Browsing by Author "Department of Biochemistry and Molecular Biology, IU School of Medicine"
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Item Advances in translational bioinformatics facilitate revealing the landscape of complex disease mechanisms(Springer (Biomed Central Ltd.), 2014) Yang, Jack Y.; Dunker, A. Keith; Liu, Jun S.; Qin, Xiang; Arabnia, Hamid R.; Yang, William; Niemierko, Andrzej; Chen, Zhongxue; Luo, Zuojie; Wang, Liangjiang; Liu, Yunlong; Xu, Dong; Deng, Youping; Tong, Weida; Yang, Mary Qu; Department of Biochemistry and Molecular Biology, IU School of MedicineAdvances of high-throughput technologies have rapidly produced more and more data from DNAs and RNAs to proteins, especially large volumes of genome-scale data. However, connection of the genomic information to cellular functions and biological behaviours relies on the development of effective approaches at higher systems level. In particular, advances in RNA-Seq technology has helped the studies of transcriptome, RNA expressed from the genome, while systems biology on the other hand provides more comprehensive pictures, from which genes and proteins actively interact to lead to cellular behaviours and physiological phenotypes. As biological interactions mediate many biological processes that are essential for cellular function or disease development, it is important to systematically identify genomic information including genetic mutations from GWAS (genome-wide association study), differentially expressed genes, bidirectional promoters, intrinsic disordered proteins (IDP) and protein interactions to gain deep insights into the underlying mechanisms of gene regulations and networks. Furthermore, bidirectional promoters can co-regulate many biological pathways, where the roles of bidirectional promoters can be studied systematically for identifying co-regulating genes at interactive network level. Combining information from different but related studies can ultimately help revealing the landscape of molecular mechanisms underlying complex diseases such as cancer.Item Alcohol consumption mediates the relationship between ADH1B and DSM-IV alcohol use disorder and criteria(Alcohol Research Documentation, 2014-07) Kilcoyne, Bari; Shmulewitz, Dvora; Meyers, Jacquelyn L.; Aharonovich, Efrat; Greenstein, Eliana; Frisch, Amos; Weizman, Abraham; Spivak, Baruch; Edenberg, Howard J.; Gelertner, Joel; Hasin, Deborah S.; Department of Biochemistry and Molecular Biology, IU School of MedicineOBJECTIVE: A single nucleotide variation in the alcohol dehydrogenase 1B (ADH1B) gene, rs1229984, produces an ADH1B enzyme with faster acetaldehyde production. This protective variant is associated with lower alcohol consumption and lower risk for alcohol use disorders (AUDs). Based on the premise that faster ADH1B kinetics decreases alcohol consumption, we formally tested if the association between ADH1B variant rs1229984 and AUDs occurs through consumption. We also tested whether the association between rs1229984 and each of the 11 Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition (DSM-IV), AUD criteria occurs through consumption. METHOD: A total of 1,130 lifetime drinkers from an Israeli household sample were assessed with a structured interview and genotyped for rs1229984 (protective allele frequency = 0.28). Logistic regression evaluated the association between rs1229984 and each phenotype (AUDs, 11 individual DSM-IV criteria). For phenotypes significantly related to rs1229984, the effect through consumption was tested with logistic regression and bootstrapping. RESULTS: ADH1B rs1229984 was significantly associated with AUDs and six criteria, with odds ratios ranging from 1.32 to 1.96. The effect through consumption was significant for these relationships, explaining 23%-74% of the total ADH1B effect. CONCLUSIONS: This is the first study to show that ADH1B rs1229984 is related to 6 of the 11 DSM-IV AUD criteria and that alcohol consumption explained a significant proportion of these associations and the association of ADH1B with AUDs. Better understanding of the relationship between ADH1B and the DSM-IV AUD criteria, including effects through consumption, will enhance our understanding of the etiologic model through which AUDs can occur.Item Antidiabetic thiazolidinediones induce ductal differentiation but not apoptosis in pancreatic cancer cells(Elsevier, 2005-02-28) Ceni, Elisabetta; Mello, Tommaso; Tarocchi, Mirko; Crabb, David W.; Caldini, Anna; Invernizzi, Pietro; Surrenti, Calogero; Milani, Stefano; Galli, Andrea; Department of Biochemistry and Molecular Biology, IU School of MedicineAIM: Thiazolidinediones (TZD) are a new class of oral antidiabetic drugs that have been shown to inhibit growth of same epithelial cancer cells. Although TZD were found to be ligands for peroxisome proliferator-activated receptor gamma (PPARgamma), the mechanism by which TZD exert their anticancer effect is presently unclear. In this study, we analyzed the mechanism by which TZD inhibit growth of human pancreatic carcinoma cell lines in order to evaluate the potential therapeutic use of these drugs in pancreatic adenocarcinoma. METHODS: The effects of TZD in pancreatic cancer cells were assessed in anchorage-independent growth assay. Expression of PPARgamma was measured by reverse-transcription polymerase chain reaction and confirmed by Western blot analysis. PPARgamma activity was evaluated by transient reporter gene assay. Flow cytometry and DNA fragmentation assay were used to determine the effect of TZD on cell cycle progression and apoptosis respectively. The effect of TZD on ductal differentiation markers was performed by Western blot. RESULTS: Exposure to TZD inhibited colony formation in a PPARgamma-dependent manner. Growth inhibition was linked to G1 phase cell cycle arrest through induction of the ductal differentiation program without any increase of the apoptotic rate. CONCLUSION: TZD treatment in pancreItem Assessment of First and Second Degree Relatives of Individuals With Bipolar Disorder Shows Increased Genetic Risk Scores in Both Affected Relatives and Young At-Risk Individuals(Wiley, 2015-10) Fullerton, Janice M.; Koller, Daniel L.; Edenberg, Howard J.; Foroud, Tatiana; Liu, Hai; Glowinski, Anne L.; McInnis, Melvin G.; Wilcox, Holly C.; Frankland, Andrew; Roberts, Gloria; Schofield, Peter R.; Mitchell, Philip B.; Nurnberger, John I.; Department of Biochemistry and Molecular Biology, IU School of MedicineRecent studies have revealed the polygenic nature of bipolar disorder (BP), and identified common risk variants associated with illness. However, the role of common polygenic risk in multiplex families has not previously been examined. The present study examined 249 European-ancestry families from the NIMH Genetics Initiative sample, comparing subjects with narrowly defined BP (excluding bipolar II and recurrent unipolar depression; n = 601) and their adult relatives without BP (n = 695). Unrelated adult controls (n = 266) were from the NIMH TGEN control dataset. We also examined a prospective cohort of young (12–30 years) offspring and siblings of individuals with BPI and BPII disorder (at risk; n = 367) and psychiatrically screened controls (n = 229), ascertained from five sites in the US and Australia and assessed with standardized clinical protocols. Thirty-two disease-associated SNPs from the PGC-BP Working Group report (2011) were genotyped and additive polygenic risk scores (PRS) derived. We show increased PRS in adult cases compared to unrelated controls (P = 3.4 × 10−5, AUC = 0.60). In families with a high-polygenic load (PRS score ≥32 in two or more subjects), PRS distinguished cases with BPI/SAB from other relatives (P = 0.014, RR = 1.32). Secondly, a higher PRS was observed in at-risk youth, regardless of affected status, compared to unrelated controls (GEE-χ2 = 5.15, P = 0.012). This report is the first to explore common polygenic risk in multiplex families, albeit using only a small number of robustly associated risk variants. We show that individuals with BP have a higher load of common disease-associated variants than unrelated controls and first-degree relatives, and illustrate the potential utility of PRS assessment in a family context.Item Chemical inhibitor targeting the replication protein A-DNA interaction increases the efficacy of Pt-based chemotherapy in lung and ovarian cancer(Elsevier, 2015-01-01) Mishra, Akaash K.; Dormi, Silvana S.; Turchi, Alaina M.; Woods, Derek S.; Turchi, John J.; Department of Biochemistry and Molecular Biology, IU School of MedicinePlatinum-based chemotherapeutics exert their therapeutic efficacy via the formation of DNA adducts which interfere with DNA replication, transcription and cell division and ultimately induce cell death. Repair and tolerance of these Pt-DNA lesions by nucleotide excision repair (NER) and homologous recombination (HR) can substantially reduce the effectiveness of therapy. Inhibition of these repair pathways, therefore, holds the potential to sensitize cancer cells to Pt treatment and increase clinical efficacy. Replication Protein A (RPA) plays essential roles in both NER and HR, along with its role in DNA replication and DNA damage checkpoint activation. Each of these functions is, in part, mediated by RPA binding to single-stranded DNA (ssDNA). Here we report the synthesis and characterization of novel derivatives of RPA small molecule inhibitors and their activity in models of epithelial ovarian cancer (EOC) and non-small cell lung cancer (NSCLC). We have synthesized analogs of our previously reported RPA inhibitor TDRL-505 and determined the structure-activity relationships. These data led us to the identification of TDRL-551, which exhibited a greater than 2-fold increase in in vitro activity. TDRL-551 showed synergy with Pt in tissue culture models of EOC and in vivo efficacy, as a single agent and in combination with platinum, in a NSCLC xenograft model. These data demonstrate the utility of RPA inhibition in EOC and NSCLC and the potential in developing novel anticancer therapeutics that target RPA-DNA interactions.Item CHOP links endoplasmic reticulum stress to NF-κB activation in the pathogenesis of nonalcoholic steatohepatitis(2015) Willy, Jeffrey A.; Young, Sara K.; Stevens, James L.; Masuoka, Howard C.; Wek, Ronald C.; Department of Biochemistry and Molecular Biology, IU School of MedicineFree fatty acid induction of inflammation and cell death is an important feature of nonalcoholic steatohepatitis (NASH) and has been associated with disruption of the endoplasmic reticulum and activation of the Unfolded Protein Response (UPR). Following chronic UPR activation, the transcription factor CHOP (GADD153/DDIT3) triggers cell death; however, the mechanisms linking the UPR or CHOP to hepatoceullular injury and inflammation in the pathogenesis of NASH are not well understood. Using HepG2 and primary human hepatocytes, we found that CHOP induces cell death and inflammatory responses following saturated free fatty acid exposure by activating NF-κB through a pathway involving IRAK2 expression, resulting in secretion of cytokines IL-8 and TNFα directly from hepatocytes. TNFα facilitates hepatocyte death upon exposure to saturated free fatty acids and secretion of both IL-8 and TNFα contribute to inflammation. Interestingly, CHOP/NF-κB signaling is not conserved in primary rodent hepatocytes. Our studies suggest that CHOP plays a vital role in the pathophysiology of NASH through induction of secreted factors that trigger inflammation and hepatocellular death via a signaling pathway specific to human hepatocytes.Item Development, validation, and comparison of four methods to simultaneously quantify L-arginine, citrulline, and ornithine in human plasma using hydrophilic interaction liquid chromatography and electrospray tandem mass spectrometry(Elsevier, 2015-11) Lai, Xianyin; Kline, Jeffrey A.; Wang, Mu; Department of Biochemistry and Molecular Biology, IU School of MedicineTo understand the role of l-arginine depletion in impaired nitric oxide synthesis in disease, it is important to simultaneously quantify arginine, citrulline, and ornithine in the plasma. Because the three amino acids are endogenous analytes, true blank matrix for them is not available. It is necessary and valuable to compare the performance of different approaches due to lack of regulatory clarity for validation. A two-step sample preparation method using methanol as protein precipitation reagent was developed in this study is used for sample preparation. Because true blank matrix for endogenous analytes is not available, water as blank matrix, 1% BSA in PBS as blank matrix, surrogate analyte, and background subtraction were designed to establish successful quantification methods. Four methods to simultaneously quantify arginine, citrulline, and ornithine in human plasma using hydrophilic interaction liquid chromatography and electrospray tandem mass spectrometry were developed, validated, and compared. The developed two-step sample preparation method using methanol as protein precipitation reagent in this study needs less time and provides higher recovery comparing with other approaches. Three of the four methods, water as blank matrix, 1% BSA in PBS as blank matrix, and surrogate analyte, have been successful in fulfilling all the criteria, while background subtraction has failed. Results of the measured concentrations in 97 human plasma samples using the three methods show that the difference between any two methods or among the three methods presents 100% of samples with less than 20% for all the three amino acids and majority of them are under 10%. The developed two-step sample preparation method using methanol as protein precipitation reagent is simple and convenient. Three of the four methods are fully validated and the validation is successful. The BSA functioned effectively as a blank matrix for these three amino acids, considering cost, data quality, matrix similarity, and practicality.Item DisProt 7.0: a major update of the database of disordered proteins(Oxford University Press, 2017-01-04) Piovesan, Damiano; Tabaro, Francesco; Micetic, Ivan; Necci, Marco; Quaglia, Federica; Oldfield, Christopher J.; Aspromonte, Maria Cristina; Davey, Norman E.; Davidovic, Radoslav; Dosztanyi, Zsuzsanna; Elofsson, Arne; Gasparini, Alessandra; Hatos, Andras; Kajava, Andrey V.; Kalmar, Lajos; Leonardi, Emanuela; Lazar, Tamas; Macedo-Ribeiro, Sandra; Macossay-Castillo, Mauricio; Meszaros, Attila; Minervini, Giovanni; Murvai, Nikoletta; Pujols, Jordi; Roche, Daniel B.; Salladini, Edoardo; Schad, Eva; Schramm, Antoine; Szabo, Beata; Tantos, Agnes; Tonello, Fiorella; Tsirigos, Konstantinos D.; Veljkovic, Nevena; Ventura, Salvador; Vranken, Wim; Warholm, Per; Uversky, Vladimir N.; Dunker, A. Keith; Longhi, Sonia; Tompa, Peter; Tosatto, Silvio C.E.; Department of Biochemistry and Molecular Biology, IU School of MedicineThe Database of Protein Disorder (DisProt, URL: www.disprot.org) has been significantly updated and upgraded since its last major renewal in 2007. The current release holds information on more than 800 entries of IDPs/IDRs, i.e. intrinsically disordered proteins or regions that exist and function without a well-defined three-dimensional structure. We have re-curated previous entries to purge DisProt from conflicting cases, and also upgraded the functional classification scheme to reflect continuous advance in the field in the past 10 years or so. We define IDPs as proteins that are disordered along their entire sequence, i.e. entirely lack structural elements, and IDRs as regions that are at least five consecutive residues without well-defined structure. We base our assessment of disorder strictly on experimental evidence, such as X-ray crystallography and nuclear magnetic resonance (primary techniques) and a broad range of other experimental approaches (secondary techniques). Confident and ambiguous annotations are highlighted separately. DisProt 7.0 presents classified knowledge regarding the experimental characterization and functional annotations of IDPs/IDRs, and is intended to provide an invaluable resource for the research community for a better understanding structural disorder and for developing better computational tools for studying disordered proteins.Item Distinctive and pervasive alterations in aqueous humor protein composition following different types of glaucoma surgery(Molecular Vision, 2015) Rosenfeld, Cyril; Price, Marianne O.; Lai, Xianyin; Witzmann, Frank A.; Price, Francis W.; Department of Biochemistry and Molecular Biology, IU School of MedicinePURPOSE: To investigate whether specific glaucoma surgeries are associated with differences in aqueous humor protein concentrations compared to eyes without filters. METHODS: In this cross-sectional study, aqueous humor samples were prospectively collected from control subjects who underwent routine cataract surgery (n=14) and from patients who had different glaucoma filters: Baerveldt aqueous shunt (n=6), Ahmed aqueous shunt (n=6), trabeculectomy (n=5), and Ex-Press trabeculectomy (n=3). Total protein concentrations were determined with Bradford assay. Tryptic digests were analyzed with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Proteins were identified with high confidence using stringent criteria and were quantitatively compared with a label-free platform. Relative protein quantities were compared across groups with ANOVA. Post hoc pair-wise comparisons were adjusted for multiple comparisons. RESULTS: Compared to the control eyes, the aqueous humor protein concentration was increased approximately tenfold in the Ahmed and Baerveldt eyes and fivefold in the trabeculectomy and Ex-Press eyes. Overall, 718 unique proteins, splice variants, or isoforms were identified. No differences in the protein concentrations were detected between the Baerveldt and Ahmed groups. Likewise, the trabeculectomy and Ex-Press groups were remarkably similar. Therefore, the aqueous shunt groups were pooled, and the trabeculectomy groups were pooled for a three-way comparison with the controls. More than 500 proteins differed significantly in relative abundance (ANOVA p<0.01) among the control, aqueous shunt, and trabeculectomy groups. Functional analyses suggested these alterations in relative protein abundance affected dozens of signaling pathways. CONCLUSIONS: Different glaucoma surgical procedures were associated with marked increases in the aqueous humor protein concentration and distinctive changes in the relative abundance of numerous proteins involved in multiple signaling pathways.Item The Ebola Virus matrix protein, VP40, requires phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) for extensive oligomerization at the plasma membrane and viral egress(Nature Publishing Group, 2016) Johnson, Kristen A.; Taghon, Geoffrey J. F.; Scott, Jordan L.; Stahelin, Robert V.; Department of Biochemistry and Molecular Biology, IU School of MedicineVP40 is one of eight proteins encoded by the Ebola Virus (EBOV) and serves as the primary matrix protein, forming virus like particles (VLPs) from mammalian cells without the need for other EBOV proteins. While VP40 is required for viral assembly and budding from host cells during infection, the mechanisms that target VP40 to the plasma membrane are not well understood. Phosphatidylserine is required for VP40 plasma membrane binding, VP40 hexamer formation, and VLP egress, However, PS also becomes exposed on the outer membrane leaflet at sites of VP40 budding, raising the question of how VP40 maintains an interaction with the plasma membrane inner leaflet when PS is flipped to the opposite side. To address this question, cellular and in vitro assays were employed to determine if phosphoinositides are important for efficient VP40 localization to the plasma membrane. Cellular studies demonstrated that PI(4,5)P2 was an important component of VP40 assembly at the plasma membrane and subsequent virus like particle formation. Additionally, PI(4,5)P2 was required for formation of extensive oligomers of VP40, suggesting PS and PI(4,5)P2 have different roles in VP40 assembly where PS regulates formation of hexamers from VP40 dimers and PI(4,5)P2 stabilizes and/or induces extensive VP40 oligomerization at the plasma membrane.