Kelly Smith | Vtu - Academia.edu (original) (raw)

Papers by Kelly Smith

Research paper thumbnail of Association between Wild Type and Mutant APC Gene Products1

Germline mutations of the APC gene are responsible for familial adenomatous polyposis, an autosom... more Germline mutations of the APC gene are responsible for familial adenomatous polyposis, an autosomal dominant inherited predisposition to coloréela! tumors. Mutation of the APC gene is also an early, if not initiating, event for sporadic coloréela!tumorigenesis. In both cases, al most all of Ihe currenlly idenlified mulalions of APC result in Ihe trun cation of Ihe prolein. In this sludy, we demonstrate thai Iruncaled APC proteins can associale with Ihe wild lype APC in vivo. Using in vitro expression and immunoprecipilalion, we show lhal Ihe first 171 residues of APC are sufficient for APC oligomerizalion and lhal Ihe first 45 amino acids of APC is necessary for Ihis interaction. These results indicale lhal most mutant APC proteins should be able lo bind lo wild lype APC protein and perhaps inactivale il in a dominant negative manner.

Research paper thumbnail of Wild-Type but not Mutant APC Associates with the Microtubule Cytoskeleton

The adenomatous polyposis coIl protein (APC) Is mutated in familial adenomatous polyposis patient... more The adenomatous polyposis coIl protein (APC) Is mutated in familial adenomatous polyposis patients as well as in sporadic colorectal tumors.

Research paper thumbnail of Expression of the APC tumor suppressor protein in oligodendroglia

Glia, 1996

Mutations of the adenomatous polyposis coli (APC) tumor suppressor gene have been linked to famil... more Mutations of the adenomatous polyposis coli (APC) tumor suppressor gene have been linked to familial polyposis, an inherited predisposition to colon cancer, and a high percentage of sporadic colon adenomas. Although this gene is best known for its role in development of bowel neoplasms, in recent studies we have found that APC mRNA levels are greatly enriched in brain compared with peripheral tissues. To help define its role in the nervous system, in this study we have determined its cellular localization immunohistochemically in adult rat brain sections and have detected intense APC immunoreactivity in oligodendrocytes. Since prominent APC immunostaining is detected in cell bodies of mature oligodendrocytes, these antibodies may provide a useful addition to available oligodendrocyte markers. Although the cellular function of APC remains undefined, previous biochemical studies have demonstrated that APC is associated with catenins, cytoplasmic proteins involved in regulating cell-cell adhesion. We propose that, in addition to its critical role in ensuring normal maturation of colonic epithelial cells, the APC tumor suppressor protein also regulates the adhesive properties of oligodendrocytes.

Research paper thumbnail of Identification of a Gene Located at Chromosome 5q21 That is Mutated in Colorectal Cancers

Research paper thumbnail of A common dominant TLR5 stop codon polymorphism abolishes flagellin signaling and is associated with susceptibility to legionnaires' disease

Journal of Immunology, 2003

Although Toll-like receptors (TLRs) are critical mediators of the immune response to pathogens, t... more Although Toll-like receptors (TLRs) are critical mediators of the immune response to pathogens, the influence of polymorphisms in this gene family on human susceptibility to infection is poorly understood. We demonstrated recently that TLR5 recognizes flagellin, a potent inflammatory stimulus present in the flagellar structure of many bacteria. Here, we show that a common stop codon polymorphism in the ligand-binding domain of TLR5 (TLR5 392STOP ) is unable to mediate flagellin signaling, acts in a dominant fashion, and is associated with susceptibility to pneumonia caused by Legionella pneumophila , a flagellated bacterium. We also show that flagellin is a principal stimulant of proinflammatory cytokine production in lung epithelial cells. Together, these observations suggest that TLR5 392STOP increases human susceptibility to infection through an unusual dominant mechanism that compromises TLR5's essential role as a regulator of the lung epithelial innate immune response.

Research paper thumbnail of The innate immune response to bacterial flagellin is mediated by Toll-like receptor 5

Nature, 2001

The innate immune system recognizes pathogen-associated molecular patterns (PAMPs) that are expre... more The innate immune system recognizes pathogen-associated molecular patterns (PAMPs) that are expressed on infectious agents, but not on the host. Toll-like receptors (TLRs) recognize PAMPs and mediate the production of cytokines necessary for the development of ...

Research paper thumbnail of The repertoire for pattern recognition of pathogens by the innate immune system is defined by cooperation between Toll-like receptors

Proceedings of The National Academy of Sciences, 2000

Toll-like receptors (TLRs) have been shown to participate in the recognition of pathogens by the ... more Toll-like receptors (TLRs) have been shown to participate in the recognition of pathogens by the innate immune system, but it is not clear how a restricted family of receptors has the capacity to recognize the wide spectrum of TLR stimuli known to exist. We report here that two members of the TLR family, TLR2 and TLR6, together coordinate macrophage activation by Gram-positive bacteria and the yeast cellwall particle, zymosan. TLR6 and TLR2 both are recruited to the macrophage phagosome, where they recognize peptidoglycan, a Gram-positive pathogen component. By contrast, TLR2 recognizes another component, bacterial lipopeptide, without TLR6. The requirement for TLR cooperation is supported by the finding that TLR2 needs a partner to activate tumor necrosis factor-␣ production in macrophages. Dimerization of the cytoplasmic domain of TLR2 does not induce tumor necrosis factor-␣ production in macrophages, whereas similar dimerization of the TLR4 cytoplasmic domain does. We show that the cytoplasmic domain of TLR2 can form functional pairs with TLR6 or TLR1, and this interaction leads to cytokine induction. Thus, the cytoplasmic tails of TLRs are not functionally equivalent, with certain TLRs requiring assembly into heteromeric complexes, whereas others are active as homomeric complexes. Finally, we show that TLR6, TLR2, and TLR1 are recruited to macrophage phagosomes that contain IgG-coated erythrocytes that do not display microbial components. The data suggest that TLRs sample the contents of the phagosome independent of the nature of the contents, and can establish a combinatorial repertoire to discriminate among the large number of pathogen-associated molecular patterns found in nature.

Research paper thumbnail of Lipocalin 2 mediates an innate immune response to bacterial infection by sequestrating iron

Nature, 2004

DMRT1 gene. As a probe, 10 ml of DOP-PCR amplification of paint E4 were used. For DMRT1 a 287-bas... more DMRT1 gene. As a probe, 10 ml of DOP-PCR amplification of paint E4 were used. For DMRT1 a 287-base-pair (bp) PstI digest of chicken DMRT1 complementary DNA, containing the DM (Doublesex and MAB-3) domain was hybridized. For both experiments the probe DNA was labelled radioactively with [ 32 P]-dATP and the BAC library screened according to the manufacturer's instructions (http:// www.genome.clemson.edu/protocols/hyb_filter.html). Positive clones were ordered from the Clemson University Genomics Institute (CUGI). For DMRT1, a final positive clone was verified by PCR using primers from conserved DMRT1 specific regions 29 . This clone was then sequenced after subcloning into a TOPO Shotgun subcloning kit (Invitrogen) according to the manufacturer's instructions. Sequences were deposited at EMBL. The DMRT1 BAC as well as the E4 specific BAC clones were labelled by standard nick translation with biotin or digoxigenin and 400-600 ng of labelled DNA were used to map the clones by FISH, as described above.

Research paper thumbnail of Evasion of Toll-like receptor 5 by flagellated bacteria

Proceedings of The National Academy of Sciences, 2005

Toll-like receptor 5 (TLR5) recognizes an evolutionarily conserved site on bacterial flagellin th... more Toll-like receptor 5 (TLR5) recognizes an evolutionarily conserved site on bacterial flagellin that is required for flagellar filament assembly and motility. The ␣ and Proteobacteria, including the important human pathogens Campylobacter jejuni, Helicobacter pylori, and Bartonella bacilliformis, require flagellar motility to efficiently infect mammalian hosts. In this study, we demonstrate that these bacteria make flagellin molecules that are not recognized by TLR5. We map the site responsible for TLR5 evasion to amino acids 89 -96 of the N-terminal D1 domain, which is centrally positioned within the previously defined TLR5 recognition site. Salmonella flagellin is strongly recognized by TLR5, but mutating residues 89 -96 to the corresponding H. pylori flaA sequence abolishes TLR5 recognition and also destroys bacterial motility. To preserve bacterial motility, ␣ and Proteobacteria possess compensatory amino acid changes in other regions of the flagellin molecule, and we engineer a mutant form of Salmonella flagellin that evades TLR5 but retains motility. These results suggest that TLR5 evasion is critical for the survival of this subset of bacteria at mucosal sites in animals and raise the intriguing possibility that flagellin receptors provided the selective force to drive the evolution of these unique subclasses of bacterial flagellins.

Research paper thumbnail of Toll-like receptor 5 recognizes a conserved site on flagellin required for protofilament formation and bacterial motility

Nature Immunology, 2003

Flagellated bacteria cause a broad range of serious gastrointestinal, urinary tract and respirato... more Flagellated bacteria cause a broad range of serious gastrointestinal, urinary tract and respiratory tract infections. In models of mucosal infection, the production of functional flagella is a bacterial virulence factor, which is required for colonization and tissue invasiveness and can elicit the recruitment of host inflammatory cells . Flagella also facilitate the infection of macrophages and epithelial cells 3,8-10 . The host targets flagella through antibodies, T cell recognition and the innate immune system 11-13 . Through TLR5, flagellin activates macrophages, dendritic cells, neutrophils and intestinal epithelial cells to produce inflammatory mediators . In addition to animals, plants and insects also have evolved innate immune recognition systems for bacterial flagellin . Plants recognize bacterial flagellin through FLS2, the flagellin sensitivity locus 2 gene product and an apparent functional equivalent of TLR5, and this recognition event triggers a systemic disease resistance response .

Research paper thumbnail of Association between Wild Type and Mutant APC Gene Products1

Germline mutations of the APC gene are responsible for familial adenomatous polyposis, an autosom... more Germline mutations of the APC gene are responsible for familial adenomatous polyposis, an autosomal dominant inherited predisposition to coloréela! tumors. Mutation of the APC gene is also an early, if not initiating, event for sporadic coloréela!tumorigenesis. In both cases, al most all of Ihe currenlly idenlified mulalions of APC result in Ihe trun cation of Ihe prolein. In this sludy, we demonstrate thai Iruncaled APC proteins can associale with Ihe wild lype APC in vivo. Using in vitro expression and immunoprecipilalion, we show lhal Ihe first 171 residues of APC are sufficient for APC oligomerizalion and lhal Ihe first 45 amino acids of APC is necessary for Ihis interaction. These results indicale lhal most mutant APC proteins should be able lo bind lo wild lype APC protein and perhaps inactivale il in a dominant negative manner.

Research paper thumbnail of Wild-Type but not Mutant APC Associates with the Microtubule Cytoskeleton

The adenomatous polyposis coIl protein (APC) Is mutated in familial adenomatous polyposis patient... more The adenomatous polyposis coIl protein (APC) Is mutated in familial adenomatous polyposis patients as well as in sporadic colorectal tumors.

Research paper thumbnail of Expression of the APC tumor suppressor protein in oligodendroglia

Glia, 1996

Mutations of the adenomatous polyposis coli (APC) tumor suppressor gene have been linked to famil... more Mutations of the adenomatous polyposis coli (APC) tumor suppressor gene have been linked to familial polyposis, an inherited predisposition to colon cancer, and a high percentage of sporadic colon adenomas. Although this gene is best known for its role in development of bowel neoplasms, in recent studies we have found that APC mRNA levels are greatly enriched in brain compared with peripheral tissues. To help define its role in the nervous system, in this study we have determined its cellular localization immunohistochemically in adult rat brain sections and have detected intense APC immunoreactivity in oligodendrocytes. Since prominent APC immunostaining is detected in cell bodies of mature oligodendrocytes, these antibodies may provide a useful addition to available oligodendrocyte markers. Although the cellular function of APC remains undefined, previous biochemical studies have demonstrated that APC is associated with catenins, cytoplasmic proteins involved in regulating cell-cell adhesion. We propose that, in addition to its critical role in ensuring normal maturation of colonic epithelial cells, the APC tumor suppressor protein also regulates the adhesive properties of oligodendrocytes.

Research paper thumbnail of Identification of a Gene Located at Chromosome 5q21 That is Mutated in Colorectal Cancers

Research paper thumbnail of A common dominant TLR5 stop codon polymorphism abolishes flagellin signaling and is associated with susceptibility to legionnaires' disease

Journal of Immunology, 2003

Although Toll-like receptors (TLRs) are critical mediators of the immune response to pathogens, t... more Although Toll-like receptors (TLRs) are critical mediators of the immune response to pathogens, the influence of polymorphisms in this gene family on human susceptibility to infection is poorly understood. We demonstrated recently that TLR5 recognizes flagellin, a potent inflammatory stimulus present in the flagellar structure of many bacteria. Here, we show that a common stop codon polymorphism in the ligand-binding domain of TLR5 (TLR5 392STOP ) is unable to mediate flagellin signaling, acts in a dominant fashion, and is associated with susceptibility to pneumonia caused by Legionella pneumophila , a flagellated bacterium. We also show that flagellin is a principal stimulant of proinflammatory cytokine production in lung epithelial cells. Together, these observations suggest that TLR5 392STOP increases human susceptibility to infection through an unusual dominant mechanism that compromises TLR5's essential role as a regulator of the lung epithelial innate immune response.

Research paper thumbnail of The innate immune response to bacterial flagellin is mediated by Toll-like receptor 5

Nature, 2001

The innate immune system recognizes pathogen-associated molecular patterns (PAMPs) that are expre... more The innate immune system recognizes pathogen-associated molecular patterns (PAMPs) that are expressed on infectious agents, but not on the host. Toll-like receptors (TLRs) recognize PAMPs and mediate the production of cytokines necessary for the development of ...

Research paper thumbnail of The repertoire for pattern recognition of pathogens by the innate immune system is defined by cooperation between Toll-like receptors

Proceedings of The National Academy of Sciences, 2000

Toll-like receptors (TLRs) have been shown to participate in the recognition of pathogens by the ... more Toll-like receptors (TLRs) have been shown to participate in the recognition of pathogens by the innate immune system, but it is not clear how a restricted family of receptors has the capacity to recognize the wide spectrum of TLR stimuli known to exist. We report here that two members of the TLR family, TLR2 and TLR6, together coordinate macrophage activation by Gram-positive bacteria and the yeast cellwall particle, zymosan. TLR6 and TLR2 both are recruited to the macrophage phagosome, where they recognize peptidoglycan, a Gram-positive pathogen component. By contrast, TLR2 recognizes another component, bacterial lipopeptide, without TLR6. The requirement for TLR cooperation is supported by the finding that TLR2 needs a partner to activate tumor necrosis factor-␣ production in macrophages. Dimerization of the cytoplasmic domain of TLR2 does not induce tumor necrosis factor-␣ production in macrophages, whereas similar dimerization of the TLR4 cytoplasmic domain does. We show that the cytoplasmic domain of TLR2 can form functional pairs with TLR6 or TLR1, and this interaction leads to cytokine induction. Thus, the cytoplasmic tails of TLRs are not functionally equivalent, with certain TLRs requiring assembly into heteromeric complexes, whereas others are active as homomeric complexes. Finally, we show that TLR6, TLR2, and TLR1 are recruited to macrophage phagosomes that contain IgG-coated erythrocytes that do not display microbial components. The data suggest that TLRs sample the contents of the phagosome independent of the nature of the contents, and can establish a combinatorial repertoire to discriminate among the large number of pathogen-associated molecular patterns found in nature.

Research paper thumbnail of Lipocalin 2 mediates an innate immune response to bacterial infection by sequestrating iron

Nature, 2004

DMRT1 gene. As a probe, 10 ml of DOP-PCR amplification of paint E4 were used. For DMRT1 a 287-bas... more DMRT1 gene. As a probe, 10 ml of DOP-PCR amplification of paint E4 were used. For DMRT1 a 287-base-pair (bp) PstI digest of chicken DMRT1 complementary DNA, containing the DM (Doublesex and MAB-3) domain was hybridized. For both experiments the probe DNA was labelled radioactively with [ 32 P]-dATP and the BAC library screened according to the manufacturer's instructions (http:// www.genome.clemson.edu/protocols/hyb_filter.html). Positive clones were ordered from the Clemson University Genomics Institute (CUGI). For DMRT1, a final positive clone was verified by PCR using primers from conserved DMRT1 specific regions 29 . This clone was then sequenced after subcloning into a TOPO Shotgun subcloning kit (Invitrogen) according to the manufacturer's instructions. Sequences were deposited at EMBL. The DMRT1 BAC as well as the E4 specific BAC clones were labelled by standard nick translation with biotin or digoxigenin and 400-600 ng of labelled DNA were used to map the clones by FISH, as described above.

Research paper thumbnail of Evasion of Toll-like receptor 5 by flagellated bacteria

Proceedings of The National Academy of Sciences, 2005

Toll-like receptor 5 (TLR5) recognizes an evolutionarily conserved site on bacterial flagellin th... more Toll-like receptor 5 (TLR5) recognizes an evolutionarily conserved site on bacterial flagellin that is required for flagellar filament assembly and motility. The ␣ and Proteobacteria, including the important human pathogens Campylobacter jejuni, Helicobacter pylori, and Bartonella bacilliformis, require flagellar motility to efficiently infect mammalian hosts. In this study, we demonstrate that these bacteria make flagellin molecules that are not recognized by TLR5. We map the site responsible for TLR5 evasion to amino acids 89 -96 of the N-terminal D1 domain, which is centrally positioned within the previously defined TLR5 recognition site. Salmonella flagellin is strongly recognized by TLR5, but mutating residues 89 -96 to the corresponding H. pylori flaA sequence abolishes TLR5 recognition and also destroys bacterial motility. To preserve bacterial motility, ␣ and Proteobacteria possess compensatory amino acid changes in other regions of the flagellin molecule, and we engineer a mutant form of Salmonella flagellin that evades TLR5 but retains motility. These results suggest that TLR5 evasion is critical for the survival of this subset of bacteria at mucosal sites in animals and raise the intriguing possibility that flagellin receptors provided the selective force to drive the evolution of these unique subclasses of bacterial flagellins.

Research paper thumbnail of Toll-like receptor 5 recognizes a conserved site on flagellin required for protofilament formation and bacterial motility

Nature Immunology, 2003

Flagellated bacteria cause a broad range of serious gastrointestinal, urinary tract and respirato... more Flagellated bacteria cause a broad range of serious gastrointestinal, urinary tract and respiratory tract infections. In models of mucosal infection, the production of functional flagella is a bacterial virulence factor, which is required for colonization and tissue invasiveness and can elicit the recruitment of host inflammatory cells . Flagella also facilitate the infection of macrophages and epithelial cells 3,8-10 . The host targets flagella through antibodies, T cell recognition and the innate immune system 11-13 . Through TLR5, flagellin activates macrophages, dendritic cells, neutrophils and intestinal epithelial cells to produce inflammatory mediators . In addition to animals, plants and insects also have evolved innate immune recognition systems for bacterial flagellin . Plants recognize bacterial flagellin through FLS2, the flagellin sensitivity locus 2 gene product and an apparent functional equivalent of TLR5, and this recognition event triggers a systemic disease resistance response .