5 research outputs found

    Peptide-Based Coacervate-Core Vesicles with Semipermeable Membranes

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    Coacervates droplets have long been considered as potential protocells to mimic living cells. However, these droplets lack a membrane and are prone to coalescence, limiting their ability to survive, interact, and organize into higher-order assemblies. This work shows that tyrosine-rich peptide conjugates can undergo liquid–liquid phase separation in a well-defined pH window and transform into stable membrane-enclosed protocells by enzymatic oxidation and cross-linking at the liquid–liquid interface. The oxidation of the tyrosine-rich peptides into dityrosine creates a semipermeable, flexible membrane around the coacervates with tunable thickness, which displays strong intrinsic fluorescence, and stabilizes the coacervate protocells against coalescence. The membranes have an effective molecular weight cut-off of 2.5 kDa, as determined from the partitioning of small dyes and labeled peptides, RNA, and polymers into the membrane-enclosed coacervate protocells. Flicker spectroscopy reveals a membrane bending rigidity of only 0.1kBT, which is substantially lower than phospholipid bilayers despite a larger membrane thickness. Finally, it is shown that enzymes can be stably encapsulated inside the protocells and be supplied with substrates from outside, which opens the way for these membrane-bound compartments to be used as molecularly crowded artificial cells capable of communication or as a vehicle for drug delivery.publishedVersio

    Activation of autophagy reverses progressive and deleterious protein aggregation in PRPF31 patient-induced pluripotent stem cell-derived retinal pigment epithelium cells

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    Introduction Mutations in pre-mRNA processing factor 31 (PRPF31), a core protein of the spliceosomal tri-snRNP complex, cause autosomal-dominant retinitis pigmentosa (adRP). It has remained an enigma why mutations in ubiquitously expressed tri-snRNP proteins result in retina-specific disorders, and so far, the underlying mechanism of splicing factors-related RP is poorly understood. Methods We used the induced pluripotent stem cell (iPSC) technology to generate retinal organoids and RPE models from four patients with severe and very severe PRPF31-adRP, unaffected individuals and a CRISPR/Cas9 isogenic control. Results To fully assess the impacts of PRPF31 mutations, quantitative proteomics analyses of retinal organoids and RPE cells were carried out showing RNA splicing, autophagy and lysosome, unfolded protein response (UPR) and visual cycle-related pathways to be significantly affected. Strikingly, the patient-derived RPE and retinal cells were characterised by the presence of large amounts of cytoplasmic aggregates containing the mutant PRPF31 and misfolded, ubiquitin-conjugated proteins including key visual cycle and other RP-linked tri-snRNP proteins, which accumulated progressively with time. The mutant PRPF31 variant was not incorporated into splicing complexes, but reduction of PRPF31 wild-type levels led to tri-snRNP assembly defects in Cajal bodies of PRPF31 patient retinal cells, altered morphology of nuclear speckles and reduced formation of active spliceosomes giving rise to global splicing dysregulation. Moreover, the impaired waste disposal mechanisms further exacerbated aggregate formation, and targeting these by activating the autophagy pathway using Rapamycin reduced cytoplasmic aggregates, leading to improved cell survival. Conclusions Our data demonstrate that it is the progressive aggregate accumulation that overburdens the waste disposal machinery rather than direct PRPF31-initiated mis-splicing, and thus relieving the RPE cells from insoluble cytoplasmic aggregates presents a novel therapeutic strategy that can be combined with gene therapy studies to fully restore RPE and retinal cell function in PRPF31-adRP patients
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