Browsing by Author "Pan, Samuel"

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Open Access
Glycosylation of gigaxonin regulates intermediate filaments: Novel molecular insights into giant axonal neuropathy: supplemental information
(2019-01-26) CHEN, PO-HAN; Smith, Timothy; Hu, Jimin; Pan, Samuel; Smith, Alexander; Lu, Annie; Chi, Jen-Tsan; Boyce, Michael
Gigaxonin (also known as KLHL16) is an E3 ligase adaptor protein that promotes the ubiquitination and degradation of intermediate filament (IF) proteins. Mutations in human gigaxonin cause the fatal neurodegenerative disease giant axonal neuropathy (GAN), in which IF proteins accumulate and aggregate in axons throughout the nervous system, impairing neuronal function and viability. Despite this pathophysiological significance, the upstream regulation and downstream effects of normal and aberrant gigaxonin function remain incompletely understood. Here, we report that gigaxonin is modified by O-linked-beta-N-acetylglucosamine (O-GlcNAc), a prevalent form of intracellular glycosylation, in a nutrient- and growth factor-dependent manner. Mass spectrometry analyses of human gigaxonin revealed nine candidate sites of O-GlcNAcylation, two of which - serine 272 and threonine 277 - are required for its ability to mediate IF turnover in novel gigaxonin-deficient human cell models that we created. Taken together, these results suggest that nutrient-responsive gigaxonin O-GlcNAcylation forms a regulatory link between metabolism and IF proteostasis. Our work may have significant implications for understanding the non-genetic modifiers of GAN phenotypes and for the optimization of gene therapy for this disease.
Open Access
Kinome screen of ferroptosis reveals a novel role of ATM in regulating iron metabolism.
(Cell death and differentiation, 2019-07-18) Chen, Po-Han; Wu, Jianli; Ding, Chien-Kuang Cornelia; Lin, Chao-Chieh; Pan, Samuel; Bossa, Nathan; Xu, Yitong; Yang, Wen-Hsuan; Mathey-Prevot, Bernard; Chi, Jen-Tsan
Ferroptosis is a specialized iron-dependent cell death that is associated with lethal lipid peroxidation. Modulation of ferroptosis may have therapeutic potential since it has been implicated in various human diseases as well as potential antitumor activities. However, much remains unknown about the underlying mechanisms and genetic determinants of ferroptosis. Given the critical role of kinases in most biological processes and the availability of various kinase inhibitors, we sought to systemically identify kinases essential for ferroptosis. We performed a forward genetic-based kinome screen against ferroptosis in MDA-MB-231 cells triggered by cystine deprivation. This screen identified 34 essential kinases involved in TNFα and NF-kB signaling. Unexpectedly, the DNA damage response serine/threonine kinase ATM (mutated in Ataxia-Telangiectasia) was found to be essential for ferroptosis. The pharmacological or genetic inhibition of ATM consistently rescued multiple cancer cells from ferroptosis triggered by cystine deprivation or erastin. Instead of the canonical DNA damage pathways, ATM inhibition rescued ferroptosis by increasing the expression of iron regulators involved in iron storage (ferritin heavy and light chain, FTH1 and FTL) and export (ferroportin, FPN1). The coordinated changes of these iron regulators during ATM inhibition resulted in a lowering of labile iron and prevented the iron-dependent ferroptosis. Furthermore, we found that ATM inhibition enhanced the nuclear translocation of metal-regulatory transcription factor 1 (MTF1), responsible for regulating expression of Ferritin/FPN1 and ferroptosis protection. Genetic depletion of MTF-1 abolished the regulation of iron-regulatory elements by ATM and resensitized the cells to ferroptosis. Together, we have identified an unexpected ATM-MTF1-Ferritin/FPN1 regulatory axis as novel determinants of ferroptosis through regulating labile iron levels.