Biology subjects

Loes, A. N.

Publications and source records attributed to Loes, A. N..

3 recordsLinked to original sources

bioRxiv2019

Evolution of multifunctionality through a pleiotropic substitution in the innate immune protein S100A9

Multifunctional proteins are evolutionary puzzles: how do proteins evolve to satisfy multiple functional constraints? S100A9 is one such multifunctional protein. It potently amplifies inflammation via Toll-like receptor 4 and is antimicrobial as part of a heterocomplex with S100A8. These two functions are seemingly regulated by proteolysis: S100A9 is readily degraded, while S100A8/S100A9 is resistant. We take an evolutionary biochemical approach to show that S100A9 evolved both functions and lost proteolytic resistance from a weakly proinflammatory, proteolytically resistant amniote ancestor. We identify a historical substitution that has pleiotropic effects on S100A9 proinflammatory activity and proteolytic resistance but has little effect on S100A8/S100A9 antimicrobial activity. We thus propose that mammals evolved S100A8/S100A9 antimicrobial and S100A9 proinflammatory activities concomitantly with a proteolytic "timer" to selectively regulate S100A9. This highlights how the same mutation can have pleiotropic effects on one functional state of a protein but not another, thus facilitating the evolution of multifunctionality.

biochemistry↗

bioRxiv2019

Identification and characterization of zebrafish Tlr4 co-receptor Md-2

The zebrafish (Danio rerio) is a powerful model organism for studies of the innate immune system. One apparent difference between human and zebrafish innate immunity is the cellular machinery for LPS-sensing. In amniotes, the protein complex formed by Toll-like receptor 4 and myeloid differentiation factor 2 (Tlr4/Md-2) recognizes the bacterial molecule lipopolysaccharide (LPS) and triggers an inflammatory response. It is believed that zebrafish have neither Md-2 nor Tlr4: Md-2 has not been identified outside of amniotes, while the zebrafish tlr4 genes appear to be paralogs, not orthologs, of amniote TLR4s. We revisited these conclusions. We identified a zebrafish gene encoding Md-2, ly96. Using single-cell RNA-Seq, we found that ly96 is transcribed in cells that also transcribe genes diagnostic for innate immune cells, including the zebrafish tlr4-like genes. Unlike amniote LY96, zebrafish ly96 expression is restricted to a small number of macrophage-like cells. In a functional assay, zebrafish Md-2 and Tlr4a form a complex that activates NF-{kappa}B signaling in response to LPS, but ly96 loss-of-function mutations gave little protection against LPS-toxicity in larval zebrafish. Finally, by analyzing the genomic context of tlr4 genes in eleven jawed vertebrates, we found that tlr4 arose prior to the divergence of teleosts and tetrapods. Thus, an LPS-sensitive Tlr4/Md-2 complex is likely an ancestral feature shared by mammals and zebrafish, rather than a de novo invention on the tetrapod lineage. We hypothesize that zebrafish retain an ancestral, low-sensitivity Tlr4/Md-2 complex that confers LPS-responsiveness to a specific subset of innate immune cells.

immunology↗

bioRxiv2019

Zinc-independent activation of Toll-like receptor 4 by S100A9

The homodimer formed by the protein S100A9 induces inflammation through Toll-like receptor 4 (TLR4), playing critical roles in both healthy and pathological innate immune responses. The molecular mechanism by which S100A9 activates TLR4 remains unknown. Previously, the interaction between purified S100A9 and TLR4 was shown to depend on Zn2+; however, the Zn2+ binding site(s) on S100A9 were not identified. Here, we investigated the role of Zn2+ binding in the pro-inflammatory activity of S100A9. We found that the S100A9 homodimer was prone to reversible, Zn2+-dependent aggregation in vitro. Using a combination of site-directed mutagenesis and Isothermal Titration Calorimetry (ITC), we identified multiple residues that contribute to Zn2+ binding in S100A9. We then used mutagenesis to construct a version of S100A9 with no detectable Zn2+ binding by either ITC or Inductively Coupled Plasma-Mass Spectrometry. This protein did not exhibit aggregation upon addition of saturating Zn2+. Further, despite the lack of Zn2+-binding, this protein was capable of activating TLR4 in a cell-based functional assay. We then modified the functional assay so the Zn2+ concentration was exceedingly low relative to the concentration of S100A9 added. Again, S100A9 was able to activate TLR4. This reveals that, despite the ability of S100A9 to bind Zn2+, S100A9 does not require Zn2+ to activate TLR4. Our work represents an important step in clarifying the nature of the interaction between S100A9 and TLR4.

biochemistry↗