Dynamic Changes in Lymphocyte Populations Establish Zebrafish as a Thymic Involution Model.

The thymus is the site of T lymphocyte development and T cell education to recognize foreign, but not self, Ags. B cells also reside and develop in the thymus, although their functions are less clear. During "thymic involution," a process of lymphoid atrophy and adipose replacement linked to sexual maturation, thymocytes decline. However, thymic B cells decrease far less than T cells, such that B cells comprise ∼1% of human neonatal thymocytes but up to ∼10% in adults. All jawed vertebrates possess a thymus, and we and others have shown zebrafish (Danio rerio) also have thymic B cells. In this article, we investigated the precise identities of zebrafish thymic T and B cells and how they change with involution. We assessed the timing and specific details of zebrafish thymic involution using multiple lymphocyte-specific, fluorophore-labeled transgenic lines, quantifying the changes in thymic T- and B-lymphocytes pre- versus postinvolution. Our results prove that, as in humans, zebrafish thymic B cells increase relative to T cells postinvolution. We also performed RNA sequencing on D. rerio thymic and marrow lymphocytes of four novel double-transgenic lines, identifying distinct populations of immature T and B cells. Collectively, this is, to our knowledge, the first comprehensive analysis of zebrafish thymic involution, demonstrating its similarity to human involution and establishing the highly genetically manipulatable zebrafish model as a template for involution studies.

Dynamic Changes in Lymphocyte Populations Establish Zebrafish as a Thymic Involution Model.

The thymus is the site of T lymphocyte development and T cell education to recognize foreign, but not self, antigens. B cells also reside and develop in the thymus, although their functions are less clear. During 'thymic involution,' a process of lymphoid atrophy and adipose replacement linked to sexual maturation, thymocytes decline. However, thymic B cells decrease far less than T cells, such that B cells comprise ~1% of human neonatal thymocytes, but up to ~10% in adults. All jawed vertebrates possess a thymus, and we and others have shown zebrafish (Danio rerio) also have thymic B cells. Here, we investigated the precise identities of zebrafish thymic T and B cells and how they change with involution. We assessed the timing and specific details of zebrafish thymic involution using multiple lymphocyte-specific, fluorophore-labeled transgenic lines, quantifying the changes in thymic T- and B-lymphocytes pre- vs. post-involution. Our results prove that, as in humans, zebrafish thymic B cells increase relative to T cells post-involution. We also performed RNA sequencing (RNA-seq) on D. rerio thymic and marrow lymphocytes of four novel double-transgenic lines, identifying distinct populations of immature T and B cells. Collectively, this is the first comprehensive analysis of zebrafish thymic involution, demonstrating its similarity to human involution, and establishing the highly genetically-manipulatable zebrafish model as a template for involution studies.

Analysis of CheW-like domains provides insights into organization of prokaryotic chemotaxis systems.

The ability to control locomotion in a dynamic environment provides a competitive advantage for microorganisms, thus driving the evolution of sophisticated regulatory systems. In total, 19 known categories of chemotaxis systems control motility mediated by flagella or Type IV pili, plus other cellular functions. A key feature that distinguishes chemotaxis systems from generic two-component regulatory systems is separation of receptor and kinase functions into distinct proteins, linked by CheW scaffold proteins. This arrangement allows for formation of varied arrays with remarkable signaling properties. We recently analyzed sequences of CheW-like domains found in CheA kinases and CheW and CheV scaffold proteins. In total, 16 Architectures of CheA, CheW, and CheV proteins contain ~94% of all CheW-like domains and form six Classes with likely functional specializations. We surveyed chemotaxis system categories and proteins containing CheW-like domains in ~1900 prokaryotic species, the most comprehensive analysis to date, revealing new insights. Co-occurrence analyses suggested that many chemotaxis systems occur in non-random combinations within species, implying synergy or antagonism. Furthermore, many Architectures of proteins containing CheW-like domains occurred predominantly with specific categories of chemotaxis systems, suggesting specialized functional interactions. We propose Class 1 (~80%) and Class 6 (~20%) CheW proteins exhibit preferences for distinct chemoreceptor structures. Furthermore, rare (~1%) Class 2 CheW proteins frequently co-occurred with methyl-accepting coiled coil proteins, which contain both receptor and kinase functions and so do not require connection via a CheW scaffold but may benefit from arrays. Last, rare multidomain CheW proteins may interact with different receptors than single-domain CheW proteins.

General strategies for using amino acid sequence data to guide biochemical investigation of protein function.

The rapid increase of '-omics' data warrants the reconsideration of experimental strategies to investigate general protein function. Studying individual members of a protein family is likely insufficient to provide a complete mechanistic understanding of family functions, especially for diverse families with thousands of known members. Strategies that exploit large amounts of available amino acid sequence data can inspire and guide biochemical experiments, generating broadly applicable insights into a given family. Here we review several methods that utilize abundant sequence data to focus experimental efforts and identify features truly representative of a protein family or domain. First, coevolutionary relationships between residues within primary sequences can be successfully exploited to identify structurally and/or functionally important positions for experimental investigation. Second, functionally important variable residue positions typically occupy a limited sequence space, a property useful for guiding biochemical characterization of the effects of the most physiologically and evolutionarily relevant amino acids. Third, amino acid sequence variation within domains shared between different protein families can be used to sort a particular domain into multiple subtypes, inspiring further experimental designs. Although generally applicable to any kind of protein domain because they depend solely on amino acid sequences, the second and third approaches are reviewed in detail because they appear to have been used infrequently and offer immediate opportunities for new advances. Finally, we speculate that future technologies capable of analyzing and manipulating conserved and variable aspects of the three-dimensional structures of a protein family could lead to broad insights not attainable by current methods.

Generalizable strategy to analyze domains in the context of parent protein architecture: A CheW case study.

Domains are the three-dimensional building blocks of proteins. An individual domain can occur in a variety of domain architectures that perform unique functions and are subject to different evolutionary selective pressures. We describe an approach to evaluate the variability in amino acid sequences of a single domain across architectural contexts. The ability to distinguish different evolutionary outcomes of one protein domain can help determine whether existing knowledge about a specific domain will apply to an uncharacterized protein, lead to insights and hypotheses about function, and guide experimental priorities. We developed and tested our approach on CheW-like domains (PF01584), which mediate protein/protein interactions and are difficult to compare experimentally. CheW-like domains occur in CheW scaffolding proteins, CheA kinases, and CheV proteins that regulate bacterial chemotaxis. We analyzed 16 domain architectures that included 94% of all CheW-like domains found in nature. We identified six Classes of CheW-like domains with presumed functional differences. CheV and most CheW proteins contained Class 1 domains, whereas some CheW proteins contained Class 6 (~20%) or Class 2 (~1%) domains instead. Most CheA proteins contained Class 3 domains. CheA proteins with multiple Hpt domains contained Class 4 domains. CheA proteins with two CheW-like domains contained one Class 3 and one Class 5. We also created SimpLogo, an innovative method for visualizing amino acid composition across large sets of multiple sequence alignments of arbitrary length. SimpLogo offers substantial advantages over standard sequence logos for comparison and analysis of related protein sequences. The R package for SimpLogo is freely available.

Predicted Functional and Structural Diversity of Receiver Domains in Fungal Two-Component Regulatory Systems.

Fungal two-component regulatory systems incorporate receiver domains into hybrid histidine kinases (HHKs) and response regulators. We constructed a nonredundant database of 670 fungal receiver domain sequences from 51 species sampled from nine fungal phyla. A much greater proportion (21%) of predicted fungal response regulators did not belong to known groups than previously appreciated. Receiver domains in Rim15 response regulators from Ascomycota and other phyla are very different from one another, as are the duplicate receiver domains in group XII HHKs. Fungal receiver domains from five known types of response regulators and 20 known types of HHKs exhibit distinct patterns of amino acids at conserved and variable positions known to be structurally and functionally important in bacterial receiver domains. We inferred structure/activity relationships from the patterns and propose multiple experimentally testable hypotheses about the mechanisms of signal transduction mediated by fungal receiver domains.

Role of Position K+4 in the Phosphorylation and Dephosphorylation Reaction Kinetics of the CheY Response Regulator.

Two-component signaling is a primary method by which microorganisms interact with their environments. A kinase detects stimuli and modulates autophosphorylation activity. The signal propagates by phosphotransfer from the kinase to a response regulator, eliciting a response. Response regulators operate over a range of time scales, corresponding to their related biological processes. Response regulator active site chemistry is highly conserved, but certain variable residues can influence phosphorylation kinetics. An Ala-to-Pro substitution (K+4, residue 113) in the Escherichia coli response regulator CheY triggers a constitutively active phenotype; however, the A113P substitution is too far from the active site to directly affect phosphochemistry. To better understand the activating mechanism(s) of the substitution, we analyzed receiver domain sequences to characterize the evolutionary role of the K+4 position. Although most featured Pro, Leu, Ile, and Val residues, chemotaxis-related proteins exhibited atypical Ala, Gly, Asp, and Glu residues at K+4. Structural and in silico analyses revealed that CheY A113P adopted a partially active configuration. Biochemical data showed that A113P shifted CheY toward a more activated state, enhancing autophosphorylation. By characterizing CheY variants, we determined that this functionality was transmitted through a hydrophobic network bounded by the ß5α5 loop and the α1 helix of CheY. This region also interacts with the phosphodonor CheAP1, suggesting that binding generates an activating perturbation similar to the A113P substitution. Atypical residues like Ala at the K+4 position likely serve two purposes. First, restricting autophosphorylation may minimize background noise generated by intracellular phosphodonors such as acetyl phosphate. Second, optimizing interactions with upstream partners may help prime the receiver domain for phosphorylation.

A Radical Reimagining of Fungal Two-Component Regulatory Systems.

Bacterial two-component regulatory systems (TCSs) mediate signal transduction by transferring phosphoryl groups between sensor kinase and response regulator proteins, sometimes using intermediary histidine-phosphotransferase (Hpt) domains to form multistep phosphorelays. Because (i) almost all known fungal sensor kinases exhibit a domain architecture characteristic of bacterial TCS phosphorelays, (ii) all known fungal Hpts are stand-alone proteins suited to shuttle between cytoplasm and nucleus, and (iii) the best-characterized fungal TCS is a canonical phosphorelay, it is widely assumed that most or all fungal TCSs function via phosphorelays. However, fungi generally encode more sensor kinases than Hpts or response regulators, leading to a disparity between putative phosphorelay inputs and outputs. The simplest resolution of this paradox is to hypothesize that most fungal sensor kinases do not participate in phosphorelays. Reimagining how fungal TCSs might function leads to multiple testable predictions.

Modulation of Response Regulator CheY Reaction Kinetics by Two Variable Residues That Affect Conformation.

Microorganisms and plants utilize two-component systems to regulate adaptive responses to changing environmental conditions. Sensor kinases detect stimuli and alter their autophosphorylation activity accordingly. Signal propagation occurs via the transfer of phosphoryl groups from upstream kinases to downstream response regulator proteins. Removal of phosphoryl groups from the response regulator typically resets the system. Members of the same protein family may catalyze phosphorylation and dephosphorylation reactions with different efficiencies, exhibiting rate constants spanning many orders of magnitude to accommodate response time scales from milliseconds to days. We previously found that variable positions one or two residues to the C-terminal side of the conserved Asp phosphorylation site (D+2) or Thr/Ser (T+1/T+2) in response regulators alter reaction kinetics by direct interaction with phosphodonor or phosphoacceptor molecules. Here, we explore the kinetic effects of amino acid substitutions at the two positions immediately C-terminal to the conserved Lys (K+1/K+2) in the model Escherichia coli response regulator CheY. We measured CheY autophosphorylation and autodephosphorylation rate constants for 27 pairs of K+1/K+2 residues that represent 84% of naturally occurring response regulators. Effects on autodephosphorylation were modest, but autophosphorylation rate constants varied by 2 orders of magnitude, suggesting that the K+1/K+2 positions influence reaction kinetics by altering the conformational spectrum sampled by CheY at equilibrium. Additional evidence supporting this indirect mechanism includes the following: the effect on autophosphorylation rate constants is independent of the phosphodonor, the autophosphorylation rate constants and dissociation constants for the phosphoryl group analog BeF3- are inversely correlated, and the K+1/K+2 positions are distant from the phosphorylation site.IMPORTANCE We have identified five variable positions in response regulators that allow the rate constants of autophosphorylation and autodephosporylation reactions each to be altered over 3 orders of magnitude in CheY. The distributions of variable residue combinations across response regulator subfamilies suggest that distinct mechanisms associated with different variable positions allow reaction rates to be tuned independently during evolution for diverse biological purposes. This knowledge could be used in synthetic-biology applications to adjust the properties (e.g., background noise and response duration) of biosensors and may allow prediction of response regulator reaction kinetics from the primary amino acid sequence.

Zebrafish B cell acute lymphoblastic leukemia: new findings in an old model.

Acute lymphoblastic leukemia (ALL) is the most common pediatric, and ninth most common adult, cancer. ALL can develop in either B or T lymphocytes, but B-lineage ALL (B-ALL) exceeds T-ALL clinically. As for other cancers, animal models allow study of the molecular mechanisms driving ALL. Several zebrafish (Danio rerio) T-ALL models have been reported, but until recently, robust D. rerio B-ALL models were not described. Then, D. rerio B-ALL was discovered in two related zebrafish transgenic lines; both were already known to develop T-ALL. Here, we report new B-ALL findings in one of these models, fish expressing transgenic human MYC (hMYC). We describe B-ALL incidence in a large cohort of hMYC fish, and show B-ALL in two new lines where T-ALL does not interfere with B-ALL detection. We also demonstrate B-ALL responses to steroid and radiation treatments, which effect ALL remissions, but are usually followed by prompt relapses. Finally, we report gene expression in zebrafish B lymphocytes and B-ALL, in both bulk samples and single B- and T-ALL cells. Using these gene expression profiles, we compare differences between the two new D. rerio B-ALL models, which are both driven by transgenic mammalian MYC oncoproteins. Collectively, these new data expand the utility of this new vertebrate B-ALL model.

Insights revealed by the co-crystal structure of the Saccharomyces cerevisiae histidine phosphotransfer protein Ypd1 and the receiver domain of its downstream response regulator Ssk1.

Two-component signaling systems are the primary means by which bacteria, archaea, and certain plants and fungi react to their environments. The model yeast, Saccharomyces cerevisiae, uses the Sln1 signaling pathway to respond to hyperosmotic stress. This pathway contains a hybrid histidine kinase (Sln1) that autophosphorylates and transfers a phosphoryl group to its own receiver domain (R1). The phosphoryl group is then transferred to a histidine phosphotransfer protein (Ypd1) that finally passes it to the receiver domain (R2) of a downstream response regulator (Ssk1). Under normal conditions, Ssk1 is constitutively and preferentially phosphorylated in the phosphorelay. Upon detecting hyperosmotic stress, Ssk1 rapidly dephosphorylates and activates the high-osmolarity glycerol (HOG) pathway, initiating a response. Despite their distinct physiological roles, both Sln1 and Ssk1 bind to Ypd1 at a common docking site. Co-crystal structures of response regulators in complex with their phosphorelay partners are scarce, leaving many mechanistic and structural details uncharacterized for systems like the Sln1 pathway. In this work, we present the co-crystal structure of Ypd1 and a near wild-type variant of the receiver domain of Ssk1 (Ssk1-R2-W638A) at a resolution of 2.80 Å. Our structural analyses of Ypd1-receiver domain complexes, biochemical determination of binding affinities for Ssk1-R2 variants, in silico free energy estimates, and sequence comparisons reveal distinctive electrostatic properties of the Ypd1/Ssk1-R2-W638A complex that may provide insight into the regulation of the Sln1 pathway as a function of dynamic osmolyte concentration.

Role of the highly conserved G68 residue in the yeast phosphorelay protein Ypd1: implications for interactions between histidine phosphotransfer (HPt) and response regulator proteins.

BACKGROUND: Many bacteria and certain eukaryotes utilize multi-step His-to-Asp phosphorelays for adaptive responses to their extracellular environments. Histidine phosphotransfer (HPt) proteins function as key components of these pathways. HPt proteins are genetically diverse, but share a common tertiary fold with conserved residues near the active site. A surface-exposed glycine at the H + 4 position relative to the phosphorylatable histidine is found in a significant number of annotated HPt protein sequences. Previous reports demonstrated that substitutions at this position result in diminished phosphotransfer activity between HPt proteins and their cognate signaling partners. RESULTS: We report the analysis of partner binding interactions and phosphotransfer activity of the prototypical HPt protein Ypd1 from Saccharomyces cerevisiae using a set of H + 4 (G68) substituted proteins. Substitutions at this position with large, hydrophobic, or charged amino acids nearly abolished phospho-acceptance from the receiver domain of its upstream signaling partner, Sln1 (Sln1-R1). An in vitro binding assay indicated that G68 substitutions caused only modest decreases in affinity between Ypd1 and Sln1-R1, and these differences did not appear to be large enough to account for the observed decrease in phosphotransfer activity. The crystal structure of one of these H + 4 mutants, Ypd1-G68Q, which exhibited a diminished ability to participate in phosphotransfer, shows a similar overall structure to that of wild-type. Molecular modelling suggests that the highly conserved active site residues within the receiver domain of Sln1 must undergo rearrangement to accommodate larger H + 4 substitutions in Ypd1. CONCLUSIONS: Phosphotransfer reactions require precise arrangement of active site elements to align the donor-acceptor atoms and stabilize the transition state during the reaction. Any changes likely result in an inability to form a viable transition state during phosphotransfer. Our data suggest that the high degree of evolutionary conservation of residues with small side chains at the H + 4 position in HPt proteins is required for optimal activity and that the presence of larger residues at the H + 4 position would cause alterations in the positioning of active site residues in the partner response regulator.

Use of restrained molecular dynamics to predict the conformations of phosphorylated receiver domains in two-component signaling systems.

Two-component signaling (TCS) is the primary means by which bacteria, as well as certain plants and fungi, respond to external stimuli. Signal transduction involves stimulus-dependent autophosphorylation of a sensor histidine kinase and phosphoryl transfer to the receiver domain of a downstream response regulator. Phosphorylation acts as an allosteric switch, inducing structural and functional changes in the pathway's components. Due to their transient nature, phosphorylated receiver domains are challenging to characterize structurally. In this work, we provide a methodology for simulating receiver domain phosphorylation to predict conformations that are nearly identical to experimental structures. Using restrained molecular dynamics, phosphorylated conformations of receiver domains can be reliably sampled on nanosecond timescales. These simulations also provide data on conformational dynamics that can be used to identify regions of functional significance related to phosphorylation. We first validated this approach on several well-characterized receiver domains and then used it to compare the upstream and downstream components of the fungal Sln1 phosphorelay. Our results demonstrate that this technique provides structural insight, obtained in the absence of crystallographic or NMR information, regarding phosphorylation-induced conformational changes in receiver domains that regulate the output of their associated signaling pathway. To our knowledge, this is the first time such a protocol has been described that can be broadly applied to TCS proteins for predictive purposes. Proteins 2016; 85:155-176. © 2016 Wiley Periodicals, Inc.