Biology subjects

Carpenter, T. S.

Publications and source records attributed to Carpenter, T. S..

3 recordsLinked to original sources

bioRxiv2025

An In Vitro BRAF Activation Assay Elucidates Molecular Mechanisms Driving the Disassembly of the Autoinhibited BRAF State

The RAF kinases (ARAF, BRAF and CRAF) are essential components of the RAS-ERK signaling pathway, which controls vital cellular processes and is frequently dysregulated in human disease. Notably, mutations that alter BRAF function are prominent drivers of human cancer and certain RASopathy disorders, making BRAF an important target for therapeutic intervention. Despite extensive research, several aspects of BRAF regulation remain unclear. In this study, we developed an in vitro BRAF activation assay using purified autoinhibited BRAF:14-3-32:MEK complexes. Our results show that fully processed, active-state KRAS alone can promote dimer-dependent BRAF activation. Moreover, we found that phosphatidylserine (PS)-containing liposomes synergized with KRAS to promote BRAF activation, achieving activity levels comparable to those observed with BRAF proteins that constitutively dimerize. In contrast, the SMP phosphatase complex had only a minimal effect on BRAF catalytic activity in this system but mediated the dephosphorylation of the negative regulatory pS365 14-3-3 binding site in a manner that was accelerated by the presence of KRAS alone or KRAS and 30% PS liposomes. Finally, we show that inhibitors blocking the BRAF RBD:KRAS interaction were able to suppress the in vitro activation of BRAF, underscoring the critical role of RAS binding in initiating the disassembly of the BRAF autoinhibited state. Thus, this assay provides valuable insights into the steps required for BRAF activation and can serve as an effective screening tool for identifying compounds that may inhibit this process and have therapeutic potential. Significance StatementBRAF is a central intermediate in RAS pathway signaling, and its activity is often elevated in human cancers and RASopathy disorders. Due to the complexity of BRAF activation, identifying compounds that sustainably inhibit BRAF function has proven difficult, emphasizing the need for a more comprehensive understanding of BRAF regulation. Here, we have developed an in vitro BRAF activation assay that elucidates key steps in this process. Our findings demonstrate that RAS binding not only recruits BRAF to the plasma membrane but initiates the disassembly of the autoinhibited monomer, which in the context of the membrane, facilitates BRAF dimerization and activation. This assay advances our understanding of BRAF regulation and provides a novel platform for drug discovery efforts targeting BRAF.

biochemistry↗

bioRxiv2025

Free energy and flexibility analysis of autoinhibited human BRAF

The RAF serine/threonine protein kinases function as direct effectors of RAS in the intracellular transmission of extracellular growth signals, and they are key targets for drug discovery given the high incidence of oncogenic mutations in RAF and other components of this signaling pathway. In its inactive state, RAF is held in an autoinhibited conformation in the cytosol through a combination of intramolecular interactions and binding to a regulatory 14-3-3 protein dimer. Activation of RAF is initiated by its interaction with membrane-localized, GTP-bound RAS, which induces conformational changes that release RAF from its autoinhibited state. However, the molecular mechanisms governing RAF activation remain incomplete, largely due to the challenges in experimentally capturing intermediate conformational states in this process. To address this gap, we developed a comprehensive all-atom model of BRAF based on existing cryo-EM structures. Using this model, we performed extensive molecular dynamics simulations to evaluate the stability and free energy landscape of autoinhibited BRAF in solution. Our analysis reveals conformational flexibility within the autoinhibited complex, suggesting that this dynamic behavior may play a role in facilitating BRAF activation upon engagement with membrane-bound RAS.

biophysics↗

bioRxiv2025

Apolipoprotein interaction induces shape remodeling and lipid phase separation in giant unilamellar vesicles

Apolipoprotein A-I (ApoA-I) - a 243-residue amphipathic protein containing an N-terminal globular domain and a primarily helical C-terminal lipid binding domain - is a principal protein component of high-density lipoprotein (HDL) or "good" cholesterol, which is an essential component of lipid homeostasis in humans. Synthesized in the liver and intestine and excreted in the blood, ApoA-I undergoes complex, cooperative, and dynamic self-assembly with membrane lipids, producing unlipi-dated (or weakly lipidated), nascent discoidal, and mature HDL states. In vitro studies demonstrate that the reconstitution of purified protein and lipids restores this cooperative self-assembly. However, the kinetic pathways by which these mesoscopic, proteolipidic assemblies form remain incompletely understood. Here, we monitor the dynamics of ApoA-I-membrane interactions through real-time monitoring of morphological changes, which ensue when ApoA-I is incubated with minimal giant unilamellar vesicles (GUVs) composed of single phospholipids or phase-separating phospholipid-cholesterol mixtures. Our fluorescence microscopy measurements reveal that the interaction initiates a gross, morphological remodeling of the parent vesicle proceeding through discrete stages involving membrane poration, solute leakage, vesiculation, and lipid-lipid phase separation. Our atomic force microscopy measurements confirm that the outcome includes discoidal nanoparticles. This qualitative phenomenology is robust and fully reproducible for different protein mutants and alleles (WT APOA-1, {Delta}49ApoA-I, ApoE-3, and ApoE-4) and other lipid mixtures (including mixtures containing phosphoserine lipids). Our molecular simulations recapitulate the essential shape changes and further reveal the composition dependence of the interactions. Together, these findings outline key steps in protein-lipid interactions that facilitate the assembly of mesoscopic reconstituted lipoproteins and nanodiscs.

biophysics↗