Spectrophotometric Study of Bridging N-Donor Ligand-Induced Supramolecular Assembly of Conjugated Zn-Trisporphyrin with a Triphenylamine Core.

This article studies the supramolecular assembly behavior of a Zn-trisporphyrin conjugate containing a triphenylamine core (1) with bridging N-donor ligands using the UV-vis spectrophotometric titration method at micromolar concentrations. Our results show that pyridine, a non-bridging ligand, formed a 3:1 open complex with 1. The corresponding binding constant was estimated to be (2.7 ± 0.15) × 1014 M-3. In contrast, bridging ligands, 4,4-bipyridine (BIPY) and 1,3-di(4-pyridyl)propane (DPYP), formed stable 3:2 double-decker complexes with 1 in solution, which collapsed to yield a 3:1 open complex when excess BIPY or DPYP was added. The binding constants for forming BIPY and DPYP double-decker complexes were estimated to be (9.26 ± 0.07) × 1027 M-4 and (3.62 ± 0.16) × 1027 M-4, respectively. The UV-vis titration profiles supported the conclusion that the degradation of the 3:2 double-decker 1∙BIPY complex is less favorable compared to that of 1∙DPYP. Consequently, the formation of the 3:1 1∙DPYP open complex proceeded more readily than that of 1∙BIPY.

Molecular Characterization of a Novel 1,3-α-3,6-Anhydro-L-Galactosidase, Ahg943, with Cold- and High-Salt-Tolerance from Gayadomonas joobiniege G7.

1,3-α-3,6-anhydro-L-galactosidase (α-neoagarooligosaccharide hydrolase) catalyzes the last step of agar degradation by hydrolyzing neoagarobiose into monomers, D-galactose, and 3,6-anhydro-Lgalactose, which is important for the bioindustrial application of algal biomass. Ahg943, from the agarolytic marine bacterium Gayadomonas joobiniege G7, is composed of 423 amino acids (47.96 kDa), including a 22-amino acid signal peptide. It was found to have 67% identity with the α-neoagarooligosaccharide hydrolase ZgAhgA, from Zobellia galactanivorans, but low identity (< 40%) with the other α-neoagarooligosaccharide hydrolases reported. The recombinant Ahg943 (rAhg943, 47.89 kDa), purified from Escherichia coli, was estimated to be a monomer upon gel filtration chromatography, making it quite distinct from other α-neoagarooligosaccharide hydrolases. The rAhg943 hydrolyzed neoagarobiose, neoagarotetraose, and neoagarohexaose into D-galactose, neoagarotriose, and neoagaropentaose, respectively, with a common product, 3,6- anhydro-L-galactose, indicating that it is an exo-acting α-neoagarooligosaccharide hydrolase that releases 3,6-anhydro-L-galactose by hydrolyzing α-1,3 glycosidic bonds from the nonreducing ends of neoagarooligosaccharides. The optimum pH and temperature of Ahg943 activity were 6.0 and 20°C, respectively. In particular, rAhg943 could maintain enzyme activity at 10°C (71% of the maximum). Complete inhibition of rAhg943 activity by 0.5 mM EDTA was restored and even, remarkably, enhanced by Ca2+ ions. rAhg943 activity was at maximum at 0.5 M NaCl and maintained above 73% of the maximum at 3M NaCl. Km and Vmax of rAhg943 toward neoagarobiose were 9.7 mg/ml and 250 µM/min (3 U/mg), respectively. Therefore, Ahg943 is a unique α-neoagarooligosaccharide hydrolase that has cold- and high-salt-adapted features, and possibly exists as a monomer.

Autophagy is required for PDAC glutamine metabolism.

Macroautophagy (autophagy) is believed to maintain energy homeostasis by degrading unnecessary cellular components and molecules. Its implication in regulating cancer metabolism recently started to be uncovered. However, the precise roles of autophagy in cancer metabolism are still unclear. Here, we show that autophagy plays a critical role in glutamine metabolism, which is required for tumor survival. Pancreatic ductal adenocarcinoma (PDAC) cells require both autophagy and typical glutamine transporters to maintain intracellular glutamine levels. Glutamine deprivation, but not that of glucose, led to the activation of macropinocytosis-associated autophagy through TFEB induction and translocation into the nucleus. In contrast, glutamine uptake increased as a compensatory response to decreased intracellular glutamine levels upon autophagy inhibition. Moreover, autophagy inhibition and glutamine deprivation did not induce cell death, while glutamine deprivation dramatically activated apoptotic cell death upon autophagy inhibition. Interestingly, the addition of α-ketoglutarate significantly rescued the apoptotic cell death caused by the combination of the inhibition of autophagy with glutamine deprivation. Our data suggest that macropinocytosis-associated autophagy is a critical process providing glutamine for anaplerosis of the TCA cycle in PDAC. Thus, targeting both autophagy and glutamine metabolism to completely block glutamine supply may provide new therapeutic approaches to treat refractory tumors.