TY  - JOUR
AB  - Upon SARS-CoV-2 infection, patients with severe forms of COVID-19 often suffer from a dysregulated immune response and hyperinflammation. Aberrant expression of cytokines and chemokines is associated with strong activation of the immunoregulatory transcription factor NF-κB, which can be directly induced by the SARS-CoV-2 protein NSP14. Here, we use NSP14 mutants and generated cells with host factor knockouts (KO) in the NF-κB signaling pathways to characterize the molecular mechanism of NSP14-induced NF-κB activation. We demonstrate that full-length NSP14 requires methyltransferase (MTase) activity to drive NF-κB induction. NSP14 WT, but not an MTase-defective mutant, is poorly expressed and inherent post-translational instability is mediated by proteasomal degradation. Binding of SARS-CoV-2 NSP10 or addition of the co-factor S-adenosylmethionine (SAM) stabilizes NSP14 and augments its potential to activate NF-κB. Using CRISPR/Cas9-engineered KO cells, we demonstrate that NSP14 stimulation of canonical NF-κB activation relies on NF-κB factor p65/RELA downstream of the NEMO/IKK complex, while c-Rel or non-canonical RelB are not required to induce NF-κB transcriptional activity. However, NSP14 overexpression is unable to induce canonical IκB kinase β (IKKβ)/NF-κB signaling and in co-immunoprecipitation assays we do not detect stable associations between NSP14 and NEMO or p65, suggesting that NSP14 activates NF-κB indirectly through its methyltransferase activity. Taken together, our data provide a framework how NSP14 can augment basal NF-κB activation, which may enhance cytokine expression in SARS-CoV-2 infected cells.
AU  - Tofaute, M.J.
AU  - Weller, B.
AU  - Graß, C.
AU  - Halder, H.
AU  - Dohai, B.S.M.
AU  - Falter-Braun, P.
AU  - Krappmann, D.
C1  - 69046
C2  - 55194
CY  - 1st Flr, 10 Queen Street Place, London, England
TI  - SARS-CoV-2 NSP14 MTase activity is critical for inducing canonical NF-κB activation.
JO  - Biosci. Rep.
VL  - 44
IS  - 1
PB  - Portland Press Ltd
PY  - 2024
SN  - 0144-8463
ER  - 

TY  - JOUR
AB  - Protein phosphatase 5 (PP5) is an evolutionary conserved serine/threonine phosphatase. Its dephosphorylation activity modulates a diverse set of cellular factors including protein kinases and the microtubule-associated tau protein involved in neurodegenerative disorders. It is auto-regulated by its heat-shock protein (Hsp90)-interacting tetratricopeptide repeat (TPR) domain and its C-terminal α-helix. In the present study, we report the identification of five specific PP5 activators [PP5 small-molecule activators (P5SAs)] that enhance the phosphatase activity up to 8-fold. The compounds are allosteric modulators accelerating efficiently the turnover rate of PP5, but do barely affect substrate binding or the interaction between PP5 and the chaperone Hsp90. Enzymatic studies imply that the compounds bind to the phosphatase domain of PP5. For the most promising compound crystallographic comparisons of the apo PP5 and the PP5-P5SA-2 complex indicate a relaxation of the auto-inhibited state of PP5. Residual electron density and mutation analyses in PP5 suggest activator binding to a pocket in the phosphatase/TPR domain interface, which may exert regulatory functions. These compounds thus may expose regulatory mechanisms in the PP5 enzyme and serve to develop optimized activators based on these scaffolds.
AU  - Haslbeck, V.*
AU  - Drazic, A.*
AU  - Eckl, J.M.*
AU  - Alte, F.*
AU  - Helmuth, M.*
AU  - Popowicz, G.M.
AU  - Rattei, T.
AU  - Braun, F.*
AU  - Weiwad, M.*
AU  - Fischer, G.*
AU  - Gemmecker, G.*
AU  - Sattler, M.
AU  - Striggow, F.*
AU  - Groll, M.*
AU  - Richter, K.*
C1  - 46359
C2  - 37733
CY  - London
TI  - Selective activators of protein phosphatase 5 target the auto-inhibitory mechanism.
JO  - Biosci. Rep.
VL  - 35
IS  - 3
PB  - Portland Press Ltd
PY  - 2015
SN  - 0144-8463
ER  -