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Peter Sutovsky - One of the best experts on this subject based on the ideXlab platform.
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ubiquitin activating Enzyme uba1 is required for sperm capacitation acrosomal exocytosis and sperm egg coat penetration during porcine fertilization
International Journal of Andrology, 2012Co-Authors: Shawn Zimmerman, Gaurishankar Manandhar, John F. Odhiambo, Chelsey Kennedy, V. Jonáková, Miriam Sutovsky, Chang-sik Park, P Maňaskovapostlerova, Peter SutovskyAbstract:Summary Protein ubiquitination is a stable, covalent post-translational modification that alters protein activity and/or targets proteins for proteolysis by the 26S proteasome. The E1-type Ubiquitin-Activating Enzyme (UBA1) is responsible for ubiquitin activation, the initial step of ubiquitin–protein ligation. Proteasomal proteolysis of ubiquitinated spermatozoa and oocyte proteins occurs during mammalian fertilization, particularly at the site of sperm acrosome contact with oocyte zona pellucida. However, it is not clear whether the substrates are solely proteins ubiquitinated during gametogenesis or if de novo ubiquitination also occurs during fertilization supported by Ubiquitin-Activating and -conjugating Enzymes present in the sperm acrosome. Along this line of inquiry, UBA1 was detected in boar sperm-acrosomal extracts by Western blotting (WB). Immunofluorescence revealed accumulation of UBA1 in the nuclei of spermatogonia, spermatocytes and spermatids, and in the acrosomal caps of round and elongating spermatids. Thiol ester assays utilizing biotinylated ubiquitin and isolated sperm acrosomes confirmed the enzymatic activity of the resident UBA1. A specific UBA1 inhibitor, PYR-41, altered the remodelling of the outer acrosomal membrane (OAM) during sperm capacitation, monitored using flow cytometry of fluorescein isothiocyanate-conjugated peanut agglutinin (FITC-PNA). Although viable and motile, the spermatozoa capacitated in the presence of PYR-41, showed significantly reduced fertilization rates during in vitro fertilization (IVF; p < 0.05). Similarly, the fertilization rate was lowered by the addition of PYR-41 directly into fertilization medium during IVF. In WB, high Mr bands, suggestive of protein ubiquitination, were detected in non-capacitated spermatozoa by antibodies against ubiquitin; WB with anti-phosphotyrosine antibodies and antibodies against acrosomal proteins SPINK2 (acrosin inhibitor) and AQN1 (spermadhesin) revealed that the capacitation-induced modification of those proteins was altered by PYR-41. In summary, it appears that de novo protein ubiquitination involving UBA1 contributes to sperm capacitation and acrosomal function during fertilization.
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Ubiquitin-Activating Enzyme (UBA1) is required for sperm capacitation, acrosomal exocytosis and sperm-egg coat penetration during porcine fertilization.
International journal of andrology, 2011Co-Authors: Shawn Zimmerman, Gaurishankar Manandhar, John F. Odhiambo, Chelsey Kennedy, V. Jonáková, P. Maňásková-postlerová, Miriam Sutovsky, Chang-sik Park, Peter SutovskyAbstract:Summary Protein ubiquitination is a stable, covalent post-translational modification that alters protein activity and/or targets proteins for proteolysis by the 26S proteasome. The E1-type Ubiquitin-Activating Enzyme (UBA1) is responsible for ubiquitin activation, the initial step of ubiquitin–protein ligation. Proteasomal proteolysis of ubiquitinated spermatozoa and oocyte proteins occurs during mammalian fertilization, particularly at the site of sperm acrosome contact with oocyte zona pellucida. However, it is not clear whether the substrates are solely proteins ubiquitinated during gametogenesis or if de novo ubiquitination also occurs during fertilization supported by Ubiquitin-Activating and -conjugating Enzymes present in the sperm acrosome. Along this line of inquiry, UBA1 was detected in boar sperm-acrosomal extracts by Western blotting (WB). Immunofluorescence revealed accumulation of UBA1 in the nuclei of spermatogonia, spermatocytes and spermatids, and in the acrosomal caps of round and elongating spermatids. Thiol ester assays utilizing biotinylated ubiquitin and isolated sperm acrosomes confirmed the enzymatic activity of the resident UBA1. A specific UBA1 inhibitor, PYR-41, altered the remodelling of the outer acrosomal membrane (OAM) during sperm capacitation, monitored using flow cytometry of fluorescein isothiocyanate-conjugated peanut agglutinin (FITC-PNA). Although viable and motile, the spermatozoa capacitated in the presence of PYR-41, showed significantly reduced fertilization rates during in vitro fertilization (IVF; p
Alan L. Schwartz - One of the best experts on this subject based on the ideXlab platform.
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Identification of a Region within the Ubiquitin-Activating Enzyme Required for Nuclear Targeting and Phosphorylation
The Journal of biological chemistry, 1997Co-Authors: Andrew G. Stephen, Aaron Ciechanover, Julie S. Trausch-azar, Patricia M. Handley-gearhart, Alan L. SchwartzAbstract:The Ubiquitin-Activating Enzyme exists as two isoforms: E1a, localized predominantly in the nucleus, and E1b, localized in the cytoplasm. Previously we generated hemagglutinin (HA) epitope-tagged cDNA constructs, HA1-E1 (epitope tag placed after the first methionine) and HA2-E1 (epitope tag placed after the second methionine) (Handley-Gearhart, P. M., Stephen, A. G., Trausch-Azar, J. S., Ciechanover, A., and Schwartz, A. L. (1994) J. Biol. Chem.269, 33171–33178), which represent the native isoforms. HA1-E1 is exclusively nuclear, whereas HA2-E1 is found predominantly in the cytoplasm. Using high resolution isoelectric focusing and SDS-polyacrylamide gel electrophoresis, we confirm that these epitope-tagged constructs HA1-E1 and HA2-E1 represent the two isoforms E1a and E1b. HA1-E1/E1a exists as one non-phosphorylated and four phosphorylated forms, and HA2-E1/E1b exists as one predominant non-phosphorylated form and two minor phosphorylated forms. We demonstrate that the first 11 amino acids are essential for phosphorylation and exclusive nuclear localization of HA1-E1. Within this region are four serine residues and a putative nuclear localization sequence (NLS; 5PLSKKRR). Removal of these four serine residues reduced phosphorylation levels by 60% but had no effect on nuclear localization of HA1-E1. Each serine residue was independently mutated to an alanine and analyzed by two-dimensional electrophoresis; only serine 4 was phosphorylated. Disruption of the basic amino acids within the NLS resulted in loss of exclusive nuclear localization and a 90–95% decrease in the phosphorylation of HA1-E1. This putative NLS was able to confer nuclear import on a non-nuclear protein in digitonin-permeabilized cells in a temperature- and ATP-dependent manner. Thus the predominant requirement for efficient phosphorylation of HA1-E1/E1a is a functional NLS, suggesting that E1a may be phosphorylated within the nucleus.
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The Ubiquitin-Activating Enzyme E1 Is Phosphorylated and Localized to the Nucleus in a Cell Cycle-dependent Manner*
The Journal of biological chemistry, 1996Co-Authors: Andrew G. Stephen, Aaron Ciechanover, Julie S. Trausch-azar, Alan L. SchwartzAbstract:Abstract The Ubiquitin-Activating Enzyme E1 exists as two isoforms, E1a (117 kDa) and E1b (110 kDa). E1a is phosphorylated, whereas E1b is not. In the present study we have demonstrated the cell cycle dependence of E1a phosphorylation: a 2-fold increase in the specific phosphorylation of E1a in G2 compared with the basal level of phosphorylation in the other stages of the cell cycle. Two-dimensional gel electrophoresis resolved E1 into the two isoforms E1a and E1b; E1a resolved further as three phosphorylated forms and one nonphosphorylated form, while E1b resolved as one nonphosphorylated form. E1a is found predominantly in the phosphorylated forms. However, the distribution of E1a among these different phosphorylated forms was not cell cycle-dependent. We next evaluated the enzymatic activity of E1 as well as its subcellular localization throughout the cell cycle. 32P-Pyrophosphate exchange activity of E1 did not vary along the cell cycle; however, the amount of ubiquitin-protein conjugates decreased by 50% in G2. Nuclear and cytosolic fractionation of cells revealed the nuclear to cytosolic ratio of phosphorylated E1a was 3-fold greater in G2 compared with the other stages of the cell cycle. Finally, purified nuclear extracts supported E1-dependent ubiquitin conjugation of exogenous substrates as did purified cytosol. However, in nuclear extracts but not in cytosol the amount of E1 activity was rate-limiting. Thus we establish nuclear E1-dependent protein ubiquitination and propose that an increase in phosphorylation of E1a in G2 functions to increase the import and/or retention of E1a in the nucleus and may modulate nuclear protein ubiquitination.
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Rescue of the complex temperature-sensitive phenotype of Chinese hamster ovary E36ts20 cells by expression of the human Ubiquitin-Activating Enzyme cDNA.
Biochemical Journal, 1994Co-Authors: P M Handley-gearhart, Aaron Ciechanover, Julie S. Trausch-azar, Alan L. SchwartzAbstract:The ubiquitin conjugation system is a multi-step pathway in which ubiquitin is activated and conjugated to acceptor proteins, one function of which is to target acceptor proteins for rapid degradation within the cell. The conjugation system is involved in many aspects of cellular functions, including the cell cycle. Several cell-cycle arrest mutant cell lines have been characterized and appear to harbour a mutant Ubiquitin-Activating Enzyme, E1, as their primary defect. One such cell line is ts20, which is derived from Chinese hamster ovary E36 cells. This cell line has been used to characterize some of the potential functions of the ubiquitin conjugation system in vivo, such as its involvement in the maturation of autophagic vacuoles. The present study describes the complete rescue of the complex ts20 phenotype following the expression of the cDNA for human E1. Stable transfectants expressing the human E1 cDNA in the CMVneo expression vector were measured for ubiquitin-conjugation activity, protein degradation and growth in culture at the nonpermissive temperature. This rescue confirms that the phenotype observed in the ts20 cells is due to a defect in the E1 Enzyme. Thus, the ts20 cell line will serve as a useful tool to delineate the functions of the ubiquitin system in vivo.
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Nuclear localization of the Ubiquitin-Activating Enzyme, E1, is cell-cycle-dependent.
The Biochemical journal, 1994Co-Authors: S J Grenfell, Aaron Ciechanover, J S Trausch-azar, P M Handley-gearhart, Alan L. SchwartzAbstract:The mechanisms that regulate ubiquitin-mediated degradation of proteins such as the mitotic cyclins at defined stages of the cell cycle are poorly understood. The initial step in the conjugation of ubiquitin to substrate proteins involves the activation of ubiquitin by the Ubiquitin-Activating Enzyme, E1. Previously we have described the subcellular localization of this Enzyme to both nuclear and cytoplasmic compartments. In the present study, we have used the 1C5 anti-E1 monoclonal antibody in immunofluorescent-microscopy and subcellular-fractionation techniques to examine the distribution of E1 during the HeLa cell cycle. E1 is both cytoskeletal and nuclear during the G1-phase. As the cells progress into S-phase, E1 is exclusively cytoskeletal and has a perinuclear distribution. During G2-phase, E1 reappears in the nucleus before breakdown of the nuclear envelope. In mitotic cells, E1 localizes to both the mitotic spindle and the cytosol, but is absent from the chromosomes. Immunoblot analysis reveals multiple forms of E1 in HeLa whole cell extract. This heterogeneity is not a result of polyubiquitination and may represent inactive pools of E1. Only the characteristic E1 doublet is able to activate ubiquitin. Cell-fractionation studies reveal a differential distribution of specific E1 isoforms throughout the cell cycle. Therefore we propose that the subcellular localization of E1 may play a role in regulating cell-cycle-dependent conjugation of ubiquitin to target proteins.
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Ubiquitin-Activating Enzyme, E1, is associated with maturation of autophagic vacuoles.
The Journal of cell biology, 1992Co-Authors: S. E. Lenk, William A. Dunn, J. S. Trausch, Aaron Ciechanover, Alan L. SchwartzAbstract:The Ubiquitin-Activating Enzyme, E1, is required for initiating a multi-step pathway for the covalent linkage of ubiquitin to target proteins. A CHO cell line containing a mutant thermolabile E1, ts20, has been shown to be defective in stress-induced degradation of proteins at restrictive temperature (Gropper et al., 1991. J. Biol. Chem. 266:3602-3610). Parental E36 cells responded to restrictive temperature by stimulating lysosome-mediated protein degradation twofold. Such a response was not observed in ts20 cells. The absence of accelerated degradation in these cells at 39.5 degrees C was accompanied by an accumulation of autolysosomes. The fractional volume of these degradative autophagic vacuoles was at least sixfold greater than that observed for either E36 cells at 30.5 degrees or 39.5 degrees C, or ts20 cells at 30.5 degrees C. These vacuoles were acidic and contained both acid phosphatase and cathepsin L, but, unlike the autolysosomes observed in E36 cells, ubiquitin-conjugated proteins were conspicuously absent. Combined, our results suggest that in ts20 cells, which are unable to generate ubiquitin-protein conjugates due to heat inactivation of E1, the formation and maturation of autophagosomes into autolysosomes is normal, but the conversion of autolysosomes into residual bodies is disrupted.
Shawn Zimmerman - One of the best experts on this subject based on the ideXlab platform.
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ubiquitin activating Enzyme uba1 is required for sperm capacitation acrosomal exocytosis and sperm egg coat penetration during porcine fertilization
International Journal of Andrology, 2012Co-Authors: Shawn Zimmerman, Gaurishankar Manandhar, John F. Odhiambo, Chelsey Kennedy, V. Jonáková, Miriam Sutovsky, Chang-sik Park, P Maňaskovapostlerova, Peter SutovskyAbstract:Summary Protein ubiquitination is a stable, covalent post-translational modification that alters protein activity and/or targets proteins for proteolysis by the 26S proteasome. The E1-type Ubiquitin-Activating Enzyme (UBA1) is responsible for ubiquitin activation, the initial step of ubiquitin–protein ligation. Proteasomal proteolysis of ubiquitinated spermatozoa and oocyte proteins occurs during mammalian fertilization, particularly at the site of sperm acrosome contact with oocyte zona pellucida. However, it is not clear whether the substrates are solely proteins ubiquitinated during gametogenesis or if de novo ubiquitination also occurs during fertilization supported by Ubiquitin-Activating and -conjugating Enzymes present in the sperm acrosome. Along this line of inquiry, UBA1 was detected in boar sperm-acrosomal extracts by Western blotting (WB). Immunofluorescence revealed accumulation of UBA1 in the nuclei of spermatogonia, spermatocytes and spermatids, and in the acrosomal caps of round and elongating spermatids. Thiol ester assays utilizing biotinylated ubiquitin and isolated sperm acrosomes confirmed the enzymatic activity of the resident UBA1. A specific UBA1 inhibitor, PYR-41, altered the remodelling of the outer acrosomal membrane (OAM) during sperm capacitation, monitored using flow cytometry of fluorescein isothiocyanate-conjugated peanut agglutinin (FITC-PNA). Although viable and motile, the spermatozoa capacitated in the presence of PYR-41, showed significantly reduced fertilization rates during in vitro fertilization (IVF; p < 0.05). Similarly, the fertilization rate was lowered by the addition of PYR-41 directly into fertilization medium during IVF. In WB, high Mr bands, suggestive of protein ubiquitination, were detected in non-capacitated spermatozoa by antibodies against ubiquitin; WB with anti-phosphotyrosine antibodies and antibodies against acrosomal proteins SPINK2 (acrosin inhibitor) and AQN1 (spermadhesin) revealed that the capacitation-induced modification of those proteins was altered by PYR-41. In summary, it appears that de novo protein ubiquitination involving UBA1 contributes to sperm capacitation and acrosomal function during fertilization.
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Ubiquitin-Activating Enzyme (UBA1) is required for sperm capacitation, acrosomal exocytosis and sperm-egg coat penetration during porcine fertilization.
International journal of andrology, 2011Co-Authors: Shawn Zimmerman, Gaurishankar Manandhar, John F. Odhiambo, Chelsey Kennedy, V. Jonáková, P. Maňásková-postlerová, Miriam Sutovsky, Chang-sik Park, Peter SutovskyAbstract:Summary Protein ubiquitination is a stable, covalent post-translational modification that alters protein activity and/or targets proteins for proteolysis by the 26S proteasome. The E1-type Ubiquitin-Activating Enzyme (UBA1) is responsible for ubiquitin activation, the initial step of ubiquitin–protein ligation. Proteasomal proteolysis of ubiquitinated spermatozoa and oocyte proteins occurs during mammalian fertilization, particularly at the site of sperm acrosome contact with oocyte zona pellucida. However, it is not clear whether the substrates are solely proteins ubiquitinated during gametogenesis or if de novo ubiquitination also occurs during fertilization supported by Ubiquitin-Activating and -conjugating Enzymes present in the sperm acrosome. Along this line of inquiry, UBA1 was detected in boar sperm-acrosomal extracts by Western blotting (WB). Immunofluorescence revealed accumulation of UBA1 in the nuclei of spermatogonia, spermatocytes and spermatids, and in the acrosomal caps of round and elongating spermatids. Thiol ester assays utilizing biotinylated ubiquitin and isolated sperm acrosomes confirmed the enzymatic activity of the resident UBA1. A specific UBA1 inhibitor, PYR-41, altered the remodelling of the outer acrosomal membrane (OAM) during sperm capacitation, monitored using flow cytometry of fluorescein isothiocyanate-conjugated peanut agglutinin (FITC-PNA). Although viable and motile, the spermatozoa capacitated in the presence of PYR-41, showed significantly reduced fertilization rates during in vitro fertilization (IVF; p
Fumio Hanaoka - One of the best experts on this subject based on the ideXlab platform.
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incubation at the nonpermissive temperature induces deficiencies in uv resistance and mutagenesis in mouse mutant cells expressing a temperature sensitive ubiquitin activating Enzyme e1
Molecular and Cellular Biology, 1997Co-Authors: Hironobu Ikehata, S Kaneda, T Seno, F Yamao, Tetsuya Ono, Fumio HanaokaAbstract:In temperature-sensitive (ts) mutants of mouse FM3A cells, the levels of mutagenesis and survival of cells treated with DNA-damaging agents have been difficult to assess because they are killed after their mutant phenotypes are expressed at the nonpermissive temperature. To avoid this difficulty, we incubated the ts mutant cells at the restrictive temperature, 39 degrees C, for only a limited period after inducing DNA damage. We used ts mutants defective in genes for Ubiquitin-Activating Enzyme (E1), DNA polymerase alpha, and p34(cdc2) kinase. Whereas the latter two showed no effect, E1 mutants were sensitized remarkably to UV light if incubated at 39 degrees C for limited periods after UV exposure. Eighty-five percent of the sensitization occurred within the first 12 h of incubation at 39 degrees C, and more than 36 h at 39 degrees C did not produce any further sensitization. Moreover, while the 39 degrees C incubation gave E1 mutants a moderate spontaneous mutator phenotype, the same treatment significantly diminished the level of UV-induced 6-thioguanine resistance mutagenesis and extended the time necessary for expression of the mutation phenotype. These characteristics of E1 mutants are reminiscent of the defective DNA repair phenotypes of Saccharomyces cerevisiae rad6 mutants, which have defects in a ubiquitin-conjugating Enzyme (E2), to which E1 is known to transfer ubiquitin. These results demonstrate the involvement of E1 in eukaryotic DNA repair and mutagenesis and provide the first direct evidence that the ubiquitin-conjugation system contributes to DNA repair in mammalian cells.
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Cloning and sequence of a functionally active cDNA encoding the mouse Ubiquitin-Activating Enzyme E1.
Gene, 1992Co-Authors: Imai N, Sumiko Kaneda, Y. Nagai, Takeshi Seno, Dai Ayusawa, Fumio Hanaoka, Fumiaki YamaoAbstract:Abstract A cDNA encoding the Ubiquitin-Activating Enzyme, El, was isolated from the mouse mammary carcinoma cell line, FM3A, and shown to complement mutant mouse cells deficient in the Enzyme. The 3495-bp cDNA encodes 1058 amino acids (aa), and shares extensive homology with the human El Enzyme at both the nucleotide and aa sequence levels.
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Complementation by a cloned human Ubiquitin-Activating Enzyme E1 of the S-phase-arrested mouse FM3A cell mutant with thermolabile E1.
Cell structure and function, 1992Co-Authors: Dai Ayusawa, Sumiko Kaneda, Fumio Hanaoka, Kaoru Sugasawa, Yoshiyasu Itoh, Hideyo Yasuda, Yasuko Murakami, Takeshi SenoAbstract:A temperature-sensitive growth mutant tsFS20 isolated from mouse FM3A cells was identified as a mutant with thermolabile Ubiquitin-Activating Enzyme E1 by transfection with a full-length cDNA encoding the human E1 Enzyme and cell-cell hybridization with an authentic E1 mutant ts85 previously isolated from FM3A cells. The resulting transformants produced thermoresistant E1 activity. Upon shift-up of temperature, asynchronously growing tsFS20 cells showed multiple points of cell-cycle arrest. At the nonpermissive temperature, tsFS20 cells that had been synchronized at the G1-S-phase progressed and accumulated in the mid-S-phase, as evidenced by the absence of G2-specific cdc2 kinase activity, while ts85 mutant cells, the widely used E1 mutant, reached the G2-phase and were arrested. Thus, the E1 mutation seemed to be involved in progression in the S-phase as well as in the G2-phase in the cell cycle. Degradation of short-lived abnormal proteins in tsFS20 cells was decreased to about 50% at the nonpermissive temperature, while the block was fully restored to the wild-type level in the transformant cells. Relevance of the unusually high incidence of the temperature-sensitive E1 mutation was discussed in terms of the E1 as a determinant of heat tolerance of cells.
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Human Ubiquitin-Activating Enzyme (E1): compensation for heat-labile mouse E1 and its gene localization on the X chromosome.
Experimental cell research, 1991Co-Authors: Mami Kudo, Tada-aki Hori, Kaoru Sugasawa, Takemi Enomoto, Fumio HanaokaAbstract:Abstract We have constructed interspecific somatic cell hybrids between a temperature-sensitive (ts) mutant cell line of mouse FM3A cells, ts85, that has a heat-labile Ubiquitin-Activating Enzyme (E1) and a human diploid fibroblast cell line, IMR-90. A hybrid clone that could grow stably at a nonpermissive temperature (39 °C) was obtained. Segregation of the hybrid cells at a permissive temperature (33 °C) gave rise to temperature-sensitive clones. The electrophoresis of extracted histones and karyotype analysis of the segregants revealed a close correlation of the ability to grow at 39 °C, the presence of uH2A (ubiquitin-H2A semihistone) at 39 °C, and the presence of the human X chromosome. One of the hybrid clones that could grow at the nonpermissive temperature contained the X chromosome as the only human chromosome. The sodium dodecyl sulfate-polyacrylamide gel electrophoretic pattern of affinity-purified E1 showed that this hybrid clone contained both human and mouse type E1. Thus we conclude that the functional gene for human E1 is located on the X chromosome.
Gaurishankar Manandhar - One of the best experts on this subject based on the ideXlab platform.
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ubiquitin activating Enzyme uba1 is required for sperm capacitation acrosomal exocytosis and sperm egg coat penetration during porcine fertilization
International Journal of Andrology, 2012Co-Authors: Shawn Zimmerman, Gaurishankar Manandhar, John F. Odhiambo, Chelsey Kennedy, V. Jonáková, Miriam Sutovsky, Chang-sik Park, P Maňaskovapostlerova, Peter SutovskyAbstract:Summary Protein ubiquitination is a stable, covalent post-translational modification that alters protein activity and/or targets proteins for proteolysis by the 26S proteasome. The E1-type Ubiquitin-Activating Enzyme (UBA1) is responsible for ubiquitin activation, the initial step of ubiquitin–protein ligation. Proteasomal proteolysis of ubiquitinated spermatozoa and oocyte proteins occurs during mammalian fertilization, particularly at the site of sperm acrosome contact with oocyte zona pellucida. However, it is not clear whether the substrates are solely proteins ubiquitinated during gametogenesis or if de novo ubiquitination also occurs during fertilization supported by Ubiquitin-Activating and -conjugating Enzymes present in the sperm acrosome. Along this line of inquiry, UBA1 was detected in boar sperm-acrosomal extracts by Western blotting (WB). Immunofluorescence revealed accumulation of UBA1 in the nuclei of spermatogonia, spermatocytes and spermatids, and in the acrosomal caps of round and elongating spermatids. Thiol ester assays utilizing biotinylated ubiquitin and isolated sperm acrosomes confirmed the enzymatic activity of the resident UBA1. A specific UBA1 inhibitor, PYR-41, altered the remodelling of the outer acrosomal membrane (OAM) during sperm capacitation, monitored using flow cytometry of fluorescein isothiocyanate-conjugated peanut agglutinin (FITC-PNA). Although viable and motile, the spermatozoa capacitated in the presence of PYR-41, showed significantly reduced fertilization rates during in vitro fertilization (IVF; p < 0.05). Similarly, the fertilization rate was lowered by the addition of PYR-41 directly into fertilization medium during IVF. In WB, high Mr bands, suggestive of protein ubiquitination, were detected in non-capacitated spermatozoa by antibodies against ubiquitin; WB with anti-phosphotyrosine antibodies and antibodies against acrosomal proteins SPINK2 (acrosin inhibitor) and AQN1 (spermadhesin) revealed that the capacitation-induced modification of those proteins was altered by PYR-41. In summary, it appears that de novo protein ubiquitination involving UBA1 contributes to sperm capacitation and acrosomal function during fertilization.
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Ubiquitin-Activating Enzyme (UBA1) is required for sperm capacitation, acrosomal exocytosis and sperm-egg coat penetration during porcine fertilization.
International journal of andrology, 2011Co-Authors: Shawn Zimmerman, Gaurishankar Manandhar, John F. Odhiambo, Chelsey Kennedy, V. Jonáková, P. Maňásková-postlerová, Miriam Sutovsky, Chang-sik Park, Peter SutovskyAbstract:Summary Protein ubiquitination is a stable, covalent post-translational modification that alters protein activity and/or targets proteins for proteolysis by the 26S proteasome. The E1-type Ubiquitin-Activating Enzyme (UBA1) is responsible for ubiquitin activation, the initial step of ubiquitin–protein ligation. Proteasomal proteolysis of ubiquitinated spermatozoa and oocyte proteins occurs during mammalian fertilization, particularly at the site of sperm acrosome contact with oocyte zona pellucida. However, it is not clear whether the substrates are solely proteins ubiquitinated during gametogenesis or if de novo ubiquitination also occurs during fertilization supported by Ubiquitin-Activating and -conjugating Enzymes present in the sperm acrosome. Along this line of inquiry, UBA1 was detected in boar sperm-acrosomal extracts by Western blotting (WB). Immunofluorescence revealed accumulation of UBA1 in the nuclei of spermatogonia, spermatocytes and spermatids, and in the acrosomal caps of round and elongating spermatids. Thiol ester assays utilizing biotinylated ubiquitin and isolated sperm acrosomes confirmed the enzymatic activity of the resident UBA1. A specific UBA1 inhibitor, PYR-41, altered the remodelling of the outer acrosomal membrane (OAM) during sperm capacitation, monitored using flow cytometry of fluorescein isothiocyanate-conjugated peanut agglutinin (FITC-PNA). Although viable and motile, the spermatozoa capacitated in the presence of PYR-41, showed significantly reduced fertilization rates during in vitro fertilization (IVF; p
