| dc.description.abstract | Eukaryotic cellular pathways are maintained and coordinated through biomolecule turnover, which includes synthesis, trafficking, degradation of cellular components and their reutilization. The process of biomolecule degradation has been shown to modulate the cellular homeostasis. Lipid degradation in mammalian cells occurs in membrane-bound organelles called lysosomes and is carried out by acid hydrolases. Mutations in the lysosomal enzymes or their defective trafficking lead to the accumulation of degradative substrates resulting in lysosome dysfunction, observed in a group (~60 types) of genetic diseases known as lysosomal storage disorders/diseases (LSDs).
In the past one decade, LSDs in India have surged and 374 cases have been reported so far, wherein 34% patients suffer from Gaucher‟s disease. In line, Gaucher‟s disease has been the most common LSD reported in the world. This disorder is caused by mutations in the gene GBA1 that encodes for lysosomal enzyme acid -glucosidase or -glucocerebrosidase (-GC). Most commonly, mutations in -GC lead to its misfolding and is subjected to ER-associated proteasomal degradation (ERAD) that impairs the trafficking of enzyme to lysosomes. This process results in accumulation of the substrate glucosylceramide causing lysosome dysfunction that lead to hepatomegaly and splenomegaly. It has been shown that few of the -GC mutants lead to the neuropathic form of the disease, which affects the central nervous system. Currently, enzyme replacement (ERT) and substrate reduction (SRT) therapies are used to cure the non-neuropathic Gaucher‟s disease. In this study, we aimed to modulate the cellular pathways that control the folding and trafficking of mutant -GC to lysosome. Cellular phosphatases/kinases are known to alter these proteostasis pathways through a post-translation modification. Thus, we studied the role of phosphatases and kinases in regulating lysosome function following their effect on Gaucher‟s disease.
The current study entitled as “Unraveling the role of cellular phosphatases in lysosome function and storage diseases” has divided into five chapters. Chapter-I outline the review of literature on lysosomes including the trafficking of lysosomal proteins, function, biogenesis
and associated diseases. Chapter-II describes the experimental procedures used in this study. Chapter-III to Chapter-V describes the experimental results and discussion.
Chapter-III: RNAi screen to identify the cellular phosphatases/kinases for enhancing the activity of lysosomes.
Lysosomal hydrolases are specialized class of enzymes that degrade various biomolecules in acidic pH of the lysosomes. These acid hydrolases turnover the intracellular and extracellular cargo received by the lysosomes through autophagy and endocytosis, respectively. Trafficking of lysosomal hydrolases involve cargo receptors such as CI-MPR (Cation-independent mannose 6-phosphate receptor), CD-MPR (Cation-dependent mannose 6-phosphate receptor), LIMP-2 (Lysosome membrane protein 2) and sortilin for their transport from Golgi to late endosomes and then the cargo transport to lysosomes. Most of these receptors utilize the post-translationally added mannose residue on the hydrolase for their vesicular transport. In pathological conditions such as LSD, a large cohort of mutant protein is unable to exit the Endoplasmic Reticulum (ER) and thus cause lysosome dysfunction. Phosphatases/kinases are known to alter the various signaling pathways transiently by de/phosphorylating the substrates respectively. We hypothesize that modulating their activity possibly alters the folding and trafficking of mutant lysosome hydrolases from the ER. Here, we have chosen to study β-GC and its mutants that are associated with Gaucher‟s disease. We have altered the activity of phosphatases/kinases by depleting the protein levels using specific pooled shRNAs against the genes.
We have performed RNAi screen of phosphatases and kinases using pooled shRNAs and measured the activity of -GC biochemically in HeLa cells. In the primary screen, we have identified 32 novel phosphatases out of 310, whose knockdown in HeLa cells improved the -GC activity by more than 1.5 times compared to control cells. Similarly, we have identified 89 out of 668 kinases and their depletion in HeLa cells enhanced the -GC activity by more than 3 folds as compared to control cells. We proceed further in evaluating the role of phosphatase hits in increasing the -GC (wild-type and mutants) activity, trafficking and lysosome function (in Chapter IV). Overall, these studies have identified new molecules, which may regulate the activity of lysosomes and possibly modulate the Gaucher‟s disease condition.
Chapter-IV: Studying the role of cellular phosphatases in restoring the lysosome function.
In this chapter, we have tested the role of phosphatase hits obtained in primary screen (Chapter III) on lysosome function/biogenesis. To validate the primary hits obtained with pooled shRNAs, we have used individual shRNAs against each phosphatase and repeated the biochemical -GC activity assay in HeLa cells both in presence and absence of a chemical inhibitor CBE (Condituriol B Epoxide, known to inhibit -GC activity). These experiments reduced the hits to 8 candidate phosphatases (DUSP14, PPP6C, SAPS3, PPA2, NT5E, PTPN20B, PPAP2B and STYXL1). To delineate their role in increasing -GC activity, we studied the trafficking of stably expressing -GC wild-type and -GC mutants (N370S) in HeLa cells. Immunofluorescence microscopy analysis showed -GCWT-mCherry primarily localized to lysosomes and its mutants localized majorly to Endoplasmic Reticulum. Interestingly, the depletion of five phosphatases enhanced the localization of -GCWT-mCherry and -GCN370S-mCherry to lysosomes. These results suggest that inhibition of activity of selected phosphatases have potential in improving the trafficking of -GC mutants to lysosomes.
To test whether phosphatase knockdown can alter the lysosome defects/activity in Gaucher‟s disease, we used primary fibroblasts derived from Gaucher‟s patients (obtained from Coriell Cell Repositories, USA) and they showed 5 - 12% of -GC activity compared to cells from healthy individuals (referred to here as wild-type cells). Upon lentiviral-mediated depletion of phosphatases in wild-type and different patient fibroblasts (having mutation N370S-V39φL, N370S-84GG, Type I-L444P or Type II-L444P), the -GC activity was enhanced to 10-20% of their respective control (scrambled shRNA-treated) cells, which is adequate to ameliorate the Gaucher‟s disease. Surprisingly, the depletion of these phosphatases in neuropathic form of the disease showed enhanced the -GC activity. Altogether, the -GC activity in Gaucher‟s patient cells is restored as equivalent to wild-type fibroblasts. Thus, our studies identified new potential candidate phosphatases, whose activity inhibition possibly provides an alternative cure for Gaucher‟s disease. In future, we would like to design the chemical inhibitors against these phosphatases those can be used for the treatment of Gaucher‟s disease. Moreover, we would like to validate the role of these phosphatases in modulating the disease condition in other LSDs.
Chapter V: Illustrating the role of pseudophosphatase STYXL1 in modulating lysosome function in Gaucher’s disease.
Phosphatases are a class of enzymes, which dephosphorylate different biomolecules based on the amino acids present in their catalytic site. Around 13.8% of cellular phosphatases are grouped into a class called pseudophosphatases and they have lost their ability to catalyze dephosphorylation due to mutations in their active site. These pseudoenzymes have been shown to act as a scaffold, competitors or direct regulators of various signaling molecules. Our RNAi screen identified (Chapter IV) a pseudophosphatase STYXL1, whose knockdown in HeLa cells enhanced the -GC trafficking and activity. To understand the mechanism governed by STYXL1, we tested the effect of its depletion (using two different shRNAs) in HeLa cells on the induction of ER stress/unfolded protein response (UPR). It has been shown that UPR is known to modulate the folding and trafficking of misfolded/unfolded proteins in the ER and acts as an adaptive and survival response to prevent their accumulation. As expected, the nuclear translocation of UPR activated transcription factors XBP1s, ATF6 and CHOP was increased in STYXL1-knockdown HeLa cells. Consistently, the transcript levels of XBP1 and BIP but not CHOP were enhanced in the STYXL1-knockdown cells. However, the treatment of STYXL1-depleted HeLa cells with a chemical inhibitor 4-PBA (attenuates ER stress) showed reduced -GC activity, suggesting an altered ER stress in the cells. In line with these results, knockdown of STYXL1 (using shRNA 1 and 2) in primary Gaucher‟s patient and wild-type fibroblasts enhanced the -GC activity (similar to the results described in Chapter IV). Moreover, primary fibroblasts derived from patients carrying N370S and L444P mutations showed reduced clustering and increased lysosome dispersion upon STYXL1-depletion. Similarly, the depletion of STYXL1 in HeLa cells also showed enhanced lysosomal distribution in addition to increased lysosome acidity and proteolytically activity compared to control cells. Overall, these results indicate an indirect role of STYXL1 on lysosome function/distribution as well as in the modulation of UPR. In future, we would like to identify the chemical inhibitors against STYXL1, which can be used as potential drug molecule to induce ER stress and also for treating Gaucher‟s disease.
Overall, our studies identified new molecular regulators, those were able to alter the lysosome function both in healthy and Gaucher‟s patient cells. These molecules have a
potential to design small molecular inhibitors against their activity that can ameliorate the diseases associate with the lysosomes. | en_US |
