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Papers by Jennifer Gormley
Protein Expression and Purification, Jan 31, 2010
Journal of leukocyte biology, 1997
Membrane cofactor protein (CD46), which normally protects autologous cells from complement lysis,... more Membrane cofactor protein (CD46), which normally protects autologous cells from complement lysis, is the human cell receptor for measles virus (MV). Interaction between MV and CD46 on monocytes can lead to suppression of monocyte activation. We have investigated the interaction between the cytoplasmic sequences of CD46 and kinases in a mouse macrophage cell line. Glutathione-S-transferase (GST) fusion proteins bearing the Cyt1 or Cyt2 alternative cytoplasmic domain of CD46 associate with macrophage kinase activity, which phosphorylates multiple proteins co-purified with the GST fusion proteins. Association with the macrophage kinase activity correlates with tyrosine phosphorylation of the CD46 cytoplasmic domains. Removing the CD46 sequences or introducing a frame-shift mutation abrogates the association with macrophage kinase activity. Renaturation studies reveal multiple kinases with apparent molecular mass of 82, 79, 58, and 50/49 kDa, which associate specifically with both CD46 ...
Henry Ford Hospital medical journal, 1992
Genetic linkage mapping and contig assembly using yeast artificial chromosome (YAC) technology fo... more Genetic linkage mapping and contig assembly using yeast artificial chromosome (YAC) technology form the basis of our strategy to clone and define the genomic structure of the pericentromeric region of chromosome 10 containing the multiple endocrine neoplasia type 2A gene. Thus far YAC walks have been initiated from five chromosome 10 pericentromeric loci including RBP3, D10S94, RET, D10Z1, and FNRB. Long range pulsed-field gel electrophoresis maps are constructed from the YACs isolated to define clone overlaps and to identify putative CpG islands. Bidirectional YAC walks are continued by rescreening the YAC library with sequence-tagged site assays developed from end-clones. Several new restriction fragment length polymorphisms and simple sequence repeat polymorphism markers have been identified from the YAC clones. In particular, two highly informative (CA)n dinucleotide repeat markers, sTCL-1 from proximal chromosome 10p (16 alleles, PIC = 0.68) and sJRH-1 from the RBP3 locus (18 a...
Genomics, 1996
2q, 7q, 8p, and 14q. Chromosome-specific STSs for 27 telomeres were identified from the 196 TYACs... more 2q, 7q, 8p, and 14q. Chromosome-specific STSs for 27 telomeres were identified from the 196 TYACs. More A YAC library enriched for telomere clones was constructed and screened for the human telomere-specific than 30,000 nucleotides derived from the TYAC vectorinsert junction regions or from regions flanking TYAC repeat sequence (TTAGGG). Altogether 196 TYAC library clones were studied: 189 new TYAC clones were microsatellites were compared to reported sequences using BLASTN. In addition to identifying homology isolated, 149 STSs were developed for 132 different TY-ACs, and 39 P1 clones were identified using 19 STSs with previously reported telomere sequences and human repeat elements, gene sequences and a number of from 16 of the TYACs. A combination of mapping methods including fluorescence in situ hybridization, so-ESTs were found to be highly homologous to the TYAC sequences. These genes include human coagulation matic cell hybrid panels, clamped homogeneous electric fields, meiotic linkage, and BLASTN sequence factor V (F5), Wee1 protein tyrosine kinase (WEE1), neurotropic protein tyrosine kinase type 2 (NTRK2), analysis was utilized to characterize the resource. Forty-five of the TYACs map to 31 specific telomere glutathione S-transferase (GST1), and b tubulin (TUBB). The TYAC/P1 resource, derivative STSs, and regions. Twenty-four linkage markers were developed and mapped within 14 proterminal regions (12 te-polymorphisms constitute an enabling resource to further studies of telomere structure and function and a lomeres and 2 terminal bands). The polymorphic markers include 12 microsatellites for 10 telomeres (1q, 2p, means for physical and genetic map integration and closure. ᭧ 1996 Academic Press, Inc. 6q, 7q, 10p, 10q, 13q, 14q, 18p, 22q) and the terminal bands of 11q and 12p. Twelve RFLP markers were identified and meiotically mapped to the telomeres of INTRODUCTION Sequence data from this article have been deposited with the In most eukaryotes the ends of linear chromosomes EMBL/GenBank Data Libraries under Accession Nos. U53008-U53019, U57699-U57704, U57849-U57876, U58872-U58879.
Biochimica et Biophysica Acta (BBA) - General Subjects, 2001
... Helms. Department of Chemistry, Laboratory for Biotechnology and BioAnalysis, and NMR Spectro... more ... Helms. Department of Chemistry, Laboratory for Biotechnology and BioAnalysis, and NMR Spectroscopy Center, Washington State University, Pullman, Washington 99164-4630, and Department of Chemistry and Biochemistry, Northern Illinois University, De Kalb, Illinois 60115. ...
Archives of Biochemistry and Biophysics, 2005
ADAMTS-4 (aggrecanase 1) is synthesized as a latent precursor protein that may require activation... more ADAMTS-4 (aggrecanase 1) is synthesized as a latent precursor protein that may require activation through removal of its prodomain before it can exert catalytic activity. We examined various proteinases as well as auto-activation under a wide range of conditions for removal of the prodomain and induction of enzymatic activity. The proprotein convertases, furin, PACE4, and PC5/6 eYciently removed the prodomain through cleavage at Arg 212 /Phe 213 , generating an active enzyme. Of a broad range of proteases evaluated, only MMP-9 and trypsin were capable of removing the prodomain. In the presence of mercuric compounds, removal of the prodomain through autocatalysis was not observed, nor was it observed at temperatures from 22 to 65°C, at ionic strengths from 0.1 to 1 M, or at acidic/neutral pH. At basic pH 8-10, removal of the prodomain by autocatalysis occurred, generating an active enzyme. In conclusion, the pro-form of ADAMTS-4 is not catalytically active and only a limited number of mechanisms mediate its N-terminal activation.
CD46 is a transmembrane complement regulatory protein widely expressed on nucleated human cells. ... more CD46 is a transmembrane complement regulatory protein widely expressed on nucleated human cells. Laboratory-adapted strains of measles virus (MV) bind to the extracellular domains of CD46 to enter human cells. The cytoplasmic portion of CD46 consists of a common juxtamembrane region and different distal sequences called Cyt1 and Cyt2. The biological functions of these cytoplasmic sequences are unknown. In this study, we show that expression of human CD46 with the Cyt1 cytoplasmic domain in mouse macrophages enhances production of nitric oxide (NO) in response to MV infection in the presence of gamma interferon (IFN-␥). Human CD46 does not increase the basal levels of NO production in mouse macrophages and does not augment NO production induced by double-stranded polyribonucleotides. Replacing the cytoplasmic domain of human CD46 with Cyt2 reduces MV and IFN-␥-induced NO production in mouse macrophages. Deleting the entire cytoplasmic domains of human CD46 does not prevent MV infection but markedly attenuates NO production in response to MV and IFN-␥. Mouse macrophages expressing a tailless human CD46 mutant are more susceptible to MV infection and produce 2 to 3 orders of magnitude more infectious virus than mouse macrophages expressing human CD46 with intact cytoplasmic domains. These results reveal a novel function of CD46 dependent on the cytoplasmic domains (especially Cyt1), which augments NO production in macrophages. These findings may have significant implications for roles of CD46 in innate immunity and MV pathogenesis.
Protein Expression and Purification, Jan 31, 2010
Janus-associated kinases (JAKs) play critical roles in cytokine signaling, and have emerged as vi... more Janus-associated kinases (JAKs) play critical roles in cytokine signaling, and have emerged as viable therapeutic targets in inflammation and oncology related diseases. To date, targeting JAK proteins with highly selective inhibitor compounds have remained elusive. We have expressed the active kinase domains for both JAK2 and JAK3 and devised purification protocols to resolve the non-, mono-(Y1007) and diphosphorylated (Y1007 and Y1008) states of JAK2 and non-and monophosphorylated states of JAK3 (Y980). An optimal purified protein yield of 20, 29 and 69 mg per 20 L cell culture was obtained for the three JAK2 forms, respectively, and 12.2 and 2.3 mg per 10 L fermentation for the two JAK3 forms allowing detailed biochemical and biophysical studies. To monitor the purification process we developed a novel HPLC activity assay where a sequential order of phosphorylation was observed whereby the first tyrosine residue was completely phosphorylated prior to phosphorylation of the tandem tyrosine residue. A Caliperbased microfluidics assay was used to determine the kinetic parameters (K m and k cat ) for each phosphorylated state, showing that monophosphorylated (Y1007) JAK2 enzyme activity increased 9-fold over that of the nonphosphorylated species, and increased an additional 6-fold for the diphosphorylated (Y1007/ Y1008) species, while phosphorylation of JAK3 resulted in a negligible increase in activity. Moreover, crystal structures have been generated for each isolated state of JAK2 and JAK3 with resolutions better than 2.4 Å. The generation of these reagents has enabled kinetic and structural characterization to inform the design of potent and selective inhibitors of the JAK family.
Protein Expression and Purification, Jan 31, 2010
Journal of leukocyte biology, 1997
Membrane cofactor protein (CD46), which normally protects autologous cells from complement lysis,... more Membrane cofactor protein (CD46), which normally protects autologous cells from complement lysis, is the human cell receptor for measles virus (MV). Interaction between MV and CD46 on monocytes can lead to suppression of monocyte activation. We have investigated the interaction between the cytoplasmic sequences of CD46 and kinases in a mouse macrophage cell line. Glutathione-S-transferase (GST) fusion proteins bearing the Cyt1 or Cyt2 alternative cytoplasmic domain of CD46 associate with macrophage kinase activity, which phosphorylates multiple proteins co-purified with the GST fusion proteins. Association with the macrophage kinase activity correlates with tyrosine phosphorylation of the CD46 cytoplasmic domains. Removing the CD46 sequences or introducing a frame-shift mutation abrogates the association with macrophage kinase activity. Renaturation studies reveal multiple kinases with apparent molecular mass of 82, 79, 58, and 50/49 kDa, which associate specifically with both CD46 ...
Henry Ford Hospital medical journal, 1992
Genetic linkage mapping and contig assembly using yeast artificial chromosome (YAC) technology fo... more Genetic linkage mapping and contig assembly using yeast artificial chromosome (YAC) technology form the basis of our strategy to clone and define the genomic structure of the pericentromeric region of chromosome 10 containing the multiple endocrine neoplasia type 2A gene. Thus far YAC walks have been initiated from five chromosome 10 pericentromeric loci including RBP3, D10S94, RET, D10Z1, and FNRB. Long range pulsed-field gel electrophoresis maps are constructed from the YACs isolated to define clone overlaps and to identify putative CpG islands. Bidirectional YAC walks are continued by rescreening the YAC library with sequence-tagged site assays developed from end-clones. Several new restriction fragment length polymorphisms and simple sequence repeat polymorphism markers have been identified from the YAC clones. In particular, two highly informative (CA)n dinucleotide repeat markers, sTCL-1 from proximal chromosome 10p (16 alleles, PIC = 0.68) and sJRH-1 from the RBP3 locus (18 a...
Genomics, 1996
2q, 7q, 8p, and 14q. Chromosome-specific STSs for 27 telomeres were identified from the 196 TYACs... more 2q, 7q, 8p, and 14q. Chromosome-specific STSs for 27 telomeres were identified from the 196 TYACs. More A YAC library enriched for telomere clones was constructed and screened for the human telomere-specific than 30,000 nucleotides derived from the TYAC vectorinsert junction regions or from regions flanking TYAC repeat sequence (TTAGGG). Altogether 196 TYAC library clones were studied: 189 new TYAC clones were microsatellites were compared to reported sequences using BLASTN. In addition to identifying homology isolated, 149 STSs were developed for 132 different TY-ACs, and 39 P1 clones were identified using 19 STSs with previously reported telomere sequences and human repeat elements, gene sequences and a number of from 16 of the TYACs. A combination of mapping methods including fluorescence in situ hybridization, so-ESTs were found to be highly homologous to the TYAC sequences. These genes include human coagulation matic cell hybrid panels, clamped homogeneous electric fields, meiotic linkage, and BLASTN sequence factor V (F5), Wee1 protein tyrosine kinase (WEE1), neurotropic protein tyrosine kinase type 2 (NTRK2), analysis was utilized to characterize the resource. Forty-five of the TYACs map to 31 specific telomere glutathione S-transferase (GST1), and b tubulin (TUBB). The TYAC/P1 resource, derivative STSs, and regions. Twenty-four linkage markers were developed and mapped within 14 proterminal regions (12 te-polymorphisms constitute an enabling resource to further studies of telomere structure and function and a lomeres and 2 terminal bands). The polymorphic markers include 12 microsatellites for 10 telomeres (1q, 2p, means for physical and genetic map integration and closure. ᭧ 1996 Academic Press, Inc. 6q, 7q, 10p, 10q, 13q, 14q, 18p, 22q) and the terminal bands of 11q and 12p. Twelve RFLP markers were identified and meiotically mapped to the telomeres of INTRODUCTION Sequence data from this article have been deposited with the In most eukaryotes the ends of linear chromosomes EMBL/GenBank Data Libraries under Accession Nos. U53008-U53019, U57699-U57704, U57849-U57876, U58872-U58879.
Biochimica et Biophysica Acta (BBA) - General Subjects, 2001
... Helms. Department of Chemistry, Laboratory for Biotechnology and BioAnalysis, and NMR Spectro... more ... Helms. Department of Chemistry, Laboratory for Biotechnology and BioAnalysis, and NMR Spectroscopy Center, Washington State University, Pullman, Washington 99164-4630, and Department of Chemistry and Biochemistry, Northern Illinois University, De Kalb, Illinois 60115. ...
Archives of Biochemistry and Biophysics, 2005
ADAMTS-4 (aggrecanase 1) is synthesized as a latent precursor protein that may require activation... more ADAMTS-4 (aggrecanase 1) is synthesized as a latent precursor protein that may require activation through removal of its prodomain before it can exert catalytic activity. We examined various proteinases as well as auto-activation under a wide range of conditions for removal of the prodomain and induction of enzymatic activity. The proprotein convertases, furin, PACE4, and PC5/6 eYciently removed the prodomain through cleavage at Arg 212 /Phe 213 , generating an active enzyme. Of a broad range of proteases evaluated, only MMP-9 and trypsin were capable of removing the prodomain. In the presence of mercuric compounds, removal of the prodomain through autocatalysis was not observed, nor was it observed at temperatures from 22 to 65°C, at ionic strengths from 0.1 to 1 M, or at acidic/neutral pH. At basic pH 8-10, removal of the prodomain by autocatalysis occurred, generating an active enzyme. In conclusion, the pro-form of ADAMTS-4 is not catalytically active and only a limited number of mechanisms mediate its N-terminal activation.
CD46 is a transmembrane complement regulatory protein widely expressed on nucleated human cells. ... more CD46 is a transmembrane complement regulatory protein widely expressed on nucleated human cells. Laboratory-adapted strains of measles virus (MV) bind to the extracellular domains of CD46 to enter human cells. The cytoplasmic portion of CD46 consists of a common juxtamembrane region and different distal sequences called Cyt1 and Cyt2. The biological functions of these cytoplasmic sequences are unknown. In this study, we show that expression of human CD46 with the Cyt1 cytoplasmic domain in mouse macrophages enhances production of nitric oxide (NO) in response to MV infection in the presence of gamma interferon (IFN-␥). Human CD46 does not increase the basal levels of NO production in mouse macrophages and does not augment NO production induced by double-stranded polyribonucleotides. Replacing the cytoplasmic domain of human CD46 with Cyt2 reduces MV and IFN-␥-induced NO production in mouse macrophages. Deleting the entire cytoplasmic domains of human CD46 does not prevent MV infection but markedly attenuates NO production in response to MV and IFN-␥. Mouse macrophages expressing a tailless human CD46 mutant are more susceptible to MV infection and produce 2 to 3 orders of magnitude more infectious virus than mouse macrophages expressing human CD46 with intact cytoplasmic domains. These results reveal a novel function of CD46 dependent on the cytoplasmic domains (especially Cyt1), which augments NO production in macrophages. These findings may have significant implications for roles of CD46 in innate immunity and MV pathogenesis.
Protein Expression and Purification, Jan 31, 2010
Janus-associated kinases (JAKs) play critical roles in cytokine signaling, and have emerged as vi... more Janus-associated kinases (JAKs) play critical roles in cytokine signaling, and have emerged as viable therapeutic targets in inflammation and oncology related diseases. To date, targeting JAK proteins with highly selective inhibitor compounds have remained elusive. We have expressed the active kinase domains for both JAK2 and JAK3 and devised purification protocols to resolve the non-, mono-(Y1007) and diphosphorylated (Y1007 and Y1008) states of JAK2 and non-and monophosphorylated states of JAK3 (Y980). An optimal purified protein yield of 20, 29 and 69 mg per 20 L cell culture was obtained for the three JAK2 forms, respectively, and 12.2 and 2.3 mg per 10 L fermentation for the two JAK3 forms allowing detailed biochemical and biophysical studies. To monitor the purification process we developed a novel HPLC activity assay where a sequential order of phosphorylation was observed whereby the first tyrosine residue was completely phosphorylated prior to phosphorylation of the tandem tyrosine residue. A Caliperbased microfluidics assay was used to determine the kinetic parameters (K m and k cat ) for each phosphorylated state, showing that monophosphorylated (Y1007) JAK2 enzyme activity increased 9-fold over that of the nonphosphorylated species, and increased an additional 6-fold for the diphosphorylated (Y1007/ Y1008) species, while phosphorylation of JAK3 resulted in a negligible increase in activity. Moreover, crystal structures have been generated for each isolated state of JAK2 and JAK3 with resolutions better than 2.4 Å. The generation of these reagents has enabled kinetic and structural characterization to inform the design of potent and selective inhibitors of the JAK family.