A Fluorogenic, Small Molecule Reporter for Mammalian Phospholipase C Isozymes

Phospholipase C isozymes (PLCs) catalyze the conversion of the membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP2) into two second messengers, inositol 1,4,5-trisphosphate and diacylglycerol. This family of enzymes are key signaling proteins that regulate the physiological responses of many extracellular stimuli such as hormones, neurotransmitters, and growth factors. Aberrant regulation of PLCs has been implicated in various diseases including cancer and Alzheimer’s disease. How, when, and where PLCs are activated under different cellular contexts are still largely unknown. We have developed a fluorogenic PLC reporter, WH-15, that can be cleaved in a cascade reaction to generate fluorescent 6-aminoquinoline. When applied in enzymatic assays with either pure PLCs or cell lysates, this reporter displays more than a 20-fold fluorescence enhancement in response to PLC activity. Under assay conditions, WH-15 has comparable Km and Vmax with the endogenous PIP2. This novel reporter will likely find broad applications that vary from imaging PLC activity in live cells to high throughput screening of PLC inhibitors

Dual Activation of Phospholipase C-ϵ by Rho and Ras GTPases

Phospholipase C-ε (PLC-ε) is a highly elaborated PLC required for a diverse set of signaling pathways. Here we use a combination of cellular assays and studies with purified proteins to show that activated RhoA and Ras isoforms directly engage distinct regions of PLC-ε to stimulate its phospholipase activity. Purified PLC-ε was activated in a guanine nucleotide- and concentration-dependent fashion by purified lipidated K-Ras reconstituted in PtdIns(4,5)P2-containing phospholipid vesicles. Whereas mutation of two critical lysine residues within the second Ras-association domain of PLC-ε prevented K-Ras-dependent activation of the purified enzyme, guanine nucleotide-dependent activation by RhoA was retained. Deletion of a loop unique to PLC-ε eliminated its activation by RhoA but not H-Ras. In contrast, removal of the autoinhibitory X/Y-linker region of the catalytic core of PLC-ε markedly activates the enzyme (Hicks, S. N., Jezyk, M. R., Gershburg, S., Seifert, J. P., Harden, T. K., and Sondek, J. (2008) Mol. Cell, 31, 383–394), but PLC-ε lacking this regulatory region retained activation by both Rho and Ras GTPases. Additive activation of PLC-ε by RhoA and K- or H-Ras was observed in intact cell studies, and this additivity was recapitulated in experiments in which activation of purified PLC-ε was quantified with PtdIns(4,5)P2-containing phospholipid vesicles reconstituted with purified, isoprenylated GTPases. A maximally effective concentration of activated RhoA also increased the sensitivity of purified PLC-ε to activation by K-Ras. These results indicate that PLC-ε can be directly and concomitantly activated by both RhoA and individual Ras GTPases resulting in diverse upstream control of signaling cascades downstream of PLC-ε

General and Versatile Autoinhibition of PLC Isozymes

Phospholipase C (PLC) isozymes are directly activated by heterotrimeric G proteins and Ras-like GTPases to hydrolyze phosphatidylinositol 4,5-bisphosphate into the second messengers diacylglycerol and inositol 1,4,5-trisphosphate. Although PLCs play central roles in myriad signaling cascades, the molecular details of their activation remain poorly understood. As described here, the crystal structure of PLC-β2 illustrates occlusion of the active site by a loop separating the two halves of the catalytic TIM barrel. Removal of this insertion constitutively activates PLC-β2 without ablating its capacity to be further stimulated by classical G protein modulators. Similar regulation occurs in other PLC members, and a general mechanism of interfacial activation at membranes is presented that provides a unifying framework for PLC activation by diverse stimuli

Prediction of Protein–Protein Interfaces on G-Protein β Subunits Reveals a Novel Phospholipase C β2 Binding Domain

Gβ subunits from heterotrimeric G-proteins directly bind diverse proteins, including effectors and regulators, to modulate a wide array of signaling cascades. These numerous interactions constrained the evolution of the molecular surface of Gβ. Though mammals contain five Gβ genes comprising two classes (Gβ1-like and Gβ5-like), plants and fungi have a single ortholog and organisms such as Caenorhabditis elegans and Drosophila melanogaster contain one copy from each class. A limited number of crystal structures of complexes containing Gβ subunits and complementary biochemical data highlight specific sites within Gβs needed for protein interactions. It is difficult to determine from these interaction sites what, if any, additional regions of the Gβ molecular surface comprise interaction interfaces essential to Gβ's role as a nexus in numerous signaling cascades. We used a comparative evolutionary approach to identify five known and eight previously-unknown putative interfaces on the surface of Gβ. We show that one such novel interface occurs between Gβ and phospholipase C β2 (PLC-β2), a mammalian Gβ interacting protein. Substitutions of residues within this Gβ-PLC-β2 interface reduce the activation of PLC-β2 by Gβ1, confirming that our de novo comparative evolutionary approach predicts previously unknown Gβ-protein interfaces. Similarly, we hypothesize the seven remaining untested novel regions contribute to putative interfaces for other Gβ interacting proteins. Finally, this comparative evolutionary approach is suitable for application to any protein involved in a significant number of protein-protein interactions

Human cardiac pericytes are susceptible to SARS-CoV-2 infection

COVID-19 is associated with serious cardiovascular complications, with incompletely understood mechanism(s). Pericytes have key functions in supporting endothelial cells and maintaining vascular integrity. We demonstrate that human cardiac pericytes are permissive to SARS-CoV-2 infection in organotypic slice and primary cell cultures. Viral entry into pericytes is mediated by endosomal proteases, and infection leads to up-regulation of inflammatory markers, vasoactive mediators, and nuclear factor kappa-B-dependent cell death. Furthermore, we present evidence of cardiac pericyte infection in COVID-19 myocarditis patients. These data demonstrate that human cardiac pericytes are susceptible to SARS-CoV-2 infection and suggest a role for pericyte infection in COVID-19

Noble gases confirm plume related mantle degassing beneath Southern Africa

Southern Africa is characterised by unusually elevated topography and abnormal heat flow. This can be explained by thermal perturbation of the mantle, but the origin of this is unclear. Geophysics has not detected a thermal anomaly in the upper mantle and there is no geochemical evidence of an asthenosphere mantle contribution to the Cenozoic volcanic record of the region. Here we show that natural CO2 seeps along the Ntlakwe-Bongwan fault within KwaZulu-Natal, South Africa, have C-He isotope systematics that support an origin from degassing mantle melts. Neon isotopes indicate that the melts originate from a deep mantle source that is similar to the mantle plume beneath Réunion, rather than the convecting upper mantle or sub-continental lithosphere. This confirms the existence of the Quathlamba mantle plume and importantly provides the first evidence in support of upwelling deep mantle beneath Southern Africa, helping to explain the regions elevation and abnormal heat flow

Acireductone Dioxygenase 1 (ARD1) Is an Effector of the Heterotrimeric G Protein Subunit in Arabidopsis

Heterotrimeric G protein complexes are conserved from plants to mammals, but the complexity of each system varies. Arabidopsis thaliana contains one Gα, one Gβ (AGB1), and at least three Gγ subunits, allowing it to form three versions of the heterotrimer. This plant model is ideal for genetic studies because mammalian systems contain hundreds of unique heterotrimers. The activation of these complexes promotes interactions between both the Gα subunit and the Gβγ dimer with enzymes and scaffolds to propagate signaling to the cytoplasm. However, although effectors of Gα and Gβ are known in mammals, no Gβ effectors were previously known in plants. Toward identifying AGB1 effectors, we genetically screened for dominant mutations that suppress Gβ-null mutant (agb1-2) phenotypes. We found that overexpression of acireductone dioxygenase 1 (ARD1) suppresses the 2-day-old etiolated phenotype of agb1-2. ARD1 is homologous to prokaryotic and eukaryotic ARD proteins; one function of ARDs is to operate in the methionine salvage pathway. We show here that ARD1 is an active metalloenzyme, and AGB1 and ARD1 both control embryonic hypocotyl length by modulating cell division; they also may contribute to the production of ethylene, a product of the methionine salvage pathway. ARD1 physically interacts with AGB1, and ARD enzymatic activity is stimulated by AGB1 in vitro. The binding interface on AGB1 was deduced using a comparative evolutionary approach and tested using recombinant AGB1 mutants. A possible mechanism for AGB1 activation of ARD1 activity was tested using directed mutations in a loop near the substrate-binding site