Biology subjects

Harms, M. J.

Publications and source records attributed to Harms, M. J..

22 records · Page 2Linked to original sources

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↗

bioRxiv2019

Transpositional scaling and niche transitions restore organ size and shape during zebrafish fin regeneration

Regenerating fish fins return to their original size and shape regardless of the nature or extent of injury. Prevailing models for this longstanding mystery of appendage regeneration speculate fin cells maintain uncharacterized positional identities that instruct outgrowth after injury. Using zebrafish, we find differential Wnt production correlates with the extent of regeneration across the caudal fin. We identify Dachshund transcription factors as markers of distal blastema cells that produce Wnt and thereby promote a pro-progenitor and -proliferation environment. We show these Dach-expressing \"niche cells\" derive from mesenchyme populating cylindrical and progressively tapered fin rays. The niche pool, and consequently Wnt, steadily dissipates as regeneration proceeds; once exhausted, ray and fin growth stops. Supported by mathematical modeling, we show longfint2 zebrafish regenerate exceptionally long fins due to a perdurant niche, representing a \"broken countdown timer\". We propose regenerated fin size is dictated by the amount of niche formed upon damage, which simply depends on the availability of intra-ray mesenchyme defined by skeletal girth at the injury site. Likewise, the fin reestablishes a tapered ray skeleton because progenitor osteoblast output reflects diminishing niche size. This \"transpositional scaling\" model contends mesenchyme-niche state transitions and positional information provided by self-restoring skeletal geometry rather than cell memories determine a regenerated fins size and shape.

developmental biology↗

bioRxiv2018

Uninterpretable interactions: epistasis as uncertainty

Epistasis is a common feature of genotype-phenotype maps. Understanding the patterns of epistasis is critical for predicting unmeasured phenotypes, explaining evolutionary trajectories, and for inferring the biological mechanisms that determine a map. One common approach is to use a linear model to decompose epistasis into specific pairwise and high-order interactions between mutations. Such interactions are then used to identify important biology or to explain how the genotype-phenotype map shapes evolution. Here we show that the coefficients extracted from such analyses are likely uninterpretable. They cannot be extracted reliably from experimental genotype-phenotype maps due to regression bias. Further, we can generate epistatic \"interactions\" indistinguishable from those in experimental maps using a completely random process. From this, we conclude that epistasis should be treated as a random, but quantifiable, variation in these maps. This perspective allows us to build predictive models with known error from a small number of measured phenotypes. It also suggests that we need mechanistic, nonlinear models to account for epistasis and decompose genotype-phenotype maps.

genetics↗

bioRxiv2017

Conservation of specificity in two low-specificity proteins

S100 proteins bind linear peptide regions of target proteins and modulate their activity. The peptide binding interface, however, has remarkably low specificity and can interact with many target peptides. It is not clear if the interface discriminates targets in a biological context, or whether biological specificity is achieved exclusively through external factors such as subcellular localization. To discriminate these possibilities, we used an evolutionary biochemical approach to trace the evolution of paralogs S100A5 and S100A6. We first used isothermal titration calorimetry to study the binding of a collection of peptides with diverse sequence, hydrophobicity, and charge to human S100A5 and S100A6. These proteins bound distinct, but overlapping, sets of peptide targets. We then studied the peptide binding properties of S100A5 and S100A6 orthologs sampled from across five representative amniote species. We found that the pattern of binding specificity was conserved along all lineages, for the last 320 million years, despite the low specificity of each protein. We next used Ancestral Sequence Reconstruction to determine the binding specificity of the last common ancestor of the paralogs. We found the ancestor bound the whole set of peptides bound by modern S100A5 and S100A6 proteins, suggesting that paralog specificity evolved by subfunctionalization. To rule out the possibility that specificity is conserved because it is difficult to modify, we identified a single historical mutation that, when reverted in human S100A5, gave it the ability to bind an S100A6-specific peptide. These results indicate that there are strong evolutionary constraints on peptide binding specificity, and that, despite being able to bind a large number of targets, the specificity of S100 peptide interfaces is indeed important for the biology of these proteins.

biochemistry↗