No abstract

The function of lysosomes relies on the ability of the lysosomal membrane to fuse with several target membranes in the cell. It is known that in lysosomal storage disorders (LSDs), lysosomal accumulation of several types of substrates is associated with lysosomal dysfunction and impairment of endocytic membrane traffic. By analysing cells from two severe neurodegenerative LSDs, we observed that cholesterol abnormally accumulates in the endolysosomal membrane of LSD cells, thereby reducing the ability of lysosomes to efficiently fuse with endocytic and autophagic vesicles. Furthermore, we discovered that soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptors (SNAREs), which are key components of the cellular membrane fusion machinery are aberrantly sequestered in cholesterol-enriched regions of LSD endolysosomal membranes. This abnormal spatial organization locks SNAREs in complexes and impairs their sorting and recycling. Importantly, reducing membrane cholesterol levels in LSD cells restores normal SNARE function and efficient lysosomal fusion. Our results support a model by which cholesterol abnormalities determine lysosomal dysfunction and endocytic traffic jam in LSDs by impairing the membrane fusion machinery, thus suggesting new therapeutic targets for the treatment of these disorders.

The hypoxia-inducible factor (HIF)-1␣ and HIF-2␣ are closely related, key transcriptional regulators of the hypoxic response, countering a low oxygen situation with the up-regulation of target genes associated with numerous processes, including vascularization and glycolysis. This involves a dual mechanism of control through both stabilization and transactivation, regulated via prolyl and asparaginyl hydroxylation. Despite high similarity with respect to protein sequence and activation pathway, a growing number of physiological and mechanistic differences between HIF-1␣ and HIF-2␣ are being reported. To further characterize this nonredundancy, the stabilization of endogenous proteins and regulation of the transactivation domains were compared in a graded oxygen environment across a series of cell lines. Although generally similar results were found, interesting and specific differences between the HIF-␣ proteins were observed within certain cell lines, such as rat adrenal PC12s, emphasizing the cell-specific nature of HIF-␣ regulation. We characterize a conserved amino acid substitution between HIF-1␣ and HIF-2␣ that contributes to the intrinsically higher FIH-1-mediated asparaginyl hydroxylation of HIF-1␣ and, hence, lower HIF-1␣ activity. In addition, our data demonstrate that the different cell lines can be classified into two distinct groups: those in which stabilization and transactivation proceed in conjunction (HeLa, 293T, and COS-1) and those cells in which HIF-␣ is stabilized prior to transactivation (PC12, HepG2, and CACO2). Interestingly, the initial stabilization of HIF-␣ prior to transactivation up-regulation predicted from in vitro derived hydroxylation data is only true for a subset of cells.In a state of hypoxia, where the demand for oxygen exceeds supply, physiological responses are mounted, including increasing the capacity for blood to carry oxygen to tissues and facilitating the production of ATP by anaerobic glycolysis. The hypoxia-inducible factors (HIFs) 4 are key transcriptional regulators of this genomic response to hypoxia in essentially all mammalian cells. HIF is composed of an oxygen-regulated HIF-␣ subunit (HIF-1␣ or HIF-2␣) and the ubiquitous aryl hydrocarbon receptor nuclear translocator (or HIF-1␤) partner protein. HIF-␣ protein turnover in normoxia is very rapid due to the inhibitory action of the HIF-␣ prolyl hydroxylases (PHDs). These oxygen-dependent enzymes hydroxylate two conserved prolyl residues within a central oxygen-dependent degradation domain of the HIF-␣ proteins, promoting binding of the Von Hippel-Lindau protein, ubiquitylation, and proteasomal degradation (1-3). Any HIF-␣ escaping this normoxic degradation is also subject to hydroxylation of a conserved asparagine within the C-terminal transactivation domain (CAD) that represses activity via abrogation of CBP/p300 recruitment (4, 5). Under hypoxic conditions, the activity of the prolyl and asparaginyl hydroxylases is inhibited, and the HIF-␣ proteins are stabilized and transcriptionally activated, leading to po...

Sanfilippo syndrome, or mucopolysaccharidosis (MPS) type III, refers to one of five autosomal recessive, neurodegenerative lysosomal storage disorders (MPS IIIA to MPS IIIE) whose symptoms are caused by the deficiency of enzymes involved exclusively in heparan sulfate degradation. The primary characteristic of MPS III is the degeneration of the central nervous system, resulting in mental retardation and hyperactivity, typically commencing during childhood. The significance of the order of events leading from heparan sulfate accumulation through to downstream changes in the levels of biomolecules within the cell and ultimately the (predominantly neuropathological) clinical symptoms is not well understood. The genes whose deficiencies cause the MPS III subtypes have been identified, and their gene products, as well as a selection of disease-causing mutations, have been characterized to varying degrees with respect to both frequency and direct biochemical consequences. A number of genetic and biochemical diagnostic methods have been developed and adopted by diagnostic laboratories. However, there is no effective therapy available for any form of MPS III, with treatment currently limited to clinical management of neurological symptoms. The availability of animal models for all forms of MPS III, whether spontaneous or generated via gene targeting, has contributed to improved understanding of the MPS III subtypes, and has provided and will deliver invaluable tools to appraise emerging therapies. Indeed, clinical trials to evaluate intrathecally-delivered enzyme replacement therapy in MPS IIIA patients, and gene therapy for MPS IIIA and MPS IIIB patients are planned or underway.

Autophagy is dependent upon lysosomes, which fuse with the autophagosome to complete the autophagic process and whose acidic interior permits the activity of their intraluminal degradative enzymes. Chloroquine (CQ) and bafilomycin A1 (BafA) each cause alkalinisation of the lumen and thus impair lysosomal function, although by distinct mechanisms. CQ diffuses into lysosomes and undergoes protonation, while BafA inhibits the ability of the vacuolar type H + -ATPase (v-ATPase) to transfer protons into the lysosome. In the present study, we examine the impact of CQ and BafA on the activity of mammalian target of rapamycin complex 1 (mTORC1), inhibition of which is an early step in promoting autophagy. We find each compound inhibits mTORC1 signalling, without affecting levels of protein components of the mTORC1 signalling pathway. Furthermore, these effects are not related to these agents' capacity to inhibit autophagy or the reduction in amino acid supply from lysosomal proteolysis. Instead, our data indicate that the reduction in mTORC1 signalling appears to be due to the accumulation of lysosomal storage material. However, there are differences in responses to these agents, for instance, in their abilities to up-regulate direct targets of transcription factor EB (TFEB), a substrate of mTORC1 that drives transcription of many lysosomal and autophagy-related genes. Nonetheless, our data imply that widely used agents that alkalinise intralysosomal pH are mimetics of acute lysosomal storage disorders (LSDs) and emphasise the importance of considering the result of CQ and BafA on mTORC1 signalling when interpreting the effects of these agents on cellular physiology.