Pathological mutations differentially affect the self-assembly and polymerisation of the innate immune system signalling adaptor molecule MyD88.

BACKGROUND: Higher-order self-assembly of proteins, or "prion-like" polymerisation, is now emerging as a simple and robust mechanism for signal amplification, in particular within the innate immune system, where the recognition of pathogens or danger-associated molecular patterns needs to trigger a strong, binary response within cells. MyD88, an important adaptor protein downstream of TLRs, is one of the most recent candidates for involvement in signalling by higher order self-assembly. In this new light, we set out to re-interpret the role of polymerisation in MyD88-related diseases and study the impact of disease-associated point mutations L93P, R196C, and L252P/L265P at the molecular level. RESULTS: We first developed new in vitro strategies to characterise the behaviour of polymerising, full-length MyD88 at physiological levels. To this end, we used single-molecule fluorescence fluctuation spectroscopy coupled to a eukaryotic cell-free protein expression system. We were then able to explore the polymerisation propensity of full-length MyD88, at low protein concentration and without purification, and compare it to the behaviours of the isolated TIR domain and death domain that have been shown to have self-assembly properties on their own. These experiments demonstrate that the presence of both domains is required to cooperatively lead to efficient polymerisation of the protein. We then characterised three pathological mutants of MyD88. CONCLUSION: We discovered that all mutations block the ability of MyD88 to polymerise fully. Interestingly, we show that, in contrast to L93P and R196C, L252P is a gain-of-function mutation, which allows the MyD88 mutant to form extremely stable oligomers, even at low nanomolar concentrations. Thus, our results shed new light on the digital "all-or-none" responses by the myddosomes and the behaviour of the oncogenic mutations of MyD88.

Methods for Expression of Recombinant Proteins Using a Pichia pastoris Cell-Free System.

Cell-free protein synthesis is a powerful tool for engineering biology and has been utilized in many diverse applications, from biosensing and protein prototyping to biomanufacturing and the design of metabolic pathways. By exploiting host cellular machinery decoupled from cellular growth, proteins can be produced in vitro both on demand and rapidly. Eukaryotic cell-free platforms are often neglected due to perceived complexity and low yields relative to their prokaryotic counterparts, despite providing a number of advantageous properties. The yeast Pichia pastoris (also known as Komagataella phaffii) is a particularly attractive eukaryotic host from which to generate cell-free extracts, due to its ability to grow to high cell densities with high volumetric productivity, genetic tractability for strain engineering, and ability to perform post-translational modifications. Here, we describe methods for conducting cell-free protein synthesis using P. pastoris as the host, from preparing the cell lysates to protocols for both coupled and linked transcription-translation reactions. By providing these methodologies, we hope to encourage the adoption of the platform by new and experienced users alike. © 2020 The Authors. Basic Protocol 1: Preparation of Pichia pastoris cell lysate Basic Protocol 2: Coupled in vitro transcription and translation Basic Protocol 3: Determining luciferase production from cell-free protein synthesis reactions Alternate Protocol 1: Linked in vitro transcription and translation Alternate Protocol 2: Quantifying HSA protein concentration Support Protocol 1: Preparation of mRNA by in vitro transcription for linked transcription and translation.