RESUMO
Antibiotic resistance is a growing public health threat that complicates the treatment of infections. ß-Lactamase enzymes, which hydrolyze the ß-lactam ring present in many common antibiotics, are a major cause of this resistance and are produced by a broad range of bacterial pathogens. Here, we developed hydrogels that degrade specifically in the presence of ß-lactamases and ß-lactamase-producing bacteria as a platform for bacteria-triggered drug delivery. A maleimide-functionalized ß-lactamase-cleavable cephalosporin was used as a crosslinker in the fabrication of hydrogels through end-crosslinked polymerization with multiarm thiol-terminated poly(ethylene glycol) macromers via Michael-type addition. We demonstrated that only hydrogels containing the responsive crosslinker were degraded by ß-lactamases and ß-lactamase-producing bacteria in vitro and in an ex vivo porcine skin infection model. Fluorescent polystyrene nanoparticles, encapsulated in the hydrogels as model cargo, were released at rates that closely tracked hydrogel wet mass loss, confirming ß-lactamase-triggered controlled cargo release. Nonresponsive hydrogels, lacking the ß-lactam crosslinker, remained stable in the presence of ß-lactamases and ß-lactamase-producing bacteria and exhibited no change in mass or nanoparticle release. Furthermore, the responsive hydrogels remained stable in non-ß-lactamase enzymes, including collagenases and lipases. These hydrogels have the potential to be used as a bacteria-triggered drug delivery system to control unnecessary exposure to encapsulated antimicrobials, which can provide effective infection treatment without exacerbating resistance.
Assuntos
Hidrogéis , beta-Lactamases , Animais , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Bactérias , Sistemas de Liberação de Medicamentos , Hidrogéis/farmacologia , Suínos , beta-Lactamas/farmacologiaRESUMO
GLUT1 is a major glucose facilitator expressed ubiquitously among tissues. Upregulation of its expression plays an important role in the development of many types of cancer and metabolic diseases. Thioredoxin-interacting protein (TXNIP) is an α-arrestin that acts as an adaptor for GLUT1 in clathrin-mediated endocytosis. It regulates cellular glucose uptake in response to both intracellular and extracellular signals via its control on GLUT1-4. In order to understand the interaction between GLUT1 and TXNIP, we generated GLUT1 lipid nanodiscs and carried out isothermal titration calorimetry and single-particle electron microscopy experiments. We found that GLUT1 lipid nanodiscs and TXNIP interact in a 1:1 ratio and that this interaction requires phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2 or PIP2).
Assuntos
Proteínas de Transporte/genética , Transportador de Glucose Tipo 1/genética , Lipídeos/genética , Fosfatidilinositol 4,5-Difosfato/química , Transporte Biológico/genética , Proteínas de Transporte/química , Clatrina/química , Endocitose/genética , Glucose/metabolismo , Transportador de Glucose Tipo 1/química , Humanos , Lipídeos/química , Fosfatidilinositol 4,5-Difosfato/genética , Transdução de SinaisRESUMO
Fatty acids are essential cellular building blocks and a major energy source. Regardless of their metabolic fate, fatty acids first need to be activated by forming a thioester with a coenzyme A group. This reaction is carried out by acyl-CoA synthetases (ACSs), of which ACSL1 (long-chain acyl-CoA synthetase 1) is an important member. Two bacterial homologues of ACSL1 crystal structures have been solved previously. One is a soluble dimeric protein, and the other is a monomeric peripheral membrane protein. The mammalian ACSL1 is a membrane protein with an N-terminal transmembrane helix. To characterize the mammalian ACSL1, we purified the full-length mouse ACSL1 and reconstituted it into lipid nanodiscs. Using enzymatic assays, mutational analysis, and cryo-electron microscopy, we show that mouse ACSL1 is active as a monomer.
