Marcin J Domagalski | University of Virginia (original) (raw)

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Papers by Marcin J Domagalski

Table S1. Kinetic parameters for 2HADHs and prominent substrates described in the literature. (XL... more Table S1. Kinetic parameters for 2HADHs and prominent substrates described in the literature. (XLSX 503 kb)

Glutamine amidotransferase (GATase) is an enzyme widespread in living organisms. It catalyzes the... more Glutamine amidotransferase (GATase) is an enzyme widespread in living organisms. It catalyzes the hydrolysis of glutamine to generate glutamic acid and ammonia. It works cooperatively with various synthases by providing ammonium nitrogen that is utilized to produce amino acids, purine and pyrimidine nucleotides, amino sugars, coenzymes and antibiotics. 1) Class-I GATase contains a characteristic Cys-His-Glu triad. The carbonyl carbon of the glutamine side chain carboxamide group is attacked by the thiolate anion of Cys, and forms a tetrahedral intermediate. Then the protonation of the amino group by His collapses this intermediate, producing a thioester intermediate and ammonia. 1) Three-dimensional structures of Class-I GATase have been reported in several species including an eukaryote, 2) ten bacteria 3)-12) and an archaeon. 13) Here, we report the crystal structure of a Class-I GATase from the hyperthermophilic archaeon Pyrococcus horikoshii OT3 (PH1346, a putative guanosine 5'-monophosphate (GMP) synthase, E.C. 6.3.5.2) 14) and its comparison with the crystal structures of anthranilate synthase from Sulfolobus solfataricus (34% identical in sequence) and GMP synthase from E. coli (37% identical in sequence). Materials and methods. The GATase from P. horikoshii (PH-GATase) was overexpressed in E. coli BL21(DE3) cells as a fusion protein with an N-terminal His tag using the pET-28a(+) vector (Novagen). The harvested cells were sonicated in 50 mM sodium phosphate buffer (pH 8.0) containing 300 mM NaCl and 10 mM imidazole and then centrifuged at 40,000 g for 30 min. The supernatant was incubated for 30 min at 353 K and centrifuged at 40,000 g for 30 min. The supernatant was batch-incubated with Ni-NTA-agarose (Qiagen) for 30 min and applied to an Econopac column (Bio-Rad, Hercules, CA). The column was washed with 50 mM sodium phosphate buffer (pH 8.0) containing 300 mM NaCl and 20 mM imidazole. Then the protein was eluted in 50 mM sodium phosphate buffer (pH 8.0) containing 300 mM NaCl and 250 mM imidazole. The protein was further purified by size exclusion chromatography with a HiLoad 26/60 Superdex 75 pg column (Amersham) equilibrated with 20 mM Tris-HCl buffer (pH 8.0) containing 100 mM NaCl. The fractions containing PH-GATase were dialyzed against 5 mM Tris-HCl (pH 8.0) and concentrated to 28.6 mg/ml in Centriprep-3 concentrators (Amicon). The purity was ascertained by SDS-PAGE and the concentration was determined with the absorbance at 280 nm. Crystallization was carried out at 278 K by the hanging-drop vapor-diffusion method using Crystal Screens 1 and 2 (Hampton Research). Each hanging-drop was

Cell, Aug 1, 2021

Lung squamous cell carcinoma (LSCC) remains a leading cause of cancer death with few therapeutic ... more Lung squamous cell carcinoma (LSCC) remains a leading cause of cancer death with few therapeutic options. We characterized the proteogenomic landscape of LSCC, providing a deeper exposition of LSCC biology with potential therapeutic implications. We identify NSD3 as an alternative driver in FGFR1-amplified tumors and low-p63 tumors overexpressing the therapeutic target survivin. SOX2 is considered undruggable, but our analyses provide rationale for exploring chromatin modifiers such as LSD1 and EZH2 to target SOX2-overexpressing tumors. Our data support complex regulation of metabolic pathways by crosstalk between post-translational modifications including ubiquitylation. Numerous immune-related proteogenomic observations suggest directions for further investigation. Proteogenomic dissection of CDKN2A mutations argue for more nuanced assessment of RB1 protein expression and phosphorylation before declaring CDK4/6 inhibition unsuccessful. Finally, triangulation between LSCC, LUAD, and HNSCC identified both unique and common therapeutic vulnerabilities. These observations and proteogenomics data resources may guide research into the biology and treatment of LSCC.

Proceedings of the Japan Academy, Series B, 2005

Glutamine amidotransferase (GATase) is an enzyme widespread in living organisms. It catalyzes the... more Glutamine amidotransferase (GATase) is an enzyme widespread in living organisms. It catalyzes the hydrolysis of glutamine to generate glutamic acid and ammonia. It works cooperatively with various synthases by providing ammonium nitrogen that is utilized to produce amino acids, purine and pyrimidine nucleotides, amino sugars, coenzymes and antibiotics. 1) Class-I GATase contains a characteristic Cys-His-Glu triad. The carbonyl carbon of the glutamine side chain carboxamide group is attacked by the thiolate anion of Cys, and forms a tetrahedral intermediate. Then the protonation of the amino group by His collapses this intermediate, producing a thioester intermediate and ammonia. 1) Three-dimensional structures of Class-I GATase have been reported in several species including an eukaryote, 2) ten bacteria 3)-12) and an archaeon. 13) Here, we report the crystal structure of a Class-I GATase from the hyperthermophilic archaeon Pyrococcus horikoshii OT3 (PH1346, a putative guanosine 5'-monophosphate (GMP) synthase, E.C. 6.3.5.2) 14) and its comparison with the crystal structures of anthranilate synthase from Sulfolobus solfataricus (34% identical in sequence) and GMP synthase from E. coli (37% identical in sequence). Materials and methods. The GATase from P. horikoshii (PH-GATase) was overexpressed in E. coli BL21(DE3) cells as a fusion protein with an N-terminal His tag using the pET-28a(+) vector (Novagen). The harvested cells were sonicated in 50 mM sodium phosphate buffer (pH 8.0) containing 300 mM NaCl and 10 mM imidazole and then centrifuged at 40,000 g for 30 min. The supernatant was incubated for 30 min at 353 K and centrifuged at 40,000 g for 30 min. The supernatant was batch-incubated with Ni-NTA-agarose (Qiagen) for 30 min and applied to an Econopac column (Bio-Rad, Hercules, CA). The column was washed with 50 mM sodium phosphate buffer (pH 8.0) containing 300 mM NaCl and 20 mM imidazole. Then the protein was eluted in 50 mM sodium phosphate buffer (pH 8.0) containing 300 mM NaCl and 250 mM imidazole. The protein was further purified by size exclusion chromatography with a HiLoad 26/60 Superdex 75 pg column (Amersham) equilibrated with 20 mM Tris-HCl buffer (pH 8.0) containing 100 mM NaCl. The fractions containing PH-GATase were dialyzed against 5 mM Tris-HCl (pH 8.0) and concentrated to 28.6 mg/ml in Centriprep-3 concentrators (Amicon). The purity was ascertained by SDS-PAGE and the concentration was determined with the absorbance at 280 nm. Crystallization was carried out at 278 K by the hanging-drop vapor-diffusion method using Crystal Screens 1 and 2 (Hampton Research). Each hanging-drop was

Horizontal gene transfer from bacteria to plants The topology of the phylogenetic tree of the GHR... more Horizontal gene transfer from bacteria to plants The topology of the phylogenetic tree of the GHRB clade does not follow the phylogeny of the tree of life derived by analysis of ribosomal RNA and other universal genes [1]. In particular, the topology of the GHRB clade suggests a possible premise for a horizontal gene transfer (HGT) from ɑ-proteobacteria to plants. A BLAST search using the sequence of hydroxyphenylpyruvate reductase from the plant Coleus blumei (HPPR_PLESU) as a query identified several closely homologous enzymes from other core angiosperms, mostly monocots and eudicots, including multiple nodulating plants. These enzymes form the homologous cluster comprising two A. thaliana hydroxypyruvate/glyoxylate reductases, HPR2 and HPR3, respectively. The highest scoring hits after plant proteins appeared to be ɑ-proteobacterial proteins, with Agrobacterium homologs in the top (55% sequence identity), suggesting that the ɑproteobacteria transferred the genes to the plants. The most similar bacterial proteins that have been studied experimentally are 2-keto-ᴅ-gluconate dehydrogenase from Gluconobacter oxydans (2KGR_GLUOX) and hydroxypyruvate/glyoxylate reductase from S. meliloti (Q92LZ4_RHIME), which also possesses activity for 2-keto-ᴅ-gluconate. In Oryza sativa, the potential HGT was followed by

Table S1. Kinetic parameters for 2HADHs and prominent substrates described in the literature. (XL... more Table S1. Kinetic parameters for 2HADHs and prominent substrates described in the literature. (XLSX 503 kb)

Glutamine amidotransferase (GATase) is an enzyme widespread in living organisms. It catalyzes the... more Glutamine amidotransferase (GATase) is an enzyme widespread in living organisms. It catalyzes the hydrolysis of glutamine to generate glutamic acid and ammonia. It works cooperatively with various synthases by providing ammonium nitrogen that is utilized to produce amino acids, purine and pyrimidine nucleotides, amino sugars, coenzymes and antibiotics. 1) Class-I GATase contains a characteristic Cys-His-Glu triad. The carbonyl carbon of the glutamine side chain carboxamide group is attacked by the thiolate anion of Cys, and forms a tetrahedral intermediate. Then the protonation of the amino group by His collapses this intermediate, producing a thioester intermediate and ammonia. 1) Three-dimensional structures of Class-I GATase have been reported in several species including an eukaryote, 2) ten bacteria 3)-12) and an archaeon. 13) Here, we report the crystal structure of a Class-I GATase from the hyperthermophilic archaeon Pyrococcus horikoshii OT3 (PH1346, a putative guanosine 5'-monophosphate (GMP) synthase, E.C. 6.3.5.2) 14) and its comparison with the crystal structures of anthranilate synthase from Sulfolobus solfataricus (34% identical in sequence) and GMP synthase from E. coli (37% identical in sequence). Materials and methods. The GATase from P. horikoshii (PH-GATase) was overexpressed in E. coli BL21(DE3) cells as a fusion protein with an N-terminal His tag using the pET-28a(+) vector (Novagen). The harvested cells were sonicated in 50 mM sodium phosphate buffer (pH 8.0) containing 300 mM NaCl and 10 mM imidazole and then centrifuged at 40,000 g for 30 min. The supernatant was incubated for 30 min at 353 K and centrifuged at 40,000 g for 30 min. The supernatant was batch-incubated with Ni-NTA-agarose (Qiagen) for 30 min and applied to an Econopac column (Bio-Rad, Hercules, CA). The column was washed with 50 mM sodium phosphate buffer (pH 8.0) containing 300 mM NaCl and 20 mM imidazole. Then the protein was eluted in 50 mM sodium phosphate buffer (pH 8.0) containing 300 mM NaCl and 250 mM imidazole. The protein was further purified by size exclusion chromatography with a HiLoad 26/60 Superdex 75 pg column (Amersham) equilibrated with 20 mM Tris-HCl buffer (pH 8.0) containing 100 mM NaCl. The fractions containing PH-GATase were dialyzed against 5 mM Tris-HCl (pH 8.0) and concentrated to 28.6 mg/ml in Centriprep-3 concentrators (Amicon). The purity was ascertained by SDS-PAGE and the concentration was determined with the absorbance at 280 nm. Crystallization was carried out at 278 K by the hanging-drop vapor-diffusion method using Crystal Screens 1 and 2 (Hampton Research). Each hanging-drop was

Cell, Aug 1, 2021

Lung squamous cell carcinoma (LSCC) remains a leading cause of cancer death with few therapeutic ... more Lung squamous cell carcinoma (LSCC) remains a leading cause of cancer death with few therapeutic options. We characterized the proteogenomic landscape of LSCC, providing a deeper exposition of LSCC biology with potential therapeutic implications. We identify NSD3 as an alternative driver in FGFR1-amplified tumors and low-p63 tumors overexpressing the therapeutic target survivin. SOX2 is considered undruggable, but our analyses provide rationale for exploring chromatin modifiers such as LSD1 and EZH2 to target SOX2-overexpressing tumors. Our data support complex regulation of metabolic pathways by crosstalk between post-translational modifications including ubiquitylation. Numerous immune-related proteogenomic observations suggest directions for further investigation. Proteogenomic dissection of CDKN2A mutations argue for more nuanced assessment of RB1 protein expression and phosphorylation before declaring CDK4/6 inhibition unsuccessful. Finally, triangulation between LSCC, LUAD, and HNSCC identified both unique and common therapeutic vulnerabilities. These observations and proteogenomics data resources may guide research into the biology and treatment of LSCC.

Proceedings of the Japan Academy, Series B, 2005

Glutamine amidotransferase (GATase) is an enzyme widespread in living organisms. It catalyzes the... more Glutamine amidotransferase (GATase) is an enzyme widespread in living organisms. It catalyzes the hydrolysis of glutamine to generate glutamic acid and ammonia. It works cooperatively with various synthases by providing ammonium nitrogen that is utilized to produce amino acids, purine and pyrimidine nucleotides, amino sugars, coenzymes and antibiotics. 1) Class-I GATase contains a characteristic Cys-His-Glu triad. The carbonyl carbon of the glutamine side chain carboxamide group is attacked by the thiolate anion of Cys, and forms a tetrahedral intermediate. Then the protonation of the amino group by His collapses this intermediate, producing a thioester intermediate and ammonia. 1) Three-dimensional structures of Class-I GATase have been reported in several species including an eukaryote, 2) ten bacteria 3)-12) and an archaeon. 13) Here, we report the crystal structure of a Class-I GATase from the hyperthermophilic archaeon Pyrococcus horikoshii OT3 (PH1346, a putative guanosine 5'-monophosphate (GMP) synthase, E.C. 6.3.5.2) 14) and its comparison with the crystal structures of anthranilate synthase from Sulfolobus solfataricus (34% identical in sequence) and GMP synthase from E. coli (37% identical in sequence). Materials and methods. The GATase from P. horikoshii (PH-GATase) was overexpressed in E. coli BL21(DE3) cells as a fusion protein with an N-terminal His tag using the pET-28a(+) vector (Novagen). The harvested cells were sonicated in 50 mM sodium phosphate buffer (pH 8.0) containing 300 mM NaCl and 10 mM imidazole and then centrifuged at 40,000 g for 30 min. The supernatant was incubated for 30 min at 353 K and centrifuged at 40,000 g for 30 min. The supernatant was batch-incubated with Ni-NTA-agarose (Qiagen) for 30 min and applied to an Econopac column (Bio-Rad, Hercules, CA). The column was washed with 50 mM sodium phosphate buffer (pH 8.0) containing 300 mM NaCl and 20 mM imidazole. Then the protein was eluted in 50 mM sodium phosphate buffer (pH 8.0) containing 300 mM NaCl and 250 mM imidazole. The protein was further purified by size exclusion chromatography with a HiLoad 26/60 Superdex 75 pg column (Amersham) equilibrated with 20 mM Tris-HCl buffer (pH 8.0) containing 100 mM NaCl. The fractions containing PH-GATase were dialyzed against 5 mM Tris-HCl (pH 8.0) and concentrated to 28.6 mg/ml in Centriprep-3 concentrators (Amicon). The purity was ascertained by SDS-PAGE and the concentration was determined with the absorbance at 280 nm. Crystallization was carried out at 278 K by the hanging-drop vapor-diffusion method using Crystal Screens 1 and 2 (Hampton Research). Each hanging-drop was

Horizontal gene transfer from bacteria to plants The topology of the phylogenetic tree of the GHR... more Horizontal gene transfer from bacteria to plants The topology of the phylogenetic tree of the GHRB clade does not follow the phylogeny of the tree of life derived by analysis of ribosomal RNA and other universal genes [1]. In particular, the topology of the GHRB clade suggests a possible premise for a horizontal gene transfer (HGT) from ɑ-proteobacteria to plants. A BLAST search using the sequence of hydroxyphenylpyruvate reductase from the plant Coleus blumei (HPPR_PLESU) as a query identified several closely homologous enzymes from other core angiosperms, mostly monocots and eudicots, including multiple nodulating plants. These enzymes form the homologous cluster comprising two A. thaliana hydroxypyruvate/glyoxylate reductases, HPR2 and HPR3, respectively. The highest scoring hits after plant proteins appeared to be ɑ-proteobacterial proteins, with Agrobacterium homologs in the top (55% sequence identity), suggesting that the ɑproteobacteria transferred the genes to the plants. The most similar bacterial proteins that have been studied experimentally are 2-keto-ᴅ-gluconate dehydrogenase from Gluconobacter oxydans (2KGR_GLUOX) and hydroxypyruvate/glyoxylate reductase from S. meliloti (Q92LZ4_RHIME), which also possesses activity for 2-keto-ᴅ-gluconate. In Oryza sativa, the potential HGT was followed by