Nietlispach, D. | bioRxiv Science

bioRxiv2024

Investigating the interactions of the cucumber mosaic virus 2b protein with the viral 1a replicase component and the cellular RNA silencing factor Argonaute 1

The cucumber mosaic virus (CMV) 2b protein is a suppressor of plant defenses and a pathogenicity determinant. Amongst the 2b proteins host targets is the RNA silencing factor Argonaute 1 (AGO1), which it binds to and inhibits. In Arabidopsis thaliana, if 2b-induced inhibition of AGO1 is too efficient it induces reinforcement of antiviral silencing by AGO2, and triggers increased resistance against aphids, CMVs insect vectors. These effects would be deleterious to CMV replication and transmission, respectively, but are moderated by the CMV 1a protein by sequestering sufficient 2b protein molecules into P-bodies to prevent excessive inhibition of AGO1. Mutant 2b protein variants were generated and red and green fluorescent protein fusions used to investigate subcellular colocalization with AGO1 and the 1a protein, and the effects of mutations on complex formation with the 1a protein and AGO1 were investigated using bimolecular fluorescence complementation and co-immunoprecipitation assays. Although we found that residues 56-60 influenced the 2b proteins interactions with the 1a protein and AGO1, it appears unlikely that any single residue or sequence domain is solely responsible. In silico predictions of intrinsic disorder within the 2b protein secondary structure were supported by circular dichroism (CD) but not by nuclear magnetic resonance (NMR) spectroscopy. Intrinsic disorder provides a plausible model to explain the 2b proteins ability to interact with AGO1, the 1a protein and other factors. However, the reasons for the conflicting conclusions provided by CD and NMR must first be resolved.

plant biology↗

bioRxiv2021

The role of water mobility in protein misfolding

The propensity for intrinsically disordered proteins to aggregate is heavily influenced by their surrounding environment. Here, we show that the mobility of the surrounding water molecules directly influences the aggregation rate of -synuclein (aSyn), a protein associated with Parkinsons disease. We observe that the addition of NaCl reduces the mobility of water, while addition of CsI increases the mobility of water. In turn, this reduces and increases the mobility of aSyn, respectively, given the change in strength and lifetime of the intermolecular forces. The reduction of aSyn mobility in the presence of NaCl ions leads to increased aggregation rates, which may be due to aggregation-competent conformations being stable for longer, thereby increasing the likelihood of establishing interactions between two adjacent monomers. In contrast, aSyn is more mobile when CsI is dissolved in the aqueous phase which leads to a reduction of successful monomeric interactions. We thus highlight the importance of the surrounding environment and describe how ion content can influence water mobility and the misfolding rate of amyloidogenic proteins, such as aSyn. By modulating the cellular environment to increase water mobility or finding small molecules to increase protein dynamics, new therapeutic targets may be found.

biophysics↗