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1.  Expression of an Epitope-Tagged Virulence Protein in Rickettsia parkeri Using Transposon Insertion 
PLoS ONE  2012;7(5):e37310.
Despite recent advances in our ability to genetically manipulate Rickettsia, little has been done to employ genetic tools to study the expression and localization of Rickettsia virulence proteins. Using a mariner-based Himar1 transposition system, we expressed an epitope-tagged variant of the actin polymerizing protein RickA under the control of its native promoter in Rickettsia parkeri, allowing the detection of RickA using commercially-available antibodies. Native RickA and epitope-tagged RickA exhibited similar levels of expression and were specifically localized to bacteria. To further facilitate protein expression in Rickettsia, we also developed a plasmid for Rickettsia insertion and expression (pRIE), containing a variant Himar1 transposon with enhanced flexibility for gene insertion, and used it to generate R. parkeri strains expressing diverse fluorescent proteins. Expression of epitope-tagged proteins in Rickettsia will expand our ability to assess the regulation and function of important virulence factors.
doi:10.1371/journal.pone.0037310
PMCID: PMC3356282  PMID: 22624012
2.  The Dynamic Distribution of CARD11 at the Immunological Synapse is Regulated by the Inhibitory Kinesin GAKIN 
Molecular cell  2010;40(5):798-809.
SUMMARY
T cell receptor (TCR) signaling to NF-κB is required for antigen-induced T cell activation. We conducted an expression-cloning screen for modifiers of CARD11, a critical adapter in antigen-receptor signaling, and identified the kinesin-3 family member GAKIN as a CARD11 inhibitor. GAKIN negatively regulates TCR signaling to NF-κB, associates with CARD11 in a signal-dependent manner, and can compete with the required signaling protein, Bcl10, for association. In addition, GAKIN dynamically localizes to the immunological synapse and regulates the redistribution of CARD11 from the central region of the synapse to a distal region. We propose that CARD11 scaffold function and occupancy at the center of the synapse are negatively regulated by GAKIN to tune the output of antigen-receptor signaling.
doi:10.1016/j.molcel.2010.11.007
PMCID: PMC3032410  PMID: 21145487
3.  Oncogenic CARD11 Mutations Induce Hyperactive Signaling By Disrupting Autoinhibition by the PKC-Responsive Inhibitory Domain† 
Biochemistry  2010;49(38):8240-8250.
The regulated activation of NF-κB by antigen receptor signaling is required for normal B and T lymphocyte activation during the adaptive immune response. Dysregulated NF-κB activation is associated with several types of lymphoma, including Diffuse Large B Cell Lymphoma (DLBCL). During normal antigen receptor signaling, the multidomain scaffold protein CARD11 undergoes a transition from a closed, inactive state to an open, active conformation that recruits several signaling proteins into a complex, leading to IKK kinase activation. This transition is regulated by the CARD11 Inhibitory Domain (ID), which participates in intramolecular interactions that prevent cofactor binding to CARD11 prior to signaling, but which is neutralized after receptor engagement by phosphorylation. Several oncogenic CARD11 mutations have been identified in DLBCL that enhance activity and that are mostly found in the Coiled-coil domain. However, the mechanisms by which these mutations cause CARD11 hyperactivity and spontaneous NF-κB activation are poorly understood. In this report, we provide several lines of evidence that oncogenic mutations F123I and L225LI induce CARD11 hyperactivity by disrupting autoinhibition by the CARD11 ID. These mutations disrupt ID-mediated intramolecular interactions, ID-dependent inhibition, and bypass the requirement for ID phosphorylation during T cell receptor signaling. Intriguingly, these mutations selectively enhance the apparent affinity of CARD11 for Bcl10, but not for other signaling proteins that are recruited to CARD11 in an ID-dependent manner during normal antigen-receptor signaling. Our results establish a mechanism that explains how DLBCL-associated mutations in CARD11 can initiate spontaneous, receptor-independent activation of NF-κB.
doi:10.1021/bi101052d
PMCID: PMC2943563  PMID: 20799731
4.  Transcriptional target-based expression cloning of immunoregulatory molecules 
Immunologic research  2010;47(1-3):172-178.
Immunologic research has benefited tremendously from expression-cloning strategies designed to isolate genes responsible for a wide variety of immunomodulatory activities, including cytokines, receptors, signaling proteins, and transcription factors. Here, we discuss the use of expression-cloning strategies that have been modified to detect cDNAs that influence gene expression as assayed by a transcriptional reporter. We summarize our experience with these screens, review important parameters, and discuss potential modifications.
doi:10.1007/s12026-009-8148-z
PMCID: PMC2892239  PMID: 20069388
Expression-cloning; Gene discovery; NF-κB; HIV-1; cDNA library
5.  Sexual dimorphism in immune response genes as a function of puberty 
BMC Immunology  2006;7:2.
Background
Autoimmune diseases are more prevalent in females than in males, whereas males have higher mortality associated with infectious diseases. To increase our understanding of this sexual dimorphism in the immune system, we sought to identify and characterize inherent differences in immune response programs in the spleens of male and female mice before, during and after puberty.
Results
After the onset of puberty, female mice showed a higher expression of adaptive immune response genes, while males had a higher expression of innate immune genes. This result suggested a requirement for sex hormones. Using in vivo and in vitro assays in normal and mutant mouse strains, we found that reverse signaling through FasL was directly influenced by estrogen, with downstream consequences of increased CD8+ T cell-derived B cell help (via cytokines) and enhanced immunoglobulin production.
Conclusion
These results demonstrate that sexual dimorphism in innate and adaptive immune genes is dependent on puberty. This study also revealed that estrogen influences immunoglobulin levels in post-pubertal female mice via the Fas-FasL pathway.
doi:10.1186/1471-2172-7-2
PMCID: PMC1402325  PMID: 16504066

Results 1-5 (5)