RESUMEN
HdeA has been shown to prevent acid-induced aggregation of proteins. With a mass of only 9.7 kDa, HdeA is one of the smallest chaperones known. Unlike other molecular chaperones, which are typically complex, multimeric ATP-dependent machines, HdeA is known to undergo an acid-induced dimer to monomer transition and functions at low pH as a disordered monomer without the need for energy factors. Thus, HdeA must possess features that allow it to bind substrates and regulate substrate affinity in a small and energy-independent package. To understand better how HdeA accomplishes this, we studied the conformational changes that accompany a shift to low pH and substrate binding. We find that the acid-induced partial unfolding and monomerization that lead to HdeA activation occur very rapidly (k >3.5 s(-1)). Activation exposes the hydrophobic dimer interface, which we found to be critical for substrate binding. We show by intramolecular FRET that the partially unfolded character of active HdeA allows the chaperone to adopt different conformations as required for the recognition and high-affinity binding of different substrate proteins. These efficient adaptations help to explain how a very small protein is rapidly activated and can bind a broad range of substrate proteins in a purely pH-regulated manner.
Asunto(s)
Proteínas de Escherichia coli/química , Chaperonas Moleculares/química , Especificidad por Sustrato , Ácidos , Sitios de Unión , Dimerización , Concentración de Iones de Hidrógeno , Unión Proteica , Conformación ProteicaRESUMEN
Research in the field of food intake regulation is gaining importance. This often includes the measurement of peptides regulating food intake. For the correct determination of a peptide's concentration, it should be stable during blood processing. However, this is not the case for several peptides which are quickly degraded by endogenous peptidases. Recently, we developed a blood processing method employing Reduced temperatures, Acidification, Protease inhibition, Isotopic exogenous controls and Dilution (RAPID) for the use in rats. Here, we have established this technique for the use in humans and investigated recovery, molecular form and circulating concentration of food intake regulatory hormones. The RAPID method significantly improved the recovery for (125)I-labeled somatostatin-28 (+39%), glucagon-like peptide-1 (+35%), acyl ghrelin and glucagon (+32%), insulin and kisspeptin (+29%), nesfatin-1 (+28%), leptin (+21%) and peptide YY3-36 (+19%) compared to standard processing (EDTA blood on ice, p <0.001). High performance liquid chromatography showed the elution of endogenous acyl ghrelin at the expected position after RAPID processing, while after standard processing 62% of acyl ghrelin were degraded resulting in an earlier peak likely representing desacyl ghrelin. After RAPID processing the acyl/desacyl ghrelin ratio in blood of normal weight subjects was 1:3 compared to 1:23 following standard processing (p = 0.03). Also endogenous kisspeptin levels were higher after RAPID compared to standard processing (+99%, p = 0.02). The RAPID blood processing method can be used in humans, yields higher peptide levels and allows for assessment of the correct molecular form.
Asunto(s)
Proteínas Sanguíneas/análisis , Técnicas de Química Analítica/métodos , Animales , Ingestión de Alimentos , Glucagón , Humanos , Insulina , Leptina , RatasRESUMEN
Pancreatic ductal adenocarcinoma (PDAC) metastasizes by neural, vascular, and local invasion routes, which limit patient survival. In nerves and vessels, SLIT2 and its ROBO receptors constitute repellent guidance cues that also direct epithelial branching. Thus, the SLIT2-ROBO system may represent a key pinch point to regulate PDAC spread. In this study, we examined the hypothesis that escaping from repellent SLIT2-ROBO signaling is essential to enable PDAC cells to appropriate their local stromal infrastructure for dissemination. Through immunohistochemical analysis, we detected SLIT2 receptors ROBO1 and ROBO4 on epithelia, nerves, and vessels in healthy pancreas and PDAC specimens, respectively. SLIT2 mRNA expression was reduced in PDAC compared with nontransformed pancreatic tissues and cell lines, suggesting a reduction in SLIT2-ROBO pathway activity in PDAC. In support of this interpretation, restoring the SLIT2 expression in SLIT2-deficient PDAC cells inhibited their bidirectional chemoattraction with neural cells, and more specifically, impaired unidirectional PDAC cell navigation along outgrowing neurites in models of neural invasion. Restoring autocrine/paracrine SLIT2 signaling was also sufficient to inhibit the directed motility of PDAC cells, but not their random movement. Conversely, RNA interference-mediated silencing of ROBO1 stimulated the motility of SLIT2-competent PDAC cells. Furthermore, culture supernatants from SLIT2-competent PDAC cells impaired migration of endothelial cells (human umbilical vein endothelial cells), whereas an N-terminal SLIT2 cleavage fragment stimulated such migration. In vivo investigations of pancreatic tumors with restored SLIT2 expression demonstrated reduced invasion, metastasis, and vascularization, with opposing effects produced by ROBO1 silencing in tumor cells or sequestration of endogenous SLIT2. Analysis of clinical specimens of PDAC showed that those with low SLIT2 mRNA expression exhibited a higher incidence and a higher fraction of tumor-infiltrated lymph nodes. Taken together, our findings argue that disrupting SLIT2-ROBO signaling in PDAC may enhance metastasis and predispose PDAC cells to neural invasion.