18 research outputs found
The novel prolyl hydroxylase-2 inhibitor caffeic acid upregulates hypoxia inducible factor and protects against hypoxia
Background & aims: Hypoxia inducible factor (HIF) is a hypoxia-associated transcription factor that has a protective role against hypoxia-induced damage. Prolyl hydroxylase-2 (PHD2) is a dioxygenase enzyme that specifically hydroxylates HIF targeting it for degradation, therefore, inhibition of the PHD2 enzyme activity acts to upregulate HIF function. This study was to identify novel PHD2 inhibitors. Methods: An established fluorescence-based PHD2 activity assay was used for inhibitors screening. Western blot and quantitative real-time PCR was used to detect the protein and mRNA levels respectively. Further animal experiment was carried out. Results: Caffeic acid was screened and identified as a novel PHD2 inhibitor. Caffeic acid treated PC12 and SH-SY5Y neuronal cell lines stabilized endogenous HIF-1α protein levels and consequently increased mRNA levels of its downstream regulated genes VEGF and EPO. Caffeic acid treatment reduced hypoxia-induced cell apoptosis and promoted HIF/BNIP3-mediated mitophagy. Moreover, animal studies indicated that caffeic acid increased the level of HIF-1α protein and mRNA levels of VEGF and EPO in the brain of mice exposed to hypoxia. Conventional brain injury markers including malondialdehyde, lactic acid and lactate dehydrogenase in the caffeic acid treated mice were shown to be reduced to the levels of the control group. Conclusions: This study suggests that caffeic acid inhibits PHD2 enzyme activity which then activates the hypoxia-associated transcription factor HIF leading to a neuroprotective effect against hypoxia
Coordination of Golgin tethering and SNARE assembly: GM130 binds syntaxin 5 in a p115-regulated manner
Membrane targeting and activation of the Lowe syndrome protein OCRL1 by rab GTPases
The X-linked disorder oculocerebrorenal syndrome of Lowe is caused by mutation of the OCRL1 protein, an inositol polyphosphate 5-phosphatase. OCRL1 is localised to the Golgi apparatus and early endosomes, and can translocate to lamellipodia upon growth factor stimulation. We show here that OCRL1 interacts with several members of the rab family of small GTPases. Strongest interaction is seen with Golgi-associated rab1 and rab6 and endosomal rab5. Point mutants defective in rab binding fail to target to the Golgi apparatus and endosomes, strongly suggesting rab interaction is required for targeting of OCRL1 to these compartments. Membrane recruitment via rab binding is required for changes in Golgi and endosomal dynamics induced by overexpression of catalytically inactive OCRL1. In vitro experiments demonstrate that rab5 and rab6 directly stimulate the 5-phosphatase activity of OCRL1. We conclude that rabs play a dual role in regulation of OCRL1, firstly targeting it to the Golgi apparatus and endosomes, and secondly, directly stimulating the 5-phosphatase activity of OCRL1 after membrane recruitment
The coiled-coil membrane protein golgin-84 is a novel rab effector required for Golgi ribbon formation
Fragmentation of the mammalian Golgi apparatus during mitosis requires the phosphorylation of a specific subset of Golgi-associated proteins. We have used a biochemical approach to characterize these proteins and report here the identification of golgin-84 as a novel mitotic target. Using cryoelectron microscopy we could localize golgin-84 to the cis-Golgi network and found that it is enriched on tubules emanating from the lateral edges of, and often connecting, Golgi stacks. Golgin-84 binds to active rab1 but not cis-Golgi matrix proteins. Overexpression or depletion of golgin-84 results in fragmentation of the Golgi ribbon. Strikingly, the Golgi ribbon is converted into mini-stacks constituting only approximately 25% of the volume of a normal Golgi apparatus upon golgin-84 depletion. These mini-stacks are able to carry out protein transport, though with reduced efficiency compared with a normal Golgi apparatus. Our results suggest that golgin-84 plays a key role in the assembly and maintenance of the Golgi ribbon in mammalian cells
The coiled-coil membrane protein golgin-84 is a novel rab effector required for Golgi ribbon formation
Fragmentation of the mammalian Golgi apparatus during mitosis requires the phosphorylation of a specific subset of Golgi-associated proteins. We have used a biochemical approach to characterize these proteins and report here the identification of golgin-84 as a novel mitotic target. Using cryoelectron microscopy we could localize golgin-84 to the cis-Golgi network and found that it is enriched on tubules emanating from the lateral edges of, and often connecting, Golgi stacks. Golgin-84 binds to active rab1 but not cis-Golgi matrix proteins. Overexpression or depletion of golgin-84 results in fragmentation of the Golgi ribbon. Strikingly, the Golgi ribbon is converted into mini-stacks constituting only approximately 25% of the volume of a normal Golgi apparatus upon golgin-84 depletion. These mini-stacks are able to carry out protein transport, though with reduced efficiency compared with a normal Golgi apparatus. Our results suggest that golgin-84 plays a key role in the assembly and maintenance of the Golgi ribbon in mammalian cells