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Ubiquilin-mediated Small Molecule Inhibition of Mammalian Target of Rapamycin Complex 1 (mTORC1) Signaling.
Coffey RT, Shi Y, Long MJ, Marr MT, Hedstrom L
(2016) J Biol Chem 291: 5221-33
MeSH Terms: Adaptor Proteins, Signal Transducing, Arginine, Carbamates, Cell Line, Tumor, Cell Proliferation, Enzyme Inhibitors, HEK293 Cells, Humans, Mechanistic Target of Rapamycin Complex 1, Multiprotein Complexes, Phosphoproteins, Phosphorylation, Protein Binding, Protein Biosynthesis, Protein Processing, Post-Translational, Ribosomal Protein S6 Kinases, Signal Transduction, TOR Serine-Threonine Kinases, Ubiquitins
Show Abstract · Added May 9, 2016
Mammalian target of rapamycin complex 1 (mTORC1) is a master regulator of cellular metabolism, growth, and proliferation. mTORC1 has been implicated in many diseases such as cancer, diabetes, and neurodegeneration, and is a target to prolong lifespan. Here we report a small molecule inhibitor (Cbz-B3A) of mTORC1 signaling. Cbz-B3A inhibits the phosphorylation of eIF4E-binding protein 1 (4EBP1) and blocks 68% of translation. In contrast, rapamycin preferentially inhibits the phosphorylation of p70(S6k) and blocks 35% of translation. Cbz-B3A does not appear to bind directly to mTORC1, but instead binds to ubiquilins 1, 2, and 4. Knockdown of ubiquilin 2, but not ubiquilins 1 and 4, decreases the phosphorylation of 4EBP1, suggesting that ubiquilin 2 activates mTORC1. The knockdown of ubiquilins 2 and 4 decreases the effect of Cbz-B3A on 4EBP1 phosphorylation. Cbz-B3A slows cellular growth of some human leukemia cell lines, but is not cytotoxic. Thus Cbz-B3A exemplifies a novel strategy to inhibit mTORC1 signaling that might be exploited for treating many human diseases. We propose that Cbz-B3A reveals a previously unappreciated regulatory pathway coordinating cytosolic protein quality control and mTORC1 signaling.
© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.
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19 MeSH Terms
Basis for a ubiquitin-like protein thioester switch toggling E1-E2 affinity.
Huang DT, Hunt HW, Zhuang M, Ohi MD, Holton JM, Schulman BA
(2007) Nature 445: 394-8
MeSH Terms: Adenosine Triphosphate, Binding Sites, Crystallography, X-Ray, Esters, Humans, Models, Molecular, NEDD8 Protein, Protein Conformation, Structure-Activity Relationship, Sulfhydryl Compounds, Ubiquitin-Activating Enzymes, Ubiquitin-Conjugating Enzymes, Ubiquitins
Show Abstract · Added March 5, 2014
Ubiquitin-like proteins (UBLs) are conjugated by dynamic E1-E2-E3 enzyme cascades. E1 enzymes activate UBLs by catalysing UBL carboxy-terminal adenylation, forming a covalent E1 throught UBL thioester intermediate, and generating a thioester-linked E2 throught UBL product, which must be released for subsequent reactions. Here we report the structural analysis of a trapped UBL activation complex for the human NEDD8 pathway, containing NEDD8's heterodimeric E1 (APPBP1-UBA3), two NEDD8s (one thioester-linked to E1, one noncovalently associated for adenylation), a catalytically inactive E2 (Ubc12), and MgATP. The results suggest that a thioester switch toggles E1-E2 affinities. Two E2 binding sites depend on NEDD8 being thioester-linked to E1. One is unmasked by a striking E1 conformational change. The other comes directly from the thioester-bound NEDD8. After NEDD8 transfer to E2, reversion to an alternate E1 conformation would facilitate release of the E2 throught NEDD8 thioester product. Thus, transferring the UBL's thioester linkage between successive conjugation enzymes can induce conformational changes and alter interaction networks to drive consecutive steps in UBL cascades.
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13 MeSH Terms
Dynamics of tissue ubiquitin pools and ubiquitin-proteasome pathway component activities during the systemic response to traumatic shock.
Patel MB, Earle SA, Majetschak M
(2007) Physiol Res 56: 547-57
MeSH Terms: Animals, Chymases, Disease Models, Animal, Femoral Fractures, Fluid Therapy, Hemorrhage, Kidney, Lung, Muscle, Skeletal, Myocardium, Proteasome Endopeptidase Complex, Serine Endopeptidases, Shock, Traumatic, Spleen, Swine, Time Factors, Ubiquitins
Show Abstract · Added June 14, 2016
Based on the biological significance of the ubiquitin-proteasome pathway (UPP) and its potential role during sepsis, burns and ischemia-reperfusion injury, we hypothesized that the systemic response to traumatic shock (TS) is accompanied by tissue-specific UPP alterations. Therefore, we studied tissue ubiquitin pools, chymotryptic- and tryptic-like proteasome peptidase activities and ubiquitin-protein ligation (UbPL) rates in skeletal muscle, heart, lung, liver, spleen and kidney using a clinically relevant porcine model (bilateral femur fracture/hemorrhage followed by fluid resuscitation). TS induced a systemic reduction of tissue-specific high molecular mass ubiquitin-protein conjugates (>50 kDa). Free ubiquitin was unaffected. The dynamic organ patterns of ubiquitin pools paralleled the typical physiological response to TS and resuscitation. Reduction of ubiquitin-protein conjugates was most pronounced in heart and lung (p<0.05 vs. control) and accompanied by significant increases in proteasome peptidase and UbPL activities in these organs. Unlike all other tissues, spleen proteasome peptidase and UbPL activities were significantly reduced 10 h after TS. These findings support the concept that the UPP could play an important role in regulation of cell functions during the early whole-body response to TS. The UPP might be a therapeutic target to improve the metabolic care after TS, particularly in the heart, lung, and spleen.
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17 MeSH Terms
Role of post-translational modifications in regulating c-Myc proteolysis, transcriptional activity and biological function.
Hann SR
(2006) Semin Cancer Biol 16: 288-302
MeSH Terms: Animals, Humans, Peptide Hydrolases, Phosphorylation, Protein Processing, Post-Translational, Protein-Serine-Threonine Kinases, Proto-Oncogene Proteins c-myc, SKP Cullin F-Box Protein Ligases, Signal Transduction, Transcription, Genetic, Ubiquitins
Show Abstract · Added March 5, 2014
The Myc proteins play a central role in cellular proliferation, differentiation, apoptosis and tumorigenesis. Although it is clear that multiple molecular mechanisms mediate these functions, it is unclear how individual mechanisms contribute and if different mechanisms work in concert or separately in mediating the diverse biological functions of c-Myc. Similarly, the role of post-translational modifications in regulating c-Myc molecular and biological properties has remained uncertain, despite over 20 years of research. In particular, phosphorylation of the N-terminal transcriptional regulatory domain has been shown to have a variety of consequences ranging from dramatic effects on apoptosis, tumorigenesis and c-Myc proteolysis to negligible effects on cellular transformation and transcriptional activity. This review attempts to provide a comprehensive and critical evaluation of the accumulated evidence to address the complex and controversial issues surrounding the role of post-translational modifications in c-Myc function, focusing on phosphorylation and ubiquitination of the N-terminal transcriptional regulatory domain. An overall model emerges that suggests phosphorylation and ubiquitination play critical roles in cell cycle progression, cell growth, apoptosis and tumorigenesis that are mediated by phosphorylation-dependent transcriptional activation of distinct sets of target genes and synchronized proteolysis.
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11 MeSH Terms
Down-regulation of UCRP and UBE2L6 in BRCA2 knocked-down human breast cells.
Tripathi MK, Chaudhuri G
(2005) Biochem Biophys Res Commun 328: 43-8
MeSH Terms: BRCA2 Protein, Breast Neoplasms, Cell Line, Cytokines, Down-Regulation, Endothelial Cells, Humans, Ubiquitin-Conjugating Enzymes, Ubiquitins
Show Abstract · Added June 14, 2013
To understand the effects of the transient ablation of BRCA2 gene expression in dividing human breast cells, we transiently knocked down BRCA2 mRNA in HMEC and other cells. Microarray analysis of mRNAs revealed the down-regulation of the mRNAs of ubiquitin cross-reacting protein (UCRP) and the E2 enzyme that help conjugating UCRP to its target proteins, namely UBE2L6 (UbcH8), in BRCA2 ablated cells. UCRP is an interferon regulated protein, involved in cell growth and cell cycle events by participating in the degradation/modulation of cell cycle regulatory proteins. Quantitative-PCR and Northern analysis confirmed down-regulation of UCRP and UBE2L6 with BRCA2 knockdown, respectively. Since UCRP and UCRPylation have critical roles in the innate immunity against viral infection and during pregnancy, our observation may indicate new roles of the BRCA2 protein.
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9 MeSH Terms
Ubiquitin-like protein Hub1 is required for pre-mRNA splicing and localization of an essential splicing factor in fission yeast.
Wilkinson CR, Dittmar GA, Ohi MD, Uetz P, Jones N, Finley D
(2004) Curr Biol 14: 2283-8
MeSH Terms: Cell Cycle, Electrophoresis, Polyacrylamide Gel, Flow Cytometry, Genes, Essential, Microscopy, Fluorescence, Mutation, Oligonucleotides, Protein Transport, RNA Splicing, Reverse Transcriptase Polymerase Chain Reaction, Ribonucleoproteins, Small Nuclear, Schizosaccharomyces, Schizosaccharomyces pombe Proteins, Two-Hybrid System Techniques, Ubiquitin-Protein Ligase Complexes, Ubiquitins
Show Abstract · Added March 5, 2014
Hub1/Ubl5 is a member of the family of ubiquitin-like proteins (UBLs). The tertiary structure of Hub1 is similar to that of ubiquitin; however, it differs from known modifiers in that there is no conserved glycine residue near the C terminus which, in ubiquitin and UBLs, is required for covalent modification of target proteins. Instead, there is a conserved dityrosine motif proximal to the terminal nonconserved amino acid. In S. cerevisiae, high molecular weight adducts can be formed in vivo from Hub1, but the structure of these adducts is not known, and they could be either covalent or noncovalent. The budding yeast HUB1 gene is not essential, but Delta hub1 mutants display defects in mating. Here, we report that fission yeast hub1 is an essential gene, whose loss results in cell cycle defects and inefficient pre-mRNA splicing. A screen for Hub1 interactors identified Snu66, a component of the U4/U6.U5 tri-snRNP splicing complex. Furthermore, overexpression of Snu66 suppresses the lethality of a hub1ts mutant. In cells lacking functional hub1, the nuclear localization of Snu66 is disrupted, suggesting that an important role for Hub1 is the correct subcellular targeting of Snu66, although our data suggest that Hub1 is likely to perform other roles in splicing as well.
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16 MeSH Terms
Three modules of zebrafish Mind bomb work cooperatively to promote Delta ubiquitination and endocytosis.
Chen W, Casey Corliss D
(2004) Dev Biol 267: 361-73
MeSH Terms: Amino Acid Sequence, Animals, DNA Primers, DNA, Complementary, Endocytosis, Fluorescent Antibody Technique, Immunoblotting, Immunohistochemistry, In Situ Hybridization, Intracellular Signaling Peptides and Proteins, Membrane Proteins, Molecular Sequence Data, Plasmids, Precipitin Tests, Receptors, Notch, Sequence Alignment, Signal Transduction, Ubiquitin-Protein Ligases, Ubiquitins, Zebrafish, Zebrafish Proteins
Show Abstract · Added April 24, 2014
Precise regulation of Notch signaling activity is critical for development of many different tissues. Here, we show that the zebrafish insertional mutation Hi904 attenuates Notch signaling, and is allelic to mind bomb. We show that Mind bomb protein displays E3 ubiquitin ligase activity in vitro and that it is associated with Delta and enhances its ubiquitination and internalization in transfected cells. Furthermore, by functional analysis of three conserved regions of Mind bomb, we show that the N-terminal half is required for Delta association, the ankyrin repeats are important for Delta internalization, and the ring fingers are required for Delta ubiquitination. Thus, the three functionally distinct modules of Mind bomb work cooperatively to regulate Notch signaling by associating with, ubiquitinating, and internalizing Delta.
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21 MeSH Terms
Fission yeast Clp1p phosphatase affects G2/M transition and mitotic exit through Cdc25p inactivation.
Wolfe BA, Gould KL
(2004) EMBO J 23: 919-29
MeSH Terms: CDC2 Protein Kinase, Cell Cycle Proteins, Fungal Proteins, G2 Phase, Mitosis, Mutation, Phosphorylation, Protein Binding, Protein Tyrosine Phosphatases, Schizosaccharomyces, Schizosaccharomyces pombe Proteins, Ubiquitin-Protein Ligases, Ubiquitins, ras-GRF1
Show Abstract · Added March 5, 2014
The Cdc14 family of phosphatases specifically reverses proline-directed phosphorylation events. In Saccharomyces cerevisiae, Cdc14p promotes Cdk1p inactivation at mitotic exit by reversing Cdk1p-dependent phosphorylations. Cdk1p is a proline-directed kinase whose activity is required in all eukaryotes for the transit into mitosis. At mitotic commitment, Cdk1p participates in its own regulation by activating the mitotic inducing phosphatase, Cdc25p, and inhibiting the opposing kinase, Wee1p. We have investigated the ability of Schizosaccharomyces pombe Clp1p, a Cdc14p homolog, to disrupt this auto-amplification loop. We show here that Clp1p is required to dephosphorylate, destabilize, and inactivate Cdc25p at the end of mitosis. Clp1p promotes recognition of Cdc25p by the anaphase-promoting complex/cyclosome, an E3 ubiquitin ligase. Failure to inactivate and destabilize Cdc25p in late mitosis delays progression through anaphase, interferes with septation initiation network signaling, and additionally advances the commitment to mitotic entry in the next cycle. This may be a widely conserved mechanism whereby Cdc14 proteins contribute to Cdk1p inactivation.
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14 MeSH Terms
A protein knockdown strategy to study the function of beta-catenin in tumorigenesis.
Cong F, Zhang J, Pao W, Zhou P, Varmus H
(2003) BMC Mol Biol 4: 10
MeSH Terms: Animals, Cell Division, Cell Line, Tumor, Colorectal Neoplasms, Cytoskeletal Proteins, Mice, Mice, Nude, Protein Engineering, Proto-Oncogene Proteins, Recombinant Fusion Proteins, Signal Transduction, Trans-Activators, Ubiquitins, Wnt Proteins, Zebrafish Proteins, beta Catenin
Show Abstract · Added March 24, 2014
BACKGROUND - The Wnt signaling pathway plays critical roles in cell proliferation and cell fate determination at many stages of development. A critical downstream target of Wnt signaling is the cytosolic beta-catenin, which is stabilized upon Wnt activation and promotes transcription of a variety of target genes including c-myc and cyclin D. Aberrant Wnt signaling, which results from mutations of either beta-catenin or adenomatous polyposis coli (APC), renders beta-catenin resistant to degradation, and has been associated with multiple types of human cancers.
RESULTS - A protein knockdown strategy was designed to reduce the cytosolic beta-catenin levels through accelerating its turnover rate. By engineering a chimeric protein with the beta-catenin binding domain of E-cadherin fused to betaTrCP ubiquitin-protein ligase, the stable beta-catenin mutant was recruited to the cellular SCF (Skp1, Cullin 1, and F-box-containing substrate receptor) ubiquitination machinery for ubiquitination and degradation. The DLD1 colon cancer cells express wild type beta-catenin at abnormally high levels due to loss of APC. Remarkably, conditional expression of betaTrCP-E-cadherin under the control of a tetracycline-repressive promoter in DLD1 cells selectively knocked down the cytosolic, but not membrane-associated subpopulation of beta-catenin. As a result, DLD1 cells were impaired in their growth and clonogenic ability in vitro, and lost their tumorigenic potential in nude mice.
CONCLUSION - We have designed a novel approach to induce degradation of stabilized/mutated beta-catenin. Our results suggest that a high concentration of cytoplasmic beta-catenin is critical for the growth of colorectal tumor cells. The protein knockdown strategy can be utilized not only as a novel method to dissect the role of oncoproteins in tumorigenesis, but also as a unique tool to delineate the function of a subpopulation of proteins localized to a specific subcellular compartment.
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16 MeSH Terms
Nuclear and cytoplasmic degradation of endogenous p53 and HDM2 occurs during down-regulation of the p53 response after multiple types of DNA damage.
Joseph TW, Zaika A, Moll UM
(2003) FASEB J 17: 1622-30
MeSH Terms: Cell Nucleus, Cysteine Endopeptidases, Cytoplasm, DNA Damage, Down-Regulation, Humans, Multienzyme Complexes, Nuclear Proteins, Proteasome Endopeptidase Complex, Proto-Oncogene Proteins, Proto-Oncogene Proteins c-mdm2, Tumor Cells, Cultured, Tumor Suppressor Protein p53, Ubiquitins
Show Abstract · Added March 5, 2014
The principal regulator of p53 stability is HDM2, an E3 ligase mediating p53 degradation via the ubiquitin-26S proteasome pathway. Until recently, the accepted model held that p53 degradation occurs exclusively on cytoplasmic proteasomes, with an absolute requirement for nuclear export of p53 via the CRM1 pathway. However, 26S proteasomes are abundant in cytosol and nucleus. Using forced overexpression of HDM2 in mutant p53 tumor cells, we previously found that p53 degradation occurs in both the nucleus and the cytoplasm. p53 null cells coexpressing export-defective p53 and HDM2 retained partial competence for p53 degradation, challenging the obligatory export model. Because the ability of local nuclear destruction might add important control in switching off the p53 pathway, we now test this notion for physiological situations in untransfected cells and determine the significance of this regulation. Despite nuclear export blockade by leptomycin B and HTLV1-Rex protein, two potent CRM1 inhibitors, nuclear degradation of endogenous wild-type p53 and HDM2 occurs during down-regulation of the p53 response. This was seen in RKO and U2OS cells recovering from all major forms of DNA damage, including UV, gamma-IR, camptothecin, or cisplatinum. Moreover, significant nuclear degradation of endogenous p53 and HDM2 occurs in isolated nuclear fractions prepared from these recovering cells. Furthermore, nuclear proteasomes efficiently degrade ubiquitinated p53 in vitro. Our data indicate that in nonlethal outcomes of cellular stress, when DNA damage has been successfully repaired and the active p53 response needs to be down-regulated quickly to resume normal homeostasis, both nuclear and cytoplasmic proteasomes are recruited to efficiently degrade the elevated p53 and HDM2 protein levels. The physiological significance of local nuclear destruction lies in the fact that it adds tighter control and speed to switching the p53 pathway off.
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14 MeSH Terms