Brooke Liang
Summer research project abstract
Evaluation of the leukocyte chemoattractant receptor CMKLR1 on T cells
Liang B; Crowder RJ; Pachynski RK
Introduction: Chemokine-like receptor 1 (CMKLR1; ChemR23) is the G-protein coupled receptor (GPCR) for the novel leukocyte chemoattractant protein, chemerin, that mediates chemotaxis. CMKLR1 expression is found to be expressed on innate immune cell subsets but has not been shown on T cells. Here, we explored whether CMKLR1 can be induced on T cells using factors abundant in the tumor microenvironment.
Methods: T cells were cultured in tumor-conditioned media, or with the cytokines IL-1, IL-10, TGF-b, TNF-a, or VEGF. In addition, T cells isolated from tumor-bearing mice were examined. Quantitative real-time PCR (qPCR) and flow cytometry were used to evaluate expression of CMKLR1 on T cells.
Results: We preliminarily show the upregulation of CMKLR1 on T cells in an NK-depleted splenocyte culture exposed to VEGF. Interestingly, NK-containing splenocyte culture identically exposed to VEGF did not show this upregulation, suggesting a role for NK cells in the regulation of CMKLR1 on T cells.
Conclusions: It would be clinically significant if CMKLR1 could be expressed on T cells, as CMKLR1-expressing T cells in conjunction with therapeutics that activate T cells could be the basis for novel cancer treatments.
Angus Toland
Summer research project abstract
A conserved amino acid motif in Lia3 is required for binding to G Quadruplex DNA
Toland AM; Chalker DL
Introduction: Tetrahymena thermophila, a free-living ciliate, is a model organism used to study the regulation of eukaryotic genomes. Ciliates use a germline micronucleus as a template to generate a somatic, polyploid, junk DNA-free macronucleus. A recently discovered protein, Lia3, has been shown to be important in targeting the elimination of DNA flanked by 5’-A5-G5-3’ sequences. These 5’-A5-G5-3’ sequences have the capacity to form G quadruplex structures in vitro and have been shown to be bound by telomerase in vivo. The goal of this project was to produce mutations in amino acid sequences within a region of ~100 amino acids conserved amongst Lia3 and functional homologs (Lia3-like or LTL proteins) also expressed in T. thermophila and observe effects on G quadruplex binding.
Methods: DNA oligomers containing mutant sequences coding for GRG310-312AAA or IYI315-317AYA were used in PCR to produce two mutant strains of Lia3 fused to maltose binding protein (Lia3-MBP) in a pMAL plasmid expressed in 10G strain Escherichia coli. The mutant proteins were then expressed in BL21 strain E. coli and induced with isopropyl β-D-1-thiogalactopyranoside. The Lia3-MBP was then isolated from induced culture via maltose elution. Purified protein was then incubated with radiolabeled G quadruplex DNA and separated by electrophoresis through a 4.5% polyacrylamide gel.
Results: The IYI315-317AYA mutant demonstrated successful G quadruplex DNA binding while the GRG310-312AAA mutant did not, indicating the amino acid sequence of the former mutation does not disrupt binding while that of the latter does. This data indicates that some, but not all, of the highly-conserved amino acids of Lia3 are critical for binding of G quadruplex DNA.
Conclusions: During this study, we demonstrated that some of the highly-conserved amino acid sequences of Lia3 are critical for G quadruplex binding. Because these sequences are found in the LTL family, which do not bind G-quadruplex DNA, it is not likely that these sequences are directly responsible for G quadruplex binding, but rather play a role in accessory binding or functional Lia3 and LTL formation (e.g. protein folding, dimerization, etc.). Additional studies will address these questions as well as determine the effect of these mutations on in vivo Lia3 function.