α2 Integrin-Dependent Suppression of Pancreatic Adenocarcinoma Cell Invasion Involves Ectodomain Regulation of Kallikrein-Related Peptidase-5

Previous reports demonstrate that the α2-integrin (α2) mediates pancreatic ductal adenocarcinoma (PDAC) cell interactions with collagens. We found that while well-differentiated cells use α2 exclusively to adhere and migrate on collagenI, poorly differentiated PDAC cells demonstrate reduced reliance on, or complete loss of, α2. Since well-differentiated PDAC lines exhibit reduced in vitro invasion and α2-blockade suppressed invasion of well-differentiated lines exclusively, we hypothesized that α2 may suppress the malignant phenotype in PDAC. Accordingly, ectopic expression of α2 retarded in vitro invasion and maintenance on collagenI exacerbated this effect. Affymetrix profiling revealed that kallikrein-related peptidase-5 (KLK5) was specifically upregulated by α2, and reduced α2 and KLK5 expression was observed in poorly differentiated PDAC cells in situ. Accordingly, well-differentiated PDAC lines express KLK5, and KLK5 blockade increased the invasion of KLK5-positive lines. The α2-cytoplasmic domain was dispensable for these effects, demonstrating that the α2-ectodomain and KLK5 coordinately regulate a less invasive phenotype in PDAC

Modification of the L1-CAM carboxy-terminus in pancreatic adenocarcinoma cells

The neural cell adhesion molecule L1 has recently been shown to be expressed in pancreatic adenocarcinoma (PDAC) cells. In this report, we demonstrate that L1 is expressed by moderately- to poorly-differentiated PDAC cells in situ, and that L1 expression is a predictor of poor patient survival. In vitro, reduced reactivity of an anti-L1 carboxy-terminus-specific antibody was observed in the more poorly differentiated fast-growing (FG) variant of the COLO357 population, versus its well-differentiated slow-growing (SG) counterpart, even though they express equivalent total L1. The carboxy-terminus of L1 mediates binding to the MAP kinase-regulating protein RanBPM and mutation of T1247/S1248 within this region attenuates the expression of malignancy associated proteins and L1-induced tumorigenicity in mice. Therefore, we reasoned that the differential epitope exposure observed might be indicative of modifications responsible for regulating these events. However, epitope mapping demonstrated that the major determinant of binding was actually N1251; mutation of T1247 and S1248, alone or together, had little effect on C20 binding. Moreover, cluster assays using CD25 ectodomain/L1 cytoplasmic domain chimeras demonstrated the N1251-dependent, RanBPM-independent stimulation of erk phosphorylation in these cells. Reactivity of this antibody also reflects the differential exposure of extracellular epitopes in these COLO357 sublines, consistent with the previous demonstration of L1 ectodomain conformation modulation by intracellular modifications. These data further support a central role for L1 in PDAC, and define a specific role for carboxy-terminal residues including N1251 in the regulation of L1 activity in PDAC cells

Architecture and self-assembly of the SARS-CoV-2 nucleocapsid protein.

The COVID-2019 pandemic is the most severe acute public health threat of the twenty-first century. To properly address this crisis with both robust testing and novel treatments, we require a deep understanding of the life cycle of the causative agent, the SARS-CoV-2 coronavirus. Here, we examine the architecture and self-assembly properties of the SARS-CoV-2 nucleocapsid (N) protein, which binds viral RNA and assembles into a filament that is packaged into new virions. We determined a 1.4 Å resolution crystal structure of this protein's N2b domain, revealing a compact, intertwined dimer very similar to that of related coronaviruses SARS-CoV and MERS-CoV. Using size exclusion chromatography and multi-angle light scattering, we find that this domain forms a dimer in solution, and that addition of the C-terminal spacer B/N3 domain mediates tetramer formation. Using hydrogen-deuterium exchange mass spectrometry, we find evidence that at least part of this putatively disordered domain is structured, potentially forming an α-helix that either self-associates or docks against the N2b domain to mediate tetramer formation. Finally, we map the locations of over 4,400 individual amino acid substitutions in the N protein from ~17,000 SARS-CoV-2 genome sequences, and find that they are strongly clustered in the protein's N2a linker domain. The nearly 300 substitutions identified within the N1b and N2b domains cluster away from their functional RNA binding and dimerization interfaces. Overall, this work reveals the architecture and self-assembly properties of a key protein in the SARS-CoV-2 life cycle. As the N protein is a common target of patient antibodies, this work will also benefit ongoing efforts to develop robust and specific serological tests, and could also benefit the analysis of patient-derived antibodies

Architecture and self-assembly of the SARS-CoV-2 nucleocapsid protein.

The COVID-2019 pandemic is the most severe acute public health threat of the twenty-first century. To properly address this crisis with both robust testing and novel treatments, we require a deep understanding of the life cycle of the causative agent, the SARS-CoV-2 coronavirus. Here, we examine the architecture and self-assembly properties of the SARS-CoV-2 nucleocapsid protein, which packages viral RNA into new virions. We determined a 1.4 Å resolution crystal structure of this protein's N2b domain, revealing a compact, intertwined dimer similar to that of related coronaviruses including SARS-CoV. While the N2b domain forms a dimer in solution, addition of the C-terminal spacer B/N3 domain mediates formation of a homotetramer. Using hydrogen-deuterium exchange mass spectrometry, we find evidence that at least part of this putatively disordered domain is structured, potentially forming an α-helix that self-associates and cooperates with the N2b domain to mediate tetramer formation. Finally, we map the locations of amino acid substitutions in the N protein from over 38,000 SARS-CoV-2 genome sequences. We find that these substitutions are strongly clustered in the protein's N2a linker domain, and that substitutions within the N1b and N2b domains cluster away from their functional RNA binding and dimerization interfaces. Overall, this work reveals the architecture and self-assembly properties of a key protein in the SARS-CoV-2 life cycle, with implications for both drug design and antibody-based testing