Methods to Monitor Ras Activation State

Kopra, Kari; Härmä, Harri

doi:10.1007/978-1-0716-1190-6_8

Cited by 3 publications

(1 citation statement)

References 23 publications

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“…Cell samples treated as above were washed twice with ice-cold PBS and harvested using centrifugation at 1500× g for 5 min at 4 • C. The protein content was extracted and quantified with NP40 lysis buffer (Beyotime Biotechnology) and a Pierce BCA Protein assay kit (Thermo Fisher Scientific), respectively, according to the manufacturer's instructions, and then further analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting to evaluate the expression of different target proteins [26,31]. For the evaluation of Rho family proteins with GTP loading [42], active forms of Rap1b (Rap1b-GTP), CDC42 (CDC42-GTP), and RAC1 (RAC1-GTP) were enriched from treated cells using an Active Rap1b Detection Kit and an Active CDC42 and RAC1 Detection Kit (Cell Signaling Technology), respectively, according to the manufacturer's instructions, and then analyzed using Western blotting. The housekeeper proteins GAPDH and Na+/K+ ATPase were selected as internal references for total and membrane protein fractions, respectively, as loading controls.…”

Section: Protein Extraction and Expression Level Evaluationmentioning

confidence: 99%

ICP4-Associated Activation of Rap1b Facilitates Herpes Simplex Virus Type I (HSV-1) Infection in Human Corneal Epithelial Cells

Zhang,

Ding,

2023

Viruses

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The strong contribution of RAS-related protein 1b (Rap1b) to cytoskeleton remodeling determines intracellular and extracellular physiological activities, including the successful infection of viruses in permissive cells, but its role in the HSV-1 life cycle is still unclear. Here, we demonstrated that the HSV-1 immediate early (IE) gene ICP4 inhibits protein kinase A (PKA) phosphorylation to induce Rap1b-activation-mediated viral infection. Rap1b activation and membrane enrichment begin at the early stage of HSV-1 infection and remain active during the proliferation period of the virus. Treating the cells with Rap1b small interfering RNA (siRNA) showed a dose-dependent decrease in viral infection levels, but no dose-dependent increase was observed after Rap1b overexpression. Further investigation indicated that the suppression of Rap1b activation derives from phosphorylated PKA and Rap1b mutants with partial or complete prenylation instead of phosphorylation, which promoted viral infection in a dose-dependent manner. Furthermore, the PKA agonist Forskolin disturbed Rap1b activation in a dose-dependent manner, accompanied by a decreasing trend in viral infection. Moreover, the HSV-1 IE gene ICP4 induced PKA dephosphorylation, leading to continuous Rap1b activation, followed by cytoskeleton rearrangement induced by cell division control protein 42 (CDC42) and Ras-related C3 botulinum toxin substrate 1 (RAC1). These further stimulated membrane-triggered physiological processes favoring virus infection. Altogether, we show the significance of Rap1b during HSV-1 infection and uncover the viral infection mechanism determined by the posttranslational regulation of the viral ICP4 gene and Rap1b host protein.

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Section: Protein Extraction and Expression Level Evaluationmentioning

confidence: 99%

ICP4-Associated Activation of Rap1b Facilitates Herpes Simplex Virus Type I (HSV-1) Infection in Human Corneal Epithelial Cells

Zhang,

Ding,

2023

Viruses

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Beyond KRAS(G12C): Biochemical and Computational Characterization of Sotorasib and Adagrasib Binding Specificity and the Critical Role of H95 and Y96

Mahran,

Kapp,

Valtonen

et al. 2024

ACS Chem. Biol.

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Mutated KRAS proteins are frequently expressed in some of the most lethal human cancers and thus have been a target of intensive drug discovery efforts for decades. Lately, KRAS-(G12C) switch-II pocket (SII−P)-targeting covalent small molecule inhibitors have finally reached clinical practice. Sotorasib (AMG-510) was the first FDA-approved covalent inhibitor to treat KRAS(G12C)-positive nonsmall cell lung cancer (NSCLC), followed soon by adagrasib (MRTX849). Both drugs target the GDP-bound state of KRAS(G12C), exploiting the strong nucleophilicity of acquired cysteine. Here, we evaluate the similarities and differences between sotorasib and adagrasib in their RAS SII−P binding by applying biochemical, cellular, and computational methods. Exact knowledge of SII−P engagement can enable targeting this site by reversible inhibitors for KRAS mutants beyond G12C. We show that adagrasib is strictly KRAS-but not KRAS(G12C)-specific due to its strong and unreplaceable interaction with H95. Unlike adagrasib, sotorasib is less dependent on H95 for its binding, making it a RAS isoform-agnostic compound, having a similar functionality also with NRAS and HRAS G12C mutants. Our results emphasize the accessibility of SII−P beyond oncogenic G12C and aid in understanding the molecular mechanism behind the clinically observed drug resistance, associated especially with secondary mutations on KRAS H95 and Y96.

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The ribosome lowers the entropic penalty of protein folding

Streit,

Bukvin,

Chan

et al. 2024

Nature

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Most proteins fold during biosynthesis on the ribosome1, and co-translational folding energetics, pathways and outcomes of many proteins have been found to differ considerably from those in refolding studies2–10. The origin of this folding modulation by the ribosome has remained unknown. Here we have determined atomistic structures of the unfolded state of a model protein on and off the ribosome, which reveal that the ribosome structurally expands the unfolded nascent chain and increases its solvation, resulting in its entropic destabilization relative to the peptide chain in isolation. Quantitative 19F NMR experiments confirm that this destabilization reduces the entropic penalty of folding by up to 30 kcal mol−1 and promotes formation of partially folded intermediates on the ribosome, an observation that extends to other protein domains and is obligate for some proteins to acquire their active conformation. The thermodynamic effects also contribute to the ribosome protecting the nascent chain from mutation-induced unfolding, which suggests a crucial role of the ribosome in supporting protein evolution. By correlating nascent chain structure and dynamics to their folding energetics and post-translational outcomes, our findings establish the physical basis of the distinct thermodynamics of co-translational protein folding.

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Methods to Monitor Ras Activation State

Cited by 3 publications

References 23 publications

ICP4-Associated Activation of Rap1b Facilitates Herpes Simplex Virus Type I (HSV-1) Infection in Human Corneal Epithelial Cells

ICP4-Associated Activation of Rap1b Facilitates Herpes Simplex Virus Type I (HSV-1) Infection in Human Corneal Epithelial Cells

Beyond KRAS(G12C): Biochemical and Computational Characterization of Sotorasib and Adagrasib Binding Specificity and the Critical Role of H95 and Y96

The ribosome lowers the entropic penalty of protein folding

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