RESUMEN
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.
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Materiales Biocompatibles , Proteínas Recombinantes de Fusión , Materiales Biocompatibles/química , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Humanos , Ingeniería de Proteínas/métodos , Animales , Ingeniería de Tejidos/métodos , Proteínas/química , Proteínas/genética , Sistemas de Liberación de Medicamentos/métodosRESUMEN
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.
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Proteínas Intrínsecamente Desordenadas , Animales , Comunicación Celular , Proteínas Intrínsecamente Desordenadas/química , Transducción de SeñalRESUMEN
For proteins, the sequence â structure â 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 â IDP/IDR ensemble â 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.
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Comunicación Celular/genética , Proteínas Intrínsecamente Desordenadas/genética , Conformación Proteica , Transducción de Señal/genética , Secuencia de Aminoácidos/genética , Humanos , Modelos Moleculares , Unión Proteica/genética , Pliegue de Proteína , Procesamiento Proteico-Postraduccional/genéticaRESUMEN
The steroid hormone ecdysone and its receptor (EcR) play critical roles in orchestrating developmental transitions in arthropods. However, the mechanism by which EcR integrates nutritional and developmental cues to correctly activate transcription remains poorly understood. Here, we show that EcR-dependent transcription, and thus, developmental timing in Drosophila, is regulated by CDK8 and its regulatory partner Cyclin C (CycC), and the level of CDK8 is affected by nutrient availability. We observed that cdk8 and cycC mutants resemble EcR mutants and EcR-target genes are systematically down-regulated in both mutants. Indeed, the ability of the EcR-Ultraspiracle (USP) heterodimer to bind to polytene chromosomes and the promoters of EcR target genes is also diminished. Mass spectrometry analysis of proteins that co-immunoprecipitate with EcR and USP identified multiple Mediator subunits, including CDK8 and CycC. Consistently, CDK8-CycC interacts with EcR-USP in vivo; in particular, CDK8 and Med14 can directly interact with the AF1 domain of EcR. These results suggest that CDK8-CycC may serve as transcriptional cofactors for EcR-dependent transcription. During the larval-pupal transition, the levels of CDK8 protein positively correlate with EcR and USP levels, but inversely correlate with the activity of sterol regulatory element binding protein (SREBP), the master regulator of intracellular lipid homeostasis. Likewise, starvation of early third instar larvae precociously increases the levels of CDK8, EcR and USP, yet down-regulates SREBP activity. Conversely, refeeding the starved larvae strongly reduces CDK8 levels but increases SREBP activity. Importantly, these changes correlate with the timing for the larval-pupal transition. Taken together, these results suggest that CDK8-CycC links nutrient intake to developmental transitions (EcR activity) and fat metabolism (SREBP activity) during the larval-pupal transition.
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Ciclina C/metabolismo , Quinasa 8 Dependiente de Ciclina/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila/crecimiento & desarrollo , Drosophila/metabolismo , Receptores de Esteroides/metabolismo , Animales , Animales Modificados Genéticamente , Ciclina C/genética , Quinasa 8 Dependiente de Ciclina/genética , Proteínas de Unión al ADN/metabolismo , Drosophila/genética , Proteínas de Drosophila/genética , Ecdisteroides/biosíntesis , Femenino , Privación de Alimentos , Regulación de la Expresión Génica , Larva/crecimiento & desarrollo , Larva/metabolismo , Mutación , Proteínas de Unión a los Elementos Reguladores de Esteroles/metabolismo , Factores de Transcripción/metabolismoRESUMEN
Intrinsically disordered proteins (IDPs) and IDP regions lack stable tertiary structure yet carry out numerous biological functions, especially those associated with signaling, transcription regulation, DNA condensation, cell division, and cellular differentiation. Both post-translational modifications (PTMs) and alternative splicing (AS) expand the functional repertoire of IDPs. Here we propose that an "IDP-based developmental toolkit," which is comprised of IDP regions, PTMs, especially multiple PTMs, within these IDP regions, and AS events within segments of pre-mRNA that code for these same IDP regions, allows functional diversification and environmental responsiveness for molecules that direct the development of complex metazoans.
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Evolución Biológica , Células Eucariotas/metabolismo , Proteínas Intrínsecamente Desordenadas/química , Proteínas Intrínsecamente Desordenadas/metabolismo , Células Procariotas/metabolismo , Células Eucariotas/química , Células Eucariotas/citología , Proteínas Intrínsecamente Desordenadas/genética , Células Procariotas/química , Células Procariotas/citología , Pliegue de Proteína , Procesamiento Proteico-PostraduccionalRESUMEN
To modulate transcription, a variety of input signals must be sensed by genetic regulatory proteins. In these proteins, flexibility and disorder are emerging as common themes. Prokaryotic regulators generally have short, flexible segments, whereas eukaryotic regulators have extended regions that lack predicted secondary structure (intrinsic disorder). Two examples illustrate the impact of flexibility and disorder on gene regulation: the prokaryotic LacI/GalR family, with detailed information from studies on LacI, and the eukaryotic family of Hox proteins, with specific insights from investigations of Ultrabithorax (Ubx). The widespread importance of structural disorder in gene regulatory proteins may derive from the need for flexibility in signal response and, particularly in eukaryotes, in protein partner selection.
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Regulación de la Expresión Génica , Proteínas de Homeodominio/química , Proteínas de Homeodominio/metabolismo , Represoras Lac/química , Represoras Lac/metabolismo , Proteínas Represoras/química , Proteínas Represoras/metabolismo , Secuencia de Aminoácidos , ADN/genética , ADN/metabolismo , Humanos , Datos de Secuencia MolecularRESUMEN
Separation of full-length protein from proteolytic products is challenging, since the properties used to isolate the protein can also be present in proteolytic products. Many separation techniques risk non-specific protein adhesion and/or require a lot of time, enabling continued proteolysis and aggregation after lysis. We demonstrate that proteolytic products aggregate for two different proteins. As a result, full-length protein can be rapidly separated from these fragments by filter flow-through purification, resulting in a substantial protein purity enhancement. This rapid approach is likely to be useful for intrinsically disordered proteins, whose repetitive sequence composition and flexible nature can facilitate aggregation.
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Bioquímica/métodos , Proteínas/aislamiento & purificación , Bioquímica/instrumentación , Proteínas Portadoras/genética , Proteínas Portadoras/aislamiento & purificación , Proteínas de Drosophila/genética , Proteínas de Drosophila/aislamiento & purificación , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/aislamiento & purificación , Proteínas Intrínsecamente Desordenadas/aislamiento & purificación , Factores de Transcripción/genética , Factores de Transcripción/aislamiento & purificaciónRESUMEN
Tumorigenic cell behaviors can be suppressed or enhanced by their physicochemical environment. As a first step toward developing materials that allow tumorigenic behaviors to be observed and manipulated, we cultured related MCF10 breast cell lines on fibers composed of the Drosophila protein Ultrabithorax (Ubx). These cell lines, originally derived from fibrocystic breast tissue, represent a continuum of tumorigenic behavior. Immortal but nontumorigenic MCF10A cells, as well as semitumorigenic MCF10AT cells, attached and spread on Ubx fibers. MCF10CA-1a cells, the most highly transformed line, secreted high concentrations of matrix metalloproteinases when cultured on Ubx materials, resulting in differences in cell attachment and cytoskeletal structure, and enabling invasive behavior. Because the mechanical and functional properties of Ubx fibers can be genetically manipulated, these materials provide a valuable tool for cancer research, allowing creation of diverse microenvironments that allow assessment of invasive, metastatic behavior.
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Neoplasias de la Mama/patología , Técnicas de Cultivo de Célula , Línea Celular Tumoral/efectos de los fármacos , Proteínas de Drosophila/química , Proteínas de Homeodominio/química , Factores de Transcripción/química , Animales , Drosophila melanogaster/química , Femenino , Humanos , Metástasis de la Neoplasia/patologíaRESUMEN
Successful design of tissue engineering scaffolds must include the ability to stimulate vascular development by incorporating angiogenic growth factors. Current approaches can allow diffusion of growth factors, incorporate active factors randomly, or can leave residual toxins. We addressed these problems by genetically fusing the gene encoding Vascular Endothelial Growth Factor (VEGF) with the Ultrabithorax (Ubx) gene to produce fusion proteins capable of self-assembly into materials. We demonstrate that VEGF-Ubx materials enhance human endothelial cell migration, prolong cell survival, and dose-dependently activate the VEGF signaling pathway. VEGF-Ubx fibers attract outgrowing sprouts in an aortic ring assay and induce vessel formation in a chicken embryo chorioallantoic membrane (CAM) assay. Collectively, these results demonstrate that the activity of VEGF remains intact in Ubx materials. This approach could provide an inexpensive and facile mechanism to stimulate and pattern angiogenesis.
Asunto(s)
Proteínas de Drosophila/genética , Proteínas de Homeodominio/genética , Morfogénesis/genética , Ingeniería de Tejidos , Factores de Transcripción/genética , Factor A de Crecimiento Endotelial Vascular/genética , Animales , Movimiento Celular/genética , Embrión de Pollo , Pollos , Proteínas de Drosophila/química , Proteínas de Homeodominio/química , Células Endoteliales de la Vena Umbilical Humana , Humanos , Neovascularización Fisiológica , Transducción de Señal , Andamios del Tejido , Factores de Transcripción/química , Factor A de Crecimiento Endotelial Vascular/químicaRESUMEN
The recombinant protein Ultrabithorax (Ubx), a Drosophila melanogaster Hox transcription factor, self-assembles into biocompatible materials in vitro that are remarkably extensible and strong. Here, we demonstrate that the strength of Ubx materials is due to intermolecular dityrosine bonds. Ubx materials auto-fluoresce blue, a characteristic of dityrosine, and bind dityrosine-specific antibodies. Monitoring the fluorescence of reduced Ubx fibers upon oxygen exposure reveals biphasic bond formation kinetics. Two dityrosine bonds in Ubx were identified by site-directed mutagenesis followed by measurements of fiber fluorescent intensity. One bond is located between the N-terminus and the homeodomain (Y4/Y296 or Y12/Y293), and another bond is formed by Y167 and Y240. Fiber fluorescence closely correlates with fiber strength, demonstrating that these bonds are intermolecular. To our knowledge, this is the first identification of specific residues that participate in dityrosine bonds in protein-based materials. The percentage of Ubx molecules harboring both bonds can be decreased or increased by mutagenesis, providing an additional mechanism to control the mechanical properties of Ubx materials. Duplication of tyrosine-containing motifs in Ubx increases dityrosine content in Ubx fibers, suggesting these motifs could be inserted in other self-assembling proteins to strengthen the corresponding materials.
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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.
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Surprisingly few transcription factors drive animal development relative to the number and diversity of final tissues and body structures. Therefore, most transcription factors must function in more than one tissue. In a famous example, members of the Hox transcription factor family are expressed in contiguous stripes along the anterior/posterior axis during animal development. Individual Hox transcription factors specify all tissues within their expression domain and thus must respond to cellular cues to instigate the correct tissue-specific gene regulatory cascade. We describe how, in the Drosophila Hox protein Ultrabithorax, intrinsically disordered regions implement, regulate and co-ordinate multiple functions, potentially enabling context-specific gene regulation. The large N-terminal disordered domain encodes most of the transcription activation domain and directly impacts DNA binding affinity by the Ubx homeodomain. Similarly, the C-terminal disordered domain alters DNA binding affinity and specificity, interaction with a Hox binding protein and strongly influences both transcription activation and repression. Phosphorylation of the N-terminal disordered domain and alternative splicing of the C-terminal disordered domain could allow the cell to both regulate and co-ordinate DNA binding, protein interactions and transcription regulation. For regulatory mechanisms relying on disorder to continue to be available when Ubx is bound to other proteins or DNA, fuzziness would need to be preserved in these macromolecular complexes. The intrinsically disordered domains in Hox proteins are predicted to be on the very dynamic end of the disorder spectrum, potentially allowing disorder to persist when Ubx is bound to proteins or DNA to regulate the function of these "fuzzy" complexes. Because both intrinsically disordered regions within Ubx have multiple roles, each region may implement several different regulatory mechanisms identified in fuzzy complexes. These intrinsic disorder-based regulatory mechanisms are likely to be critical for allowing Ubx to sense tissue identity and respond by implementing a context-specific gene regulatory cascade.
Asunto(s)
Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Proteínas de Homeodominio/química , Proteínas de Homeodominio/metabolismo , Factores de Transcripción/química , Factores de Transcripción/metabolismo , AnimalesRESUMEN
Although yeast two-hybrid experiments are commonly used to identify protein interactions, the frequent occurrence of false negatives and false positives hampers data interpretation. Using both yeast one-hybrid and two-hybrid experiments, we have identified potential sources of these problems: the media preparation protocol and the source of the yeast nitrogen base may not only impact signal range but also effect whether a result appears positive or negative. While altering media preparation may optimize signal differences for individual experiments, media preparation must be reported in detail to replicate studies and accurately compare results from different experiments.
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Incorporation of nanoparticles during the hierarchical self-assembly of protein-based materials can impart function to the resulting composite materials. Herein we demonstrate that the structure and nanoparticle distribution of composite fibers are sensitive to the method of nanoparticle addition and the physicochemical properties of both the nanoparticle and the protein. Our model system consists of a recombinant enhanced green fluorescent protein-Ultrabithorax (EGFP-Ubx) fusion protein and luminescent CdSe-ZnS core-shell quantum dots (QDs), allowing us to optically assess the distribution of both the protein and nanoparticle components within the composite material. Although QDs favorably interact with EGFP-Ubx monomers, the relatively rough surface morphology of composite fibers suggests EGFP-Ubx-QD conjugates impact self-assembly. Indeed, QDs templated onto EGFP-Ubx film post-self-assembly can be subsequently drawn into smooth composite fibers. Additionally, the QD surface charge impacts QD distribution within the composite material, indicating that surface charge plays an important role in self-assembly. QDs with either positively or negatively charged coatings significantly enhance fiber extensibility. Conversely, QDs coated with hydrophobic moieties and suspended in toluene produce composite fibers with a heterogeneous distribution of QDs and severely altered fiber morphology, indicating that toluene severely disrupts Ubx self-assembly. Understanding factors that impact the protein-nanoparticle interaction enables manipulation of the structure and mechanical properties of composite materials. Since proteins interact with nanoparticle surface coatings, these results should be applicable to other types of nanoparticles with similar chemical groups on the surface.
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Materiales Biocompatibles/síntesis química , Materiales Biomiméticos/síntesis química , Proteínas Fluorescentes Verdes/metabolismo , Puntos Cuánticos , Proteínas Recombinantes de Fusión/metabolismo , Materiales Biocompatibles/análisis , Materiales Biomiméticos/análisis , Compuestos de Cadmio/química , Clonación Molecular , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Escherichia coli , Transferencia Resonante de Energía de Fluorescencia , Proteínas Fluorescentes Verdes/química , Proteínas Fluorescentes Verdes/genética , Proteínas de Homeodominio/química , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Interacciones Hidrofóbicas e Hidrofílicas , Luminiscencia , Microfibrillas/química , Nanopartículas/química , Plásmidos , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Compuestos de Selenio/química , Electricidad Estática , Propiedades de Superficie , Factores de Transcripción/química , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transformación Bacteriana , Compuestos de Zinc/químicaRESUMEN
The development of protein-based materials with diverse mechanical properties will facilitate the realization of a broad range of potential applications. The recombinant Drosophila melanogaster transcription factor Ultrabithorax self-assembles under mild conditions in aqueous buffers into extremely extensible materials. By controlling fiber diameter, both the mechanism of extension and the magnitude of the mechanical properties can be varied. Narrow Ultrabithorax fibers (diameter <10 µm) extend elastically, whereas the predominantly plastic deformation of wide fibers (diameter >15 µm) reflects the increase in breaking strain with increasing diameter, apparently due to a change in structure. The breaking stress/strain of the widest fibers resembles that of natural elastin. Intermediate fibers display mixed properties. Fiber bundles retain the mechanical properties of individual fibers but can withstand much larger forces. Controlling fiber size and generating fiber superstructures is a facile way to manipulate the mechanical characteristics of protein fibers and rationally engineer macroscale protein-based materials with desirable properties.
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Proteínas de Drosophila/fisiología , Proteínas de Homeodominio/fisiología , Fenómenos Mecánicos , Proteínas/ultraestructura , Factores de Transcripción/fisiología , Animales , Materiales Biocompatibles , Drosophila melanogaster , Ensayo de MaterialesRESUMEN
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.
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Enfermedad , Salud , Proteínas de Homeodominio/metabolismo , Factores de Transcripción/metabolismo , Animales , Crecimiento y Desarrollo , Humanos , Proteínas Intrínsecamente Desordenadas/metabolismoRESUMEN
The use of proteins as monomers for materials assembly enables customization of chemical, physical, and functional properties. However, natural materials-forming proteins are difficult to produce as recombinant protein monomers and require harsh conditions to initiate assembly. We have generated materials using the recombinant transcription factor Ultrabithorax, a Drosophila melanogaster protein not known or anticipated to form extended oligomers in vivo. Ultrabithorax self-assembles at the air-water interface into nanoscale fibers, which further associate to form macroscale films, sheets, ropes, and tethered encapsulates. These materials self-adhere, allowing construction of more complex architectures. The Ultrabithorax sequence contains two regions capable of generating materials, only one of which contains motifs found in elastomeric proteins. However, both minimal regions must be included to produce robust materials. Relative to other protein-based materials, Ultrabithorax assembles at significantly reduced concentrations, on faster timescales, and under gentler conditions, properties that facilitate future materials engineering and functionalization.
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Materiales Biocompatibles/química , Materiales Biocompatibles/metabolismo , Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Proteínas de Homeodominio/química , Proteínas de Homeodominio/metabolismo , Factores de Transcripción/química , Factores de Transcripción/metabolismo , Animales , Western Blotting , ADN/genética , ADN/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Tejido Elástico/química , Sustancias Macromoleculares/química , Ensayo de Materiales , Dominios y Motivos de Interacción de Proteínas , Estrés MecánicoRESUMEN
Rice University has created a team-taught interactive biology course for nonmajors with a focus on cutting edge biology in the news-advances in biotechnology, medicine, and science policy, along with the biological principles and methodology upon which these advances are based. The challenges inherent to teaching current topics were minimized by team-teaching the course, providing knowledgeable and enthusiastic lecturers for every topic while distributing the effort required to update material. Postdoctoral associates and advanced graduate students served as lecturers, providing an opportunity for them to develop their teaching skills and learn to communicate effectively with nonscientists on newsworthy topics related to their research. Laboratory tours, in-class demonstrations, and mock-ups helped lecturers convey surprisingly advanced ideas with students who lacked a strong theoretical or practical science background. A faculty member and co-coordinator administer the class, organize class activities, and mentor the speakers on teaching techniques and lecture design. Course design, lecture topics, hands-on activities, and approaches to successfully solve the difficulties inherent to team teaching are discussed. Course evaluations reflect student involvement in, and enjoyment of, the class.
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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.
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Proteínas de Drosophila/metabolismo , Regulación de la Expresión Génica , Proteínas de Homeodominio/metabolismo , Mutación , Factores de Transcripción/metabolismo , Activación Transcripcional , Animales , Animales Modificados Genéticamente , Proteínas de Drosophila/genética , Drosophila melanogaster , Proteínas de Homeodominio/genética , Fenotipo , Factores de Transcripción/genéticaRESUMEN
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.