Lau, A. — Page 2 | bioRxiv Science

bioRxiv2018

Structural basis of Cullin-2 RING E3 ligase regulation by the COP9 signalosome

Cullin-Ring E3 Ligases (CRLs) regulate a multitude of cellular pathways through specific substrate receptors. The COP9 signalosome (CSN) deactivates CRLs by removing NEDD8 (N8) from activated Cullins. The structure of stable CSN-CRL can be used to understand this mechanism of regulation. Here we present the first structures of the neddylated and deneddylated CSN-CRL2 complexes by combining single particle cryo-electron microscopy (cryo-EM) with chemical cross-linking mass spectrometry (MS). These structures reveal a conserved mechanism of CSN activation, consisting of conformational clamping of the CRL2 substrate by CSN2/CSN4, release of the catalytic CSN5/CSN6 heterodimer and finally activation of the CSN5 deneddylation machinery. Using hydrogen deuterium exchange-MS we show that CRL2 binding and conformational activation of CSN5/CSN6 occur in a neddylation-independent manner. The presence of NEDD8 is required to activate the CSN5 active site. Overall, by synergising cryo-EM with MS, we identified novel sensory regions of the CSN that mediate its stepwise activation mechanism and provide a framework for better understanding the regulatory mechanism of other Cullin family members.\n\nOne sentence summaryStructure and dynamics of the CSN-CRL2 complexes assessed by cryo-electron microscopy and structural mass spectrometry.

biochemistry↗

bioRxiv2018

NetrinG1/NGL-1 Axis promotes pancreatictumorigenesis through cancer associated fibroblastderived nutritional supply and immunosuppression

Pancreatic ductal adenocarcinoma (PDAC) has a poor 5-year survival rate and lacks effective therapeutics. Therefore, it is of paramount importance to identify new targets. Using multi-plex data from patient tissue, three-dimensional co-culturing in vitro assays, and orthotopic murine models, we identified Netrin G1 (NetG1) as a promoter of PDAC tumorigenesis. NetG1+ cancer-associated fibroblasts (CAFs) supported PDAC survival, through a NetG1 mediated effect on glutamate/glutamine metabolism. NetG1+ CAFs were intrinsically immunosuppressive and inhibited NK cell mediated killing of tumor cells. These pro-tumor functions were controlled by a signaling circuit downstream to NetG1, which was comprised of AKT/4E-BP1, p38/FRA1, vesicular glutamate transporter 1, and glutamine synthetase. Finally blocking NetG1 with a neutralizing antibody stunted in vivo tumorigenesis, suggesting NetG1 as potential target in PDAC. SignificancePDAC is a devastating disease lacking effective therapies. A major hallmark of PDAC is desmoplasia, characterized by the expansion of CAFs and their extracellular matrix, creating a unique microenvironment that limits blood-supplied nutrition and is highly immunosuppressive. A better understanding of the role of CAFs in PDAC may lead to the identification of new targets for therapeutic intervention. Here, we uncovered roles for NetG1 in CAFs to promote tumorigenesis. NetG1 was important for two major CAF functions: the metabolic support of PDAC cells and the intrinsic immunosuppressive capacity of CAFs. Our results helped clarify the role that CAFs play in PDAC, by defining CAF phenotypes through NetG1 expression. Moreover, we established a link between CAF driven metabolism and their intrinsic immunosuppressive capacity, and identified a signaling circuit that governs NetG1 functions. Finally, we demonstrated the therapeutic potential of inhibiting NetG1 in vivo by limiting tumorigenesis in mice with a neutralizing antibody, illustrating that targeting stromal NetG1 could be an attractive therapeutic approach.

cancer biology↗