Nieznanska, H. | bioRxiv Science

bioRxiv2025

Liquid-Liquid Phase Separation-mediated formation of amyloid fibrils from DcpS scavenger enzymes.

Decapping Scavenger (DcpS) enzyme was initially identified by its ability to hydrolyze the cap structure resulting from mRNA decay. Human DcpS is an established target for acute myeloid leukemia (AML) and hepatic metastasis. Recently, the protein has been linked to neuronal development regulation and implicated in certain developmental neurological disorders. Here we demonstrate for the first time that DcpS undergoes misfolding in vitro, leading to the formation of amyloid-like fibrils. Fibrillization was observed for human and nematode (C. elegans) DcpS using transmission electron microscope (TEM) imaging, Thioflavin T (ThT) fluorescence assay, Fourier-transform infrared (FT-IR) spectroscopy, circular dichroism (CD) spectroscopy, differential scanning fluorimetry (DSF), and dynamic light scattering (DLS). Additionally, the DcpSINS15 insertional mutant linked to the Al-Raquad syndrome, exhibited accelerated fibril aggregation kinetics compared to the wild type protein. Moreover, we show that the DcpS species investigated in this study undergo liquid-liquid phase separation (LLPS) prior to amyloid-formation. We propose that the LLPS phase transition underlies the intricate kinetics (e.g. lack of a clearly-resolved lag phase) of the misfolding process. As the physiological implications of the here-reported propensity of DcpS to lose its biological function through the coupled LLPS-fibrillization transition remain to be elucidated, this work lays the groundwork for further studies on this phenomenon.

molecular biology↗

bioRxiv2023

OMA1 protease eliminates arrested protein import intermediates upon depolarization of the inner mitochondrial membrane.

Most mitochondrial proteins originate from the cytosol and require active transport into the organelle. Such precursor proteins must be largely unfolded to pass through translocation channels in mitochondrial membranes. Misfolding of transported proteins can result in their arrest and translocation failure. Arrested proteins block further import, disturbing mitochondrial functions and cellular proteostasis. Cellular responses to translocation failure have been defined in yeast. To discover molecular mechanisms that resolve failed import events in human cells, we developed the translocase clogging model using a fusion protein with a rigid domain. The mechanism we uncover differs significantly from these described in fungi, where ATPase-driven extraction of blocked protein is directly coupled with proteasomal processing. We found human cells to rely primarily on mitochondrial factors to clear translocation channel blockage. The mitochondrial membrane depolarization triggered proteolytic cleavage of the stalled protein, which involved mitochondrial protease OMA1. The cleavage allowed releasing the protein fragment that blocked the translocase. The released fragment was further cleared in the cytosol by the valosin containing protein (VCP)/p97 and proteasome.

molecular biology↗