Project description:Primary outcome(s): Measure auto-lysosome function in the cytoplasm by electron microscopy to examine the changes in the number of autophagy.
Project description:The accumulation of DNA in the cytosol serves as a key immunostimulatory signal associated with infections, cancer and genomic damage. Cytosolic DNA triggers immune responses by activating the cGAS/STING pathway. The binding of DNA to the cytosolic enzyme cGAMP synthase (cGAS), activates its enzymatic activity, leading to the synthesis of a second messenger, cyclic[G(2’,5’)pA(3’,5’)] (2’3’-cGAMP). 2’3’-cGAMP, a cyclic dinucleotide (CDN), activates the protein ‘stimulator of interferon genes’ (STING), which in turn activates the transcription factors IRF3 and NF-κB promoting the transcription of genes encoding type I interferons and other cytokines and mediators that stimulate a broader immune response. Exogenous 2’3’-cGAMP produced by malignant cells and other CDNs, including CDNs produced by bacteria and synthetic CDNs used in cancer immunotherapy, must traverse the cell membrane to activate STING in target cells. How these charged CDNs pass through the lipid bilayer is unknown. Here we used a genome-wide CRISPR interference screen to identify the reduced folate carrier SLC19A1, a folate-organic phosphate antiporter, as the major transporter for CDNs. CDN uptake and functional responses are inhibited by depleting SLC19A1 from human cells and enhanced by overexpressing SLC19A1. In both human cell lines and primary cells ex vivo, CDN uptake is inhibited by folates, as well as two medications approved for treatment of inflammatory diseases, sulfasalazine and the antifolate methotrexate. The identification of SLC19A1 as the major transporter of CDNs into cells has implications for the immunotherapeutic treatment of cancer, host responsiveness to CDN-producing pathogenic microorganisms, and potentially in certain inflammatory diseases.
Project description:We retrospectively investigated sera, obtained from 47 patients after the complete cure of liver cancer, using follow-up proteome analysis for 8 years, and found a protein, whose expression increased over time, peaked at the definitive diagnosis of bone metastases by bone scintigraphy, and tapered after the start of subsequent treatment. Fractions obtained by the reverse-phase HPLC of this protein were digested with trypsin and examined by LC-MS/MS analysis. The data were analyzed with a Mascot database search. As a result, the mass of fragment 576-628 of the C-terminal region of alpha-fibrinogen precursor was 5,805, which corresponded to that of the target peak. On the other hand, the mass of region 576-629 (with Val at the C-terminal) was 5,904, a mass value 99 higher than the target peak. This corresponds to a peak detected in the serum sample at a mass value 100 higher than the target peak. This peak with a mass value 100 higher was the same as the target peak in terms of chromatographic behavior, indicating that this peak had a protein sequence similar to that of the target peak. The isoelectric point (pI) of the fragment of region 576-628 was 8.07. This is consistent with the fact that the target peak indicates the behavior of a basic material in ion exchange. Thus, we concluded that the target peak 5,813 was a fragment of C-terminal region 576-628 of the alpha-fibrinogen precursor.
