Otterpohl K.L.,University of Nebraska Medical Center |
Otterpohl K.L.,Childrens Health Research Center |
Gould K.A.,University of Nebraska Medical Center
PLoS ONE | Year: 2017
The Rad50 Interacting Protein 1 (Rint1) influences cellular homeostasis through maintenance of endoplasmic reticulum, Golgi and centrosome integrity and regulation of vesicle transport, autophagy and the G2/M checkpoint. Rint1 has been postulated to function as a tumor suppressor as well as an oncogene, with its role depending perhaps upon the precise cellular and/or experimental context. In humans, heterozygosity for germline missense variants in RINT1 have, in some studies, been associated with increased risk of both breast and Lynch syndrome type cancers. However, it is not known if these germline variants represent loss of function alleles or gain of function alleles. Based upon these findings, as well as our initial consideration of Rint1 as a potential candidate for Mom5, a genetic modifier of intestinal tumorigenesis in ApcMin/+ mice, we sought to explicitly examine the impact of Rint1 on tumorigenesis in ApcMin/+ mice. However, heterozygosity for a knockout of Rint1 had no impact on tumorigenesis in Rint1+/-; ApcMin/+ mice. Likewise, we found no evidence to suggest that the remaining Rint1 allele was lost somatically in intestinal tumors in ApcMin/+ mice. Interestingly, in contrast to what has been observed in Rint1+/- mice on a mixed genetic background, Rint1+/- mice on a pure C57BL/6J background did not show spontaneous tumor development. We also evaluated colorectal cancer data available in the COSMIC and ONCOMINE databases and found that RINT1 overexpression, as well as the presence of somatic missense mutations in RINT1 were associated with colorectal cancer development. In vitro evaluation of two missense variants in RINT1 suggested that such variants do have the potential to impact RINT1 function. © 2017 Otterpohl, Gould.This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Morriswood B.,Medical University of Vienna |
Havlicek K.,Medical University of Vienna |
Demmel L.,Medical University of Vienna |
Yavuz S.,Medical University of Vienna |
And 8 more authors.
Eukaryotic Cell | Year: 2013
The trypanosomes are a family of parasitic protists of which the African trypanosome, Trypanosoma brucei, is the best characterized. The complex and highly ordered cytoskeleton of T. brucei has been shown to play vital roles in its biology but remains difficult to study, in large part owing to the intractability of its constituent proteins. Existing methods of protein identification, such as bioinformatic analysis, generation of monoclonal antibody panels, proteomics, affinity purification, and yeast two-hybrid screens, all have drawbacks. Such deficiencies-troublesome proteins and technical limitations-are common not only to T. brucei but also to many other protists, many of which are even less well studied. Proximity-dependent biotin identification (BioID) is a recently developed technique that allows forward screens for interaction partners and near neighbors in a native environment with no requirement for solubility in nonionic detergent. As such, it is extremely well suited to the exploration of the cytoskeleton. In this project, BioID was adapted for use in T. brucei. The trypanosome bilobe, a discrete cytoskeletal structure with few known protein components, represented an excellent test subject. Use of the bilobe protein TbMORN1 as a probe resulted in the identification of seven new bilobe constituents and two new flagellum attachment zone proteins. This constitutes the first usage of BioID on a largely uncharacterized structure, and demonstrates its utility in identifying new components of such a structure. This remarkable success validates BioID as a new tool for the study of unicellular eukaryotes in particular and the eukaryotic cytoskeleton in general. © 2013, American Society for Microbiology. All Rights Reserved.
Mdaki K.S.,Childrens Health Research Center |
Larsen T.D.,Childrens Health Research Center |
Wachal A.L.,Childrens Health Research Center |
Schimelpfenig M.D.,University of South Dakota |
And 6 more authors.
American Journal of Physiology - Heart and Circulatory Physiology | Year: 2016
Offspring of diabetic pregnancies are at risk of cardiovascular disease at birth and throughout life, purportedly through fuel-mediated influences on the developing heart. Preventative measures focus on glycemic control, but the contribution of additional offenders, including lipids, is not understood. Cellular bioenergetics can be influenced by both diabetes and hyperlipidemia and play a pivotal role in the pathophysiology of adult cardiovascular disease. This study investigated whether a maternal high-fat diet, independently or additively with diabetes, could impair fuel metabolism, mitochondrial function, and cardiac physiology in the developing offspring's heart. Sprague-Dawley rats fed a control or high-fat diet were administered placebo or streptozotocin to induce diabetes during pregnancy and then delivered offspring from four groups: control, diabetes exposed, diet exposed, and combination exposed. Cardiac function, cellular bioenergetics (mitochondrial stress test, glycolytic stress test, and palmitate oxidation assay), lipid peroxidation, mitochondrial histology, and copy number were determined. Diabetes-exposed offspring had impaired glycolytic and respiratory capacity and a reduced proton leak. High-fat diet-exposed offspring had increased mitochondrial copy number, increased lipid peroxidation, and evidence of mitochondrial dysfunction. Combination-exposed pups were most severely affected and demonstrated cardiac lipid droplet accumulation and diastolic/systolic cardiac dysfunction that mimics that of adult diabetic cardiomyopathy. This study is the first to demonstrate that a maternal high-fat diet impairs cardiac function in offspring of diabetic pregnancies through metabolic stress and serves as a critical step in understanding the role of cellular bioenergetics in developmentally programmed cardiac disease. © 2016 the American Physiological Society.
Roux K.J.,Childrens Health Research Center
Current protocols in protein science / editorial board, John E. Coligan ... [et al.] | Year: 2013
BioID is a unique method to screen for physiologically relevant protein interactions that occur in living cells. This technique harnesses a promiscuous biotin ligase to biotinylate proteins based on proximity. The ligase is fused to a protein of interest and expressed in cells, where it biotinylates proximal endogenous proteins. Because it is a rare protein modification in nature, biotinylation of these endogenous proteins by BioID fusion proteins enables their selective isolation and identification with standard biotin-affinity capture. Proteins identified by BioID are candidate interactors for the protein of interest. BioID can be applied to insoluble proteins, can identify weak and/or transient interactions, and is amenable to temporal regulation. Initially applied to mammalian cells, BioID has potential application in a variety of cell types from diverse species. Copyright © 2013 John Wiley & Sons, Inc.
Li L.,Childrens Health Research Center
Nature Cell Biology | Year: 2016
Aberrant Notch signalling has been linked to many cancers including choroid plexus (CP) tumours, a group of rare and predominantly paediatric brain neoplasms. We developed animal models of CP tumours, by inducing sustained expression of Notch1, that recapitulate properties of human CP tumours with aberrant NOTCH signalling. Whole-transcriptome and functional analyses showed that tumour cell proliferation is associated with Sonic Hedgehog (Shh) in the tumour microenvironment. Unlike CP epithelial cells, which have multiple primary cilia, tumour cells possess a solitary primary cilium as a result of Notch-mediated suppression of multiciliate differentiation. A Shh-driven signalling cascade in the primary cilium occurs in tumour cells but not in epithelial cells. Lineage studies show that CP tumours arise from monociliated progenitors in the roof plate characterized by elevated Notch signalling. Abnormal SHH signalling and distinct ciliogenesis are detected in human CP tumours, suggesting the SHH pathway and cilia differentiation as potential therapeutic avenues. © 2016 Nature Publishing Group
Roux K.J.,Childrens Health Research Center |
Roux K.J.,University of South Dakota
Cellular and Molecular Life Sciences | Year: 2013
Various methods have been established for the purpose of identifying and characterizing protein-protein interactions (PPIs). This diverse toolbox provides researchers with options to overcome challenges specific to the nature of the proteins under investigation. Among these techniques is a category based on proximity-dependent labeling of proteins in living cells. These can be further partitioned into either hypothesis-based or unbiased screening methods, each with its own advantages and limitations. Approaches in which proteins of interest are fused to either modifying enzymes or receptor sequences allow for hypothesis-based testing of protein proximity. Protein crosslinking and BioID (proximity-dependent biotin identification) permit unbiased screening of protein proximity for a protein of interest. Here, we evaluate these approaches and their applications in living eukaryotic cells. © 2013 Springer Basel.
Roux K.J.,Childrens Health Research Center |
Roux K.J.,University of South Dakota |
Kim D.I.,Childrens Health Research Center |
Burke B.,Singapore Institute of Medical Biology
Current Protocols in Protein Science | Year: 2013
BioID is a unique method to screen for physiologically relevant protein interactions that occur in living cells. This technique harnesses a promiscuous biotin ligase to biotinylate proteins based on proximity. The ligase is fused to a protein of interest and expressed in cells, where it biotinylates proximal endogenous proteins. Because it is a rare protein modification in nature, biotinylation of these endogenous proteins by BioID fusion proteins enables their selective isolation and identification with standard biotin-affinity capture. Proteins identified by BioID are candidate interactors for the protein of interest. BioID can be applied to insoluble proteins, can identify weak and/or transient interactions, and is amenable to temporal regulation. Initially applied to mammalian cells, BioID has potential application in a variety of cell types from diverse species. © 2013 by John Wiley & Sons, Inc.
Booze M.L.,Childrens Health Research Center |
Hansen J.M.,Brigham Young University |
Vitiello P.F.,Childrens Health Research Center |
Vitiello P.F.,University of South Dakota
Free Radical Biology and Medicine | Year: 2016
Thiol switches are important regulators of cellular signaling and are coordinated by several redox enzyme systems including thioredoxins, peroxiredoxins, and glutathione. Thioredoxin-1 (Trx1), in particular, is an important signaling molecule not only in response to redox perturbations, but also in cellular growth, regulation of gene expression, and apoptosis. The active site of this enzyme is a highly conserved C-G-P-C motif and the redox mechanism of Trx1 is rapid which presents a challenge in determining specific substrates. Numerous in vitro approaches have identified Trx1-dependent thiol switches; however, these findings may not be physiologically relevant and little is known about Trx1 interactions in vivo. In order to identify Trx1 targets in vivo, we generated a transgenic mouse with inducible expression of a mutant Trx1 transgene to stabilize intermolecular disulfides with protein substrates. Expression of the Trx1 “substrate trap” transgene did not interfere with endogenous thioredoxin or glutathione systems in brain, heart, lung, liver, and kidney. Following immunoprecipitation and proteomic analysis, we identified 41 homeostatic Trx1 interactions in perinatal lung, including previously described Trx1 substrates such as members of the peroxiredoxin family and collapsin response mediator protein 2. Using perinatal hyperoxia as a model of oxidative injury, we found 17 oxygen-induced interactions which included several cytoskeletal proteins which may be important to alveolar development. The data herein validates this novel mouse model for identification of tissue- and cell-specific Trx1-dependent pathways that regulate physiological signals in response to redox perturbations. © 2016 Elsevier Inc.
Floen M.J.,University of South Dakota |
Forred B.J.,Childrens Health Research Center |
Bloom E.J.,Childrens Health Research Center |
Vitiello P.F.,University of South Dakota |
Vitiello P.F.,Childrens Health Research Center
Free Radical Biology and Medicine | Year: 2014
The most common form of newborn chronic lung disease, bronchopulmonary dysplasia (BPD), is thought to be caused by oxidative disruption of lung morphogenesis, which results in decreased pulmonary vasculature and alveolar simplification. Although cellular redox status is known to regulate cellular proliferation and differentiation, redox-sensitive pathways associated with these processes in developing pulmonary epithelium are unknown. Redox-sensitive pathways are commonly regulated by cysteine thiol modifications. Therefore two thiol oxidoreductase systems, thioredoxin and glutathione, were chosen to elucidate the roles of these pathways on cell death. Studies herein indicate that thiol oxidation contributes to cell death through impaired activity of glutathione-dependent and thioredoxin (Trx) systems and altered signaling through redox-sensitive pathways. Free thiol content decreased by 71% with hyperoxic (95% oxygen) exposure. Increased cell death was observed during oxygen exposure when either the Trx or the glutathione-dependent system was pharmacologically inhibited with aurothioglucose (ATG) or buthionine sulfoximine, respectively. However, inhibition of the Trx system yielded the smallest decrease in free thiol content (1.44% with ATG treatment vs 21.33% with BSO treatment). Although Trx1 protein levels were unchanged, Trx1 function was impaired during hyperoxic treatment as indicated by progressive cysteine oxidation. Overexpression of Trx1 in H1299 cells utilizing an inducible construct increased cell survival during hyperoxia, whereas siRNA knockdown of Trx1 during oxygen treatment reduced cell viability. Overall, this indicated that a comparatively small pool of proteins relies on Trx redox functions to mediate cell survival in hyperoxia, and the protective functions of Trx1 are progressively lost by its oxidative inhibition. To further elucidate the role of Trx1, potential Trx1 redox protein-protein interactions mediating cytoprotection and cell survival pathways were determined by utilizing a substrate trap (mass action trapping) proteomics approach. With this method, known Trx1 targets were detected, including peroxiredoxin-1 as well as novel targets, including two HSP90 isoforms (HSP90AA1 and HSP90AB1). Reactive cysteines within the structure of HSP90 are known to modulate its ATPase-dependent chaperone activity through disulfide formation and S-nitrosylation. Whereas HSP90 expression is unchanged at the protein level during hyperoxic exposure, siRNA knockdown significantly increased hyperoxic cell death by 2.5-fold, indicating cellular dependence on HSP90 chaperone functions in response to hyperoxic exposure. These data support the hypothesis that hyperoxic impairment of Trx1 has a negative impact on HSP90-oxidative responses critical to cell survival, with potential implications for pathways implicated in lung development and the pathogenesis of BPD. © 2014 Elsevier Inc.
PubMed | Childrens Health Research Center
Type: Journal Article | Journal: PloS one | Year: 2017
Mitochondria play a fundamental role in the regulation of cell death during accumulation of oxidants. High concentrations of atmospheric oxygen (hyperoxia), used clinically to treat tissue hypoxia in premature newborns, is known to elicit oxidative stress and mitochondrial injury to pulmonary epithelial cells. A consequence of oxidative stress in mitochondria is the accumulation of peroxides which are detoxified by the dedicated mitochondrial thioredoxin system. This system is comprised of the oxidoreductase activities of peroxiredoxin-3 (Prx3), thioredoxin-2 (Trx2), and thioredoxin reductase-2 (TrxR2). The goal of this study was to understand the role of the mitochondrial thioredoxin system and mitochondrial injuries during hyperoxic exposure. Flow analysis of the redox-sensitive, mitochondrial-specific fluorophore, MitoSOX, indicated increased levels of mitochondrial oxidant formation in human adenocarcinoma cells cultured in 95% oxygen. Increased expression of Trx2 and TrxR2 in response to hyperoxia were not attributable to changes in mitochondrial mass, suggesting that hyperoxic upregulation of mitochondrial thioredoxins prevents accumulation of oxidized Prx3. Mitochondrial oxidoreductase activities were modulated through pharmacological inhibition of TrxR2 with auranofin and genetically through shRNA knockdown of Trx2 and Prx3. Diminished Trx2 and Prx3 expression was associated with accumulation of mitochondrial superoxide; however, only shRNA knockdown of Trx2 increased susceptibility to hyperoxic cell death and increased phosphorylation of apoptosis signal-regulating kinase-1 (ASK1). In conclusion, the mitochondrial thioredoxin system regulates hyperoxic-mediated death of pulmonary epithelial cells through detoxification of oxidants and regulation of redox-dependent apoptotic signaling.