The regulation of transmembrane metalloproteinases and their functions

Wei, ShuoMembers 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.University of Delaware, Department of Biological SciencesM.S

Whole-genome sequencing identifies I-SceI-mediated transgene integration sites in <i>Xenopus tropicalis snai2:eGFP</i> line

AbstractTransgenesis 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 tropicalissnai2snai2:eGFPXenopus tropicali

b). &amp;quot;Precise Marine DGPS Positioning Using P code and High Performance C/A Code Technologies

Geomatics Engineering where they are responsible for teaching and research related to positioning, navigation and hydrography. They have been involved with GPS development and applications since the early 80s and are the authors of numerous related papers. Messrs. Gang Lu, Bryan Townsend and Congyu Liu are research associates and MSc candidate, respectively, in the same Department. They are working in the area of GPS attitude determination, Loran-C/GPS integration, and GPS kinematic positioning, respectively. Mr. Lu completed his M.Sc. at The University of Calgary in 1991. Mr. Rob Hare is an hydrographer with the Canadian Hydrographic Service (Pacific Region) where he is involved with special projects such as GPS applications in hydrography. He holds a BSc i

Cdk5 inhibitory peptide (CIP) inhibits Cdk5/p25 activity induced by high glucose in pancreatic beta cells and recovers insulin secretion from p25 damage.

Cdk5/p25 hyperactivity has been demonstrated to lead to neuron apoptosis and degenerations. Chronic exposure to high glucose (HG) results in hyperactivity of Cdk5 and reduced insulin secretion. Here, we set out to determine whether abnormal upregulation of Cdk5/p25 activity may be induced in a pancreatic beta cell line, Min6 cells. We first confirmed that p25 were induced in overexpressed p35 cells treated with HG and increased time course dependence. Next, we showed that no p25 was detected under short time HG stimulation (4-12 hrs), however was detectable in the long exposure in HG cells (24 hrs and 48 hrs). Cdk5 activity in the above cells was much higher than low glucose treated cells and resulted in more than 50% inhibition of insulin secretion. We confirmed these results by overexpression of p25 in Min6 cells. As in cortical neurons, CIP, a small peptide, inhibited Cdk5/p25 activity and restored insulin secretion. The same results were detected in co-infection of dominant negative Cdk5 (DNCdk5) with p25. CIP also reduced beta cells apoptosis induced by Cdk5/p25. These studies indicate that Cdk5/p25 hyperactivation deregulates insulin secretion and induces cell death in pancreatic beta cells and suggests that CIP may serve as a therapeutic agent for type 2 diabetes

Diphthamide deficiency promotes association of eEF2 with p53 to induce p21 expression and neural crest defects

Abstract Diphthamide is a modified histidine residue unique for eukaryotic translation elongation factor 2 (eEF2), a key ribosomal protein. Loss of this evolutionarily conserved modification causes developmental defects through unknown mechanisms. In a patient with compound heterozygous mutations in Diphthamide Biosynthesis 1 (DPH1) and impaired eEF2 diphthamide modification, we observe multiple defects in neural crest (NC)-derived tissues. Knockin mice harboring the patient’s mutations and Xenopus embryos with Dph1 depleted also display NC defects, which can be attributed to reduced proliferation in the neuroepithelium. DPH1 depletion facilitates dissociation of eEF2 from ribosomes and association with p53 to promote transcription of the cell cycle inhibitor p21, resulting in inhibited proliferation. Knockout of one p21 allele rescues the NC phenotypes in the knockin mice carrying the patient’s mutations. These findings uncover an unexpected role for eEF2 as a transcriptional coactivator for p53 to induce p21 expression and NC defects, which is regulated by diphthamide modification

CIP restores insulin secretion after inhibition by elevated Cdk5 activity.

<p>Min 6 cells were transiently infected with EV, CIP, p25, p25/CIP, and p25/DNcdk5 constructs respectively. After 48 hours infection, cells were washed and starved for 2 hrs. Then, cells were stimulated by 3 mM and 25 mM glucose respectively for 2 hrs. Insulin secreted into the supernatant was measured using LINCO, ELISA Kit. CIP increased the insulin secretion after inhibition by p25 (compared p25/CIP to p25), (* P<0.01; ** P<0.05). Data represent mean ± SE of three experiments.</p

CIP can rescued high glucose treatment induced apoptosis in overexpressed p35 Min6 cells.

<p>A Cells infected with p35 were apoptotic at both 25 mM and 50 mM glucose concentrations (lanes 5 and 6). Cells with co-transfected CIP however, showed a reduced level of apoptosis (lane 7), and the same result can be see in the cells treated with roscovitine (lane 8). The control cells at HG (50 mM) showed a strong cleaved caspase 3 band which indicated that very high glucose is indeed cytotoxic. Interestingly, there is no apoptosis markers in the cells infected with p35 and treated with 3 mM glucose (lane 4). B The apoptosis data quantified from A, (* P<0.01). Data represent mean ± SE of three experiments.</p