Biology subjects

Harms, M. J.

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

At least 19 recordsLinked to original sources

bioRxiv2024

Applying computational protein design to engineer affibodies for affinity-controlled delivery of vascular endothelial growth factor and platelet-derived growth factor

Effective angiogenesis requires the coordinated presentation of vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF), which play competing roles in stimulating vascular outgrowth and stabilization. Dysregulation in VEGF and PDGF secretion are implicated in abnormal vascular structures, poor vessel stability, and inadequate tissue repair. Current biomaterial delivery vehicles for these proteins have a limited ability to precisely control the kinetics of protein release, preventing systematic exploration of their temporal effects. Here, we combined yeast surface display and computational protein design to engineer eight VEGF-specific and PDGF-specific protein binders called affibodies with a broad range of affinities (dissociation constants = 2.23-9260 nM) for affinity-controlled protein release. Starting from one VEGF- and one PDGF-specific affibody discovered from yeast surface display, computational modeling was used to select disruptive mutations to create VEGF-specific affibodies with lower affinities for VEGF and expand the affinity range of PDGF-specific affibodies. In both cases, the specificity of the affibodies for their target protein was either maintained or enhanced. Soluble protein-specific affibodies modulated protein bioactivity as evidenced by changes in VEGF-induced endothelial cell proliferation and luminescent output of a PDGF-responsive fibroblast cell line. Affibody-conjugated polyethylene glycol maleimide (PEG-mal) hydrogels sustained VEGF and PDGF release compared to hydrogels without affibodies, enabling tunability of protein release over 7 days. VEGF and PDGF released from affibody-conjugated hydrogels exhibited higher bioactivity compared to proteins released from hydrogels without affibodies, highlighting the potential of these engineered affinity interactions to prolong protein bioactivity. This work underscores the power of computational protein design to enhance biomaterial functionality, creating a platform for tunable protein delivery to support angiogenesis and tissue repair.

bioengineering↗

bioRxiv2024

Changing expression system alters oligomerization and proinflammatory activity of recombinant human S100A9

S100A9 is a Damage Associated Molecular Pattern (DAMP) that activates the innate immune system via Toll-like receptor 4 (TLR4). Despite many years of study, the mechanism of activation remains unknown. To date, much of the biochemical characterization of S100A9 has been performed using recombinant S100A9 expressed in E. coli (S100A9ec). TLR4 is the canonical receptor for LPS, a molecule found in the outer membrane of E. coli, raising the possibility of artifacts due to LPS contamination. Here we report characterization of LPS-free recombinant S100A9 expressed in insect cells (S100A9in). We show that S100A9in does not activate TLR4. This difference does not appear to be due to LPS contamination, protein misfolding, purification artifacts, or differences in phosphorylation. We show instead that S100A9in adopts an altered oligomeric state compared to S100A9ec. Disrupting oligomer formation with the E. coli disaggregase SlyD restores activity to S100A9in. Our results also indicate that the oligomeric state of S100A9 is a major factor in its ability to activate TLR4 and that this can be altered in unexpected ways by the recombinant expression system used to produce the protein.

biochemistry↗

bioRxiv2024

Zebrafish do not have calprotectin

The protein heterodimer calprotectin and its component proteins, S100A8 and S100A9, play important antibacterial and proinflammatory roles in the mammalian innate immune response. Gaining mechanistic insights into the regulation and biological function of calprotectin will help facilitate patient diagnostics and therapy and further our understanding of the host-microbe interface. Recent literature has identified zebrafish s100a10b as zebrafish calprotectin based on sequence similarity, genomic context, and transcriptional upregulation during the immune response to bacterial infections. The field would benefit from expanding the breadth of calprotectin studies into a zebrafish innate immunity model. Here, we carefully evaluated the possibility that zebrafish possess a calprotectin. We found that zebrafish do not possess an ortholog of mammalian S100A8 or S100A9. We then identified four zebrafish s100 proteins-- including s100a10b--that are expressed in immune cells and upregulated during the immune response. We recombinantly expressed and purified these proteins and measured the antimicrobial and proinflammatory characteristics. We found that none of the zebrafish proteins exhibited activity comparable to mammalian calprotectin. Our work demonstrates conclusively that zebrafish have no ortholog of calprotectin, and the most plausible candidate proteins have not convergently evolved similar functions.

immunology↗

bioRxiv2024

S100A9 interacts with a dynamic region on CD14 to activate Toll-like receptor 4.

S100A9 is a Damage Associated Molecular Pattern (DAMP) that activates inflammatory pathways via Toll-like receptor 4 (TLR4). This activity plays important homeostatic roles in tissue repair, but can also contribute to inflammatory diseases. The mechanism of activation is unknown. Here, we follow up on a previous observation that the protein CD14 is an important co-receptor that enables S100A9 to activate TLR4. Using cell-based functional assays and a combination of mutations and pharmocological perturbations, we found that CD14 must be membrane bound to potentiate TLR4 activation by S100A9. Additionally, S100A9 is sensitive to inhibitors of pathways downstream of TLR4 internalization. Together, this suggests that S100A9 induces activity via CD14-dependent internalization of TLR4. We then used mutagenesis, structural modeling, and in vitro binding experiments to establish that S100A9 binds to CD14s N-terminus in a region that overlaps with, but is not identical to, the region where CD14 binds its canonical ligand, lipopolysaccharide (LPS). In molecular dynamics simulations, this region of the protein is dynamic, allowing it to reorganize to recognize both S100A9 (a soluble protein) and LPS (a small hydrophobic molecule). Our work is the first attempt at a molecular characterization of the S100A9/CD14 interaction, bringing us one step closer to unraveling the full mechanism by which S100A9 activates TLR4/MD-2.

biochemistry↗

bioRxiv2024

Navigating contradictions: Salmonella Typhimurium chemotactic responses to conflicting chemoeffector signals show parity with bacterial growth benefits

Motile bacteria sense and avoid deleterious stimuli in their environment through chemorepulsion, a behavior that helps them locate permissive ecological niches. In the gut, indole is a bacteriostatic compound produced by the microbiota and is thought to act as a chemorepellent for invading pathogens, thereby protecting the host against infection. The principal reservoir of intestinal indole is fecal matter, a complex biological material that contains both attractant and repellent stimuli. Whether indole in its natural context is sufficient for pathogen chemorepulsion or host protection has remained unknown. Using an intestinal explant system, we show that while pure indole indeed suppresses an infection advantage mediated through chemotaxis for the enteric pathogen Salmonella enterica serovar Typhimurium, this effect is abolished in the presence of other chemoeffectors present in feces, including the chemoattractant L-Serine (L-Ser), in a manner dependent on the chemoreceptor Tsr. Live imaging reveals that although S. Typhimurium is repelled by pure indole, the pathogen is actually strongly attracted to human fecal matter despite its high indole content, and that this response is mediated by Tsr, which simultaneously senses both indole and L-Ser. Fecal attraction is conserved across diverse Enterobacteriaceae species that harbor Tsr orthologues, including Escherichia coli, Citrobacter koseri, Enterobacter cloacae, and clinical isolates of non-typhoidal Salmonella. In a defined system of fecal chemoeffectors, we find that L-Ser and other fecal chemoattractants override indole chemorepulsion, but the magnitude of bacterial chemoattraction is controlled by indole levels. Together, these findings suggest that indole in its native context is not protective against enteric infection and that indole taxis actually benefits pathogens during infection by locating niches with low competitor density. Our study highlights the limitations of applying single-effector studies in predicting bacterial behavior in natural environments, where chemotaxis is shaped by the integration of multiple, often opposing, chemical signals.

microbiology↗

bioRxiv2023

Bacterial vampirism mediated through chemotaxis

Bacteria of the family Enterobacteriaceae are associated with gastrointestinal (GI) bleeding and bacteremia and are a leading cause of death, from sepsis, for individuals with inflammatory bowel diseases. The bacterial behaviors and mechanisms underlying why these bacteria are prone to bloodstream entry remains poorly understood. Herein, we report that clinical isolates of non-typhoidal Salmonella enterica serovars, Escherichia coli, and Citrobacter koseri are rapidly attracted toward sources of human serum. To simulate GI bleeding, we utilized a custom injection-based microfluidics device and found that femtoliter volumes of human serum are sufficient to induce the bacterial population to swim toward and aggregate at the serum source. This response is orchestrated through chemotaxis, and a major chemical cue driving chemoattraction is L-serine, an amino acid abundant in serum that is recognized through direct binding by the chemoreceptor Tsr. We report the first crystal structures of Salmonella Typhimurium Tsr in complex with L-serine and identify a conserved amino acid recognition motif for L-serine shared among Tsr orthologues. By mapping the phylogenetic distribution of this chemoreceptor we found Tsr to be widely conserved among Enterobacteriaceae and numerous World Health Organization priority pathogens associated with bloodstream infections. Lastly, we find that Enterobacteriaceae use human serum as a source of nutrients for growth and that chemotaxis and the chemoreceptor Tsr provides a competitive advantage for migration into enterohaemorrhagic lesions. We term this bacterial behavior of taxis toward serum, colonization of hemorrhagic lesions, and the consumption of serum nutrients, as "bacterial vampirism" which may relate to the proclivity of Enterobacteriaceae for bloodstream infections.

microbiology↗

bioRxiv2023

Simultaneous Mapping of DNA Binding and Nucleosome Positioning with SpLiT-ChEC

The organization of chromatin - including the positions of nucleosomes and the binding of other proteins to DNA - helps define transcriptional profiles in eukaryotic organisms. While techniques like ChIP-Seq and MNase-Seq can map protein-DNA and nucleosome localization separately, assays designed to simultaneously capture nucleosome positions and protein-DNA interactions can produce a detailed picture of the chromatin landscape. Most assays that monitor chromatin organization and protein binding rely on antibodies, which often exhibit nonspecific binding, and/or the addition of bulky adducts to the DNA-binding protein being studied, which can affect their expression and activity. Here, we describe SpyCatcher Linked Targeting of Chromatin Endogenous Cleavage (SpLiT-ChEC), where a 13-amino acid SpyTag peptide, appended to a protein of interest, serves as a highly-specific targeting moiety for in situ enzymatic digestion. The SpyTag/SpyCatcher system forms a covalent bond, linking the target protein and a co-expressed MNase-SpyCatcher fusion construct. SpyTagged proteins are expressed from endogenous loci, whereas MNase-SpyCatcher expression is induced immediately before harvesting cultures. MNase is activated with high concentrations of calcium, which primarily digests DNA near target protein binding sites. By sequencing the DNA fragments released by targeted MNase digestion, we found that this method recovers information on protein binding and proximal nucleosome positioning. SpLiT-ChEC provides precise temporal control that we anticipate can be used to monitor chromatin under various conditions and at distinct points in the cell cycle.

genomics↗

bioRxiv2023

The importance of input sequence set to consensus-derived proteins and their relationship to reconstructed ancestral proteins

A protein sequence encodes its energy landscape - all the accessible conformations, energetics, and dynamics. The evolutionary relationship between sequence and landscape can be probed phylogenetically by compiling a multiple sequence alignment of homologous sequences and generating common ancestors via Ancestral Sequence Reconstruction or a consensus protein containing the most common amino acid at each position. Both ancestral and consensus proteins are often more stable than their extant homologs - questioning the differences and suggesting that both approaches serve as general methods to engineer thermostability. We used the Ribonuclease H family to compare these approaches and evaluate how the evolutionary relationship of the input sequences affects the properties of the resulting consensus protein. While the overall consensus protein is structured and active, it neither shows properties of a well-folded protein nor has enhanced stability. In contrast, the consensus protein derived from a phylogenetically-restricted region is significantly more stable and cooperatively folded, suggesting that cooperativity may be encoded by different mechanisms in separate clades and lost when too many diverse clades are combined to generate a consensus protein. To explore this, we compared pairwise covariance scores using a Potts formalism as well as higher-order couplings using singular value decomposition (SVD). We find the SVD coordinates of a stable consensus sequence are close to coordinates of the analogous ancestor sequence and its descendants, whereas the unstable consensus sequences are outliers in SVD space.

biophysics↗

bioRxiv2022

Disentangling contact and ensemble epistasis in a riboswitch

Mutations introduced into macromolecules often exhibit epistasis, where the effect of one mutation alters the effect of another. Knowledge of the mechanisms that lead to epistasis is important for understanding how macromolecules work and evolve, as well as for effective macromolecular engineering. Here we investigate the interplay between "contact epistasis" (epistasis arising from physical interactions between mutated residues) and "ensemble epistasis" (epistasis that occurs when a mutation redistributes the conformational ensemble of a macromolecule, thus changing the effect of the second mutation). We argue that the two mechanisms can be distinguished in allosteric macromolecules by measuring epistasis at differing allosteric effector concentrations. Contacts give rise to epistasis in the microscopic equilibrium constants describing the conformational ensemble. Ensemble epistasis manifests in thermodynamic observables, such as the energy of ligand binding or enzyme activation, that depend on the concentration of allosteric effector. Using this framework, we experimentally investigated the origins of epistasis in three mutant cycles introduced into the adenine riboswitch aptamer domain. We found evidence for both contact and ensemble epistasis in all cycles. Further, we found that the two mechanisms of epistasis can interact with each other. For example, in one mutant cycle we observe contact epistasis of 6 kcal/mol attenuated by the ensemble to only 1.5 kcal/mol in the final thermodynamic observable. Finally, our work yields simple heuristics for identifying contact and ensemble epistasis using limited experimental measurements. Statement of significanceMutations to protein or RNA molecules often have different effects when introduced individually versus together. To understand and engineer biological macromolecules, we must identify the mechanistic origins of this phenomenon. Here, we measured the interplay between direct, physical interactions between mutations ("contact epistasis") and indirect interactions mediated by conformational ensembles ("ensemble epistasis"). We introduced pairs of mutations into an RNA molecule that transitions between several different conformations. We found epistasis arising from both contacts and the ensemble, and that the two mechanisms could synergize with one another. Our work reveals that one must consider the effects of mutations on multiple conformations to understand epistasis and suggests a few rules-of-thumb for disentangling contact and ensemble epistasis in other macromolecules.

biophysics↗

bioRxiv2022

An experimental demonstration of ensemble epistasis in the lac repressor

Epistatic, non-additive, interactions between mutations reveal the functional architecture of living systems, strongly shape evolution, and present a difficult challenge for bioengineers. Interpreting and modeling epistasis requires knowledge of the mechanisms that bring it about. We recently argued that "ensemble epistasis" could be a generic mechanism for epistasis between mutations introduced into a single macromolecule. Because proteins exist as ensembles of interconverting conformations, a mutation could induce epistasis by subtly altering ensemble composition and thus the effects of subsequent mutations. Here we show experimentally that the thermodynamic ensemble does indeed yield high magnitude epistasis in the lac repressor. We observed two- and three-way epistasis in DNA binding, with magnitudes as large or larger than the individual effects of mutations. This biophysical effect propagated to substantial epistasis in gene expression in vivo. As predicted in previous theoretical work, IPTG concentration tunes the magnitude of ensemble epistasis. Further, our observations could all be captured with a rigorous mathematical model of the lac repressor ensemble. Given that conformational ensembles are unavoidable features of macromolecules, we expect this is a ubiquitous and underappreciated cause of intramolecular epistasis.

biophysics↗

bioRxiv2022

Higher-order interactions shape microbial interactions as microbial community complexity increases

Non-pairwise interactions, or higher-order interactions (HOIs), in microbial communities have been claimed to explain the emergent features in microbiomes. Yet, the re-organization of microbial interactions between pairwise cultures and larger communities remains largely unexplored from a molecular perspective but is central to our understanding and further manipulation of microbial communities. Here, we used a bottom-up approach to investigate microbial interaction mechanisms from pairwise cultures up to 4-species communities from a simple microbiome (Hafnia alvei, Geotrichum candidum, Pencillium camemberti and Escherichia coli). Specifically, we characterized the interaction landscape for each species combination involving E. coli by identifying E. colis interaction-associated genes using an RB-TnSeq-based interaction assay. We observed a deep reorganization of the interaction-associated genes, with very few 2-species interactions conserved all the way up to a 4-species community and the emergence of multiple HOIs. We further used a quantitative genetics strategy to decipher how 2-species interactions were quantitatively conserved in higher community compositions. Epistasis-based analysis revealed that, of the interactions that are conserved at all levels of complexity, 82% follow an additive pattern. Altogether, we demonstrate the complex architecture of microbial interactions even within a simple microbiome, and provide a mechanistic and molecular explanation of HOIs.

microbiology↗

bioRxiv2022

Evolution avoids a pathological stabilizing interaction in the immune protein S100A9

Stability constrains evolution. While much is known about constraints on destabilizing mutations, less is known about the constraints on stabilizing mutations. We recently identified a mutation in the innate immune protein S100A9 that provides insight into such constraints. When introduced into human S100A9, M63F simultaneously increases the stability of the protein and disrupts its natural ability to activate Toll-like receptor 4. Using chemical denaturation, we found that M63F stabilizes a calcium-bound conformation of hS100A9. We then used NMR to solve the structure of the mutant protein, revealing that the mutation distorts the hydrophobic binding surface of hS100A9, explaining its deleterious effect on function. Hydrogen deuterium exchange (HDX) experiments revealed stabilization of the region around M63F in the structure, notably Phe37. In the structure of the M63F mutant, the Phe37 and Phe63 sidechains are in contact, plausibly forming an edge-face {nu}-stack. Mutating Phe37 to Leu abolished the stabilizing effect of M63F as probed by both chemical denaturation and HDX. It also restored the biological activity of S100A9 disrupted by M63F. These findings reveal that Phe63 creates a "molecular staple" with Phe37 that stabilizes a non-functional conformation of the protein, thus disrupting function. Using a bioinformatic analysis, we found that S100A9 proteins from different organisms rarely have Phe at both positions 37 and 63, suggesting that avoiding a pathological stabilizing interaction indeed constrains S100A9 evolution. This work highlights an important evolutionary constraint on stabilizing mutations: they must avoid inappropriately stabilizing non-functional protein conformations. SIGNIFICANCE STATEMENTProtein stability constrains protein evolution. While much is known about evolutionary constraints on destabilizing mutations, less is known about the constraints on stabilizing mutations. We recently found a mutation to an innate immune protein that increases its stability but disrupts its function. Here we show, through careful biophysical and functional studies, that this mutation stabilizes a nonfunctional form of the protein through a direct interaction with another amino acid. We find that specific amino acids can be tolerated at each of the interacting positions individually, but not at both simultaneously. This pattern has been conserved over millions of years of evolution. Our work highlights an underappreciated evolutionary constraint on stabilizing mutations: they must avoid inappropriately stabilizing non-functional protein conformations.

biophysics↗

bioRxiv2020

Ensemble epistasis: thermodynamic origins of non-additivity between mutations

Non-additivity between mutations--epistasis--profoundly shapes evolution. It can be difficult to understand its mechanistic origins. Here we show that "ensemble epistasis" is likely a universal feature of macromolecules. Using a simple analytical model, we found that epistasis arises when two conditions are met: 1) a macro-molecule populates at least three structures and 2) mutations have differential effects on a least two of the inactive structures. To explore the relative magnitude of ensemble epistasis, we performed a virtual deep-mutational scan of the allosteric Ca2+ signaling protein S100A4. We found that 27% of mutation pairs gave ensemble epistasis with a magnitude on the order of thermal fluctuations, 1 kT. We observed many forms of epistasis: magnitude, sign, and reciprocal sign epistasis. Depending on the effector concentration, the same mutation pair could even exhibit different forms of epistasis. The ubiquity of ensembles in biology and its pervasiveness in our dataset suggests that ensemble epistasis may be a universal mechanism of epistasis. Significance statementAddressing the mechanistic origins of evolutionary unpredictability is critical to understanding how mutations combine to determine phenotype. Here we lay the theoretical foundations and investigate the plausibility of a potentially universal mechanism of unpredictability in macromolecules. Macromolecules often adopt a set of interchanging structures, called a thermodynamic ensemble. Mutations can change the relative population of each structure, introducing unpredictability in the mapping between genotype and phenotype. The conditions under which we expect this to arise are common in macromolecules, suggesting that this form of unpredictability may be pervasive in evolution. We conclude that the thermodynamic ensemble bakes unpredictability into biology and that future attempts to address it might incorporate this mechanistic insight.

biophysics↗

bioRxiv2020

Exploring the evolutionary history of kinetic stability in the alpha-lytic protease family

In addition to encoding the final tertiary fold and stability, the primary sequence of a protein encodes the folding trajectory and kinetic barriers that determines the speed of folding. How these kinetic barriers are encoded by the sequence is not well understood. Here, we use evolutionary sequence variation in the alpha-lytic protease (LP) protein family to probe the relationship between sequence and energy landscape. LP has an unusual energy landscape: the native state of LP is not the most thermodynamically favored conformation and, instead, it remains folded due to a large kinetic barrier preventing unfolding. In order to fold, LP utilizes an N-terminal pro region of similar size to the protease itself that functions as a folding catalyst. Once folded, the pro region is removed, and the native state does not unfold on a biologically relevant timescale. Without the pro region, LP folds on the order of millennia. A phylogenetic search uncovers LP homologs with a wide range of pro-region sizes, including some with no pro region at all. In the resulting phylogenetic tree, these homologs cluster by pro-region size. Homologs naturally lacking pro regions are thermodynamically stable, fold much faster than LP, yet retain the same fold as LP. Key amino acids thought to contribute to LPs extreme kinetic stability are lost in these homologs, further supporting their role in kinetic stability. This study highlights how the entire energy landscape plays an important role in determining the evolutionary pressures on and changes to the protein sequence.

biophysics↗

bioRxiv2020

Learning Peptide Recognition Rules for a Low-Specificity Protein

Many proteins interact with short linear regions of target proteins. For some proteins, however, it is difficult to identify a well-defined sequence motif that defines its target peptides. To overcome this difficulty, we used supervised machine learning to train a model that treats each peptide as a collection of easily-calculated biochemical features rather than as an amino acid sequence. As a test case, we dissected the peptide-recognition rules for human S100A5 (hA5), a low-specificity calcium binding protein. We trained a Random Forest model against a recently released, high-throughput phage display dataset collected for hA5. The model identifies hydrophobicity and shape complementarity, rather than polar contacts, as the primary determinants of peptide binding specificity in hA5. We tested this hypothesis by solving a crystal structure of hA5 and through computational docking studies of diverse peptides onto hA5. These structural studies revealed that peptides exhibit multiple binding modes at the hA5 peptide interface--all of which have few polar contacts with hA5. Finally, we used our trained model to predict new, plausible binding targets in the human proteome. This revealed a fragment of the protein -1-syntrophin binds to hA5. Our work helps better understand the biochemistry and biology of hA5, as well as demonstrating how high-throughput experiments coupled with machine learning of biochemical features can reveal the determinants of binding specificity in low-specificity proteins.

biophysics↗

bioRxiv2020

Were ancestral proteins less specific?

Some have hypothesized that ancestral proteins were, on average, less specific than their descendants. If true, this would provide a universal axis along which to organize protein evolution and suggests that reconstructed ancestral proteins may be uniquely powerful tools for protein engineering. Ancestral sequence reconstruction studies are one line of evidence used to support this hypothesis. Previously, we performed such a study, investigating the evolution of peptide binding specificity for the paralogs S100A5 and S100A6. The modern proteins appeared more specific than their last common ancestor (ancA5/A6), as each paralog bound a subset of the peptides bound by ancA5/A6. In the current study, we revisit this transition, using quantitative phage display to measure the interactions of 19,194 random peptides with human S100A5, S100A6, and ancA5/A6. This unbiased screen reveals a different picture. While S100A5 and S100A6 do indeed bind to a subset of the peptides recognized by ancA5/A6, they also acquired new peptide partners outside of the set recognized by ancA5/A6. Our previous work showed that ancA5/A6 had lower specificity than its descendants when measured against biological targets; our new work shows that ancA5/A6 has similar specificity to the modern proteins when measured against a random set of peptide targets. This demonstrates that altered biological specificity does not necessarily indicate altered intrinsic specificity, and sounds a cautionary note for using ancestral reconstruction studies with biological targets as a means to infer global evolutionary trends in specificity.

evolutionary biology↗

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↗