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bioRxiv2023

A progesterone derivative linked to a stable phospholipid activates breast cancer cell response without leaving the cell membrane

In hormone-responsive breast cancer cells, progesterone (P4) has been shown to act via its nuclear receptor (PR), a ligand-activated transcription factor. A small fraction of PR is palmitoylated and anchored to the cell membrane (mbPR) forming a complex with estrogen receptor alpha (ER). Upon hormone exposure, either directly or via interaction with ER mbPR activates the SRC/RAS/ERK kinase pathway leading to phosphorylation of PR by ERK. Kinase activation is essential for P4 gene regulation, as the ERK and MSK1 kinases are recruited by the PR to its genomic binding sites and trigger chromatin remodeling. An interesting open question is whether activation of mbPR can result in gene regulation in the absence of ligand binding to intracellular PR. This matter has been investigated in the past using P4 attached to serum albumin, but the attachment is leaky and albumin can be endocytosed and degraded, liberating P4. Here we propose a more stringent approach to address this issue by ensuring attachment of P4 to the cell membrane via covalent binding to a stable phospholipid. This strategy identifies the actions of P4 independent of hormone binding to intracellular PR. We found that a membrane-attached progestin can activate mbPR, the ERK signalling pathway leading to PR phosphorylation, initial gene regulation and entry into the cell cycle, in the absence of detectable intracellular progestin.

genomics↗

bioRxiv2020

The extent of protein hydration dictates the preference for heterogeneous or homogeneous nucleation generating either parallel or antiparallel β-sheet α-synuclein aggregates

-Synuclein amyloid self-assembly is the hallmark of a number of neurodegenerative disorders, including Parkinsons disease, although there is still very limited understanding about the factors and mechanisms that trigger this process. Primary nucleation has been observed to be initiated in vitro at hydrophobic/hydrophilic interfaces by heterogeneous nucleation generating parallel {beta}-sheet aggregates, although no such interfaces have yet been identified in vivo. In this work, we have discovered that -synuclein can self-assemble into amyloid aggregates by homogeneous nucleation, without the need of an active surface, and with a preference for an antiparallel {beta}-sheet arrangement. This particular structure has been previously proposed to be distinctive of stable toxic oligomers and we here demonstrate that it indeed represents the most stable structure of the preferred amyloid pathway triggered by homogeneous nucleation under limited hydration conditions, including those encountered inside -synuclein droplets generated by liquid-liquid phase separation. In addition, our results highlight the key role that water plays not only in modulating the transition free energy of amyloid nucleation, and thus governing the initiation of the process, but also in dictating the type of preferred primary nucleation and the type of amyloid polymorph generated depending on the extent of protein hydration. These findings are particularly relevant in the context of in vivo -synuclein aggregation where the protein can encounter a variety of hydration conditions in different cellular microenvironments, including the vicinity of lipid membranes or the interior of membraneless compartments, which could lead to the formation of remarkably different amyloid polymorphs by either heterogeneous or homogeneous nucleation.

biophysics↗