Kieffer-Jaquinod, S. | bioRxiv Science

bioRxiv2023

Oxidation-sensitive cysteines drive IL-38 amyloid formation

Cytokines of the interleukin (IL)-1 family are widely expressed in epithelial surfaces, including the epidermis, where they play a key role in the maintenance of barrier integrity and host defense. A recent report associated the IL-1 family member IL-33 with stress granules (SGs) in epithelial cells. Formation of SGs is promoted by the aggregation of proteins harboring low complexity regions (LCRs). In this study, using computational analyses, we predicted the presence of LCRs in six of the eleven IL-1 family members. Among these, IL-38 contained a long LCR and localized to Ras GTPase-activating protein binding protein 1 (G3BP1) positive SGs, as well as to G3BP1 negative intracellular protein condensates in keratinocytes exposed to oxidative stress (OS). In addition, we identified two highly aggregation-prone amyloid core (AC) motifs in the IL-38 LCR and detected the formation of amyloid IL-38 aggregates in response to OS in cells and in vitro. Disulfide bond mapping, in silico modelling and the analysis of specific cysteine mutants supported a model in which specific oxidation-sensitive cysteines act as redox switches to modify the conformation of IL-38 and thus the surface exposure of its ACs, shuttling it from a soluble state into biomolecular condensates. Finally, the presence of IL-38 granules in human epidermal layers highly exposed to environmental OS suggests that oxidation-induced formation of amyloid aggregates, as a previously unrecognized intrinsic biological property of IL-38, may be physiologically relevant at this epithelial barrier.

cell biology↗

bioRxiv2022

Three-dimensional envelope and subunit interactions of the plastid-encoded RNA polymerase from Sinapis alba

RNA polymerases (RNAPs) involved in gene transcription are found in all living organisms with degrees of complexity ranging from single polypeptide chains to multimeric enzymes. In the chloroplasts, the nuclear-encoded RNA polymerase and the plastid-encoded RNA polymerase (PEP) are both involved in the selective transcription of the plastid genome. The PEP is a prokaryotic-type multimeric RNAP found in different states depending on light stimuli and cell identity. One of its active states requires the assembly of nuclear-encoded PEP-Associated Proteins (PAPs) on the catalytic core, producing a complex of more than 900 kDa regarded as essential for chloroplast biogenesis. A purification procedure compatible with structural analysis was used to enrich the native PEP from Sinapis alba chloroplasts. Mass spectrometry (MS)-based proteomic analysis identified the core components, the PAPs and additional members, and coupled to cross-linking (XL-MS) provided initial structural information about the relative position of PEP subunits. Sequence alignments of the catalytic core subunits across various chloroplasts of the green lineage and prokaryotes combined with structural data show that the overall shape of the catalytic core and the residues essential for the catalytic activity are conserved. However, variations are observed at the surface of the core, some of them corresponding to PAP binding sites as suggested by XL-MS experiments. Using negative stain electron microscopy, the PEP 3D envelope was calculated. 3D classification shows that the protrusions which we attribute to the PAPs are firmly associated with the catalytic core. Overall, the shape of the S. alba PEP envelope is different from that of RNAPs.

biochemistry↗