Browsing by Author "Lu, Congyu"
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Item The regulation of transmembrane metalloproteinases and their functions(University of Delaware, 2018) Lu, CongyuMembers of the metzincin superfamily of metalloproteinases, including the disintegrin and metalloproteinases (ADAMs) and the matrix metalloproteinases (MMPs), have long been associated with diseases such as cancer and arthritis. Among all the metzincin metalloproteinases, transmembrane metzincins such as transmembrane ADAMs and transmembrane MMPs, have emerged as new targets for cancer therapy. ☐ Previous research in our lab suggests that ADAM9, which is highly expressed in many types of solid tumors, promotes colorectal cancer (CRC) progression. To understand the mechanism of action for ADAM9, I carried out RNA-seq in HCT116 human CRC cells with ADAM9 knockdown. A total of 351 differentially expressed genes (DEGs), including 93 up-regulated genes and 258 down-regulated genes, were identified. My enrichment analysis revealed that MAPK signaling pathway, p53 signaling pathway and FoxO signaling pathway, are significantly enriched. These pathways may mediate the function of ADAM9 in CRC progression. ☐ My bioinformatics analysis predicts the C-terminal lysine residue in the cytoplasmic tail of several ADAMs may be ubiquitinated to target these ADAMs for proteasome-mediated degradation. One of these ADAMs, ADAM12, has been implicated in the pathology of malignant tumors, such as breast cancer. To test if the C-terminal K903 residue is important for ADAM12 turnover, I generated the K903R mutant, as well as a mutant with the whole cytoplasmic tail deleted. I found that the levels of both mutants are elevated as compared with wild-type ADAM12. My findings indicate that the most C-terminal lysine residue of ADAM12 is likely involved in its degradation. ☐ The third part of this research concerns a cross-regulation of transmembrane metalloproteinases by cysteine proteases. We found that treatment of both normal and neuroblastoma cells with K777, a cysteine protease inhibitor, leads to post-transcriptional accumulation of MMP14. Surprisingly, MMP14 substrates also accumulate in the cells, suggesting that the activity of this transmembrane MMP is actually inhibited. We therefore hypothesize that the accumulation of MMP14 upon K777 treatment is due to reduced autocleavage. Consistent with this hypothesis, a protease-dead mutant of MMP14 does not accumulate after K777 treatment. These results unveil a possible crosstalk between cysteine proteases and transmembrane metalloproteinases, which warrants further investigation.Item Whole-genome sequencing identifies I-SceI-mediated transgene integration sites in Xenopus tropicalis snai2: eGFP line(G3: Genes | Genomes | Genetics, 2022-02-16) Wang, Jian; Lu, Congyu; Wei, ShuoTransgenesis with the meganuclease I-SceI is a safe and efficient method, but the underlying mechanisms remain unclear due to the lack of information on transgene localization. Using I-SceI, we previously developed a transgenic Xenopus tropicalis line expressing enhanced green fluorescent protein driven by the neural crest-specific snai2 promoter/enhancer, which is a powerful tool for studying neural crest development and craniofacial morphogenesis. Here we carried out whole-genome shotgun sequencing for the snai2: eGFP embryos to identify the transgene integration sites. With a 19x sequencing coverage, we estimated that 6 copies of the transgene were inserted into the X. tropicalis genome in the hemizygous transgenic embryos. Two transgene integration loci adjacent to each other were identified in a non-coding region on Chromosome 1, possibly as a result of duplication after a single transgene insertion. Interestingly, genomic DNA at the boundaries of the transgene integration loci contains short sequences homologous to the I-SceI recognition site, suggesting that the integration was not random but probably mediated by sequence homology. To our knowledge, our work represents the first genome-wide sequencing study on a transgenic organism generated with I-SceI, which is useful for evaluating the potential genetic effects of I-SceI-mediated transgenesis and further understanding the mechanisms underlying this transgenic method.