The Bithorax Complex (BX-C) homeobox proteins specify segmental identities along the anterior-posterior axis during Drosophila embryogenesis. Differential expression of the BX-C genes abd-A and Abd-B distinguishes abdominal from thoracic adipocytes, yet the mechanism regulating this heterogeneity remains poorly understood. Here, we identify cis-regulatory elements (CREs) and transcription factors that direct abdominal-specific expression of abd-A and Abd-B in the larval fat body. Fine-mapping analyses identified a 627-bp CRE within the Abd-B locus and a ~6-kb CRE within the abd-A locus sufficient to drive heterogeneous expression. Yeast one-hybrid screening combined with functional analyses identified Lola, Lolal, and Combgap as key repressors of Abd-B, whereas Piragua (Prg) and Seven up (Svp) function as transcriptional activators, indicating that adipocyte heterogeneity in postembryonic adipose tissue is actively regulated. In turn, lola and prg are repressed by Abd-B, whereas lolal and svp are activated, forming a feedback circuit further modulated by Wnt signaling, which promotes lola and lolal expression while repressing svp. CUT&RUN analyses suggest that these interactions are direct, with dTCF/Pan and Abd-B occupancy detected at target loci. Together, our findings define a transcriptional circuit that regulates Abd-B gene transcription to pattern adipose tissue and may establish the developmental basis of fat depot specialization.

Adipocytes play essential roles in lipid metabolism and energy homeostasis, with regional differences affecting their functions and disease susceptibility. However, the mechanisms underlying this regional heterogeneity remain unclear. Here we demonstrate that the Bithorax Complex (BX-C) genes, specifically abdominal A (abd-A) and Abdominal B (Abd-B), define regional differences in Drosophila larval adipocytes. Abdominal adipocytes, expressing abd-A and Abd-B exhibit unique characteristics compared to thoracic adipocytes, with active Wnt/Wingless signaling further amplifying these regional differences. Depleting abd-A and Abd-B in adipocytes delays larval-pupal transition, causes pupal lethality, and attenuates the expression of Wnt/Wg target genes, thereby dampening Wnt signaling-induced lipid mobilization. Additionally, Wnt signaling enhances the transcription of abd-A and Abd-B, establishing a feedforward loop that reinforces the interplay between Wnt signaling and BX-C genes. These findings reveal how the cell-autonomous expression of BX-C genes defines adipocyte heterogeneity, a process further modulated by Wnt signaling in Drosophila larvae.

The Drosophila intrinsically disordered protein Ultrabithorax (Ubx) undergoes a series of phase transitions, beginning with noncovalent interactions between apparently randomly organized monomers, and evolving over time to form increasingly ordered coacervates. This assembly process ends when specific dityrosine covalent bonds lock the monomers in place, forming macroscale materials. Inspired by this hierarchical, multistep assembly process, we analyzed the impact of protein concentration, assembly time, and subphase composition on the early, noncovalent stages of Ubx assembly, which are extremely sensitive to their environment. We discovered that in low salt buffers, we can generate a new type of Ubx material from early coacervates using 5-fold less protein, and 100-fold less assembly time. Comparison of the new materials with standard Ubx fibers also revealed differences in the extent of wrinkling on the fiber surface. A new image analysis technique based on autocorrelation of scanning electron microscopy (SEM) images was developed to quantify these structural differences. These differences extend to the molecular level: new materials form more dityrosine covalent cross-links per monomer, but without requiring the specific tyrosine residues necessary for crosslinking previously established materials. We conclude that varying the assembly conditions represents a facile and inexpensive process for creating new materials. Most new biopolymers are created by changing the composition of the monomers or the method used to drive assembly. In contrast, in this study we used the same monomers and assembly approach, but altered the assembly time and chemical environment to create a new material with unique properties.

The ability to add bioactivities, such as cell signaling or ligand recognition, to biomaterials has generated the potential to include multiple bioactivities into a single material. In some cases, it is desirable to localize these activities to different areas of the biomaterial, creating functional patterns. While photolithography and 3D printing have been effective techniques for patterning functions in many materials, patterning remains a challenge in materials composed of protein, in part due to how these materials are artificially assembled. Protein fibers are often produced from protein films that co-acervate at the air-water interface. This chapter describes methods to leverage this coacervation process to pattern materials, using the Drosophila melanogaster Hox protein Ultrabithorax (Ubx) as a model self-assembling protein. Through gene fusion, Ubx and a functional protein are produced as a single polypeptide, capable of both forming materials and performing the activity of interest. This functionality is retained in the final materials. In this chapter, we describe how to use multiple Ubx fusion proteins to not only imbue the final materials with multiple functions, but also to create macroscale patterns of the appended proteins in fibrous protein-based materials. These patterned materials include striped fibers, bifunctional-faced fibers, gradient fibers, and core-shell fibers.

Leave-one-out green fluorescent proteins (LOO_GFPs) have a reduced quantum yield relative to the parent protein and form fluorescent oligomers in the unbound state. Immobilizing LOO_GFPs in materials composed of the Drosophila protein Ultrabithorax (Ubx) via gene fusion increased the fluorescent signal, significantly stabilized the biosensor, and prevented oligomerization into fluorescent aggregates, which has the potential to elevate the sensor's noise well above the signal. Interactions between LOO_GFP and Ubx hampered analyte rebinding. By optimizing the concentrations of LOO_GFP, salt, and detergent in the assay, the signal to noise ratio for the biosensor increased fourfold. These modified fibers represent the first incorporation of a protein complementation assay into protein-based materials, as well as the first incorporation, via gene fusion, of a heterodimeric functional protein into materials composed of a different self-assembling protein. This study highlights the advantages and identifies potential pitfalls associated with protein immobilization in materials.

Adipocytes distributed throughout the body play crucial roles in lipid metabolism and energy homeostasis. Regional differences among adipocytes influence normal function and disease susceptibility, but the mechanisms driving this regional heterogeneity remain poorly understood. Here, we report a genetic crosstalk between the Bithorax Complex ( BX-C ) genes and Wnt/Wingless signaling that orchestrates regional differences among adipocytes in Drosophila larvae. Abdominal adipocytes, characterized by the exclusive expression of abdominal A ( abd-A ) and Abdominal B ( Abd-B ), exhibit distinct features compared to thoracic adipocytes, with Wnt signaling further amplifying these disparities. Depletion of BX-C genes in adipocytes reduces fat accumulation, delays larval-pupal transition, and eventually leads to pupal lethality. Depleting Abd-A or Abd-B reduces Wnt target gene expression, thereby attenuating Wnt signaling-induced lipid mobilization. Conversely, Wnt signaling stimulated abd-A transcription, suggesting a feedforward loop that amplifies the interplay between Wnt signaling and BX-C in adipocytes. These findings elucidate how the crosstalk between cell-autonomous BX-C gene expression and Wnt signaling define unique metabolic behaviors in adipocytes in different anatomical regions of fat body, delineating larval adipose tissue domains.

Proteins implement many useful functions, including binding ligands with unparalleled affinity and specificity, catalyzing stereospecific chemical reactions, and directing cell behavior. Incorporating proteins into materials has the potential to imbue devices with these desirable traits. This review highlights recent advances in creating active materials by genetically fusing a self-assembling protein to a functional protein. These fusion proteins form materials while retaining the function of interest. Key advantages of this approach include elimination of a separate functionalization step during materials synthesis, uniform and dense coverage of the material by the functional protein, and stabilization of the functional protein. This review focuses on macroscale materials and discusses (i) multiple strategies for successful protein fusion design, (ii) successes and limitations of the protein fusion approach, (iii) engineering solutions to bypass any limitations, (iv) applications of protein fusion materials, including tissue engineering, drug delivery, enzyme immobilization, electronics, and biosensing, and (v) opportunities to further develop this useful technique.

The leave-one-out (LOO) green fluorescent protein (GFP) approach to biosensor design combines computational protein design with split protein reconstitution. LOO-GFPs reversibly fold and gain fluorescence upon encountering the target peptide, which can be redefined by computational design of the LOO site. Such an approach can be used to create reusable biosensors for the early detection of emerging biological threats. Enlightening biophysical inferences for nine LOO-GFP biosensor libraries are presented, with target sequences from dengue, influenza, or HIV, replacing beta strands 7, 8, or 11. An initially low hit rate was traced to components of the energy function, manifesting in the over-rewarding of over-tight side chain packing. Also, screening by colony picking required a low library complexity, but designing a biosensor against a peptide of at least 12 residues requires a high-complexity library. This double-bind was solved using a “piecemeal” iterative design strategy. Also, designed LOO-GFPs fluoresced in the unbound state due to unwanted dimerization, but this was solved by fusing a fully functional prototype LOO-GFP to a fiber-forming protein, Drosophila ultrabithorax, creating a biosensor fiber. One influenza hemagglutinin biosensor is characterized here in detail, showing a shifted excitation/emission spectrum, a micromolar affinity for the target peptide, and an unexpected photo-switching ability.

Leave-One-Out Green Fluorescent Proteins (LOO_GFPs) unite a peptide sensor and detection method in a single biocompatible molecule. In LOO_GFPs, one β-strand of the GFP protein is removed and the cavity is re-engineered to specifically bind a desired analyte. Unfortunately, LOO_GFPs have a reduced quantum yield relative to the parent protein, and form fluorescent oligomers in the unbound state, reducing signal-to-noise. Immobilizing LOO_GFPs in materials composed of the Drosophila protein Ultrabithorax (Ubx) via gene fusion both increased the fluorescent signal and prevented oligomerization, substantially reducing background noise. These fibers represent the first incorporation of aheterodimeric protein into materials via gene fusion. Interactions between LOO_GFP and Ubx that hampered analyte rebinding were mitigated by optimizing salt and detergent concentrations in the assay. The result is a useful first-generation fluorescent biosensor, immobilized in and stabilized by robust protein fibers. This study highlights the advantages and identifies potential pitfalls associated with protein immobilization in materials.

Signaling pathways allow cells to detect and respond to a wide variety of chemical (e.g. Ca2+ or chemokine proteins) and physical stimuli (e.g., sheer stress, light). Together, these pathways form an extensive communication network that regulates basic cell activities and coordinates the function of multiple cells or tissues. The process of cell signaling imposes many demands on the proteins that comprise these pathways, including the abilities to form active and inactive states, and to engage in multiple protein interactions. Furthermore, successful signaling often requires amplifying the signal, regulating or tuning the response to the signal, combining information sourced from multiple pathways, all while ensuring fidelity of the process. This sensitivity, adaptability, and tunability are possible, in part, due to the inclusion of intrinsically disordered regions in many proteins involved in cell signaling. The goal of this collection is to highlight the many roles of intrinsic disorder in cell signaling. Following an overview of resources that can be used to study intrinsically disordered proteins, this review highlights the critical role of intrinsically disordered proteins for signaling in widely diverse organisms (animals, plants, bacteria, fungi), in every category of cell signaling pathway (autocrine, juxtacrine, intracrine, paracrine, and endocrine) and at each stage (ligand, receptor, transducer, effector, terminator) in the cell signaling process. Thus, a cell signaling pathway cannot be fully described without understanding how intrinsically disordered protein regions contribute to its function. The ubiquitous presence of intrinsic disorder in different stages of diverse cell signaling pathways suggest that more mechanisms by which disorder modulates intra- and inter-cell signals remain to be discovered.

For proteins, the sequence → structure → function paradigm applies primarily to enzymes, transmembrane proteins, and signaling domains. This paradigm is not universal, but rather, in addition to structured proteins, intrinsically disordered proteins and regions (IDPs and IDRs) also carry out crucial biological functions. For these proteins, the sequence → IDP/IDR ensemble → function paradigm applies primarily to signaling and regulatory proteins and regions. Often, in order to carry out function, IDPs or IDRs cooperatively interact, either intra- or inter-molecularly, with structured proteins or other IDPs or intermolecularly with nucleic acids. In this IDP/IDR thematic collection published in Cell Communication and Signaling, thirteen articles are presented that describe IDP/IDR signaling molecules from a variety of organisms from humans to fruit flies and tardigrades (“water bears”) and that describe how these proteins and regions contribute to the function and regulation of cell signaling. Collectively, these papers exhibit the diverse roles of disorder in responding to a wide range of signals as to orchestrate an array of organismal processes. They also show that disorder contributes to signaling in a broad spectrum of species, ranging from micro-organisms to plants and animals.

During animal development, HOX transcription factors determine the fate of developing tissues to generate diverse organs and appendages. The power of these proteins is striking: mis-expressing a HOX protein causes homeotic transformation of one body part into another. During development, HOX proteins interpret their cellular context through protein interactions, alternative splicing, and post-translational modifications to regulate cell proliferation, cell death, cell migration, cell differentiation, and angiogenesis. Although mutation and/or mis-expression of HOX proteins during development can be lethal, changes in HOX proteins that do not pattern vital organs can result in survivable malformations. In adults, mutation and/or mis-expression of HOX proteins disrupts their gene regulatory networks, deregulating cell behaviors and leading to arthritis and cancer. On the molecular level, HOX proteins are composed of DNA binding homeodomain, and large regions of unstructured, or intrinsically disordered, protein sequence. The primary roles of HOX proteins in arthritis and cancer suggest that mutations associated with these diseases in both the structured and disordered regions of HOX proteins can have substantial functional effects. These insights lead to new questions critical for understanding and manipulating HOX function in physiological and pathological conditions.

Proteins can bind, release, and detect molecules with unparalleled specificity. Incorporating proteins into materials can confer the ability to recognize specific ligands. We have leveraged the ability of materials composed of the Drosophila transcription factor Ultrabithorax (Ubx), a partially intrinsically disordered protein, to bind ligands, with the goals of delivering DNA to cells and creating biosensors. DNA binding and delivery is accomplished via the Ubx homeodomain, which retains its ability to recognize specific DNA sequences in Ubx materials. Ubx materials reversibly bind DNAs harboring the Ubx target DNA sequence and can protect these DNAs from degradation. DNA sequences with multiple binding sites have a slower rate of release from Ubx fibers. Ubx fibers can deliver DNA to transform E. coli, sustaining DNA release for up to 60 days.To build a biosensor, an additional protein (LOO8GFP) is incorporated into the materials via gene fusion. In this approach, the loo8gfp gene is placed adjacent to the ubx gene without intervening stop codons. When transformed into bacteria, this fused gene produces a LOO8GFP‐Ubx fusion protein. The intrinsically disordered regions of Ubx enable LOO8GFP incorporation without disrupting materials assembly or structure. The proof‐of‐concept sensor system is leave‐one‐out GFP (LOO8GFP), in which the 8th β‐strand has been ‘left out’ of circularly permuted GFP. Without this β‐strand the GFP is unable to fold properly and is not fluorescent. Fluorescence can be reconstituted with the addition of the left‐out peptide, making this construct a self‐reporting biosensor for the missing piece of its sequence. However, LOO8GFP can oligomerize, which (i) hampers ligand binding and (ii) leads to fluorescence in the unbound state. Ubx offers a transparent support system for LOO8GFP that maintains LOO8GFP stability and binding affinity while preventing LOO8GFP aggregation. To assess the utility of this composite material, we are examining assembly of LOO8GFP‐Ubx fibers, measuring binding affinities, and quantifying on‐ and off‐rates for the left‐out peptides. In the long term, the LOO8GFP‐Ubx system can be engineered to bind and detect other peptide sequences. Thus, Ubx materials can be engineered for variety of potential functions and uses based on incorporating bioactive DNAs and/or proteins.

During animal development, Hox transcription factors cause similar regions of the body to acquire different fates and drive the creation of organs and appendages unique to each region. DNA binding in Hox proteins is accomplished via a 60 aa homeodomain. The extreme conservation of homeodomain sequence and DNA binding ability in vitro belies the diverse and critical activities of full-length Hox proteins in vivo. This conundrum, termed the “Hox paradox” suggests that sequences outside the homeodomain, which are largely intrinsically disordered, must somehow diversify DNA recognition. Within the disordered regions of the Drosophila melanogaster Hox protein Ultrabithorax (Ubx), mutation of short, evolutionarily conserved motifs profoundly influences DNA binding, and can either significantly decrease or increase affinity. Comparison of fluorescence and heat capacity measurements as well as electron microscopy data for wild-type and mutant Ubx proteins suggests that Ubx adopts an ensemble of conformations bounded by two structures, a “closed” state in which the short motifs bind the homeodomain and sterically preclude DNA interaction, and an “open” state in which the homeodomain is exposed and able to recognize DNA. Mutations either enhance or inhibit DNA binding by shifting the structural ensemble. This model provides a mechanism by which DNA binding by Hox transcription factors can be regulated by multiple environmental cues such as phosphorylation, protein interactions, and alternative splicing. Sequence comparisons within the Drosophila Hox protein family suggest that this model may also explain the phenomenon of posterior prevalence, in which ectopic expression of a posteriorly expressed Hox protein can subvert the fate of a more anterior region of the body, but anterior Hox proteins cannot alter the fate of a more posterior body region.

Linking changes in amino acid sequences to the evolution of transcription regulatory domains is often complicated by the low sequence complexity and high mutation rates of intrinsically disordered protein regions. For the Hox transcription factor Ultrabithorax (Ubx), conserved motifs distributed throughout the protein sequence enable direct comparison of specific protein regions, despite variations in the length and composition of the intervening sequences. In cell culture, the strength of transcription activation by Drosophila melanogaster Ubx correlates with the presence of a predicted helix within its activation domain. Curiously, this helix is not preserved in species more divergent than flies, suggesting the nature of transcription activation may have evolved. To determine whether this helix contributes to Drosophila Ubx function in vivo, wild-type and mutant proteins were ectopically expressed in the developing wing and the phenotypes evaluated. Helix mutations alter Drosophila Ubx activity in the developing wing, demonstrating its functional importance in vivo. The locations of activation domains in Ubx orthologues were identified by testing the ability of truncation mutants to activate transcription in yeast one-hybrid assays. In Ubx orthologues representing 540 million years of evolution, the ability to activate transcription varies substantially. The sequence and the location of the activation domains also differ. Consequently, analogous regions of Ubx orthologues change function over time, and may activate transcription in one species, but have no activity, or even inhibit transcription activation in another species. Unlike homeodomain-DNA binding, the nature of transcription activation by Ubx has substantially evolved.

Split fluorescent proteins have been engineered for various purposes, in each case signaling their spontaneous reconstitution by fluorescence. By combining split protein reconstitution and computational protein design, we have constructed a circularly permuted and truncated variant of green fluorescent protein (GFP) in which the seventh beta strand has been left out and the sites around it computationally designed to accommodate a peptide from influenza hemagglutinin. We call this a "leave-one-out" GFP biosensor (LOO-GFP). A LOO-GFP was designed using DEEdesign, selected by plate screening a bacterial library in the presence of the influenza peptide target, and was found to have seven point mutations. But binding was weak (9μM) and was at the expense of stability. The weakened, partially folded protein aggregated in the absence of its target. Furthermore, the aggregated biosensor fluoresced more than its monomeric peptide-bound form. In this work, the LOO-GFP was rationally redesigned to fold more robustly and bind the target tighter. Modeling of the GFP folding pathway suggested that one of the seven mutations, F83W, interfered with the closing of the beta barrel. Mutating this residue back to a F indeed, along with several other rationally justified changes followed by re-screening, produced several biosensor sequences with slower unfolding rates, a positive binding signal, and higher chromophore maturation efficiency. We also observed a blue-shift in the excitation spectrum, and lower background fluorescence in the unbound state. Kd was unchanged. In parallel experiments, LOO-GFP biosensors were genetically fused to fibers formed by the Drosophila protein ultrabithorax (Ubx), and were found to be absent any background fluorescence in the unbound state, but recovered fluorescence when exposed to the target peptide. Implications for the design of biosensing materials are discussed.

The development of functionalized materials is needed to enable diverse applications. Protein-based materials are typically biocompatible and biodegradable and can exhibit a wide variety of useful mechanical properties. Most importantly, gene fusion enables facile incorporation of active proteins into the materials. However, many protocols rely on denaturing conditions to stimulate materials formation. These conditions would be expected to inactivate any appended functional proteins. This chapter describes methods to create protein fibers and films in a mild aqueous buffer near neutral pH. This facile, inexpensive single-pot approach to materials assembly does not require any special equipment. Also included in this chapter are methods to fuse fibers to form fiber bundles, and to use fibers for cell culture. Although these methods were developed to generate materials from the Drosophila Hox transcription factor Ultrabithorax, they may also work for other self-assembling proteins, many of which have sequence features in common with Ubx.

The development of materials with diverse mechanical and functional properties enables a broad range of applications. For materials composed of protein, well‐established molecular biology techniques can theoretically be used to alter the mechanical and functional properties of materials. However, repetition of amino acid sequences in the constituent proteins can complicate engineering the mechanical properties. Furthermore, most protein‐based materials are assembled under denaturing conditions, thus precluding incorporation of full‐length proteins in an active state. Our lab discovered that the Drosophila melanogaster transcription factor Ultrabithorax (Ubx) hierarchically self‐assembles into novel, ordered biomaterials, ranging from nanoscale fibrils to macroscale fibers, films, meshes. These materials are stabilized by specific dityrosine bonds that spontaneously form during assembly. The location of the participating tyrosines in unique sequence motifs enables the reliable tuning of the materials by mutagenesis to be either weaker or stronger. Because Ubx materials rapidly form in mild, aqueous buffers, a surprisingly wide variety of proteins can be incorporated via gene fusion without harming either materials assembly or the function of the appended protein. As one example, when Ubx is fused to angiogenic growth factors, the resulting fibers control cell signaling and cell behavior in vitro , and can instigate and guide blood vessel formation in vivo . Ubx materials provide an inexpensive and facile platform for customizing materials for a variety of applications. Support or Funding Information NSF Ted Nash Long Life Foundation This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .