Browsing by Author "Nijhawan, Deepak"
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Item Bayesian Spatial Analysis of High Throughput Sequencing Data(2021-05-01T05:00:00.000Z) Zhang, Minzhe; Mendell, Joshua T.; Nijhawan, Deepak; Zhan, Xiaowei; Xie, Yang; Xiao, GuanghuaThe past decade has witnessed the development and wide use of high-throughput sequencing data in biology. The recent advancement of RNA Sequencing (RNA-Seq) coupled with other molecular technologies such as methylated RNA immunoprecipitation (MeRIP) and spatial barcoding has delivered more specialized platform to investigate certain cellular process and spatial molecular profiling. However, the development of associated analysis tools capable of accommodating the unique features of these new sequencing technologies is still lacking or unsatisfied. For the past few years, I have been devoting to the methodology development of MeRIP-Seq and spatial molecular profiling data. The proposed BaySeqPeak and BOOST-GP methods demonstrated good accuracy, sensitivity and robustness in identifying methylated RNA region and spatial variable genes in both the simulation study and real data analysis.Item Biochemical Pathways in Apoptosis(2005-05-03) Nijhawan, Deepak; Wang, XiaodongCaspases are a family of proteases that once activated execute apoptosis, a cellular suicide pathway. Activated caspases have a unique property to cleave and activate themselves. Once the first caspase is activated, it generates a chain reaction resulting in robust caspase activity and rapid death. The central question in apoptosis is to understand how the first caspase is activated. To address this question, we present an assay that recapitulates de novo caspase activation in vitro. We describe how this assay was used to purify three proteins that were sufficient to reconstitute caspase activation in vitro: Apaf-1, cytochrome c, and caspase-9. The mechanism of caspase activation in vivo, however, is complicated by these proteins subcelluar localization. In the living cell, Apaf-1 and caspase-9 are cytoplasmic whereas cytochrome c is mitochondria, however, during apoptosis, cytochrome is released to the cytoplasm. In vitro, cytochrome c induces the formation of a stable Apaf-1/caspase-9 complex and caspase-9 autoactivation suggesting that cytochrome c release from the mitochondria to the cytosol is the rate-limiting event that leads to caspase activation. This conclusion shifted the focus of our studies upstream from what initiates caspase activation to what triggers cytochrome c release. The second part of the dissertation uses a biochemical approach to identify how cytochrome c release is regulated after exposure to ultraviolet light. Ultraviolet light irradiation of HeLa cells triggers an apoptotic response mediated by mitochondria. Biochemical analysis of such a response revealed that the initial step leading to cytochrome c release is the complete disappearance of the mRNA of Mcl-1, an anti-apoptotic member of the Bcl-2 family. This event leads to the elimination of Mcl-1 protein from cells due to the short half-life of its protein. The block or delay of Mcl-1 disappearance by either proteasome inhibitors or Mcl-1 over-expression prevents subsequent steps of this apoptotic pathway including the translocation of Bax and Bcl-xL from cytosol to mitochondria and dephosphorylation of BimEL on mitochondria. These sequential events lead to the oligomerization of Bax and Bak on the mitochondria, cytochrome c release and caspase activation.Item Ceramide-Induced Alternative Translocation of TM4SF20(2015-10-29) Lee, Ching En; Pfeiffer, Julie K.; McKnight, Steven L.; Kahn, Jeffrey; Nijhawan, Deepak; Ye, JinThe polytopic membrane protein TM4SF20 (transmembrane 4 L6 family 20) is a protein containing four transmembrane helices that inhibits the Regulated Intramembrane Proteolysis (RIP) of the transcriptional factor CREB3L1 (cAMP response element binding protein 3-like 1), a transcription factor synthesized as a membrane-bound precursor. CREB3L1 RIP is induced by several stimuli: ER stress, viral infections, the chemotherapeutic drug, doxorubicin, and the sphingolipid, ceramide. Additionally, TGF-β (transforming growth factor-β), a cytokine known to stimulate collagen production, induces the proteolytic activation of CREB3L1 in human A549 cells through inhibition of TM4SF20 expression, which normally inhibits RIP of CREB3L1. We also find that the TM4SF20 regulation of CREB3L1 RIP is regulated by ceramide. In this study we find that ceramide can regulate the ability of first transmembrane domain of TM4SF20 to determine its orientation in the membrane. Under normal conditions, TM4SF20 is synthesized as a protein that inhibits the cleavage of CREB3L1 when TRAM2 (translocation associated membrane protein 2) is associated with the ER translocon. Excess ceramide dissociates TRAM2 from the ER translocon such that the N-terminus of TM4SF20 can no longer be forced by the first transmembrane domain to function as a signal peptide. Under excess ceramide conditions, TM4SF20 adopts a completely opposite topology and allows the cleavage of CREB3L1 to proceed. We have designated this novel mechanism for transmembrane protein regulation as "alternative translocation."Item Characterization of Ubiquitin Ligase Targeting by Anticancer Sulfonamides(2020-08-01T05:00:00.000Z) Ting, Tabitha Chung-Yan; De Martino, George; Nijhawan, Deepak; Yu, Hongtao; DeBose-Boyd, Russell A.Aryl sulfonamides are small molecules that are selectively toxic to a subset of human cancer cell lines. Clinical trials of the aryl sulfonamide indisulam have resulted in modest clinical activity against a subset of solid tumors. Recent work revealed that indisulam recruits the RNA binding protein RBM39 to DCAF15, a component of the CRL4-DCAF15 E3 ubiquitin ligase. This recruitment results in RBM39 ubiquitination and degradation, leading to splicing defects and cancer cell death (Han et al., 2017; Uehara et al., 2017). The mechanism of action of sulfonamides is similar to that of immunomodulatory drugs (IMiDs), which recruit substrates to the closely related CRL4-CRBN E3 ubiquitin ligase for ubiquitination. Known for their roles in inhibiting embryonic development and cancer cell growth, IMiDs exert their pleiotropic effects by targeting a variety of substrate proteins to the CRL4-CRBN E3. Despite major advances in our understanding of aryl sulfonamides, it is unclear whether sulfonamides also target multiple substrates or modulate the endogenous function of the CRL4-DCAF15 E3 ligase. This dissertation describes our efforts to define the requirements for RBM39 ubiquitination, identify other substrates that are recruited to the CRL4-DCAF15 E3 ligase, and further our understanding of the cellular consequences of indisulam treatment. In Chapters 2 and 3, we define the components required for RBM39 ubiquitination using a combination of in vitro and in vivo techniques. In Chapters 4 and 5, we identify putative endogenous substrates and a previously undescribed neo-substrate recruited to the CRL4-DCAF15 for ubiquitination. In Chapter 6, we characterize the cellular consequences of indisulam treatment and neo-substrate degradation. In aggregate, this work aims to contribute to our understanding of the sulfonamide mechanism of action and the field of targeted protein degradation.Item Chemical Footprinting of Polymeric Structure of hnRNPA2 Low Complexity Domain(2016-05-24) Xiang, Siheng; Nijhawan, Deepak; Rosen, Michael K.; Yu, Hongtao; Kliewer, Steven A.; McKnight, Steven L.Many DNA and RNA regulatory proteins contain polypeptide domains that are unstructured when analyzed in cell lysates. These domains are typified by an over-representation of a limited number of amino acids and have been termed prion-like, intrinsically disordered or low complexity domains. These low complexity sequences have been shown to induce phase transition in low salt buffer. When incubated at high concentration, certain of these low complexity domains polymerize into labile, amyloid-like fibers. I developed a chemical footprinting method to probe solvent accessible residues in the low complexity domain polymers. By acetylating protein side chains with N-acetylimidazole, and comparing the acetylation in native and denatured conformation by use of SILAC mass spectrometry, I generated an NAI footprint for hnRNPA2 polymers. I deployed this footprinting technique to probe the structure of the native hnRNPA2 protein present in isolated nuclei, and offered evidence that its low complexity domain exists in a similar conformation as that described for recombinant polymers of the protein. To study the structure of the low complexity sequence in liquid-like droplets, I systematically mutated individual tyrosine or phenylalanine residues to serine, assayed the ratio of these mutants that partitioned into the droplet phase, and compared the results with their abilities to grow polymeric fibers from wild-type seeds. The same region which contained mutations impeding fiber growth were found to display decreased partitioning into liquid-like droplets. Additionally, the NAI footprint of hnRNPA2 in these liquid-like droplets appeared to be similar to the footprint found in fibers. These observations suggest that the hnRNPA2 low complexity domain adopts a similar structure in amyloid-like fibers and liquid-like droplets. Combining these results, my studies favor the perspective that cross-beta polymerization commonly drives the formation of hydrogels, the retention of low complexity domains trapped by hydrogels, the formation of liquid-like droplets, the partitioning of low complexity domains into existing liquid-like droplets, and the formation and maturation of RNA granules. In other words, my results provide evidence that the involvement of low complexity domains in the formation of RNA granules, liquid-like droplets and hydrogels all rely on one in the same phenomenon - cross-beta polymerization.Item Discovery, Biological Profiling and Mechanistic Studies of Three Novel Antimalarials(2021-05-01T05:00:00.000Z) Lawong, Aloysus Kome; Nijhawan, Deepak; MacMillan, John; Ready, Joseph M.; Wetzel, Dawn; Phillips, Margaret A.Emerging resistance of the malaria causing Plasmodium parasite to current first-line therapies underscores the need for new antimalarial agents with broad ranging activity against multiple stages of the parasite. No new chemical class of antimalarials has been introduced into clinical practice since 1996. Overcoming emerging drug resistance requires new drugs with novel modes of action. With the aim of identifying new classes of antimalarials, I completed a phenotypic high throughput screen of two synthetic chemistry libraries against erythrocytic stage P. falciparum and compiled a portfolio of chemically novel validated antiplasmodials (ALCHM1 - 18). Herein I describe the discovery and characterization of three prioritized scaffolds: a tetrazole-based ALCHM3 series, an azetidine amide ALCHM17 series and a piperidine carboxamide ALCHM18 series. I report here on the biological profiling, mechanistic characterization and potential as next-generation anti-malarial agents of these three previously unreported scaffolds. ALCHM3 is a novel chemical series, with fast kill kinetics that targets the historically druggable heme polymerization pathway. The fast kill azetidine amide ALCHM17 series, is a novel scaffold with inhibitory activity in the pre-erythrocytic and erythrocytic stages, and the first azetidine scaffold with Pfcarl associated resistance. The piperidine carboxamide ALCHM18 is a proteasome β5 subunit-selective inhibitor, with a moderate rate of kill, species selectivity, strong starting in vitro and in vivo ADME properties, and potential to become the first proteasome inhibiting preclinical candidate for malaria treatment.Item Elucidating the Target and Selectivity of a Non-Small Cell Lung Cancer Toxin(2019-07-11) Madhusudhan, Nikhil; Tu, Benjamin; Nijhawan, Deepak; DeBerardinis, Ralph J.; McFadden, David G.; Mishra, PrashantSelective toxicity among cancer cells of the same lineage is a hallmark of targeted therapies, and so identifying compounds that impair proliferation selectively is an important strategy in drug development. Here, we report the discovery of the quinazoline dione compound (QDC), a molecule that exhibits selective toxicity across 100 non-small cell lung cancer (NSCLC) lines. Using photoreactive probes, we found that the QDC targets mitochondrial Complex I. We investigated the selective toxicity of Complex I inhibition across NSCLC and found that a high baseline content of aspartate formed via reductive glutamine metabolism promotes resistance to mitochondrial inhibition. Altered metabolism is an important feature of cancer and there is significant interest in understanding how differences in tumor metabolism can be exploited for therapy. NSCLC cells subject to mitochondrial inhibition use reductive carboxylation to meet aspartate demand, which suggests that targeting reductive aspartate synthesis would enhance clinical response to Complex I inhibitors.Item Elucidation of the Mechanism of Action of a Cell Line Selective Toxin(2019-01-31) Theodoropoulos, Panayotis Christos; Yu, Hongtao; Nijhawan, Deepak; Brown, Michael S.; Ready, Joseph M.; Chen, Zhijian J.A hallmark of targeted cancer therapies is selective toxicity among cancer cell lines. We evaluated results from a viability screen of over 200,000 small molecules to identify two chemical series, oxalamides and benzothiazoles, that were selectively toxic to the same four of 12 lung cancer cell lines at low nanomolar concentrations. Sensitive cell lines expressed cytochrome P450 (CYP) 4F11, which metabolized the compounds into irreversible stearoyl CoA desaturase (SCD) inhibitors. SCD has been recognized as a promising biological target in cancer and metabolic disease. However, SCD is essential to sebocytes, and accordingly SCD inhibitors cause skin toxicity. Mouse sebocytes were unable to activate the benzothiazoles or oxalamides into SCD inhibitors, providing a therapeutic window for inhibiting SCD in vivo. We thus offer a strategy to target SCD in cancer by taking advantage of high CYP expression in a subset of tumors.Item Immunotherapy: a new era in cancer treatment(2015-07-10) Nijhawan, DeepakItem Investigating the Mechanism and Mode of Action of Golgi Toxins(December 2023) Cervantes, Christopher Luis; Liszczak, Glen; Posner, Bruce A.; Wang, Fei; Nijhawan, DeepakAuxin-inducible forward genetics uncovered point mutations within Golgi Brefeldin A Resistant Guanine Nucleotide Exchange Factor 1 or GBF1 following lethal dose selection with a synthetic disubstituted pyrimidine toxin called Golgitox (GTOX). Resistant clones were also cross-resistant to the fungal toxin, Brefeldin A (BFA), and synthetic GBF1 inhibitor, Golgicide A (GCA). Like BFA and GCA, GTOX triggered Golgi disassembly via GBF1. Given that BFA is a reported molecular glue, we profiled Gbf1-Arf interactions in 293T cell lysates pre-treated with Golgi toxin. Both GTOX and GCA promoted GBF1-dependent interactions with Arfs 4 and 5, whereas BFA also interacted with Arf1. GBF1 domain mapping revealed that the HUS-SEC7-HDS1 domains were sufficient for promoting GTOX-dependent engagement with Arfs 4 and 5. Meanwhile, structural activity relationship studies showed that modifying the methyl group on the benzimidazole ring preserved GTOX activity and interactions between Gbf1 and Arfs 4 and 5. To assess which Arfs regulate BFA and GTOX cytotoxicities, genome-wide CRISPR/Cas9 compound enrichment screens were carried out, which identified ARF4 as being the most enriched hit. Next, we validated that ARF4 loss-of-function partially confers resistance to BFA and GTOX. Next, we asked whether GTOX preferentially interacts with ARF4-GDP versus ARF4-GTP. We found that exogenous ARF4 T31N (GDP-locked mutant) sensitized HCT116 scramble control cells 4-fold to GTOX, interacted with Gbf1 just as well as WT Arf4, but failed to rescue Arf4-mediated BiP retrieval. Collectively, these results suggest that the Gbf1-GTOX-Arf4-GDP complex is functionally inactive but deleterious to cell viability. Taken together, GTOX may act as a molecular glue to suppress GBF1 functions through downstream effector substrates like Arfs 4 and 5.Item Investigation of the Discoipyrroles and Other Marine Bacteria Derived Natural Products(2017-07-17) Colosimo, Dominic Andrew; Bruick, Richard K.; MacMillan, John; Ready, Joseph M.; Nijhawan, DeepakNatural products are a rich source of scientific innovation. These chemical compounds are canonically celebrated as biomedical tools or synthetic chemistry feats. Discovery of new chemical compounds has benefited from the study of natural product biosynthesis, or the methods by which they are constructed. Additionally, these studies have advanced the fields of ecology, industrial chemistry, and protein biophysics. This work will demonstrate how the discoipyrrole family of natural products inspired novel biomedical and biosynthetic discovery. In particular, their biosynthesis features unique mechanisms that are independent of protein catalysts, a growing trend found in natural products. Unrelated to the discoipyrroles, the manipulation of biosynthesis pathways using the methodology known as ribosome engineering will be discussed. This work demonstrates how the discovery and development of bioactive natural products can be enhanced by biosynthetic interrogations.Item Metabolic Regulation of Protein Phosphorylation and Acetylation(2018-07-12) Zhang, Menglu; Kohler, Jennifer J.; Tu, Benjamin; Nijhawan, Deepak; Yu, HongtaoCelluar metabolism can influence phosphorylation and acetylation modifications on proteins as part of an intricate network of cellular and organismal regulation. We have investigated one molecular mechanism through which protein phosphorylation and acetylation can be regulated based on metabolic status and how metabolic enzymes are regulated by nutrient availability. In the first part of this study, we report that a simple enzyme involved in acetate utilization, Acetyl-CoA synthetase 2 (ACSS2), promotes systemic fat storage and utilization through selective regulation of genes involved in lipid metabolism. We reveal that mice lacking ACSS2 exhibit a significant reduction in body weight and hepatic steatosis in a diet-induced obesity model. ACSS2 deficiency reduces dietary lipid absorption by the intestine, and perturbs repartitioning and utilization of triglycerides from adipose tissue to the liver due to lowered expression of lipid transporters and fatty acid oxidation genes. In this manner, ACSS2 promotes the systemic storage or metabolism of fat according to the fed or fasted state. Targeting ACSS2 may therefore offer therapeutic benefit for the treatment of fatty liver disease. We also report that ACSS2 may play a critical role in the development of pancreatic cancer. We have demonstrated that ACSS2 expression in a KRas-driven mouse model of pancreatic ductal adenocarcinoma (PDAC) showed that ACSS2 was absent in normal pancreatic tissue but expressed at very high levels in precancerous lesions of PDAC. The absence of ACSS2 in mouse pancreatic cancer models reduced the tumor burdens, and ACSS2 expression is correlated with tumor size. These data indicate that ACSS2 has a potential function in the development of PDAC. The experiments reported in the first two chapters of this thesis were performed in close collaboration with a former postdoc in the lab, Dr. Zhiguang Huang. In the second part of the study, we report that methylation of Protein Phosphatase 2A (PP2A) may play a critical role in regulating cell growth and autophagy. We have reconstituted the methylation activity of leucine carboxyl methyltransferase 1 (LCMT-1) in vitro and determined the kinetic parameters of LCMT-1-catalyzed methylation of PP2A. We reveal that LCMT-1 might be a "SAM sensor" as it is very sensitive to the SAM/SAH ratio. Methionine deprivation study in cell lines revealed that methionine depletion boosts PP2A demethylation. We further conducted a high-throughput screen to identify potent and specific small molecule inhibitors of LCMT-1.Item Modulating SIRP-alpha, a Myeloid-Specific Immune Checkpoint, for Immunotherapeutic Treatment of Malignancy(2016-04-01) Ring, Nan Guo; Weissman, Irving; Laccetti, Andrew; Nijhawan, Deepak; Wang, RichardBACKGROUND: CD47 is a "don't-eat-me" signal upregulated by many types of cancer that signals through its receptor SIRPα to inhibit macrophage-mediated destruction. Recent studies have demonstrated the efficacy of anti-CD47 blockade in synergizing with traditional monoclonal antibodies (mAbs) to enhance phagocytosis of cancer cells. We proposed targeting SIRPα as an alternative strategy and as a bridge to creating a single format bispecific macrophage-enhancing antibody (BiME). OBJECTIVE: The focus of this project was to test the therapeutic efficacy of SIRPα-blockade in combination treatment with other anti-tumor mAbs and in a single agent format as BiMEs. METHODS: We created KWAR23, a SIRPα-blocking monoclonal antibody, and from it, we derived several BiMEs in the dual-variable-domain immunoglobulin format to target CD20, Her2, and CD70. We tested these agents in high-throughput phagocytosis assays. We have also begun testing the therapeutic efficacy of KWAR23 in combination with rituximab in a xenograft mouse model of B cell lymphoma. RESULTS: KWAR23 significantly enhanced the efficacy and potency of phagocytosis of mAb-opsonized cancer cells when compared to the mAbs alone in B cell lymphoma and breast adenocarcinoma. BiMEs resulted in greater efficacy of phagocytosis of cancer cells when compared to their parent mAbs for B cell lymphoma, breast adenocarcinoma, and renal cell carcinoma. SIRPα-blockade in combination with rituximab also appeared to significantly reduce tumor burden in mice engrafted with a B cell lymphoma cell line. CONCLUSIONS: The therapeutic role of check-point inhibitors has become increasingly apparent and important in the treatment of cancer in recent years, but the role played by macrophages remains unclear. We have now demonstrated multiple strategies for targeting the CD47-SIRPα pathway to harness macrophages in antibody-mediated cell killing. This research may further benefit the field of immunotherapy as we learn more about the intricate interplay between innate and adaptive immunity in cancer biology.Item On Sulfur Sensing in Saccharomyces cerevisiae(December 2021) Johnson, Zane Miller; Nijhawan, Deepak; De Martino, George; Yu, Hongtao; Tu, BenjaminThe unique chemistry available to sulfur compared to oxygen, such as the ability to exist in numerous oxidation states and greater nucleophilicity, makes many of the biochemical reactions requisite for cellular life possible. As a result of this critical importance, organisms have developed several mechanisms for sensing and maintaining levels of sulfur-containing metabolites. In the yeast Saccharomyces cerevisiae, regulation of sulfur metabolism can be distilled down to the actions of two proteins; the F-box protein Met30, and the transcriptional coactivator Met4. Met30 belongs to the family of SCF (Skp1-Cul1-F-box protein) E3 ubiquitin ligases, and negatively regulates the transcriptional activity of the master transcriptional activator of sulfur metabolism genes, Met4, via oligo-ubiquitination when sulfur metabolite levels are high. When yeast are starved of sulfur, Met30 ceases to ubiquitinate Met4, releasing it to be deubiquitinated and transcriptionally active to boost levels of a network of sulfur metabolic genes known as the MET regulon to restore sulfur metabolite levels. While the molecular activities of both Met30 and Met4 have been extensively studied over the last two decades, the biochemical basis for sulfur-sensing by the Met30 E3 ligase has remained unknown. Herein, I reveal the biochemical details by which Met30, the master regulator of sulfur metabolism, senses the availability of sulfur metabolites to modulate its E3 ligase activity to regulate sulfur metabolism in yeast. Utilizing a combination of yeast genetics and biochemical assays, I show that Met30 uses redox-active cysteine residues in its C-terminal WD-40 repeat region to modulate binding between itself and its substrate Met4 in accordance with the availability of sulfur metabolites. These insights represent significant advances in the understanding of sulfur metabolic regulation in yeast.Item The Role of CK1 in Necroptosis and Confirmation of Amyloid-Like Fibers in Necroptosis Using 2D SDD-AGE(2020-05-01T05:00:00.000Z) Addams, Sarah Elizabeth; Nijhawan, Deepak; Cobb, Melanie H.; Tu, Benjamin; Wang, ZhigaoNecroptosis is a regulated necrotic cell death pathway, mediated by a supermolecular complex called the necrosome, which contains receptor-interacting protein kinase 1 and 3 (RIPK1, RIPK3) and mixed-lineage kinase domain-like protein (MLKL). Phosphorylation of human RIPK3 at serine 227 (S227) has been shown to be required for downstream MLKL binding and necroptosis progression. Tandem immunoprecipitation of RIPK3 reveals that casein kinase 1 (CK1) family proteins associate with the necrosome upon necroptosis induction, and this interaction depends on the kinase activity of RIPK3. In addition, CK1 proteins colocalize with RIPK3 puncta during necroptosis. Importantly, CK1 proteins directly phosphorylate RIPK3 at S227 in vitro and in vivo. Loss of CK1 proteins abolishes S227 phosphorylation and blocks necroptosis. Furthermore, a RIPK3 mutant with mutations in the CK1 recognition motif fails to be phosphorylated at S227, does not bind or phosphorylate MLKL, and is unable to activate necroptosis. These results strongly suggest that CK1 proteins are necrosome components which are responsible for RIPK3-S227 phosphorylation. During necroptosis, RIPK1 and RIPK3 are known to form an amyloid fiber. During our lab's investigation into the necroptosis signaling pathway, an amyloid fiber containing MLKL was observed. Based on size, the MLKL fiber appeared to represent a previously undescribed complex. However, the possibility this was the result of either degradation or dissociation of some of the proteins during electrophoresis could not be ruled out. To address this, I developed a protocol based on semi-denaturing detergent agarose gel electrophoresis (SDD-AGE) which allows detection of the SDS-resistant amyloid-like fibers in the cell extracts without purification. Performing second dimension of SDD-AGE determined that the size heterogeneity previously noted was due to a distinct amyloid species. This method allows fast, qualitative confirmation that the amyloid or amyloid-like fibers are not partially dissociating during the SDD-AGE process, and is not limited to use in the necroptosis field.Item Toxic PRn Poly-Dipeptides Encoded by the C9orf72 Repeat Expansion Block Nuclear Import and Export(2017-03-02) Shi, Kevin; Nijhawan, Deepak; McKnight, Steven L.; Rizo-Rey, José; Rosen, Michael K.Expansion of the (GGGGCC)n hexanucleotide repeat within the first intron of the C9orf72 gene is the mutation that leads to the most prevalent heritable form of amyotrophic lateral sclerosis (ALS). The expanded repeat is aberrantly transcribed from both sense and anti-sense strands relative to the C9orf72 gene, and both transcripts are translated in an ATG-independent manner to yield five distinct poly-dipeptides. Expression of either the glycine:arginine (GRn) or proline:arginine (PRn) poly-dipeptide in Drosophila leads to neurodegneration of the eye, and when introduced into culturing medium, synthetic forms of both peptides lead to the death of human cells. We recently identified PRn poly-dipeptide interacting partners in vivo using an unbiased proteomics approach, demonstrating that the nuclear pore complex (NPC) as a major binding target. The biological significance of the PRn peptide-NPC interaction became apparent when we observed major defects in both mature mRNA export to the cytoplasm and protein import into the nucleus after cells were treated with PRn. The functional nucleocytoplasmic transport defect caused by PRn peptide was due to binding of the peptide to the central channel of the NPC as visualized by super-resolution microscopy. The NPC central channel is comprised of phenylalanine-glycine (FG) domain nucleoporins, and these proteins are essential for the NPC's active transport and passive size-exclusion permeability barrier functions. Purified FG domains of Nup54 and Nup98, both identified in our proteomics study as PRn targets, polymerized into structurally labile, cross-β sheet fibers under physiological conditions. Several lines of evidence suggested that the polymerized state of FG domains is relevant to intact NPC. First, PRn peptides only bound to polymerized Nup54 and Nup98 and not to the soluble versions of either protein, potentially representing how PRn binds to the FG-rich central channel. Second, the aliphatic alcohol 1,6-hexanediol (HD) selectively disrupted the permeability barrier of the NPC, while 2,5-hexanediol had no effect. Similarly, 1,6-HD can effectively solubilize Nup FG polymers in vitro, while 2,5-HD had no effect on polymer stability. Finally, PRn binding to the NPC abrogated the disruption of the permeability barrier by 1,6-HD. Similarly, PRn also protected the Nup FG polymers from solubilization by 1,6-HD. Our study has elucidated a major mechanism by which the C9orf72 expansion associated PRn poly-dipeptide inhibits the transport of macromolecules in and out of the nucleus, leading to a major disruption of cellular physiology. Our results support a model in which the FG domains of the NPC exist in equilibrium between the polymerized and unpolymerized states. By binding to polymerized FG domains and stabilizing them, the PRn peptide is understood to shift the equilibrium toward the polymerized state, with consequent blockage of nuclear transport. The aliphatic alcohol 1,6-hexanediol has the opposite effect, shifting the equilibrium toward the unstructured state of FG domains and disrupting the permeability barrier. The effects of the PRn peptide and aliphatic alcohols on nuclear pore function represent extreme conditions. More subtle changes in the equilibrium between structured and unstructured FG domains may regulate nuclear pore function in living cells.