Many lysosomal storage disease-associated mutant enzymes exhibit compromised endoplasmic reticulum (ER) folding and therefore are subjected to degradation instead of being trafficked to the lysosome, where they normally degrade their substrates. This leads to lysosomal substrate accumulation and thus, pathology.
In Specific Aim 1 we aspire to identify proteostasis network components responsible for (1) folding wild type and Gaucher disease-associated mutant ?-glucocerebrosidases (GCs) in the ER, (2) trafficking them through the Golgi and on to the lysosome, (3) stabilizing/activating them in the lysosome, and (4) degrading misfolded GC in the ER by immunoisolating the GC interactome (antibody recognizes the extreme C-terminus and is thus conformation and mutant insensitive) and identifying the proteins by mass spectrometry. Mutant ?- glucocerebrosidases exhibit overlapping interactomes with distinct members because they accumulate in various subcellular compartments. GC interacting partners, prioritized on the basis of 4 methods, will be RNAi depleted in L444P GC cells to discern what extent GC proteostasis is perturbed using the intact cell GC activity assay, endo H sensitivity and immunofluorescence colocalization. Several novel GC proteostasis network components have already been identified, allowing several hypotheses regarding the GC proteostasis network to be tested. Simply lowering the growth temperature of patient-derived cells to 300C, inducing the unfolded protein response (UPR), or increasing ER Ca2+ concentration with ryanodine antagonists enhances mutant enzyme folding, trafficking and function enabling the interactome of adapted cells vs. controls to be determined to test numerous hypotheses about mechanism, including whether these results extend to other lysosomal storage diseases.
In Specific Aim 2, we explore which genetically-encodable, small molecule regulated UPR- associated transcription factors individually and in combination can enhance mutant lysosomal enzyme folding, trafficking and function. Understanding the transcriptome upregulated by these transcription factors and combinations affording heterodimeric transcription factors cross referenced to the upregulated proteome in proteostasis network adapted cells will provide additional mechanistic insight into the enhancement in proteostasis. Unregulated overexpression of XBP1s restores L444P GC proteostasis motivating the development of aryl oxime ether IRE1 activators that will be tested in patient-derived cells from multiple lysosomal storage diseases to discern efficacy. IRE1 Inhibitors should be useful to cancer researchers.
This research defines the protein homeostasis network that can be altered to enhance mutant lysosomal storage disease-associated enzyme folding, trafficking and function. Adapting the protein homeostasis network with small molecules offers the possibility of ameliorating multiple lysosomal storage diseases of similar etiology by fixing the folding and trafficking of the mutant enzyme instead of replacing the enzyme-the current standard of care, which is expensive, disease specific and not useful for alleviating neuropathic lysosomal storage diseases. Small molecule adaptors of cellular protein homeostasis used in combination with pharmacologic chaperones that bind to and stabilize a particular protein are envisioned to be a very promising future approach for treating loss-of-function diseases, of which the lysosomal storage diseases of focus herein are one class.
|Baranczak, Aleksandra; Liu, Yu; Connelly, Stephen et al. (2015) A fluorogenic aryl fluorosulfate for intraorganellar transthyretin imaging in living cells and in Caenorhabditis elegans. J Am Chem Soc 137:7404-14|
|Cho, Younhee; Zhang, Xin; Pobre, Kristine Faye R et al. (2015) Individual and collective contributions of chaperoning and degradation to protein homeostasis in E. coli. Cell Rep 11:321-33|
|Chen, John J; Genereux, Joseph C; Qu, Song et al. (2014) ATF6 activation reduces the secretion and extracellular aggregation of destabilized variants of an amyloidogenic protein. Chem Biol 21:1564-74|
|Hebert, Daniel N; Lamriben, Lydia; Powers, Evan T et al. (2014) The intrinsic and extrinsic effects of N-linked glycans on glycoproteostasis. Nat Chem Biol 10:902-10|
|Cooley, Christina B; Ryno, Lisa M; Plate, Lars et al. (2014) Unfolded protein response activation reduces secretion and extracellular aggregation of amyloidogenic immunoglobulin light chain. Proc Natl Acad Sci U S A 111:13046-51|
|Tan, Yun Lei; Genereux, Joseph C; Pankow, Sandra et al. (2014) ERdj3 is an endoplasmic reticulum degradation factor for mutant glucocerebrosidase variants linked to Gaucher's disease. Chem Biol 21:967-76|
|Shoulders, Matthew D; Ryno, Lisa M; Cooley, Christina B et al. (2013) Broadly applicable methodology for the rapid and dosable small molecule-mediated regulation of transcription factors in human cells. J Am Chem Soc 135:8129-32|
|Ong, Derrick Sek Tong; Wang, Ya-Juan; Tan, Yun Lei et al. (2013) FKBP10 depletion enhances glucocerebrosidase proteostasis in Gaucher disease fibroblasts. Chem Biol 20:403-15|
|Shoulders, Matthew D; Ryno, Lisa M; Genereux, Joseph C et al. (2013) Stress-independent activation of XBP1s and/or ATF6 reveals three functionally diverse ER proteostasis environments. Cell Rep 3:1279-92|
|Orwig, Susan D; Tan, Yun Lei; Grimster, Neil P et al. (2011) Binding of 3,4,5,6-tetrahydroxyazepanes to the acid-?-glucosidase active site: implications for pharmacological chaperone design for Gaucher disease. Biochemistry 50:10647-57|
Showing the most recent 10 out of 21 publications