The 3′-Phosphoadenosine 5′-Phosphosulfate Transporters, PAPST1 and 2, Contribute to the Maintenance and Differentiation of Mouse Embryonic Stem Cells

Recently, we have identified two 3′-phosphoadenosine 5′-phosphosulfate (PAPS) transporters (PAPST1 and PAPST2), which contribute to PAPS transport into the Golgi, in both human and Drosophila. Mutation and RNA interference (RNAi) of the Drosophila PAPST have shown the importance of PAPST-dependent sulfation of carbohydrates and proteins during development. However, the functional roles of PAPST in mammals are largely unknown. Here, we investigated whether PAPST-dependent sulfation is involved in regulating signaling pathways required for the maintenance of mouse embryonic stem cells (mESCs), differentiation into the three germ layers, and neurogenesis. By using a yeast expression system, mouse PAPST1 and PAPST2 proteins were shown to have PAPS transport activity with an apparent Km value of 1.54 µM or 1.49 µM, respectively. RNAi-mediated knockdown of each PAPST induced the reduction of chondroitin sulfate (CS) chain sulfation as well as heparan sulfate (HS) chain sulfation, and inhibited mESC self-renewal due to defects in several signaling pathways. However, we suggest that these effects were due to reduced HS, not CS, chain sulfation, because knockdown of mouse N-deacetylase/N-sulfotransferase, which catalyzes the first step of HS sulfation, in mESCs gave similar results to those observed in PAPST-knockdown mESCs, but depletion of CS chains did not. On the other hand, during embryoid body formation, PAPST-knockdown mESCs exhibited abnormal differentiation, in particular neurogenesis was promoted, presumably due to the observed defects in BMP, FGF and Wnt signaling. The latter were reduced as a result of the reduction in both HS and CS chain sulfation. We propose that PAPST-dependent sulfation of HS or CS chains, which is regulated developmentally, regulates the extrinsic signaling required for the maintenance and normal differentiation of mESCs

Analysis ofDrosophilaGlucuronyl C5-Epimerase

exterior, apartment tower, July-Sept. 198

Heparan Sulfate Structure Affects Autophagy, Lifespan, Responses to Oxidative Stress, and Cell Degeneration in Drosophila parkin Mutants

Autophagy is a catabolic process that provides cells with energy and molecular building blocks during nutritional stress. Autophagy also removes misfolded proteins and damaged organelles, a critical mechanism for cellular repair. Earlier work demonstrated that heparan sulfate proteoglycans, an abundant class of carbohydrate-modified proteins found on cell surfaces and in the extracellular matrix, suppress basal levels of autophagy in several cell types during development in Drosophila melanogaster. In studies reported here, we examined the capacity of heparan sulfate synthesis to influence events affected by autophagy, including lifespan, resistance to reactive oxygen species (ROS) stress, and accumulation of ubiquitin-modified proteins in the brain. Compromising heparan sulfate synthesis increased autophagy-dependent processes, evident by extended lifespan, increased resistance to ROS, and reduced accumulation of ubiquitin-modified proteins in the brains of ROS exposed adults. The capacity of altering heparan sulfate biosynthesis to protect cells from injury was also evaluated in two different models of neurodegeneration, overexpression of Presenilin and parkin mutants. Presenilin overexpression in the retina produces cell loss, and compromising heparan sulfate biosynthesis rescued retinal patterning and size abnormalities in these animals. parkin is the fly homolog of human PARK2, one of the genes responsible for juvenile onset Parkinson’s Disease. Parkin is involved in mitochondrial surveillance and compromising parkin function results in degeneration of both flight muscle and dopaminergic neurons in Drosophila. Altering heparan sulfate biosynthesis suppressed flight muscle degeneration and mitochondrial dysmorphology, indicating that activation of autophagy-mediated removal of mitochondria (mitophagy) is potentiated in these animals. These findings provide in vivo evidence that altering the levels of heparan sulfate synthesis activates autophagy and can provide protection from a variety of cellular stressors