[P007] Poster

12:10 PM - 1:00 PM JST(3:10 AM - 4:00 AM UTC)

[P04-481] Exploring usable and unusable protein sequences based on the “neutrality” of amino acid homorepeats (PolyX)

○Yukihiro Murase¹, Hisao Moriya¹ (1. Okayama Univ. (Japan))

Keywords:

amino acid homorepeat,overexpression,protein cytotoxicity,protein evolution

How biological systems distinguish between “usable” and “unusable” protein sequences within the vast amino acid sequence space remains largely unresolved. In this study, we focused on simple homorepeat sequences consisting of ten identical amino acids (PolyX) as a model to probe the fundamental constraints on sequence usability. The frequent occurrence of a given PolyX in proteomes suggests that it is functionally tolerated or benign, whereas absent or rare PolyX sequences may have been evolutionarily eliminated due to toxicity.
To test this hypothesis, we systematically evaluated the “neutrality”–an integrated measure of harmful versus beneficial properties–of PolyX by overexpressing fluorescent protein fusions containing PolyX in Saccharomyces cerevisiae. Our results revealed clear amino acid–dependent biases in PolyX neutrality. PolyX sequences composed of hydrophilic and negatively charged amino acids exhibited low toxicity, whereas those composed of hydrophobic and positively charged amino acids were highly toxic. Notably, these trends closely matched the frequency and repeat length of PolyX sequences in the yeast proteome, suggesting that proteome composition is shaped by sequence-dependent toxicity.
Furthermore, similar neutrality patterns were observed when using different fluorescent protein and in experiments conducted in Escherichia coli, indicating that the toxicity of PolyX is conserved regardless of fusion partners and across both prokaryotic and eukaryotic systems. Interestingly, several PolyX sequences (E, S, N, and Q) exhibited toxicity-mitigating effects. Mechanistic investigations using Western blotting and transcriptome analysis revealed that PolyE suppresses protein aggregation and reduces the expression of stress-response genes.
Our findings suggest that PolyX neutrality defines the “compatible sequence space” of proteins, where certain sequences like PolyE act as evolutionary buffers to maintain proteostasis. This study provides a quantitative framework for understanding how physicochemical constraints on protein solubility shape the evolution of proteomes and offers a new metric for rational protein design.