Browsing by Subject "Carrier Proteins"
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Item Cell-type-specific eQTL of primary melanocytes facilitates identification of melanoma susceptibility genes(Cold Spring Harbor Laboratory Press, 2018-11) Zhang, Tongwu; Choi, Jiyeon; Kovacs, Michael A.; Shi, Jianxin; Xu, Mai; Goldstein, Alisa M.; Trower, Adam J.; Bishop, D. Timothy; Iles, Mark M.; Duffy, David L.; MacGregor, Stuart; Amundadottir, Laufey T.; Law, Matthew H.; Loftus, Stacie K.; Pavan, William J.; Brown, Kevin M.; Epidemiology, School of Public HealthMost expression quantitative trait locus (eQTL) studies to date have been performed in heterogeneous tissues as opposed to specific cell types. To better understand the cell-type-specific regulatory landscape of human melanocytes, which give rise to melanoma but account for <5% of typical human skin biopsies, we performed an eQTL analysis in primary melanocyte cultures from 106 newborn males. We identified 597,335 cis-eQTL SNPs prior to linkage disequilibrium (LD) pruning and 4997 eGenes (FDR < 0.05). Melanocyte eQTLs differed considerably from those identified in the 44 GTEx tissue types, including skin. Over a third of melanocyte eGenes, including key genes in melanin synthesis pathways, were unique to melanocytes compared to those of GTEx skin tissues or TCGA melanomas. The melanocyte data set also identified trans-eQTLs, including those connecting a pigmentation-associated functional SNP with four genes, likely through cis-regulation of IRF4 Melanocyte eQTLs are enriched in cis-regulatory signatures found in melanocytes as well as in melanoma-associated variants identified through genome-wide association studies. Melanocyte eQTLs also colocalized with melanoma GWAS variants in five known loci. Finally, a transcriptome-wide association study using melanocyte eQTLs uncovered four novel susceptibility loci, where imputed expression levels of five genes (ZFP90, HEBP1, MSC, CBWD1, and RP11-383H13.1) were associated with melanoma at genome-wide significant P-values. Our data highlight the utility of lineage-specific eQTL resources for annotating GWAS findings, and present a robust database for genomic research of melanoma risk and melanocyte biology.Item Critical Role of the mTOR Pathway in Development and Function of Myeloid-Derived Suppressor Cells in lal−/− Mice(Elsevier B.V., 2014-02) Ding, Xinchun; Du, Hong; Yoder, Mervin C.; Yan, Cong; Department of Pathology and Laboratory Medicine, IU School of MedicineLysosomal acid lipase (LAL) is essential for the hydrolysis of cholesteryl esters and triglycerides to generate cholesterol and free fatty acids in cellular lysosomes. Ablation of the lal gene (lal−/−) systemically increased expansion of cluster of differentiation molecule 11b (CD11b), lymphocyte antigen 6G (Ly6G) myeloid-derived suppressor cells (MDSCs) that caused myeloproliferative neoplasms in mice. Study of lal−/− bone marrow Ly6G+ MDSCs via transcriptional profiling showed increases in mammalian target of rapamycin (mTOR) signaling pathway transcripts. Injection of mTOR pharmacologic inhibitors into lal−/− mice significantly reduced bone marrow myelopoiesis and systemic CD11b+Ly6G+ cell expansion. Rapamycin treatment of lal−/− mice stimulated a shift from immature CD11b+Ly6G+ cells to CD11b+ single-positive cells in marrow and tissues and partially reversed the increased cell proliferation, decreased apoptosis, increased ATP synthesis, and increased cell cycling of bone marrow CD11b+Ly6G+ cells obtained from lal−/− mice. Pharmacologic and siRNA suppression of mTOR, regulatory-associated protein of mTOR, rapamycin-insensitive companion of mTOR, and Akt1 function corrected CD11b+Ly6G+ cell in lal−/− mice development from Lin− progenitor cells and reversed the immune suppression on T-cell proliferation and function in association with decreased reactive oxygen species production, and recovery from impairment of mitochondrial membrane potential compared with control mutant cells. These results indicate a crucial role of LAL-regulated mTOR signaling in the production and function of CD11b+Ly6G+ cells. The mTOR pathway may serve as a novel target to modulate the emergence of MDSCs in those pathophysiologic states in which these cells play an immunosuppressive role.Item Endoplasmic Reticulum Stress Activates the Inflammasome via NLRP3- and Caspase-2-Driven Mitochondrial Damage(Elsevier, 2015-09-15) Bronner, Denise N.; Abuaita, Basel H.; Chen, Xiaoyun; Fitzgerald, Katherine A.; Nuñez, Gabriel; He, Yongqun; Yin, Xiao-Ming; O’Riordan, Mary X.D.; Department of Pathology and Laboratory Medicine, IU School of MedicineEndoplasmic reticulum (ER) stress is observed in many human diseases, often associated with inflammation. ER stress can trigger inflammation through nucleotide-binding domain and leucine-rich repeat containing (NLRP3) inflammasome, which might stimulate inflammasome formation by association with damaged mitochondria. How ER stress triggers mitochondrial dysfunction and inflammasome activation is ill defined. Here we have used an infection model to show that the IRE1α ER stress sensor regulates regulated mitochondrial dysfunction through an NLRP3-mediated feed-forward loop, independently of ASC. IRE1α activation increased mitochondrial reactive oxygen species, promoting NLRP3 association with mitochondria. NLRP3 was required for ER stress-induced cleavage of caspase-2 and the pro-apoptotic factor, Bid, leading to subsequent release of mitochondrial contents. Caspase-2 and Bid were necessary for activation of the canonical inflammasome by infection-associated or general ER stress. These data identify an NLRP3-caspase-2-dependent mechanism that relays ER stress to the mitochondria to promote inflammation, integrating cellular stress and innate immunity.Item Ferroxitosis: a cell death from modulation of oxidative phosphorylation and PKM2-dependent glycolysis in melanoma(Impact Journals, LLC, 2014-12-30) Lakhter, Alexander J.; Hamilton, James; Dagher, Pierre C.; Mukkamala, Suresh; Hato, Takashi; Dong, X. Charlie; Mayo, Lindsey D.; Harris, Robert A.; Shekhar, Anantha; Ivan, Mircea; Brustovetsky, Nickolay; Naidu, Samisubbu R.; Department of Dermatology, IU School of MedicineReliance on glycolysis is a characteristic of malignancy, yet the development of resistance to BRAF inhibitors in melanoma is associated with gain of mitochondrial function. Concurrent attenuation of oxidative phosphorylation and HIF-1α/PKM2-dependent glycolysis promotes a non-apoptotic, iron- and oxygen-dependent cell death that we term ferroxitosis. The redox cycling agent menadione causes a robust increase in oxygen consumption, accompanied by significant loss of intracellular ATP and rapid cell death. Conversely, either hypoxic adaptation or iron chelation prevents menadione-induced ferroxitosis. Ectopic expression of K213Q HIF-1α mutant blunts the effects of menadione. However, knockdown of HIF-1α or PKM2 restores menadione-induced cytotoxicity in hypoxia. Similarly, exposure of melanoma cells to shikonin, a menadione analog and a potential PKM2 inhibitor, is sufficient to induce ferroxitosis under hypoxic conditions. Collectively, our findings reveal that ferroxitosis curtails metabolic plasticity in melanoma.Item Kalirin : novel role in osteocyte function(2013) Wayakanon, Kornchanok; Bruzzaniti, Angela; Windsor, Lester Jack; Chu, Tien-Min Gabriel; Cho, Sopanis D.; Cook, Norman Blaine, 1954-Communication between bone cells is important for the maintenance of bone mass. Although osteocytes are deeply embedded within the mineralized matrix, they are essential for the regulation of osteoblast and osteoclast functions. However, the intracellular proteins that control the morphology and function of osteocytes, and their ability to communicate with other bone cells are still unknown. Kalirin is a novel multi-domain GTP exchange factor (GEF) protein that activates the RhoGTPases. Recently, we found that 14 week old female Kalirin knockout (Kal-KO) mice exhibit a 45% decrease in trabecular bone density and have significantly lower cortical area, perimeter, thickness and polar cross-sectional moment of inertia (-12.6%, -7.2%, -7.6% and -21.9%, respectively) than WT mice. Kalirin was found to be expressed in osteoclasts and osteoblasts but its expression and function in osteocytes is currently unclear. We examined the role of Kalirin on the morphology and function of osteocytes. Primary osteocytes were isolated by sequential collagenase digestions from long bones (femurs and tibias) of 10-week old WT and Kal-KO mice. Immunofluorescent staining revealed Kalirin was localized to the perinuclear region of primary osteocytes and MLO-Y4 cells, and was detected along the cytoplasmic processes of primary osteocytes. We also examined primary osteocytes isolated from the long bones of Kal-KO and WT mice for changes in the length and number of cytoplasmic processes. Kal-KO osteocytes were found to express significantly fewer cytoplasmic processes per cell (3.3±0.21) than WT osteocytes (4.7±0.3). In addition, the cytoplasmic processes of Kal-KO osteocytes were shorter (79.5±4.6 µm) than those observed for WT osteocytes (85.4±3.6 µm) (p <0.01). Quantitative PCR revealed the expression of mRNA for the three major Kalirin isoforms (Kal-7, Kal-9, Kal-12) in primary osteocytes and in MLO-Y4 cells. Moreover, the mRNA levels of osteoprotegerin (OPG) and SOST, which are important for controlling osteoclast differentiation and Wnt signaling leading to bone formation, respectively, were reduced in Kal-KO osteocytes. Next, the role of Kalirin in osteocyte morphology and function was further examined. Treatment of MLO-Y4 cells for 5 days with nerve growth factor, which is known to activate Kalirin in neurons, or over-expression of the Ser-Thr kinase domain of Kal-12, promoted cytoplasmic process elongation and upregulated phosphorylated ERK and RhoA levels. Together, these results suggest that Kalirin controls osteocyte morphology and function in part by regulating cytoskeletal remodeling and the activity of ERK and RhoA. Furthermore, Kalirin may control the bone remodeling cycle by regulating osteocyte signaling to osteoclasts and osteoblasts.Item Levels of the E2 interacting protein TopBP1 modulate papillomavirus maintenance stage replication(Elsevier, 2015-04) Kanginakudru, Sriramana; DeSmet, Marsha; Thomas, Yanique; Morgan, Iain M.; Androphy, Elliot J.; Department of Dermatology, IU School of MedicineThe evolutionarily conserved DNA topoisomerase II beta-binding protein 1 (TopBP1) functions in DNA replication, DNA damage response, and cell survival. We analyzed the role of TopBP1 in human and bovine papillomavirus genome replication. Consistent with prior reports, TopBP1 co-localized in discrete nuclear foci and was in complex with papillomavirus E2 protein. Similar to E2, TopBP1 is recruited to the region of the viral origin of replication during G1/S and early S phase. TopBP1 knockdown increased, while over-expression decreased transient virus replication, without affecting cell cycle. Similarly, using cell lines harboring HPV-16 or HPV-31 genome, TopBP1 knockdown increased while over-expression reduced viral copy number relative to genomic DNA. We propose a model in which TopBP1 serves dual roles in viral replication: it is essential for initiation of replication yet it restricts viral copy number.Item Mutations in the CDP-Choline Pathway for Phospholipid Biosynthesis Bypass the Requirement for an Essential Phospholipid Transfer Protein(Cell Press, 1991) Cleves, Ann E.; McGee, Todd P.; Whitters, Eric A.; Champion, Kathleen M.; Aitken, Jacqueline R.; Dowhan, William; Goebl, Mark; Bankaitis, Vytas A.; Biochemistry and Molecular Biology, School of MedicineSEC14p is the yeast phosphatidylinositol (PI)/phosphatidylcholine (PC) transfer protein, and it effects an essential stimulation of yeast Golgi secretory function. We now report that the SEC14p localizes to the yeast Golgi and that the SEC14p requirement can be specifically and efficiently bypassed by mutations in any one of at least six genes. One of these suppressor genes was the structural gene for yeast choline kinase (CKI), disruption of which rendered the cell independent of the normally essential SEC14p requirement. The antagonistic action of the CKI gene product on SEC14p function revealed a previously unsuspected influence of biosynthetic activities of the CDP-choline pathway for PC biosynthesis on yeast Golgi function and indicated that SEC14p controls the phospholipid content of yeast Golgi membranes in vivo.Item Peptidoglycan recognition protein 3 and Nod2 synergistically protect mice from dextran sodium sulfate-induced colitis(The American Association of Immunologists, 2014-09-15) Jing, Xuefang; Park, Shin Yong; Núñez, Gabriel; Dziarski, Roman; Gupta, Dipika; Department of Medicine, IU School of MedicineAberrant immune response and changes in the gut microflora are the main causes of inflammatory bowel disease (IBD). Peptidoglycan recognition proteins (Pglyrp1, Pglyrp2, Pglyrp3, and Pglyrp4) are bactericidal innate immunity proteins that maintain normal gut microbiome, protect against experimental colitis, and are associated with IBD in humans. Nucleotide-binding oligomerization domain 2 (Nod2) is an intracellular bacterial sensor and may be required for maintaining normal gut microbiome. Mutations in Nod2 are strongly associated with Crohn's disease, but the causative mechanism is not understood, and the role of Nod2 in ulcerative colitis is not known. Because IBD is likely caused by variable multiple mutations in different individuals, in this study, we examined the combined role of Pglyrp3 and Nod2 in the development of experimental colitis in mice. We demonstrate that a combined deficiency of Pglyrp3 and Nod2 results in higher sensitivity to dextran sodium sulfate-induced colitis compared with a single deficiency. Pglyrp3(-/-)Nod2(-/-) mice had decreased survival and higher loss of body weight, increased intestinal bleeding, higher apoptosis of colonic mucosa, elevated expression of cytokines and chemokines, altered gut microbiome, and increased levels of ATP in the colon. Increased sensitivity to dextran sodium sulfate-induced colitis in Pglyrp3(-/-)Nod2(-/-) mice depended on increased apoptosis of intestinal epithelium, changed gut microflora, and elevated ATP. Pglyrp3 deficiency contributed colitis-predisposing intestinal microflora and increased intestinal ATP, whereas Nod2 deficiency contributed higher apoptosis and responsiveness to increased level of ATP. In summary, Pglyrp3 and Nod2 are both required for maintaining gut homeostasis and protection against colitis, but their protective mechanisms differ.Item SH3 Domain-Containing Protein 2 Plays a Crucial Role at the Step of Membrane Tubulation during Cell Plate Formation(American Society of Plant Biologists, 2017-06) Ahn, Gyeongik; Kim, Hyeran; Kim, Dae Heon; Hanh, Hong; Yoon, Youngdae; Singaram, Indira; Wijesinghe, Kaveesha J.; Johnson, Kristen A.; Zhuang, Xiaohong; Liang, Zizhen; Stahelin, Robert V.; Jiang, Liwen; Cho, Wonhwa; Kang, Byung-Ho; Hwang, Inhwan; Biochemistry and Molecular Biology, School of MedicineDuring cytokinesis in plants, trans-Golgi network-derived vesicles accumulate at the center of dividing cells and undergo various structural changes to give rise to the planar cell plate. However, how this conversion occurs at the molecular level remains elusive. In this study, we report that SH3 Domain-Containing Protein 2 (SH3P2) in Arabidopsis thaliana plays a crucial role in converting vesicles to the planar cell plate. SH3P2 RNAi plants showed cytokinesis-defective phenotypes and produced aggregations of vesicles at the leading edge of the cell plate. SH3P2 localized to the leading edge of the cell plate, particularly the constricted or curved regions of the cell plate. The BAR domain of SH3P2 induced tubulation of vesicles. SH3P2 formed a complex with dynamin-related protein 1A (DRP1A) and affected DRP1A accumulation to the cell plate. Based on these results, we propose that SH3P2 functions together with DRP1A to convert the fused vesicles to tubular structures during cytokinesis.Item Studies on triadin and insights into its role in Ca²⁺Release(1999) Kobayashi, Yvonne Monique