The scaffold RhoGAP protein ARHGAP8/BPGAP1 synchronizes Rac and Rho signaling to facilitate cell migration.

Rho GTPases regulate cell morphogenesis and motility under the tight control of guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). However, the underlying mechanism(s) that coordinate their spatiotemporal activities, whether separately or together, remain unclear. We show that a prometastatic RhoGAP, ARHGAP8/BPGAP1, binds to inactive Rac1 and localizes to lamellipodia. BPGAP1 recruits the RacGEF Vav1 under epidermal growth factor (EGF) stimulation and activates Rac1, leading to polarized cell motility, spreading, invadopodium formation, and cell extravasation and promotes cancer cell migration. Importantly, BPGAP1 down-regulates local RhoA activity, which influences Rac1 binding to BPGAP1 and its subsequent activation by Vav1. Our results highlight the importance of BPGAP1 in recruiting Vav1 and Rac1 to promote Rac1 activation for cell motility. BPGAP1 also serves to control the timing of Rac1 activation with RhoA inactivation via its RhoGAP activity. BPGAP1, therefore, acts as a dual-function scaffold that recruits Vav1 to activate Rac1 while inactivating RhoA to synchronize both Rho and Rac signaling in cell motility. As epidermal growth factor receptor (EGFR), Vav1, RhoA, Rac1, and BPGAP1 are all associated with cancer metastasis, BPGAP1 could provide a crucial checkpoint for the EGFR-BPGAP1-Vav1-Rac1-RhoA signaling axis for cancer intervention.

Structural basis for p50RhoGAP BCH domain-mediated regulation of Rho inactivation.

Spatiotemporal regulation of signaling cascades is crucial for various biological pathways, under the control of a range of scaffolding proteins. The BNIP-2 and Cdc42GAP Homology (BCH) domain is a highly conserved module that targets small GTPases and their regulators. Proteins bearing BCH domains are key for driving cell elongation, retraction, membrane protrusion, and other aspects of active morphogenesis during cell migration, myoblast differentiation, and neuritogenesis. We previously showed that the BCH domain of p50RhoGAP (ARHGAP1) sequesters RhoA from inactivation by its adjacent GAP domain; however, the underlying molecular mechanism for RhoA inactivation by p50RhoGAP remains unknown. Here, we report the crystal structure of the BCH domain of p50RhoGAP Schizosaccharomyces pombe and model the human p50RhoGAP BCH domain to understand its regulatory function using in vitro and cell line studies. We show that the BCH domain adopts an intertwined dimeric structure with asymmetric monomers and harbors a unique RhoA-binding loop and a lipid-binding pocket that anchors prenylated RhoA. Interestingly, the ß5-strand of the BCH domain is involved in an intermolecular ß-sheet, which is crucial for inhibition of the adjacent GAP domain. A destabilizing mutation in the ß5-strand triggers the release of the GAP domain from autoinhibition. This renders p50RhoGAP active, thereby leading to RhoA inactivation and increased self-association of p50RhoGAP molecules via their BCH domains. Our results offer key insight into the concerted spatiotemporal regulation of Rho activity by BCH domain-containing proteins.

Structural Basis of SARS-CoV-2- and SARS-CoV-Receptor Binding and Small-Molecule Blockers as Potential Therapeutics.

Over the past two decades, deadly coronaviruses, with the most recent being the severe acute respiratory syndrome-related coronavirus-2 (SARS-CoV-2) 2019 pandemic, have majorly challenged public health. The path for virus invasion into humans and other hosts is mediated by host-pathogen interactions, specifically virus-receptor binding. An in-depth understanding of the virus-receptor binding mechanism is a prerequisite for the discovery of vaccines, antibodies, and small-molecule inhibitors that can interrupt this interaction and prevent or cure infection. In this review, we discuss the viral entry mechanism, the known structural aspects of virus-receptor interactions (SARS-CoV-2 S/humanACE2, SARS-CoV S/humanACE2, and MERS-CoV S/humanDPP4), the key protein domains and amino acid residues involved in binding, and the small-molecule inhibitors and other drugs that have (as of June 2020) exhibited therapeutic potential. Specifically, we review the potential clinical utility of two transmembrane serine protease 2 (TMPRSS2)-targeting protease inhibitors, nafamostat mesylate and camostat mesylate, as well as two novel potent fusion inhibitors and the repurposed Ebola drug, remdesivir, which is specific to RNA-dependent RNA polymerase, against human coronaviruses, including SARS-CoV-2.

Structural Basis of SARS-CoV-2 and SARS-CoV Antibody Interactions.

The 2019 coronavirus pandemic remains a major public health concern. Neutralizing antibodies (nAbs) represent a cutting-edge antiviral strategy. We focus here on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and SARS-CoV, and discuss current progress in antibody research against rampant SARS-CoV-2 infections. We provide a perspective on the mechanisms of SARS-CoV-2-derived nAbs, comparing these with existing SARS-CoV-derived antibodies. We offer insight into how these antibodies cross-react and cross-neutralize by analyzing available structures of spike (S) glycoprotein-antibody complexes. We also propose ways of adopting antibody-based strategies - such as cocktail antibody therapeutics against SARS-CoV-2 - to overcome the possible resistance of currently identified mutants and mitigate possible antibody-dependent enhancement (ADE) pathologies. This review provides a platform for the progression of antibody and vaccine design against SARS-CoV-2, and possibly against future coronavirus pandemics.

Isthmin is a novel vascular permeability inducer that functions through cell-surface GRP78-mediated Src activation.

AIMS: Isthmin (ISM) is a recently identified 60 kDa secreted angiogenesis inhibitor. Two cell-surface receptors for ISM have been defined, the high-affinity glucose-regulated protein 78 kDa (GRP78) and the low-affinity αvß5 integrin. As αvß5 integrin plays an important role in pulmonary vascular permeability (VP) and ISM is highly expressed in mouse lung, we sought to clarify the role of ISM in VP. METHODS AND RESULTS: Recombinant ISM (rISM) dose-dependently enhances endothelial monolayer permeability in vitro and local dermal VP when administered intradermally in mice. Systemic rISM administration through intravenous injection leads to profound lung vascular hyperpermeability but not in other organs. Mechanistic investigations using molecular, biochemical approaches and specific chemical inhibitors revealed that ISM-GRP78 interaction triggers a direct interaction between GRP78 and Src, leading to Src activation and subsequent phosphorylation of adherens junction proteins and loss of junctional proteins from inter-endothelial junctions, resulting in enhanced VP. Dynamic studies of Src activation, VP and apoptosis revealed that ISM induces VP directly via Src activation while apoptosis contributes indirectly only after prolonged treatment. Furthermore, ISM is significantly up-regulated in lipopolysaccharide (LPS)-treated mouse lung. Blocking cell-surface GRP78 by systemic infusion of anti-GRP78 antibody significantly attenuates pulmonary vascular hyperpermeability in LPS-induced acute lung injury (ALI) in mice. CONCLUSION: ISM is a novel VP inducer that functions through cell-surface GRP78-mediated Src activation as well as induction of apoptosis. It induces a direct GRP78-Src interaction, leading to cytoplasmic Src activation. ISM contributes to pulmonary vascular hyperpermeability of LPS-induced ALI in mice.