Shanti P Gangwar - Academia.edu (original) (raw)

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Papers by Shanti P Gangwar

One enzyme which catalyzes the last step of the formation of the hormone retinoic acid from vitam... more One enzyme which catalyzes the last step of the formation of the hormone retinoic acid from vitamin A (retinol) is retinal dehydrogenase type II (RalDH2). RalDH2, expressed in the Escherichia coli BL21(DE3) strain, was puri®ed and crystallized using ammonium sulfate as a precipitant. These crystals belong to the space group P2 1 2 1 2 1 (a = 108, b = 150, c = 168 A Ê , = = = 90).

Although the pathogenesis of schizophrenia (SCZ) is proposed to involve alterations of neural cir... more Although the pathogenesis of schizophrenia (SCZ) is proposed to involve alterations of neural circuits via synaptic dysfunction, the underlying molecular mechanisms remain poorly understood. Recent exome sequencing studies of SCZ have uncovered numerous single-nucleotide variants (SNVs); however, the majority of these SNVs have unknown functional consequences, leaving their disease relevance uncertain. Filling this knowledge gap requires systematic application of quantitative and scalable assays to assess known and novel biological functions of genes. Here we demonstrate loss-of-function effects of multiple rare coding SNVs found in SCZ subjects in the GIT1 (G protein-coupled receptor kinase interacting ArfGAP 1) gene using functional cell-based assays involving coexpression of GIT1 and PAK3 (p21 protein (Cdc42/Rac)-activated kinase 3). Most notably, a GIT1-R283W variant reported in four independent SCZ cases was defective in activating PAK3 as well as MAPK (mitogen-activated protein kinase). Similar functional deficits were found for a de novo SCZ variant GIT1-S601N. Additional assays revealed deficits in the capacity of GIT1-R283W to stimulate PAK phosphorylation in cultured hippocampal neurons. In addition, GIT1-R283W showed deficits in the induction of GAD1 (glutamate decarboxylase 1) protein expression. Extending these functional assays to 10 additional rare GIT1 variants revealed the existence of an allelic series with the majority of the SCZ case variants exhibiting loss of function toward MAPK activation in a manner correlated with loss of PAK3 activation. Taken together, we propose that rare variants in GIT1, along with other genetic and environmental factors, cause dysregulation of PAK3 leading to synaptic deficits in SCZ.

ESX-1-secreted protein regulator (EspR; Rv3849) is a key regulator in Mycobacterium tuberculosis ... more ESX-1-secreted protein regulator (EspR; Rv3849) is a key regulator in Mycobacterium tuberculosis that delivers bacterial proteins into the host cell during infection. EspR binds directly to the Rv3616c-Rv3614c promoter and activates transcription and secretes itself from the bacterial cell by the ESX-1 system. The three-dimensional structure of EspR will aid in understanding the mechanisms by which it binds to the Rv3616c-Rv3614c promoter and is involved in transcriptional activation. This study will significantly aid in the development of EspR-based therapeutics against M. tuberculosis. The full-length EspR gene from M. tuberculosis (H37Rv strain) was cloned and overexpressed as a soluble protein in Escherichia coli. The protein was purified by affinity chromatography using His-tagged protein followed by size-exclusion chromatography. EspR was crystallized using polyethylene glycol 3350 as precipitant. The crystals diffracted to 3.2 Å resolution using synchrotron radiation of wavelength 0.97625 Å . The crystal belonged to space group P3 1 21 and contained three monomers in the asymmetric unit. Native and heavy-atom-derivatized data sets were collected from EspR crystals for use in ab initio structure-solution techniques.

The human transporter associated with antigen processing (TAP) protein belongs to the ATP-binding... more The human transporter associated with antigen processing (TAP) protein belongs to the ATP-binding cassette (ABC) transporter superfamily and is formed by the heterodimerization of TAP1 and TAP2 subunits. TAP selectively pumps cytosolic peptides into the lumen of the endoplasmic reticulum in an ATP-dependent manner. The catalytic cycle of the ATPase domain of TAP is not understood at the molecular level. The structures of catalytic intermediates of the ATPase domain of TAP will contribute to the understanding of the chemical mechanism of ATP hydrolysis. In order to understand this mechanism, the ATPase domain of human TAP1 (NBD1) was expressed and purified, crystallized in nucleotide-free and transition-state complex forms and X-ray crystallographic studies were performed. The NBD1 protein was crystallized (i) in the nucleotide-free apo form; (ii) in complex with ADP–Mg 2+ , mimicking the product-bound state; (iii) in complex with vanadate–ADP–Mg 2+ , mimicking the ATP-bound state; and (iv) in complex with azide–ADP–Mg 2+ , also mimicking the ATP-bound state. X-ray diffraction data sets were collected for apo and complexed NBD1 using an in-house X-ray diffraction facility at a wavelength of 1.5418 A ˚. The apo and complexed NBD1 crystals belonged to the primitive hexagonal space group P6 2 , with one monomer in the asymmetric unit. Here, the crystallization, data collection and preliminary crystallographic analysis of apo and complexed NBD1 are reported.

The Ergp55 protein belongs to the Ets family of transciption factors. The Ets transcription facto... more The Ergp55 protein belongs to the Ets family of transciption factors. The Ets transcription factors are involved in various developmental processes and the regulation of cancer metabolism. They contain a highly similar DNA-binding domain known as the ETS domain and have diverse functions in oncogenesis and physiology. The Ets transcription factors differ in their DNA-binding preference at the ETS site and the mechanisms by which they target genes are not clearly understood. To understand its DNA-binding mechanism, the ETS domain of Ergp55 was expressed and purified. The ETS domain was crystallized in the native form and in complex forms with DNA sequences from the E74 and cfos promoters. An X-ray diffraction data set was collected from an ETS-cfos DNA complex crystal at a wavelength of 0.9725 Å on the BM14 synchrotron beamline at the ESRF, France. The ETS-cfos DNA complex crystal belonged to space group C222 1 , with four molecules in the asymmetric unit. For structure analysis, initial phases for the ETS-cfos DNA complex were obtained by the molecular-replacement technique with Phaser in the CCP4 suite using the coordinates of Fli-1 protein (PDB entry 1fli) and cfos DNA (PDB entry 1bc7) as search models. Structure analysis of the ETS-cfos DNA complex may possibly explain the DNA-binding specificity and its mechanism of interaction with the ETS domain of Ergp55.

Background: The Ergp55 protein belongs to Ets family of transcription factor. The Ets proteins ar... more Background: The Ergp55 protein belongs to Ets family of transcription factor. The Ets proteins are highly conserved in their DNA binding domain and involved in various development processes and regulation of cancer metabolism. To study the structure and DNA binding autoinhibition mechanism of Ergp55 protein, we have produced full length and smaller polypeptides of Ergp55 protein in E. coli and characterized using various biophysical techniques.

The CarD protein is highly expressed in mycobacterial strains under basal conditions and is trans... more The CarD protein is highly expressed in mycobacterial strains under basal conditions and is transcriptionally induced during multiple types of genotoxic stress and starvation. The CarD protein binds the subunit of RNA polymerase and influences gene expression. The disruption of interactions between CarD and the subunit of RNA polymerase has a significant effect on mycobacterial survival, resistance to stress and pathogenesis. To understand the structure of CarD and its interaction with the subunit of RNA polymerase, Mycobacterium tuberculosis CarD (MtbCarD) and the Thermus aquaticus RNA polymerase subunit were recombinantly expressed and purified. Secondary-structure analysis using circular-dichroism spectroscopy indicated that MtbCarD contains 6060% -helix, 607% -sheet and $33% random-coil structure. The C-terminal domain of MtbCarD (CarD 83-161 ) was crystallized and its X-ray structure was determined at 2.1 Å resolution. CarD 83-161 forms a distorted Y-shaped structure containing bundles of three helices connected by a loop. The residues forming the distorted Y-shaped structure are highly conserved in CarD sequences from other mycobacterial species. Comparison of the CarD 83-161 structure with the recently determined full-length M. tuberculosis and T. thermophilus CarD crystal structures revealed structural differences in residues 141-161 of the C-terminal domain of the CarD 83-161 structure. The structural changes in the CarD 83-161 structure occurred owing to proteolysis and crystallization artifacts.

One enzyme which catalyzes the last step of the formation of the hormone retinoic acid from vitam... more One enzyme which catalyzes the last step of the formation of the hormone retinoic acid from vitamin A (retinol) is retinal dehydrogenase type II (RalDH2). RalDH2, expressed in the Escherichia coli BL21(DE3) strain, was puri®ed and crystallized using ammonium sulfate as a precipitant. These crystals belong to the space group P2 1 2 1 2 1 (a = 108, b = 150, c = 168 A Ê , = = = 90).

Although the pathogenesis of schizophrenia (SCZ) is proposed to involve alterations of neural cir... more Although the pathogenesis of schizophrenia (SCZ) is proposed to involve alterations of neural circuits via synaptic dysfunction, the underlying molecular mechanisms remain poorly understood. Recent exome sequencing studies of SCZ have uncovered numerous single-nucleotide variants (SNVs); however, the majority of these SNVs have unknown functional consequences, leaving their disease relevance uncertain. Filling this knowledge gap requires systematic application of quantitative and scalable assays to assess known and novel biological functions of genes. Here we demonstrate loss-of-function effects of multiple rare coding SNVs found in SCZ subjects in the GIT1 (G protein-coupled receptor kinase interacting ArfGAP 1) gene using functional cell-based assays involving coexpression of GIT1 and PAK3 (p21 protein (Cdc42/Rac)-activated kinase 3). Most notably, a GIT1-R283W variant reported in four independent SCZ cases was defective in activating PAK3 as well as MAPK (mitogen-activated protein kinase). Similar functional deficits were found for a de novo SCZ variant GIT1-S601N. Additional assays revealed deficits in the capacity of GIT1-R283W to stimulate PAK phosphorylation in cultured hippocampal neurons. In addition, GIT1-R283W showed deficits in the induction of GAD1 (glutamate decarboxylase 1) protein expression. Extending these functional assays to 10 additional rare GIT1 variants revealed the existence of an allelic series with the majority of the SCZ case variants exhibiting loss of function toward MAPK activation in a manner correlated with loss of PAK3 activation. Taken together, we propose that rare variants in GIT1, along with other genetic and environmental factors, cause dysregulation of PAK3 leading to synaptic deficits in SCZ.

ESX-1-secreted protein regulator (EspR; Rv3849) is a key regulator in Mycobacterium tuberculosis ... more ESX-1-secreted protein regulator (EspR; Rv3849) is a key regulator in Mycobacterium tuberculosis that delivers bacterial proteins into the host cell during infection. EspR binds directly to the Rv3616c-Rv3614c promoter and activates transcription and secretes itself from the bacterial cell by the ESX-1 system. The three-dimensional structure of EspR will aid in understanding the mechanisms by which it binds to the Rv3616c-Rv3614c promoter and is involved in transcriptional activation. This study will significantly aid in the development of EspR-based therapeutics against M. tuberculosis. The full-length EspR gene from M. tuberculosis (H37Rv strain) was cloned and overexpressed as a soluble protein in Escherichia coli. The protein was purified by affinity chromatography using His-tagged protein followed by size-exclusion chromatography. EspR was crystallized using polyethylene glycol 3350 as precipitant. The crystals diffracted to 3.2 Å resolution using synchrotron radiation of wavelength 0.97625 Å . The crystal belonged to space group P3 1 21 and contained three monomers in the asymmetric unit. Native and heavy-atom-derivatized data sets were collected from EspR crystals for use in ab initio structure-solution techniques.

The human transporter associated with antigen processing (TAP) protein belongs to the ATP-binding... more The human transporter associated with antigen processing (TAP) protein belongs to the ATP-binding cassette (ABC) transporter superfamily and is formed by the heterodimerization of TAP1 and TAP2 subunits. TAP selectively pumps cytosolic peptides into the lumen of the endoplasmic reticulum in an ATP-dependent manner. The catalytic cycle of the ATPase domain of TAP is not understood at the molecular level. The structures of catalytic intermediates of the ATPase domain of TAP will contribute to the understanding of the chemical mechanism of ATP hydrolysis. In order to understand this mechanism, the ATPase domain of human TAP1 (NBD1) was expressed and purified, crystallized in nucleotide-free and transition-state complex forms and X-ray crystallographic studies were performed. The NBD1 protein was crystallized (i) in the nucleotide-free apo form; (ii) in complex with ADP–Mg 2+ , mimicking the product-bound state; (iii) in complex with vanadate–ADP–Mg 2+ , mimicking the ATP-bound state; and (iv) in complex with azide–ADP–Mg 2+ , also mimicking the ATP-bound state. X-ray diffraction data sets were collected for apo and complexed NBD1 using an in-house X-ray diffraction facility at a wavelength of 1.5418 A ˚. The apo and complexed NBD1 crystals belonged to the primitive hexagonal space group P6 2 , with one monomer in the asymmetric unit. Here, the crystallization, data collection and preliminary crystallographic analysis of apo and complexed NBD1 are reported.

The Ergp55 protein belongs to the Ets family of transciption factors. The Ets transcription facto... more The Ergp55 protein belongs to the Ets family of transciption factors. The Ets transcription factors are involved in various developmental processes and the regulation of cancer metabolism. They contain a highly similar DNA-binding domain known as the ETS domain and have diverse functions in oncogenesis and physiology. The Ets transcription factors differ in their DNA-binding preference at the ETS site and the mechanisms by which they target genes are not clearly understood. To understand its DNA-binding mechanism, the ETS domain of Ergp55 was expressed and purified. The ETS domain was crystallized in the native form and in complex forms with DNA sequences from the E74 and cfos promoters. An X-ray diffraction data set was collected from an ETS-cfos DNA complex crystal at a wavelength of 0.9725 Å on the BM14 synchrotron beamline at the ESRF, France. The ETS-cfos DNA complex crystal belonged to space group C222 1 , with four molecules in the asymmetric unit. For structure analysis, initial phases for the ETS-cfos DNA complex were obtained by the molecular-replacement technique with Phaser in the CCP4 suite using the coordinates of Fli-1 protein (PDB entry 1fli) and cfos DNA (PDB entry 1bc7) as search models. Structure analysis of the ETS-cfos DNA complex may possibly explain the DNA-binding specificity and its mechanism of interaction with the ETS domain of Ergp55.

Background: The Ergp55 protein belongs to Ets family of transcription factor. The Ets proteins ar... more Background: The Ergp55 protein belongs to Ets family of transcription factor. The Ets proteins are highly conserved in their DNA binding domain and involved in various development processes and regulation of cancer metabolism. To study the structure and DNA binding autoinhibition mechanism of Ergp55 protein, we have produced full length and smaller polypeptides of Ergp55 protein in E. coli and characterized using various biophysical techniques.

The CarD protein is highly expressed in mycobacterial strains under basal conditions and is trans... more The CarD protein is highly expressed in mycobacterial strains under basal conditions and is transcriptionally induced during multiple types of genotoxic stress and starvation. The CarD protein binds the subunit of RNA polymerase and influences gene expression. The disruption of interactions between CarD and the subunit of RNA polymerase has a significant effect on mycobacterial survival, resistance to stress and pathogenesis. To understand the structure of CarD and its interaction with the subunit of RNA polymerase, Mycobacterium tuberculosis CarD (MtbCarD) and the Thermus aquaticus RNA polymerase subunit were recombinantly expressed and purified. Secondary-structure analysis using circular-dichroism spectroscopy indicated that MtbCarD contains 6060% -helix, 607% -sheet and $33% random-coil structure. The C-terminal domain of MtbCarD (CarD 83-161 ) was crystallized and its X-ray structure was determined at 2.1 Å resolution. CarD 83-161 forms a distorted Y-shaped structure containing bundles of three helices connected by a loop. The residues forming the distorted Y-shaped structure are highly conserved in CarD sequences from other mycobacterial species. Comparison of the CarD 83-161 structure with the recently determined full-length M. tuberculosis and T. thermophilus CarD crystal structures revealed structural differences in residues 141-161 of the C-terminal domain of the CarD 83-161 structure. The structural changes in the CarD 83-161 structure occurred owing to proteolysis and crystallization artifacts.