C. elegans LIN-66 mediates EIF-3/eIF3-dependent protein translation via a cold-shock domain.

Protein translation initiation is a conserved process involving many proteins acting in concert. The 13 subunit eukaryotic initiation factor 3 (eIF3) complex is essential for assembly of the pre-initiation complex that scans mRNA and positions ribosome at the initiation codon. We previously reported that a gain-of-function (gf) mutation affecting the G subunit of the Caenorhabditis elegans eIF3 complex, eif-3.g(gf), selectively modulates protein translation in the ventral cord cholinergic motor neurons. Here, through unbiased genetic suppressor screening, we identified that the gene lin-66 mediates eif-3.g(gf)-dependent protein translation in motor neurons. LIN-66 is composed largely of low-complexity amino acid sequences with unknown functional domains. We combined bioinformatics analysis with in vivo functional dissection and identified a cold-shock domain in LIN-66 critical for its function. In cholinergic motor neurons, LIN-66 shows a close association with EIF-3.G in the cytoplasm. The low-complexity amino acid sequences of LIN-66 modulate its subcellular pattern. As cold-shock domains function broadly in RNA regulation, we propose that LIN-66 mediates stimulus-dependent protein translation by facilitating the interaction of mRNAs with EIF-3.G.

Structured and disordered regions of Ataxin-2 contribute differently to the specificity and efficiency of mRNP granule formation.

Ataxin-2 (ATXN2) is a gene implicated in spinocerebellar ataxia type II (SCA2), amyotrophic lateral sclerosis (ALS) and Parkinsonism. The encoded protein is a therapeutic target for ALS and related conditions. ATXN2 (or Atx2 in insects) can function in translational activation, translational repression, mRNA stability and in the assembly of mRNP-granules, a process mediated by intrinsically disordered regions (IDRs). Previous work has shown that the LSm (Like-Sm) domain of Atx2, which can help stimulate mRNA translation, antagonizes mRNP-granule assembly. Here we advance these findings through a series of experiments on Drosophila and human Ataxin-2 proteins. Results of Targets of RNA Binding Proteins Identified by Editing (TRIBE), co-localization and immunoprecipitation experiments indicate that a polyA-binding protein (PABP) interacting, PAM2 motif of Ataxin-2 may be a major determinant of the mRNA and protein content of Ataxin-2 mRNP granules. Experiments with transgenic Drosophila indicate that while the Atx2-LSm domain may protect against neurodegeneration, structured PAM2- and unstructured IDR- interactions both support Atx2-induced cytotoxicity. Taken together, the data lead to a proposal for how Ataxin-2 interactions are remodelled during translational control and how structured and non-structured interactions contribute differently to the specificity and efficiency of RNP granule condensation as well as to neurodegeneration.

Bladder cancer therapy using a conformationally fluid tumoricidal peptide complex.

Partially unfolded alpha-lactalbumin forms the oleic acid complex HAMLET, with potent tumoricidal activity. Here we define a peptide-based molecular approach for targeting and killing tumor cells, and evidence of its clinical potential (ClinicalTrials.gov NCT03560479). A 39-residue alpha-helical peptide from alpha-lactalbumin is shown to gain lethality for tumor cells by forming oleic acid complexes (alpha1-oleate). Nuclear magnetic resonance measurements and computational simulations reveal a lipid core surrounded by conformationally fluid, alpha-helical peptide motifs. In a single center, placebo controlled, double blinded Phase I/II interventional clinical trial of non-muscle invasive bladder cancer, all primary end points of safety and efficacy of alpha1-oleate treatment are reached, as evaluated in an interim analysis. Intra-vesical instillations of alpha1-oleate triggers massive shedding of tumor cells and the tumor size is reduced but no drug-related side effects are detected (primary endpoints). Shed cells contain alpha1-oleate, treated tumors show evidence of apoptosis and the expression of cancer-related genes is inhibited (secondary endpoints). The results are especially encouraging for bladder cancer, where therapeutic failures and high recurrence rates create a great, unmet medical need.

Size Determination of Protein Oligomers/Aggregates Using Diffusion NMR Spectroscopy.

Diffusion-ordered spectroscopy (DOSY) is a widely used NMR technique for the identification of different chemical moieties/compounds contained in mixtures and has been successfully employed for the separation of small molecules based on hydrodynamic radii. Herein we show that DOSY can also be applied for the size determination of larger biomolecules such as proteins and protein oligomers/aggregates. Proof-of-principle is first shown with a cross-linked oligomeric protein mixture where the hydrodynamic volumes of each component are estimated and subsequently verified with size-exclusion HPLC and SDS polyacrylamide gel electrophoresis. We then determine the sizes of protein oligomers contained in a protein solution subjected under amyloid fibrillogenesis conditions. These studies aim to provide insight into the kinetics behind protein aggregation involved in amyloidosis as well as to determine the hydrodynamic radii of proteins within the mixture.