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1.  Evaluation of the Fluids Mixing Enclosure System for Life Science Experiments During a Commercial Caenorhabditis elegans Spaceflight Experiment 
The Student Spaceflight Experiments Program (SSEP) is a United States national science, technology, engineering, and mathematics initiative that aims to increase student interest in science by offering opportunities to perform spaceflight experiments. The experiment detailed here was selected and flown aboard the third SSEP mission and the first SSEP mission to the International Space Station (ISS). Caenorhabditis elegans is a small, transparent, self-fertilizing hermaphroditic roundworm that is commonly used in biological experiments both on Earth and in Low Earth Orbit. Past experiments have found decreased expression of mRNA for several genes whose expression can be controlled by the FOXO transcription factor DAF-16. We flew a daf-16 mutant and control worms to determine if the effects of spaceflight on C. elegans are mediated by DAF-16. The experiment used a Type Two Fluids Mixing Enclosure (FME), developed by Nanoracks LLC, and was delivered to the ISS aboard the SpaceX Dragon and returned aboard the Russian Soyuz. The short time interval between experiment selection and the flight rendered preflight experiment verification tests impossible. In addition, published research regarding the viability of the FME in life science experiments was not available. The experiment was therefore structured in such a way as to gather the needed data. Here we report that C. elegans can survive relatively short storage and activation in the FME but cannot produce viable populations for post-flight analysis on extended missions. The FME appears to support short-duration life science experiments, potentially on supply or crew exchange missions, but not on longer ISS expeditions. Additionally, the flown FME was not properly activated, reportedly due to a flaw in training procedures. We suggest that a modified transparent FME could prevent similar failures in future flight experiments.
PMCID: PMC3684985  PMID: 23794777
Commercial Spaceflight; Space Life Sciences; C. elegans; Fluids Mixing Enclosure; DAF-16; FOXO
2.  Nutrient-driven O-GlcNAc cycling influences autophagic flux and neurodegenerative proteotoxicity 
Autophagy  2013;9(4):604-606.
O-GlcNAcylation is an abundant post-translational modification implicated in human neurodegenerative diseases. We showed that loss-of-function of OGT (O-linked GlcNAc transferase) alleviated, while loss of OGA (O-GlcNAc selective β-N-acetyl-D-glucosaminidase) enhanced, the proteotoxicity of C. elegans neurodegenerative disease models including tauopathy, β-amyloid peptide and polyglutamine expansion. The O-GlcNAc cycling mutants act, in part, by altering insulin signaling, proteasome activity and autophagy. In mutants lacking either of these enzymes of O-GlcNAc cycling, there is a striking accumulation of GFP::LGG-1 (C. elegans homolog of Atg8 and LC3) and increased phosphatidylethanolamine (PE)-modified GFP::LGG-1 upon starvation. We speculate that O-GlcNAc cycling is a key nutrient-responsive regulator of autophagic flux acting at multiple levels including direct modification of BECN1 and BCL2.
PMCID: PMC3627674  PMID: 23328586
neurodegeneration; proteotoxicity; O-GlcNAc; proteostasis; autophagy; proteasome; insulin signaling
3.  O-GlcNAc cycling shows neuroprotective potential in C. elegans models of neurodegenerative disease 
Worm  2013;2(4):e27043.
C. elegans has proven to be an excellent organism in which to model human neurodegenerative disease.1–7 The worm’s simple nervous system, lineage, and neural maps, easily scored movement phenotypes, and robust forward and reverse genetics make it optimal for studying age-dependent processes on a reasonable time scale. A popular approach has been the introduction of transgenes expressing GFP-tagged proteotoxic human proteins into neurons leading to visible aggregation or movement phenotypes.2,4,6,8–13 In addition, the maintenance of proteostasis networks has been extensively studied using the power of worm genetics.8–13 These networks include genes involved in insulin-like signaling, the heat shock response, the response to hypoxia, and mTOR and AMPK pathways linked to aging.14 Another pathway with suggestive links to neurodegeneration is the O-GlcNAc cycling pathway, a nutrient-dependent post-translational modification known to be altered in brains from patients with Alzheimer disease.15–19 In this commentary, we summarize our recent findings showing that viable mutants of O-GlcNAc cycling in C. elegans dramatically alter the neurotoxicity of four distinct C. elegans models of neurodegenerative disease.7 Mutants in O-GlcNAc cycling alter the toxicity of mutant tau, polyglutamine expansion reporters, and amyloid β-peptide. The findings further suggest that O-GlcNAc cycling acts at many steps in the lifecycle of aggregation-prone targets. The C. elegans system is likely to continue to provide insights into this complex problem. The involvement of O-GlcNAc cycling in the maintenance of proteostasis raises the possibility of targeting the enzymes catalyzing this critical post-translational modification for therapeutic intervention.
PMCID: PMC3917942  PMID: 24744983
O-GlcNAc; apoptosis; glycobiology; neurodegeneration; proteostasis; signaling; transcription
4.  Thyroid hormone receptor β suppresses SV40-mediated tumorigenesis via novel nongenomic actions 
Accumulated evidence suggests that thyroid hormone receptor β (TRβ) could function as a tumor suppressor, but the detailed mechanisms by which TRβ inhibits tumorigenesis are not fully understood. The present studies explored the mechanisms by which TRβ acted to inhibit thyroid tumor development mediated by simian virus-40 (SV40). In mouse xenograft models, SV40 large T antigen (SV40Tag)-immortalized human thyroid epithelial (HTori) cells rapidly induced tumors, but the tumor development was totally blocked by TRβ stably expressed in HTori cells. Previous studies showed that the SV40Tag oncoprotein binds to and inactivates tumor suppressors p53 and retinoblastoma protein (Rb), thereby inducing tumorigenesis. Here we showed that one of the mechanisms by which TRβ suppressed tumor development was by competing with p53 and Rb for binding to SV40Tag. The interaction of TRβ with SV40Tag led to reactivation of Rb to inhibit cell cycle progression. TRβ- SV40Tag interaction also resulted in reactivating p53 to increase the expression of Pten, thus attenuating PI3K-AKT signaling to decrease cell proliferation and to induce apoptosis. The present study uncovered a novel action of TRβ as a tumor suppressor initiated via interfering with the recruitment of Rb and p53 by SV40Tag oncoprotein through protein-protein interaction, thereby acting to block tumor development.
PMCID: PMC3433110  PMID: 22957312
Thyroid hormone receptor; tumor suppressor; tumorigenesis; thyroid hormone; xenograft models
5.  Epigenetics gets Sweeter: O-GlcNAc joins the ‘Histone Code’ 
Chemistry & biology  2010;17(12):1272-1274.
O-GlcNAcylation has now been added to the growing list of histone modifications making up the multifaceted ‘histone-code’ (Sakabe et al., 2010). The sites of O-GlcNAc-histone modification hint at a role in chromatin remodeling, thus adding to mounting evidence that O-GlcNAc cycling sits atop a robust regulatory network maintaining higher-order chromatin structure and epigenetic memory.
PMCID: PMC3010756  PMID: 21168762
6.  O-GlcNAc cycling: Emerging Roles in Development and Epigenetics 
The nutrient-sensing hexosamine signaling pathway modulates the levels of O-linked N-acetylglucosamine (O-GlcNAc) on key targets impacting cellular signaling, protein turnover and gene expression. O-GlcNAc cycling may be deregulated in neurodegenerative disease, cancer, and diabetes. Studies in model organisms demonstrate that the O-GlcNAc transferase (OGT/Sxc) is essential for Polycomb group (PcG) repression of the homeotic genes, clusters of genes responsible for the adult body plan. Surprisingly, from flies to man, the O-GlcNAcase (OGA, MGEA5) gene is embedded within the NK cluster, the most evolutionarily ancient of three homeobox gene clusters regulated by PcG repression. PcG repression also plays a key role in maintaining stem cell identity, recruiting the DNA methyltransferase machinery for imprinting, and in X-chromosome inactivation. Intriguingly, the Ogt gene resides near the Xist locus in vertebrates and is subject to regulation by PcG-dependent X-inactivation. OGT is also an enzymatic component of the human dosage compensation complex. These ‘evo-devo’ relationships linking O-GlcNAc cycling to higher order chromatin structure provide insights into how nutrient availability may influence the epigenetic regulation of gene expression. O-GlcNAc cycling at promoters and PcG repression represent concrete mechanisms by which nutritional information may be transmitted across generations in the intra-uterine environment. Thus, the nutrient-sensing hexosamine signaling pathway may be a key contributor to the metabolic deregulation resulting from prenatal exposure to famine, or the ‘vicious cycle’ observed in children of mothers with type-2 diabetes and metabolic disease.
PMCID: PMC2917487  PMID: 20488252
7.  The Hexosamine Signaling Pathway: O-GlcNAc cycling in feast or famine 
Biochimica et biophysica acta  2009;1800(2):80.
The enzymes of O-GlcNAc cycling couple the nutrient-dependent synthesis of UDP-GlcNAc to O-GlcNAc modification of Ser/Thr residues of key nuclear and cytoplasmic targets. This series of reactions culminating in O-GlcNAcylation of targets has been termed the Hexosamine Signaling Pathway (HSP). The evolutionarily ancient enzymes of O-GlcNAc cycling have co-evolved with other signaling effecter molecules; they are recruited to their targets by many of the same mechanisms used to organize canonic kinase-dependent signaling pathways. This co-recruitment of the enzymes of O-GlcNAc cycling drives a binary switch impacting pathways of anabolism and growth (nutrient uptake) and catabolic pathways (nutrient sparing and salvage). The Hexosamine Signaling Pathway (HSP) has thus emerged as a versatile cellular regulator modulating numerous cellular signaling cascades influencing growth, metabolism, cellular stress, circadian rhythm, and host-pathogen interactions. In mammals, the nutrient-sensing HSP has been harnessed to regulate such cell-specific functions as neutrophil migration, and activation of B-cells and T-cells. This review summarizes the diverse approaches being used to examine O-GlcNAc cycling. It will emphasize the impact O-GlcNAcylation has upon signaling pathways that may be become deregulated in diseases of the immune system, diabetes mellitus, cancer, cardiovascular disease, and neurodegenerative diseases.
PMCID: PMC2815088  PMID: 19647043
8.  O-GlcNAc cycling: Implications for Neurodegenerative disorders 
The dynamic post-translational modification of proteins by O-linked N-Acetylglucosamine (O-GlcNAc), termed O-GlcNAcylation, is an important mechanism for modulating cellular signaling pathways. O-GlcNAcylation impacts transcription, translation, organelle trafficking, proteasomal degradation and apoptosis. O-GlcNAcylation has been implicated in the etiology of several human diseases including type-2 diabetes and neurodegeneration. This review describes the pair of enzymes responsible for the cycling of this post-translational modification: O-GlcNAc transferase (OGT) and β-N-acetylglucosaminidase (OGA), with a focus on the function of their structural domains. We will also highlight the important processes and substrates regulated by these enzymes, with an emphasis on the role of O-GlcNAc as a nutrient sensor impacting insulin signaling and the cellular stress response. Finally, we will focus attention on the many ways by which O-GlcNAc cycling may affect the cellular machinery in the neuroendocrine and central nervous systems.
PMCID: PMC2757632  PMID: 19782947
Glycosylation; O-GlcNAc transferase; O-GlcNAcase
9.  Elevated O-GlcNAc-dependent signaling through inducible mOGT expression selectively triggers apoptosis 
Amino Acids  2010;40(3):885-893.
O-linked N-acetylglucosamine transferase (OGT) catalyzes O-GlcNAc addition to numerous cellular proteins including transcription and nuclear pore complexes and plays a key role in cellular signaling. One differentially spliced isoform of OGT is normally targeted to mitochondria (mOGT) but is quite cytotoxic when expressed in cells compared with the ncOGT isoform. To understand the basis of this selective cytotoxicity, we constructed a fully functional ecdysone-inducible GFP–OGT. Elevated GFP–OGT expression induced a dramatic increase in intracellular O-GlcNAcylated proteins. Furthermore, enhanced OGT expression efficiently triggered programmed cell death. Apoptosis was dependent upon the unique N-terminus of mOGT, and its catalytic activity. Induction of mOGT expression triggered programmed cell death in every cell type tested including INS-1, an insulin-secreting cell line. These studies suggest that deregulated activity of the mitochondrially targeted mOGT may play a role in triggering the programmed cell death observed with diseases such as diabetes mellitus and neurodegeneration.
PMCID: PMC3040817  PMID: 20824293
O-GlcNAc; O-linked N-acetylglucosamine transferase; Apoptosis
10.  Karyopherin β3: a new cellular target for the HPV-16 E5 oncoprotein 
Epidemiological and experimental studies have shown that high-risk human papillomaviruses (HPVs) are the causative agents of cervical cancer worldwide, and that HPV-16 is associated with more than half of these cases. In addition to the well-characterized E6 and E7 oncoproteins of HPV-16, recent evidence increasingly has implicated the HPV-16 E5 protein (16E5) as an important mediator of oncogenic transformation. Since 16E5 has no known intrinsic enzymatic activity, its effects on infected cells are most likely mediated by interactions with various cellular proteins and/or its documented association with lipid rafts. In the present study, we describe a new cellular target that binds to 16E5 in COS cells and in stable human ectocervical cell lines. This target is karyopherin β3, a member of the nuclear import receptor family with critical roles in the nuclear import of ribosomal proteins and in the secretory pathway.
PMCID: PMC2773212  PMID: 18455505
HPV-16; E5 oncoprotein; karyopherin β3
11.  A convenient synthesis of the C-1- phosphonate analogue of UDP-GlcNAc and its evaluation as an inhibitor of O-linked GlcNAc transferase (OGT) 
Carbohydrate research  2007;343(2):189-195.
The C-1-phosphonate analogue of UDP-GlcNAc has been synthesized using an α-configured C-1-aldehyde as a key intermediate. Addition of the anion of diethyl phosphate to the aldehyde produced the hydroxyphosphonate. The configuration of this key intermediate was determined by x-ray crystallography. Deoxygenation, coupling of the resulting phosphonic acid with UMP and deprotection gave the target molecule as a di-sodium salt. This analogue had no detectable activity as an inhibitor of (OGT).
PMCID: PMC2243073  PMID: 18039537
12.  Nuclear Receptor Corepressor Is a Novel Regulator of Phosphatidylinositol 3-Kinase Signaling▿  
Molecular and Cellular Biology  2007;27(17):6116-6126.
The nuclear receptor corepressor (NCoR) regulates the activities of DNA-binding transcription factors. Recent observations of its distribution in the extranuclear compartment raised the possibility that it could have other cellular functions in addition to transcription repression. We previously showed that phosphatidylinositol 3-kinase (PI3K) signaling is aberrantly activated by a mutant thyroid hormone β receptor (TRβPV, hereafter referred to as PV) via physical interaction with p85α, thus contributing to thyroid carcinogenesis in a mouse model of follicular thyroid carcinoma (TRβPV/PV mouse). Since NCoR is known to modulate the actions of TRβ mutants in vivo and in vitro, we asked whether NCoR regulates PV-activated PI3K signaling. Remarkably, we found that NCoR physically interacted with and competed with PV for binding to the C-terminal SH2 (Src homology 2) domain of p85α, the regulatory subunit of PI3K. Confocal fluorescence microscopy showed that both NCoR and p85α were localized in the nuclear as well as in the cytoplasmic compartments. Overexpression of NCoR in thyroid tumor cells of TRβPV/PV mouse reduced PI3K signaling, as indicated by the decrease in the phosphorylation of its immediate downstream effector, p-AKT. Conversely, lowering cellular NCoR by siRNA knockdown in tumor cells led to overactivated p-AKT and increased cell proliferation and motility. Furthermore, NCoR protein levels were significantly lower in thyroid tumor cells than in wild-type thyrocytes, allowing more effective binding of PV to p85α to activate PI3K signaling and thus contributing to tumor progression. Taken together, these results indicate that NCoR, via protein-protein interaction, is a novel regulator of PI3K signaling and could serve to modulate thyroid tumor progression.
PMCID: PMC1952145  PMID: 17606624
13.  Aberrant accumulation of PTTG1 induced by a mutated thyroid hormone β receptor inhibits mitotic progression 
Journal of Clinical Investigation  2006;116(11):2972-2984.
Overexpression of pituitary tumor–transforming 1 (PTTG1) is associated with thyroid cancer. We found elevated PTTG1 levels in the thyroid tumors of a mouse model of follicular thyroid carcinoma (TRβPV/PV mice). Here we examined the molecular mechanisms underlying elevated PTTG1 levels and the contribution of increased PTTG1 to thyroid carcinogenesis. We showed that PTTG1 was physically associated with thyroid hormone β receptor (TRβ) as well as its mutant, designated PV. Concomitant with thyroid hormone–induced (T3-induced) degradation of TRβ, PTTG1 proteins were degraded by the proteasomal machinery, but no such degradation occurred when PTTG1 was associated with PV. The degradation of PTTG1/TRβ was activated by the direct interaction of the liganded TRβ with steroid receptor coactivator 3 (SRC-3), which recruits proteasome activator PA28γ. PV, which does not bind T3, could not interact directly with SRC-3/PA28γ to activate proteasome degradation, resulting in elevated PTTG1 levels. The accumulated PTTG1 impeded mitotic progression in cells expressing PV. Our results unveil what we believe to be a novel mechanism by which PTTG1, an oncogene, is regulated by the liganded TRβ. The loss of this regulatory function in PV led to an aberrant accumulation of PTTG1 disrupting mitotic progression that could contribute to thyroid carcinogenesis.
PMCID: PMC1592548  PMID: 17039256
14.  Endoplasmic Reticulum-Localized Human Papillomavirus Type 16 E5 Protein Alters Endosomal pH but Not trans-Golgi pH 
Journal of Virology  2005;79(9):5839-5846.
The human papillomavirus type 16 (HPV-16) E5 protein is a small, hydrophobic polypeptide that is expressed in virus-infected keratinocytes and alters receptor signaling pathways, apoptotic responses, and endosomal pH. Despite its ability to inhibit endosomal acidification, the HPV-16 E5 protein is found predominantly in the endoplasmic reticulum (ER), suggesting that its effect may be indirect and perhaps global. To determine whether E5 alters the pHs of additional intracellular compartments, we transduced human keratinocytes with a codon-optimized E5 vector and then quantified endosomal and trans-Golgi pHs using sensitive, compartment-specific, ratiometric pHluorin constructs. E5 protein increased endosomal pH from 5.9 to 6.9 but did not affect the normal trans-Golgi pH of 6.3. Confirming the lack of alteration in trans-Golgi pH, we observed no alterations in the acidification-dependent processing of the proH3 protein. C-terminal deletions of E5, which retained normal expression and localization in the ER, were defective for endosomal alkalization. Thus, E5 does not uniformly alkalinize intracellular compartments, and its C-terminal 10 amino acids appear to mediate interactions with critical ER targets that modulate proton pump function and/or localization.
PMCID: PMC1082759  PMID: 15827198
15.  Crp79p, Like Mex67p, Is an Auxiliary mRNA Export Factor in Schizosaccharomyces pombe 
Molecular Biology of the Cell  2002;13(8):2571-2584.
The export of mRNA from the nucleus to the cytoplasm involves interactions of proteins with mRNA and the nuclear pore complex. We isolated Crp79p, a novel mRNA export factor from the same synthetic lethal screen that led to the identification of spMex67p in Schizosaccharomyces pombe. Crp79p is a 710-amino-acid-long protein that contains three RNA recognition motif domains in tandem and a distinct C-terminus. Fused to green fluorescent protein (GFP), Crp79p localizes to the cytoplasm. Like Mex67p, Crp79-GFP binds poly(A)+ RNA in vivo, shuttles between the nucleus and the cytoplasm, and contains a nuclear export activity at the C-terminus that is Crm1p-independent. All of these properties are essential for Crp79p to promote mRNA export. Crp79p import into the nucleus depends on the Ran system. A domain of spMex67p previously identified as having a nuclear export activity can functionally substitute for the nuclear export activity at the C-terminus of Crp79p. Although both Crp79p and spMex67p function to export mRNA, Crp79p does not substitute for all of spMex67p functions and probably is not a functional homologue of spMex67p. We propose that Crp79p is a nonessential mRNA export carrier in S. pombe.
PMCID: PMC117926  PMID: 12181330
16.  Calreticulin Is a Receptor for Nuclear Export 
The Journal of Cell Biology  2001;152(1):127-140.
In previous work, we used a permeabilized cell assay that reconstitutes nuclear export of protein kinase inhibitor (PKI) to show that cytosol contains an export activity that is distinct from Crm1 (Holaska, J.M., and B.M. Paschal. 1995. Proc. Natl. Acad. Sci. USA. 95: 14739–14744). Here, we describe the purification and characterization of the activity as calreticulin (CRT), a protein previously ascribed to functions in the lumen of the ER. We show that cells contain both ER and cytosolic pools of CRT. The mechanism of CRT-dependent export of PKI requires a functional nuclear export signal (NES) in PKI and involves formation of an export complex that contains RanGTP. Previous studies linking CRT to downregulation of steroid hormone receptor function led us to examine its potential role in nuclear export of the glucocorticoid receptor (GR). We found that CRT mediates nuclear export of GR in permeabilized cell, microinjection, and transfection assays. GR export is insensitive to the Crm1 inhibitor leptomycin B in vivo, and it does not rely on a leucine-rich NES. Rather, GR export is facilitated by its DNA-binding domain, which is shown to function as an NES when transplanted to a green fluorescent protein reporter. CRT defines a new export pathway that may regulate the transcriptional activity of steroid hormone receptors.
PMCID: PMC2193655  PMID: 11149926
nucleocytoplasmic transport; Ran GTPase; nuclear pore complex; protein export; hormone receptor
17.  Mex67p of Schizosaccharomyces pombe Interacts with Rae1p in Mediating mRNA Export 
Molecular and Cellular Biology  2000;20(23):8767-8782.
We identified the Schizosaccharomyces pombe mex67 gene (spmex67) as a multicopy suppressor of rae1-167 nup184-1 synthetic lethality and the rae1-167 ts mutation. spMex67p, a 596-amino-acid-long protein, has considerable sequence similarity to the Saccharomyces cerevisiae Mex67p (scMex67p) and human Tap. In contrast to scMEX67, spmex67 is essential for neither growth nor nuclear export of mRNA. However, an spmex67 null mutation (Δmex67) is synthetically lethal with the rae1-167 mutation and accumulates poly(A)+ RNA in the nucleus. We identified a central region (149 to 505 amino acids) within spMex67p that associates with a complex containing Rae1p that complements growth and mRNA export defects of the rae1-167 Δmex67 synthetic lethality. This region is devoid of RNA-binding, N-terminal nuclear localization, and the C-terminal nuclear pore complex-targeting regions. The (149–505)-green fluorescent protein (GFP) fusion is found diffused throughout the cell. Overexpression of spMex67p inhibits growth and mRNA export and results in the redistribution of the diffused localization of the (149–505)-GFP fusion to the nucleus and the nuclear periphery. These results suggest that spMex67p competes for essential mRNA export factor(s). Finally, we propose that the 149–505 region of spMex67p could act as an accessory factor in Rae1p-dependent transport and that spMex67p participates at various common steps with Rae1p export complexes in promoting the export of mRNA.
PMCID: PMC86506  PMID: 11073978

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