAD reporter activity. (A–C) WT or mutant CSAD-(−1,895/+60)-luc construct (0.2 μg), β-gal expression construct (0.05 μg), and 0.1 μg (+) or 0.2 μg (++) of HNF4α and/or SHP expression plasmids were cotransfected into AML12 cells. The total amount of transfected plasmid DNA in each condition was adjusted by pcDNA3.0 plasmid. Luciferase and β-gal activities were measured 48 h later. *Statistical significance versus WT-Luc control + pcDNA3.0. #Statistical significance versus WT-Luc + HNF4α. All results are shown as mean ± SD of triplicate assays. (D, E) CSAD mRNA and protein expression in liver-specific SHP KO mice and controls. Results of mRNA expression were expressed as mean ± SE (n = 3–4). *Statistical significance versus WT. Densitometry was performed using ImageJ software and normalized to loading controls.

Article Snippet: Luciferase Reporter Assay Mouse CSAD promoter fragments were generated by PCR and cloned into PGL3-basic vector (Promega, Madison, WI, USA).

Techniques: Activity Assay, Mutagenesis, Construct, Expressing, Transfection, Plasmid Preparation, Luciferase, Software