A mutation in a mild form of galactosialidosis impairs dimerization of the protective protein and renders it unstable.
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The lysosomal disorder galactosialidosis is caused by deficiency of the protective protein in the absence of which the activities of the enzymes beta-galactosidase and neuraminidase are reduced. Aside from its protective function towards the two glycosidases, this protein has cathepsin A-like activity. A point mutation in the protective protein gene, resulting in the substitution of Phe412 with Val in the gene product, was identified in two unrelated patients with the late infantile form of the disease. Expression in COS-1 cells of a protective protein cDNA with the base substitution resulted in the synthesis of a mutant protein that lacks cathepsin A-like activity. The newly made mutant precursor was shown to be partially retained in the endoplasmic reticulum. Only a fraction is transported to the lysosomes where it is degraded soon after proteolytic processing into the mature two-chain form. Since the mutant precursor, contrary to the wild type protein, does not form homodimers, the dimerization process might be a condition for the proper targeting and stable conformation of the protective protein. These results clarify the mechanism underlying the combined deficiency in these patients, and give new insight into the structure-function relationship of the wild type protein. (+info)
Endocytic mechanisms for targeted drug delivery.
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Advances in the delivery of targeted drug systems have evolved to enable highly regulated site specific localization to subcellular organelles. Targeting therapeutics to individual intracellular compartments has resulted in benefits to therapies associated with these unique organelles. Endocytosis, a mechanism common to all cells in the body, internalizes macromolecules and retains them in transport vesicles which traffic along the endolysosomal scaffold. An array of vesicular internalization mechanisms exist, therefore understanding the key players specific to each pathway has allowed researchers to bioengineer macromolecular complexes for highly specialized delivery. Membrane specific receptors most frequently enter the cell through endocytosis following the binding of a high affinity ligand. High affinity ligands interact with membrane receptors, internalize in membrane bound vesicles, and traffic through cells in different manners to allow for accumulation in early endosomal fractions or lysosomally associated fractions. Although most drug delivery complexes aim to avoid lysosomal degradation, more recent studies have shown the clinical utility in directed protein delivery to this environment for the enzymatic release of therapeutics. Targeting nanomedicine complexes to the endolysosomal pathway has serious potential for improving drug delivery for the treatment of lysosomal storage diseases, cancer, and Alzheimer's disease. Although several issues remain for receptor specific targeting, current work is investigating a synthetic receptor approach for high affinity binding of targeted macromolecules. (+info)
Lysosomal dysfunction results in altered energy balance.
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The mucopolysaccharidosis (MPS) type VII mouse was originally described as the adipose storage deficiency mouse because of its extreme lean phenotype of unknown etiology. Here, we show that adipose storage deficiency and lower leptin levels are common to five different lysosomal storage diseases (LSDs): MPSI, MPSIIIB, MPSVII, Niemann-Pick type A/B, and infantile neuronal ceroid lipofuscinosis. Elevated circulating pro-inflammatory proteins (VCAM1 and MCP1) were found in multiple LSDs. Multiple anti-inflammatory strategies (dexamethasone, MCP1 deficiency, M3 expression) failed to alter adiposity in LSD animals. All of the models had normal or greater caloric intake and lower to normal metabolic rate, fasting plasma glucose, non-esterified fatty acids, cholesterol, and triglycerides. Triglycerides were lower in the livers of MPSI mice, and the trend was lower in the muscle. Lipid absorption and processing in MPSI mice were indistinguishable from those in normal mice following oral gavage of olive oil. The increased lean mass of MPSI and MPSIIIB mice suggests a shift in adipose triglycerides to lysosomal storage. In agreement, MPSI livers had a similar total caloric content but reduced caloric density, indicating a shift in energy from lipids to proteins/carbohydrates (lysosomal storage). Enzyme replacement therapy normalized the caloric density within 48 h without reducing total caloric content. This was due to an increase in lipids. Recycling of stored material is likely reduced or nonexistent. Therefore, to maintain homeostasis, energy is likely diverted to synthesis at the expense of typical energy storage depots. Thus, these diseases will serve as important tools in studying the role of lysosome function in metabolism and obesity. (+info)
A block of autophagy in lysosomal storage disorders.
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Most lysosomal storage disorders (LSDs) are caused by deficiencies of lysosomal hydrolases. While LSDs were among the first inherited diseases for which the underlying biochemical defects were identified, the mechanisms from enzyme deficiency to cell death are poorly understood. Here we show that lysosomal storage impairs autophagic delivery of bulk cytosolic contents to lysosomes. By studying the mouse models of two LSDs associated with severe neurodegeneration, multiple sulfatase deficiency (MSD) and mucopolysaccharidosis type IIIA (MPSIIIA), we observed an accumulation of autophagosomes resulting from defective autophagosome-lysosome fusion. An impairment of the autophagic pathway was demonstrated by the inefficient degradation of exogenous aggregate-prone proteins (i.e. expanded huntingtin and mutated alpha-synuclein) in cells from LSD mice. This impairment resulted in massive accumulation of polyubiquitinated proteins and of dysfunctional mitochondria which are the putative mediators of cell death. These data identify LSDs as 'autophagy disorders' and suggest the presence of common mechanisms in the pathogenesis of these and other neurodegenerative diseases. (+info)
Update on treatment of lysosomal storage diseases.
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Lysosomal storage diseases (LSDs) are a large group of disorders caused by a deficiency of specific enzymes responsible for the degradation of substances present in lysosomes. In the past few years, treatments for LSDs were non specific and could only cope with signs and symptoms of the diseases. A successful therapeutic approach to LSDs should instead address to the underlying causes of the diseases, thus helping the degradation of the accumulated metabolites in the various organs, and at the same time preventing their further deposition. One way is to see to an available source of the deficient enzyme: bone marrow transplantation, enzyme replacement therapy and gene therapy are based on this rationale. The purpose of substrate reduction therapy is to down regulate the formation of the lysosomal substance to a rate at which the residual enzyme activity can catabolize the stored and de novo produced lysosomal substrate. Chemical chaperone therapy is based on small molecules able to bind and stabilize the misfolded enzymes. This paper offers a historical overview on the therapeutic strategies for LSDs. (+info)
ER and oxidative stresses are common mediators of apoptosis in both neurodegenerative and non-neurodegenerative lysosomal storage disorders and are alleviated by chemical chaperones.
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It is estimated that more than 40 different lysosomal storage disorders (LSDs) cumulatively affect one in 5000 live births, and in the majority of the LSDs, neurodegeneration is a prominent feature. Neuronal ceroid lipofuscinoses (NCLs), as a group, represent one of the most common (one in 12,500 births) neurodegenerative LSDs. The infantile NCL (INCL) is the most devastating neurodegenerative LSD, which is caused by inactivating mutations in the palmitoyl-protein thioesterase-1 (PPT1) gene. We previously reported that neuronal death by apoptosis in INCL, and in the PPT1-knockout (PPT1-KO) mice that mimic INCL, is at least in part caused by endoplasmic reticulum (ER) and oxidative stresses. In the present study, we sought to determine whether ER and oxidative stresses are unique manifestations of INCL or they are common to both neurodegenerative and non-neurodegenerative LSDs. Unexpectedly, we found that ER and oxidative stresses are common manifestations in cells from both neurodegenerative and non-neurodegenerative LSDs. Moreover, all LSD cells studied show extraordinary sensitivity to brefeldin-A-induced apoptosis, which suggests pre-existing ER stress conditions. Further, we uncovered that chemical disruption of lysosomal homeostasis in normal cells causes ER stress, suggesting a cross-talk between the lysosomes and the ER. Most importantly, we found that chemical chaperones that alleviate ER and oxidative stresses are also cytoprotective in all forms of LSDs studied. We propose that ER and oxidative stresses are common mediators of apoptosis in both neurodegenerative and non-neurodegenerative LSDs and suggest that the beneficial effects of chemical/pharmacological chaperones are exerted, at least in part, by alleviating these stress conditions. (+info)
Lysosomal storage diseases as disorders of autophagy.
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The cellular turnover of proteins and organelles requires cooperation between the autophagic and the lysosomal degradation pathways. A crucial step in this process is the fusion of the autophagosome with the lysosome. In our study we demonstrate that in Lysosomal Storage Disorders (LSDs) accumulation of undegraded substrates in lysosomes, due to deficiency of specific lysosomal enzymes, impairs the fusion between autophagosomes and lysosomes. This, in turn, leads to a progressive accumulation of poly-ubiquitinated protein aggregates and of dysfunctional mitochondria. These findings suggest that neurodegeneration in LSDs may share some mechanisms with late-onset neurodegenerative disorders in which the accumulation of protein aggregates is a prominent feature. (+info)
Effect of lysosomal storage on bis(monoacylglycero)phosphate.
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BMP [bis(monoacylglycero)phosphate] is an acidic phospholipid and a structural isomer of PG (phosphatidylglycerol), consisting of lysophosphatidylglycerol with an additional fatty acid esterified to the glycerol head group. It is thought to be synthesized from PG in the endosomal/lysosomal compartment and is found primarily in multivesicular bodies within the same compartment. In the present study, we investigated the effect of lysosomal storage on BMP in cultured fibroblasts from patients with eight different LSDs (lysosomal storage disorders) and plasma samples from patients with one of 20 LSDs. Using ESI-MS/MS (electrospray ionization tandem MS), we were able to demonstrate either elevations or alterations in the individual species of BMP, but not of PG, in cultured fibroblasts. All affected cell lines, with the exception of Fabry disease, showed a loss of polyunsaturated BMP species relative to mono-unsaturated species, and this correlated with the literature reports of lysosomal dysfunction leading to elevations of glycosphingolipids and cholesterol in affected cells, processes thought to be critical to the pathogenesis of LSDs. Plasma samples from patients with LSDs involving storage in macrophages and/or with hepatomegaly showed an elevation in the plasma concentration of the C(18:1)/C(18:1) species of BMP when compared with control plasmas, whereas disorders involving primarily the central nervous system pathology did not. These results suggest that the release of BMP is cell/tissue-specific and that it may be useful as a biomarker for a subset of LSDs. (+info)