Browsing by Author "Sardiello, Marco"

Now showing 1 - 4 of 4
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    A Rapid and Sensitive Method for Measuring NAcetylglucosaminidase Activity in Cultured Cells
    (Public Library of Science, 2013) Mauri, Victor; Lotfi, Parisa; Segatori, Laura; Sardiello, Marco
    A rapid and sensitive method to quantitatively assess N-acetylglucosaminidase (NAG) activity in cultured cells is highly desirable for both basic research and clinical studies. NAG activity is deficient in cells from patients with Mucopolysaccharidosis type IIIB (MPS IIIB) due to mutations in NAGLU, the gene that encodes NAG. Currently available
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    CLN8 is an endoplasmic reticulum cargo receptor that regulates lysosome biogenesis
    (Springer Nature, 2018) di Ronza, Alberto; Bajaj, Lakshya; Sharma, Jaiprakash; Sanagasetti, Deepthi; Lotfi, Parisa; Adamski, Carolyn Joy; Collette, John; Palmieri, Michela; Amawi, Abdallah; Popp, Lauren; Chang, Kevin Tommy; Meschini, Maria Chiara; Leung, Hon-Chiu Eastwood; Segatori, Laura; Simonati, Alessandro; Sifers, Richard Norman; Santorelli, Filippo Maria; Sardiello, Marco
    Organelle biogenesis requires proper transport of proteins from their site of synthesis to their target subcellular compartment1,2,3. Lysosomal enzymes are synthesized in the endoplasmic reticulum (ER) and traffic through the Golgi complex before being transferred to the endolysosomal system4,5,6, but how they are transferred from the ER to the Golgi is unknown. Here, we show that ER-to-Golgi transfer of lysosomal enzymes requires CLN8, an ER-associated membrane protein whose loss of function leads to the lysosomal storage disorder, neuronal ceroid lipofuscinosis 8 (a type of Batten disease)7. ER-to-Golgi trafficking of CLN8 requires interaction with the COPII and COPI machineries via specific export and retrieval signals localized in the cytosolic carboxy terminus of CLN8. CLN8 deficiency leads to depletion of soluble enzymes in the lysosome, thus impairing lysosome biogenesis. Binding to lysosomal enzymes requires the second luminal loop of CLN8 and is abolished by some disease-causing mutations within this region. Our data establish an unanticipated example of an ER receptor serving the biogenesis of an organelle and indicate that impaired transport of lysosomal enzymes underlies Batten disease caused by mutations in CLN8.
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    Peroxisomal biogenesis is genetically and biochemically linked to carbohydrate metabolism in Drosophila and mouse
    (Public Library of Science, 2017) Wangler, Michael F.; Chao, Yu-Hsin; Bayat, Vafa; Giagtzoglou, Nikolaos; Shinde, Abhijit Babaji; Putluri, Nagireddy; Coarfa, Cristian; Donti, Taraka; Graham, Brett H.; Faust, Joseph E.; McNew, James A.; Moser, Ann; Sardiello, Marco; Baes, Myriam; Bellen, Hugo J.
    Peroxisome biogenesis disorders (PBD) are a group of multi-system human diseases due to mutations in the PEX genes that are responsible for peroxisome assembly and function. These disorders lead to global defects in peroxisomal function and result in severe brain, liver, bone and kidney disease. In order to study their pathogenesis we undertook a systematic genetic and biochemical study of Drosophila pex16 and pex2 mutants. These mutants are short-lived with defects in locomotion and activity. Moreover these mutants exhibit severe morphologic and functional peroxisomal defects. Using metabolomics we uncovered defects in multiple biochemical pathways including defects outside the canonical specialized lipid pathways performed by peroxisomal enzymes. These included unanticipated changes in metabolites in glycolysis, glycogen metabolism, and the pentose phosphate pathway, carbohydrate metabolic pathways that do not utilize known peroxisomal enzymes. In addition, mutant flies are starvation sensitive and are very sensitive to glucose deprivation exhibiting dramatic shortening of lifespan and hyperactivity on low-sugar food. We use bioinformatic transcriptional profiling to examine gene co-regulation between peroxisomal genes and other metabolic pathways and we observe that the expression of peroxisomal and carbohydrate pathway genes in flies and mouse are tightly correlated. Indeed key steps in carbohydrate metabolism were found to be strongly co-regulated with peroxisomal genes in flies and mice. Moreover mice lacking peroxisomes exhibit defective carbohydrate metabolism at the same key steps in carbohydrate breakdown. Our data indicate an unexpected link between these two metabolic processes and suggest metabolism of carbohydrates could be a new therapeutic target for patients with PBD.
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    TFEB regulates lysosomal proteostasis
    (Oxford University Press, 2013) Song, Wensi; Wang, Fan; Savini, Marzia; Ake, Ashley; di Ronza, Alberto; Sardiello, Marco; Segatori, Laura
    Loss-of-function diseases are often caused by destabilizing mutations that lead to protein misfolding and degradation. Modulating the innate protein homeostasis (proteostasis) capacity may lead to rescue of native folding of the mutated variants, thereby ameliorating the disease phenotype. In lysosomal storage disorders (LSDs), a number of highly prevalent alleles have missense mutations that do not impair the enzyme's catalytic activity but destabilize its native structure, resulting in the degradation of the misfolded protein. Enhancing the cellular folding capacity enables rescuing the native, biologically functional structure of these unstable mutated enzymes. However, proteostasis modulators specific for the lysosomal system are currently unknown. Here, we investigate the role of the transcription factor EB (TFEB), a master regulator of lysosomal biogenesis and function, in modulating lysosomal proteostasis in LSDs. We show that TFEB activation results in enhanced folding, trafficking and lysosomal activity of a severely destabilized glucocerebrosidase (GC) variant associated with the development of Gaucher disease (GD), the most common LSD. TFEB specifically induces the expression of GC and of key genes involved in folding and lysosomal trafficking, thereby enhancing both the pool of mutated enzyme and its processing through the secretory pathway. TFEB activation also rescues the activity of a β-hexosaminidase mutant associated with the development of another LSD, Tay–Sachs disease, thus suggesting general applicability of TFEB-mediated proteostasis modulation to rescue destabilizing mutations in LSDs. In summary, our findings identify TFEB as a specific regulator of lysosomal proteostasis and suggest that TFEB may be used as a therapeutic target to rescue enzyme homeostasis in LSDs.