scholarly journals Involvement of the DNA Repair Protein hHR23 inp53Degradation

2003 ◽  
Vol 23 (24) ◽  
pp. 8960-8969 ◽  
Author(s):  
Sandra Glockzin ◽  
Francois-Xavier Ogi ◽  
Arnd Hengstermann ◽  
Martin Scheffner ◽  
Christine Blattner
Keyword(s):  
Dna Repair ◽  
26S Proteasome ◽  
Polyubiquitin Chains ◽  
Proteolytic Pathway ◽  
Repair Protein ◽  
Uba Domain ◽  
Dna Repair Protein ◽  
The Stability

ABSTRACT The stability of the tumor suppressor protein p53 is regulated via the ubiquitin-proteasome-dependent proteolytic pathway. Like most substrates of this pathway, p53 is modified by the attachment of polyubiquitin chains prior to proteasome-mediated degradation. However, the mechanism(s) involved in the delivery of polyubiquitylated p53 molecules to the proteasome are currently unclear. Here, we show that the human DNA repair protein hHR23 binds to polyubiquitylated p53 via its carboxyl-terminal ubiquitin-associated (Uba) domain shielding p53 from deubiquitylation in vitro and in vivo. In addition, downregulation of hHR23 expression within cells by RNA interference results in accumulation of p53. Since the Ubl domain of hHR23 has been shown to interact with the 26S proteasome, we propose that hHR23 is intrinsically involved in the delivery of polyubiquitylated p53 molecules to the proteasome. In this model, the Uba domain of hHR23 binds to polyubiquitin chains formed on p53 and protects them from deubiquitylation, while the Ubl domain delivers the polyubiquitylated p53 molecules to the proteasome.

scholarly journals Modulation of nitrosourea resistance in myeloid leukemias

Blood ◽  
1988 ◽  
Vol 71 (5) ◽  
pp. 1487-1494 ◽  
Author(s):  
SL Gerson ◽  
JE Trey
Keyword(s):  
Dna Repair ◽  
Therapeutic Index ◽  
Myelogenous Leukemia ◽  
Leukemic Cells ◽  
Biochemical Modulation ◽  
Repair Protein ◽  
Myeloid Leukemias ◽  
Dna Repair Protein ◽  
Dna Alkyltransferase

Abstract Drug resistance in myeloid leukemias may be mediated by an increased capacity to repair chemotherapy-induced DNA damage. Some tumor cell lines that are resistant to nitrosoureas contain the DNA repair protein O6-alkylguanine-DNA alkyltransferase (alkyltransferase). This protects cells by removing cytotoxic, nitrosourea-induced O6-alkylguanine adducts. We measured the level of alkyltransferase activity in myeloid leukemic cells freshly obtained from patients to determine whether the alkyltransferase was an important factor in nitrosourea resistance in these cells and whether inactivation of this protein could sensitize leukemic cells to nitrosoureas. Myeloid leukemic cells from patients with acute nonlymphocytic leukemia and chronic myelogenous leukemia had higher levels of alkyltransferase than did myeloid precursors from normal donors (P less than .01). This difference did not appear to be due to the state of differentiation of the leukemic or normal cells. To show that this repair protein mediated nitrosourea resistance in leukemic cells, cells were treated with the modified base O6- methylguanine to selectively and irreversibly inactivate the alkyltransferase and then exposed to 1,3-bis (2-chloroethyl)-1- nitrosourea (BCNU). An 18-hour incubation in 0.5 mmol/L O6- methylguanine caused an 87% +/- 3.6% decrease in alkyltransferase activity in leukemic cells and a 73% +/- 8.6% decrease in normal myeloid precursors. After treatment with O6-methylguanine, clonogenic leukemic cells from ten different donors became much more sensitive to BCNU, with a decrease in the dose needed to reduce colony survival by 50% (LD50) of 6.3 +/- 1.4-fold. A lesser effect was seen on CFU-GM, BFU- E, and CFU-GEM where the LD50 decreased two- to threefold. These studies show that nitrosourea resistance in myeloid leukemic cells can be abrogated by inactivation of the DNA repair protein O6-alkylguanine- DNA alkyltransferase. This method of biochemical modulation of DNA repair will sensitize leukemic cells to nitrosoureas in vitro and has the potential of increasing the therapeutic index of nitrosoureas in this disease.

scholarly journals HSP27 Is a Ubiquitin-Binding Protein Involved in I-κBα Proteasomal Degradation

2003 ◽  
Vol 23 (16) ◽  
pp. 5790-5802 ◽  
Author(s):  
Arnaud Parcellier ◽  
Elise Schmitt ◽  
Sandeep Gurbuxani ◽  
Daphné Seigneurin-Berny ◽  
Alena Pance ◽  
...  
Keyword(s):  
Direct Interaction ◽  
Cell Types ◽  
26S Proteasome ◽  
Necrosis Factor Alpha ◽  
Polyubiquitin Chains ◽  
Ubiquitinated Proteins ◽  
Activation Of Transcription ◽  
Ubiquitin Proteasome

ABSTRACT HSP27 is an ATP-independent chaperone that confers protection against apoptosis through various mechanisms, including a direct interaction with cytochrome c. Here we show that HSP27 overexpression in various cell types enhances the degradation of ubiquitinated proteins by the 26S proteasome in response to stressful stimuli, such as etoposide or tumor necrosis factor alpha (TNF-α). We demonstrate that HSP27 binds to polyubiquitin chains and to the 26S proteasome in vitro and in vivo. The ubiquitin-proteasome pathway is involved in the activation of transcription factor NF-κB by degrading its main inhibitor, I-κBα. HSP27 overexpression increases NF-κB nuclear relocalization, DNA binding, and transcriptional activity induced by etoposide, ΤNF-α, and interleukin 1β. HSP27 does not affect I-κBα phosphorylation but enhances the degradation of phosphorylated I-κBα by the proteasome. The interaction of HSP27 with the 26S proteasome is required to activate the proteasome and the degradation of phosphorylated I-κBα. A protein complex that includes HSP27, phosphorylated I-κBα, and the 26S proteasome is formed. Based on these observations, we propose that HSP27, under stress conditions, favors the degradation of ubiquitinated proteins, such as phosphorylated I-κBα. This novel function of HSP27 would account for its antiapoptotic properties through the enhancement of NF-κB activity.

Structure of a human DNA repair protein UBA domain that interacts with HIV-1 Vpr

10.1038/4220 ◽  
1998 ◽  
Vol 5 (12) ◽  
pp. 1042-1047 ◽  
Author(s):  
Thorsten Dieckmann ◽  
Elizabeth S. Withers-Ward ◽  
Mark A. Jarosinski ◽  
Chuan-Fa Liu ◽  
Irvin S. Y. Chen ◽  
...  
Keyword(s):  
Dna Repair ◽  
Human Dna ◽  
Repair Protein ◽  
Uba Domain ◽  
Dna Repair Protein ◽  
Hiv 1

scholarly journals A Chemical Genetics Analysis of the Roles of Bypass Polymerase DinB and DNA Repair Protein AlkB in Processing N2-Alkylguanine Lesions In Vivo

PLoS ONE ◽  
2014 ◽  
Vol 9 (4) ◽  
pp. e94716 ◽  
Author(s):  
Nidhi Shrivastav ◽  
Bogdan I. Fedeles ◽  
Deyu Li ◽  
James C. Delaney ◽  
Lauren E. Frick ◽  
...  
Keyword(s):  
Dna Repair ◽  
Chemical Genetics ◽  
Repair Protein ◽  
Dna Repair Protein ◽  
Bypass Polymerase

scholarly journals Ime2, a Meiosis-Specific Kinase in Yeast, Is Required for Destabilization of Its Transcriptional Activator, Ime1

2002 ◽  
Vol 22 (7) ◽  
pp. 2047-2056 ◽  
Author(s):  
Noga Guttmann-Raviv ◽  
Sabine Martin ◽  
Yona Kassir
Keyword(s):  
Feedback Loop ◽  
Kinase Activity ◽  
Transcriptional Activator ◽  
26S Proteasome ◽  
Positive Regulators ◽  
Atpase Subunits ◽  
The Stability ◽  
Diploid Cells

ABSTRACT In the budding yeast Saccharomyces cerevisiae, entry into meiosis and its successful completion depend on two positive regulators, Ime1 and Ime2. Ime1 is a transcriptional activator that is required for transcription of IME2, a serine/threonine protein kinase. We show that in vivo Ime2 associates with Ime1, that in vitro Ime2 phosphorylates Ime1, and that in living cells the stability of Ime1 depends on Ime2. Diploid cells with IME2 deleted show an increase in the level of Ime1, whereas haploid cells overexpressing IME2 show a decrease in the stability of Ime1. Furthermore, the level of Ime1 depends on the kinase activity of Ime2. Using a mutation in one of the ATPase subunits of the proteasome, RPT2, we demonstrate that Ime1, amino acids 270 to 360, is degraded by the 26S proteasome. We also show that Ime2 itself is an extremely unstable protein whose expression in vegetative cultures is toxic. We propose that a negative-feedback loop ensures that the activity of Ime1 will be restricted to a narrow window.

scholarly journals Modulation of nitrosourea resistance in myeloid leukemias

Blood ◽  
1988 ◽  
Vol 71 (5) ◽  
pp. 1487-1494 ◽  
Author(s):  
SL Gerson ◽  
JE Trey
Keyword(s):  
Dna Repair ◽  
Therapeutic Index ◽  
Myelogenous Leukemia ◽  
Leukemic Cells ◽  
Biochemical Modulation ◽  
Repair Protein ◽  
Myeloid Leukemias ◽  
Dna Repair Protein ◽  
Dna Alkyltransferase

Drug resistance in myeloid leukemias may be mediated by an increased capacity to repair chemotherapy-induced DNA damage. Some tumor cell lines that are resistant to nitrosoureas contain the DNA repair protein O6-alkylguanine-DNA alkyltransferase (alkyltransferase). This protects cells by removing cytotoxic, nitrosourea-induced O6-alkylguanine adducts. We measured the level of alkyltransferase activity in myeloid leukemic cells freshly obtained from patients to determine whether the alkyltransferase was an important factor in nitrosourea resistance in these cells and whether inactivation of this protein could sensitize leukemic cells to nitrosoureas. Myeloid leukemic cells from patients with acute nonlymphocytic leukemia and chronic myelogenous leukemia had higher levels of alkyltransferase than did myeloid precursors from normal donors (P less than .01). This difference did not appear to be due to the state of differentiation of the leukemic or normal cells. To show that this repair protein mediated nitrosourea resistance in leukemic cells, cells were treated with the modified base O6- methylguanine to selectively and irreversibly inactivate the alkyltransferase and then exposed to 1,3-bis (2-chloroethyl)-1- nitrosourea (BCNU). An 18-hour incubation in 0.5 mmol/L O6- methylguanine caused an 87% +/- 3.6% decrease in alkyltransferase activity in leukemic cells and a 73% +/- 8.6% decrease in normal myeloid precursors. After treatment with O6-methylguanine, clonogenic leukemic cells from ten different donors became much more sensitive to BCNU, with a decrease in the dose needed to reduce colony survival by 50% (LD50) of 6.3 +/- 1.4-fold. A lesser effect was seen on CFU-GM, BFU- E, and CFU-GEM where the LD50 decreased two- to threefold. These studies show that nitrosourea resistance in myeloid leukemic cells can be abrogated by inactivation of the DNA repair protein O6-alkylguanine- DNA alkyltransferase. This method of biochemical modulation of DNA repair will sensitize leukemic cells to nitrosoureas in vitro and has the potential of increasing the therapeutic index of nitrosoureas in this disease.

scholarly journals OTUB1 non-catalytically regulates the stability of the E2 ubiquitin conjugating enzyme UBE2E1

2018 ◽  
Author(s):  
Nagesh Pasupala ◽  
Marie E. Morrow ◽  
Lauren T. Que ◽  
Barbara A. Malynn ◽  
Averil Ma ◽  
...  
Keyword(s):  
Catalytic Mechanism ◽  
Polyubiquitin Chains ◽  
Protein Ubiquitination ◽  
Ubiquitin Conjugating Enzyme ◽  
E2 Enzymes ◽  
The Stability ◽  
In Cells ◽  
Conjugating Enzyme

AbstractOTUB1 is a deubiquitinating enzyme that cleaves K48-linked polyubiquitin chains and also regulates ubiquitin signaling through a unique, non-catalytic mechanism. OTUB1 binds to a subset of E2 ubiquitin conjugating enzymes and inhibits their activity by trapping the E2~ubiquitin thioester and preventing ubiquitin transfer. The same set of E2s stimulate the deubiquitinating activity of OTUB1 when the E2 is not charged with ubiquitin. Previous studies have shown that, in cells, OTUB1 binds to members of the UBE2D (UBCH5) and UBE2E families, as well as to UBC13 (UBE2N). Cellular roles have been identified for the interaction of OTUB1 with UBC13 and members of the UBE2D family, but not for UBE2E E2 enzymes. We report here a novel role for OTUB1-E2 interactions in modulating E2 protein ubiquitination. We find that depletion of OTUB1 dramatically destabilizes the E2 conjugating enzyme UBE2E1 (UBE2E1) in cells and that this effect is independent of the catalytic activity of OTUB1 but depends on the ability of OTUB1 to bind to UBE2E1. We show that OTUB1 suppresses UBE2E1 autoubiquitinationin vitroand in cells, thereby preventing UBE2E1 from being targeted to the proteasome for degradation. Taken together, we have found a new role for OTUB1 in rescuing specific E2s from degradationin vivo.

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