RESUMEN
We describe a small molecule ligand ACA-14 (2-hydroxy-5-{[(2-phenylcyclopropyl) carbonyl] amino} benzoic acid) as an initial lead for the development of direct inhibitors of KRAS, a notoriously difficult anticancer drug target. We show that the compound binds to KRAS near the switch regions with affinities in the low micromolar range and exerts different effects on KRAS interactions with binding partners. Specifically, ACA-14 impedes the interaction of KRAS with its effector Raf and reduces both intrinsic and SOS-mediated nucleotide exchange rates. Likely as a result of these effects, ACA-14 inhibits signal transduction through the MAPK pathway in cells expressing mutant KRAS and inhibits the growth of pancreatic and colon cancer cells harboring mutant KRAS. We thus propose compound ACA-14 as a useful initial lead for the development of broad-acting inhibitors that target multiple KRAS mutants and simultaneously deplete the fraction of GTP-loaded KRAS while abrogating the effector-binding ability of the already GTP-loaded fraction.
RESUMEN
Staphylococcus aureus can target a variety of tissues, causing life-threatening infections. The basis for this diversity stems from the microorganism's ability to spread in the vascular system throughout the body. To survive in blood, S. aureus coats itself with a fibrinogen (Fg)/fibrin shield. The protective shield is assembled by the coordinated actions of a number of Fg-binding bacterial proteins that manipulate the host's blood coagulation system. Several of the Fg binders appear redundant, sharing similar functional motifs. This observation led us to screen for the presence of novel proteins with significant amino acid identities to von Willebrand factor-binding protein (vWbp), a key component in the shield assembly machinery. One identified protein showed significant sequence identity with the C-terminal region of vWbp, and we consequently named it vWbp homologous protein (vhp). The vhp gene lies within a cluster of genes that encode other virulence factors in S. aureus. Although each isolate only contains one copy of the vhp gene, S. aureus has at least three distinct alleles, vhpA, B, and C, that are present in the core genome. All three vhp isoforms bind Fg with high affinity, targeting a site located in the D fragment of Fg. We further identified an â¼79 amino acid-long, conserved segment within the C-terminal region of vWbp that shares high sequence identities (54 to 67%) with the vhps and binds soluble Fg with high affinity. Further analysis of this conserved motif and the intact vhps revealed intriguing differences in the Fg binding behavior, perhaps suggesting that these proteins have similar but discrete functions in the shield assembly. IMPORTANCE The life-threatening diseases caused by multidrug-resistant Staphylococcus aureus strains are a worldwide medical problem due to treatment limitations and the lack of an effective vaccine. The ability of S. aureus to coat itself with a protective fibrinogen (Fg)/fibrin shield allows the organism to survive in blood and to disseminate and cause invasive diseases. This process represents a promising target for novel antistaphylococcal treatment strategies but is incompletely understood. S. aureus expresses a number of Fg-binding proteins. Some of these proteins have apparently redundant functions. Proteins with similar functions often share a structural or functional motif with each other. In this study, we identified a protein homologous to the C-terminal of von Willebrand factor-binding protein (vWbp), a key contributor in the Fg shield assembly that also binds Fg. Further analysis allowed us to identify a common Fg-binding motif.
Asunto(s)
Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Proteínas Portadoras/metabolismo , Fibrinógeno/metabolismo , Staphylococcus aureus/química , Factor de von Willebrand/metabolismo , Proteínas Portadoras/genética , Unión Proteica , Infecciones Estafilocócicas/microbiología , Staphylococcus aureus/genética , Staphylococcus aureus/metabolismo , Factores de VirulenciaRESUMEN
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.
Asunto(s)
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
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.
RESUMEN
Understanding gene regulation by Hox transcription factors requires understanding the forces that underlie DNA binding by these proteins. Electrophoretic mobility shift analysis (EMSA) not only allows measurement of protein affinity and cooperativity but also permits visualization of differently migrating protein-DNA complexes, including complexes with different compositions or complexes with identical compositions yet assembled in different geometries. Furthermore, protein activity can be measured, allowing correction of binding constants for the percentage of protein that is properly folded and capable of binding DNA. Protocols for measuring protein activity and the equilibrium DNA-binding dissociation constant (K d) are provided. This versatile assay system can be adjusted based on specific needs to measure other parameters, including the kinetic association and dissociation constants (k a and k d) and the formation of heterologous protein-protein interactions.
Asunto(s)
ADN/metabolismo , Ensayo de Cambio de Movilidad Electroforética , Proteínas de Homeodominio/metabolismo , Animales , Drosophila melanogaster , Oligonucleótidos/metabolismo , Unión ProteicaRESUMEN
Intrinsically disordered proteins are anticipated to be more prone to aggregation than folded, stable proteins. Chemical additives included in the buffer can help maintain proteins in a soluble, monomeric state. However, the array of chemicals that impact protein solubility is staggering, precluding iterative testing of chemical conditions during purification. Herein, we describe a filter-based aggregation assay to rapidly identify chemical additives that maintain solubility for a protein of interest. A hierarchical approach to buffer selection is provided, in which the type of chemical which best improves solubility is first determined, followed by identifying the optimal chemical and its most effective concentration. Finally, combinations of chemical additives can be assessed if necessary. Although this assay can be applied to purified protein, partially purified protein, or aggregated protein, this protocol specifically details the use of this assay for crude cell lysate. This approach allows identification of solubility-promoting buffers prior to the initial protein purification.