RESUMEN
Host protein folding stress responses can play important roles in RNA virus replication and evolution. Prior work suggested a complicated interplay between the cytosolic proteostasis stress response, controlled by the transcriptional master regulator heat shock factor 1 (HSF1), and human immunodeficiency virus-1 (HIV-1). We sought to uncouple HSF1 transcription factor activity from cytotoxic proteostasis stress and thereby better elucidate the proposed role(s) of HSF1 in the HIV-1 lifecycle. To achieve this objective, we used chemical genetic, stress-independent control of HSF1 activity to establish whether and how HSF1 influences HIV-1 replication. Stress-independent HSF1 induction decreased both the total quantity and infectivity of HIV-1 virions. Moreover, HIV-1 was unable to escape HSF1-mediated restriction over the course of several serial passages. These results clarify the interplay between the host's heat shock response and HIV-1 infection and motivate continued investigation of chaperones as potential antiviral therapeutic targets.
Asunto(s)
Respuesta al Choque Térmico , Proteostasis , Factores de Transcripción del Choque Térmico/genética , Factores de Transcripción del Choque Térmico/metabolismo , Humanos , Chaperonas Moleculares , Replicación ViralRESUMEN
Classically, the unfolded protein response (UPR) safeguards secretory pathway proteostasis. The most ancient arm of the UPR, the IRE1-activated spliced X-box binding protein 1 (XBP1s)-mediated response, has roles in secretory pathway maturation beyond resolving proteostatic stress. Understanding the consequences of XBP1s activation for cellular processes is critical for elucidating mechanistic connections between XBP1s and development, immunity, and disease. Here, we show that a key functional output of XBP1s activation is a cell type-dependent shift in the distribution of N-glycan structures on endogenous membrane and secreted proteomes. For example, XBP1s activity decreased levels of sialylation and bisecting GlcNAc in the HEK293 membrane proteome and secretome, while substantially increasing the population of oligomannose N-glycans only in the secretome. In HeLa cell membranes, stress-independent XBP1s activation increased the population of high-mannose and tetraantennary N-glycans, and also enhanced core fucosylation. mRNA profiling experiments suggest that XBP1s-mediated remodeling of the N-glycome is, at least in part, a consequence of coordinated transcriptional resculpting of N-glycan maturation pathways by XBP1s. The discovery of XBP1s-induced N-glycan structural remodeling on a glycome-wide scale suggests that XBP1s can act as a master regulator of N-glycan maturation. Moreover, because the sugars on cell-surface proteins or on proteins secreted from an XBP1s-activated cell can be molecularly distinct from those of an unactivated cell, these findings reveal a potential new mechanism for translating intracellular stress signaling into altered interactions with the extracellular environment.
Asunto(s)
Polisacáridos/metabolismo , Proteína 1 de Unión a la X-Box/metabolismo , Línea Celular , Línea Celular Tumoral , Células HEK293 , Células HeLa , Humanos , Manosa/metabolismo , Proteoma/metabolismo , Transducción de Señal/fisiología , Transcripción Genética/fisiología , Respuesta de Proteína Desplegada/fisiologíaRESUMEN
Proteostasis in the cytosol is governed by the heat shock response. The master regulator of the heat shock response, heat shock factor 1 (HSF1), and key chaperones whose levels are HSF1-regulated have emerged as high-profile targets for therapeutic applications ranging from protein misfolding-related disorders to cancer. Nonetheless, a generally applicable methodology to selectively and potently inhibit endogenous HSF1 in a small molecule-dependent manner in disease model systems remains elusive. Also problematic, the administration of even highly selective chaperone inhibitors often has the side effect of activating HSF1 and thereby inducing a compensatory heat shock response. Herein, we report a ligand-regulatable, dominant negative version of HSF1 that addresses these issues. Our approach, which required engineering a new dominant negative HSF1 variant, permits dosable inhibition of endogenous HSF1 with a selective small molecule in cell-based model systems of interest. The methodology allows us to uncouple the pleiotropic effects of chaperone inhibitors and environmental toxins from the concomitantly induced compensatory heat shock response. Integration of our method with techniques to activate HSF1 enables the creation of cell lines in which the cytosolic proteostasis network can be up- or down-regulated by orthogonal small molecules. Selective, small molecule-mediated inhibition of HSF1 has distinctive implications for the proteostasis of both chaperone-dependent globular proteins and aggregation-prone intrinsically disordered proteins. Altogether, this work provides critical methods for continued exploration of the biological roles of HSF1 and the therapeutic potential of heat shock response modulation.
Asunto(s)
Proteínas de Unión al ADN/antagonistas & inhibidores , Bibliotecas de Moléculas Pequeñas/metabolismo , Factores de Transcripción/antagonistas & inhibidores , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Citometría de Flujo , Regulación de la Expresión Génica/efectos de los fármacos , Células HEK293 , Factores de Transcripción del Choque Térmico , Humanos , Immunoblotting , Análisis por Micromatrices , Reacción en Cadena en Tiempo Real de la Polimerasa , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacología , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
The molecular architecture of the mature N-glycome is dynamic, with consequences for both normal and pathologic processes. Elucidating cellular mechanisms that modulate the N-linked glycome is, therefore, crucial. The unfolded protein response (UPR) is classically responsible for maintaining proteostasis in the secretory pathway by defining levels of chaperones and quality control proteins. Here, we employ chemical biology methods for UPR regulation to show that stress-independent activation of the UPR's XBP1s transcription factor also induces a panel of N-glycan maturation-related enzymes. The downstream consequence is a distinctive shift toward specific hybrid and complex N-glycans on N-glycoproteins produced from XBP1s-activated cells, which we characterize by mass spectrometry. Pulse-chase studies attribute this shift specifically to altered N-glycan processing, rather than to changes in degradation or secretion rates. Our findings implicate XBP1s in a new role for N-glycoprotein biosynthesis, unveiling an important link between intracellular stress responses and the molecular architecture of extracellular N-glycoproteins.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Polisacáridos/química , Factores de Transcripción/metabolismo , Respuesta de Proteína Desplegada , Proteínas de Unión al ADN/química , Electroforesis en Gel de Poliacrilamida , Células HEK293 , Humanos , Polisacáridos/fisiología , Biosíntesis de Proteínas , Reacción en Cadena en Tiempo Real de la Polimerasa , Factores de Transcripción del Factor Regulador X , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Factores de Transcripción/química , Proteína 1 de Unión a la X-BoxRESUMEN
N(5)-CAIR synthetase, an essential enzyme in microorganisms, converts 5-aminoimidazole ribonucleotide (AIR) and bicarbonate to N(5)-CAIR with the aid of ATP. Previous X-ray crystallographic analyses of Aspergillus clavatus N(5)-CAIR synthetase postulated that R271, H273, and K353 were important for bicarbonate binding and for catalysis. As reported here, site-directed mutagenesis of these residues revealed that R271 and H273 are, indeed, critical for bicarbonate binding and catalysis whereas all K353 mutations, even ones conservative in nature, are inactive. Studies on the R271K mutant protein revealed cooperative substrate inhibition for ATP with a Ki of 1.2 mM. Kinetic investigation of the H273A mutant protein indicated that it was cooperative with respect to AIR; however, this effect was not seen in either the wild-type or any of the other mutant proteins. Cooperative ATP-dependent inhibition of wild-type N(5)-CAIR synthetase was also detected with ATP displaying a Ki of 3.3 mM. Taken together, these results indicate that N(5)-CAIR synthetase operates maximally within a narrow concentration of ATP.
Asunto(s)
Aminoimidazol Carboxamida/análogos & derivados , Ligasas/genética , Ribonucleótidos/biosíntesis , Adenosina Trifosfato/metabolismo , Secuencia de Aminoácidos , Bicarbonatos/metabolismo , Sitios de Unión/genética , Catálisis , Dominio Catalítico/genética , Escherichia coli/enzimología , Cinética , Ligasas/metabolismo , Modelos Moleculares , Mutagénesis Sitio-DirigidaRESUMEN
Several thieno[2,3-d]pyrimidinediones have been synthesized and examined for antibacterial activity against a range of gram-positive and gram-negative pathogens. Two compounds displayed potent activity (2-16 mg/L) against multi-drug resistant gram-positive organisms, including methicillin resistant, vancomycin-intermediate, vancomycin-resistant Staphylococcus aureus (MRSA, VISA, VRSA) and vancomycin-resistant enterococci (VRE). Only one of these agents possessed moderate activity (16-32 mg/L) against gram-negative strains. An examination of the cytotoxicity of these agents revealed that they displayed low toxicity (40-50 mg/L) against mammalian cells and very low hemolytic activity (2-7%). Taken together, these studies suggest that thieno[2,3-d]pyrimidinediones are interesting scaffolds for the development of novel gram-positive antibacterial agents.
Asunto(s)
Antibacterianos/química , Antibacterianos/farmacología , Bacterias/efectos de los fármacos , Pirimidinonas/química , Pirimidinonas/farmacología , Animales , Antibacterianos/síntesis química , Antibacterianos/toxicidad , Hemólisis/efectos de los fármacos , Ratones , Células 3T3 NIH , Pirimidinonas/síntesis química , Pirimidinonas/toxicidad , OvinosRESUMEN
The enormous success of antibiotics is seriously threatened by the development of resistance to most of the drugs available on the market. Thus, novel antibiotics are needed that are less prone to bacterial resistance and are directed toward novel biological targets. Antimicrobial peptides (AMPs) have attracted considerable attention due to their unique mode of action and broad spectrum activity. However, these agents suffer from liability to proteases and the high cost of manufacturing has impeded their development. Previously, we have reported on a novel class of benzophenone-based antibiotics and early studies suggested that these agents might target the bacterial membrane. In this study, we present our work on the mechanism of action of these novel membrane targeted antibiotics. These compounds have good affinities to polyanionic components of the cell wall such as lipoteichoic acid (LTA) and lipopolysaccharide (LPS). We found that these agents release potassium ions from treated bacteria; thus, resulting in disruption of the bacterial membrane potential. Benzophenone-based membrane targeted antibiotics (BPMTAs) cause membrane disruption in synthetic lipid vesicles that mimic Gram-positive or Gram-negative bacteria. The compounds display no hemolytic activity up to a concentration that is 100 times the MIC values and they are capable of curing mice of a lethal MRSA infection. Repeated attempts to develop a mutant resistant to these agents has failed. Taken together, BPMTAs represent a promising new class of membrane-targeted antibacterial agents.
Asunto(s)
Antibacterianos/química , Antibacterianos/uso terapéutico , Benzofenonas/química , Benzofenonas/uso terapéutico , Farmacorresistencia Bacteriana , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Infecciones Estafilocócicas/tratamiento farmacológico , Animales , Antibacterianos/farmacología , Benzofenonas/farmacología , Hemólisis/efectos de los fármacos , Humanos , Liposomas/metabolismo , Staphylococcus aureus Resistente a Meticilina/metabolismo , Ratones , Potasio/metabolismo , Ovinos , Infecciones Estafilocócicas/metabolismoRESUMEN
There is much interest in designing molecular sized containers that influence and facilitate chemical reactions within their nanocavities. On top of the advantages of improved yield and selectivity, the studies of reactions in confinement also give important clues that extend our basic understanding of chemical processes. We report here, the synthesis and self-assembly of an expanded bis-urea macrocycle to give crystals with columnar channels. Constructed from two C-shaped phenylethynylene units and two urea groups, the macrocycle affords a large pore with a diameter of â¼9 Å. Despite its increased size, the macrocycles assemble into columns with high fidelity to afford porous crystals. The porosity and accessibility of these channels have been demonstrated by gas adsorption studies and by the uptake of coumarin to afford solid inclusion complexes. Upon UV-irradiation, these inclusion complexes facilitate the conversion of coumarin to its anti-head-to-head (HH) photodimer with high selectivity. This is contrary to what is observed upon the solid-state irradiation of coumarin, which affords photodimers with low selectivity and conversion.
Asunto(s)
Alquinos/química , Cumarinas/química , Compuestos Macrocíclicos/química , Fenetilaminas/química , Urea/química , Dimerización , Procesos FotoquímicosRESUMEN
Pyridine macrocycles with no cavities assembled into close packed columns yet absorbed guests including hydrogen, carbon dioxide, and iodine.
Asunto(s)
Compuestos Macrocíclicos/síntesis química , Piridinas/química , Urea/química , Dióxido de Carbono/química , Cristalografía por Rayos X , Hidrógeno/química , Yodo/química , Compuestos Macrocíclicos/química , Modelos Moleculares , Estructura MolecularRESUMEN
Second-generation self-assembling bis-urea macrocycles were designed that form columnar structures in the solid state. The new macrocycles were constructed from more flexible building blocks yielding greater solubility and a more efficient synthesis. In addition, heteroatoms in the form of ether oxygens were incorporated in the walls of the macrocycles to provide additional recognition sites for guest encapsulation. We observed reduced fidelity of the stacking motif and in some cases the intermolecular urea-urea hydrogen bonds were disrupted by the formation of intramolecular hydrogen bonds. We also observed new offset assembly motifs that maintained the urea-urea interaction. These results suggest that the stacking of the arene units in the rigid first-generation systems was an important factor in guiding the formation of the columnar stacks.
Asunto(s)
Compuestos Macrocíclicos/química , Urea/análogos & derivados , Urea/química , Cristalografía por Rayos X , Enlace de Hidrógeno , Compuestos Macrocíclicos/síntesis química , Modelos Moleculares , Estructura Molecular , Método de Montecarlo , Solubilidad , Urea/síntesis químicaRESUMEN
Changing an ether to a ketone within the framework of a bis-urea macrocycle has little effect on the supramolecular assembly of this building block into porous crystals but introduces a triplet sensitizer into the framework that dramatically alters the photochemical reactions of included guests.
Asunto(s)
Fotoquímica , Éteres/química , Cetonas/química , Modelos MolecularesRESUMEN
This article studies the origins of selectivity for the [2+2] cycloadditions of alpha,beta-unsaturated ketones within a porous crystalline host. The host, formed by the self-assembly of a bis-urea macrocycle, contains accessible channels of approximately 6 A diameter and forms stable inclusion complexes with a variety of cyclic and acyclic alpha,beta-unsaturated ketone derivatives. Host 1 crystals provide a robust confined reaction environment for the highly selective [2+2] cycloaddition of 3-methyl-2-cyclopentenone, 2-cyclohexenone, and 2-methyl-2-cyclopentenone, forming their respective exo head-to-tail dimers in high conversion. The products are readily extracted from the self-assembled host and the crystalline host can be efficiently recovered and reused. Molecular modeling studies indicate that the origin of the observed selectivity is due to the excellent match between the size and shape of these guests to dimensions of the host channel and to the preorganization of neighboring enones into favorable reaction geometries. Small substrates, such as acrylic acid and methylvinylketone, were bound by the host and were protected from photoreactions. Larger substrates, such as 4,4-dimethyl-2-cyclohexenone and mesityl oxide, do not undergo selective [2+2] cycloaddition reactions. In an effort to understand these differences in reactivity, we examined these host-guest complexes by thermogravimetric analysis (TGA), NMR, powder X-ray diffraction (PXRD) and molecular modeling.
Asunto(s)
Ciclohexanonas/química , Compuestos Heterocíclicos/química , Cetonas/química , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Estructura Molecular , Porosidad , Difracción de PolvoRESUMEN
We report a highly selective 2 + 2 cycloaddition of 2-cyclohexenone in the presence of self-assembled bisurea macrocycles that yields the head-to-tail photodimer. The reaction proceeds with high conversion and with decreased incidence of secondary photorearrangement. Furthermore, the product can be extracted from the assembly, and the solid assembly is readily recovered and reused, much like a zeolite.