Highlights d Phosphoproteomics analysis of SARS-CoV-2-infected cells uncovers signaling rewiring d Infection promotes host p38 MAPK cascade activity and shutdown of mitotic kinases d Infection stimulates CK2-containing filopodial protrusions with budding virus d Kinase activity analysis identifies potent antiviral drugs and compounds

The kinase Raf-1 can be activated by treatment of cells with mitogens and by the protein kinase C (PKC) activator 12-O-tetradecanoyl-phorbol-13-acetate (TPA) (reviewed in refs 1,2). Activated Raf-1 triggers a protein kinase cascade by direct phosphorylation of MAP kinase kinase, resulting in phosphorylation of ternary complex factor and Jun by MAP kinase. Here we investigate the molecular mechanism and biological consequences of PKC alpha-mediated Raf-1 activation in NIH3T3 fibroblasts. PKC alpha directly phosphorylates and activates Raf-1 both in vitro and in vivo. PKC alpha induces Raf-1 phosphorylation at several sites, including a serine residue at position 499. Mutation of serine at this position or at residue 259 does not abrogate Raf-1 stimulation by a combination of Ras plus the src tyrosine kinase Lck, but severely impedes Raf-1 activation by PKC alpha. Consistent with such a direct interaction is the observation that Raf-1 and PKC alpha cooperate in the transformation of NIH3T3 cells. The Ser499 phosphorylation site is necessary for this synergism.

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The NADPH and oxygen-dependent conversion of (2 S)-naringenin to genistein catalyzed by a microsomal preparation from elicitor-treated soybean cell suspension cultures has been resolved into two steps. In the first step (2s)-naringenin is converted to a product (P-2) which yields genistein in a second step. The chemical behaviour of P-2 and its ultraviolet and mass spectral data are consistent with a 2-hydroxyisoflavanone structure. The conversion of (2 S)-naringenin to P-2 requires NADPH, oxygen and cytochrome P-450. The participation of cytochrome P-450 was demonstrated by CO inhibition of the reaction and its partial reversal by light, and by inhibition with typical cytochrome P-450 inhibitors. On a Percoll gradient the membrane fraction which catalyzes P-2 formation coincides with marker enzymes for the endoplasmic reticulum and with the position of cytochrome P-450. Enzymatic activity for conversion of P-2 to genistein is mainly present in the supernatant of the 160000 x g fraction. This reaction, formally a dehydration, does not require NADPH or oxygen.Isoflavonoids comprise a large group of natural products. A recent survey by Ingham lists 510 naturally occurring isoflavonoids [l]. Isoflavonoids can have estrogenic, insecticidal, piscicidal and antimicrobial properties [2, 31. Antimicrobial isoflavonoids can act as phytoalexins in the defence of plants against potential pathogens [4].Detailed studies with radioactively labelled precursors have proved that isoflavones originate from a chalcone/ flavanone precursor by a 2,3-migration of the B-ring [5, 61, which has recently been shown to be an intramolecular rearrangement [28]. The finding that (-) (2 S)-5,7,4-trihydroxyflavanone [(2S)-naringenin] was stereospecifically incorporated into biochanin A in Cicer arietinum seedlings demonstrated that very probably the (2s)-flavanone and not the chalcone is the substrate for the rearrangement to isoflavone [7]. It was also shown that dihydrokaempferol is not a precursor for isoflavones in Cicer seedlings [8].The enzymology of the rearrangement of flavanone to isoflavone remained unknown until recently, when we were able to demonstrate the conversion of (2 S)-naringenin to genistein with a microsomal preparation from elicitorchallenged soybean cell cultures in the presence of NADPH and oxygen [9]. We now present a more detailed report on this enzyme system.

The exact role of innate immune cells upon infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and their contribution to the formation of the corona virus-induced disease (COVID)-19 associated cytokine storm is not yet fully understood. We show that human in vitro differentiated myeloid dendritic cells (mDC) as well as M1 and M2 macrophages are susceptible to infection with SARS-CoV-2 but are not productively infected. Furthermore, infected mDC, M1-, and M2 macrophages show only slight changes in their activation status. Surprisingly, none of the infected innate immune cells produced the pro-inflammatory cytokines interleukin (IL)−6, tumor necrosis factor (TNF)-α, or interferon (IFN)−α. Moreover, even in co-infection experiments using different stimuli, as well as non-influenza (non-flu) or influenza A (flu) viruses, only very minor IL-6 production was induced. In summary, we conclude that mDC and macrophages are unlikely the source of the first wave of cytokines upon infection with SARS-CoV-2.

Four cytochrome P450-dependent enzymes, among them dihydroxypterocarpan 6a-hydroxylase (D6aH), are specifically involved in the elicitor-inducible biosynthesis of glyceollins, the phytoalexins of soybean. Here we report that CYP93A1 cDNA, which we isolated previously from elicitor-induced soybean cells, codes for a protein with D6aH activity. Analysis of the catalytic properties of recombinant CYP93A1 expressed in yeast, its NADPH dependency, stereoselectivity and high substrate affinity confirmed that D6aH is the physiological function of CYP93A1. It thus represents the first isoflavonoidspecific CYP to be characterized at the molecular level. In elicitor-treated soybean cells producing phytoalexins, increases in D6aH activity were correlated with elevated transcript levels which indicates that expression of the enzyme is regulated at the level of transcription. Therefore, CYP93A1 cDNA can be used as a specific molecular marker for the inducible defense response against pathogen attack.z 1998 Federation of European Biochemical Societies.

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Vascular endothelial growth factor (VEGF) is an angiogenic growth factor with a target‐cell specificity highly restricted to vascular endothelial cells. Recombinant baculovirus were constructed for the production of two different forms of the human VEGF protein in insect cells. VEGF165 and VEGF121 proteins produced by Sf158 cells underwent a similar processing compared with mammalian cells, including efficient glycosylation, formation of a disulfide‐linked dimer and secretion into the media. Only one of these forms, VEGF165 had a high affinity for heparin and this characteristic was used to purify this form to homogenicity by a two‐step heparin‐affinity chromatcgraphy. The biological activity of the purified 43‐kDa homodimer was demonstrated by high‐affinity binding to VEGF receptors, and by the induction of DNA synthesis in vascular endothelial cells. A positive angiogenic activity in vivo was demonstrated by the day‐13 chorioallantoic‐membrane assay. The mitogenic potency of VEGF121 for human umbilical vein endothelial cells was very similar compared to VEGF165. These results demonstrate that an angiogenic growth factor whose normal processing requires glycosylation and disulfide‐bridge formation can be efficiently expressed in high concentration (up to 5 μg/ml) in insects cells.