@article{MTMT:2015348, title = {Ubiquitylation of Drosophila p54/Rpn10/S5a Regulates Its Interaction with the UBA-UBL Polyubiquitin Receptors}, url = {https://m2.mtmt.hu/api/publication/2015348}, author = {Lipinszki, Zoltán and Kovács, Levente and Deák, Péter and Udvardy, Andor}, doi = {10.1021/bi3001006}, journal-iso = {BIOCHEMISTRY-US}, journal = {BIOCHEMISTRY}, volume = {51}, unique-id = {2015348}, issn = {0006-2960}, abstract = {Analysis of the in vivo ubiquitylation of the p54/Rpn10 polyubiquitin receptor subunit of the Drosophila 26S proteasome revealed that the site of ubiquitylation is the C-terminal cluster of lysines, which is conserved in higher eukaryotes. Extraproteasomal p54 was extensively multiubiquitylated, but only very modest modification was detected in the proteasome-assembled subunit. Ubiquitylation of p54 seriously jeopardizes one of its most important functions, i.e., the interaction of its ubiquitin-interacting motifs with the ubiquitin-like domain of Dsk2 and Rad23 extraproteasomal polyubiquitin receptors. This modification of p54 supports the previous notion that p54 is a shuttling subunit of the 26S proteasome with a specific extraproteasomal function. This assumption is supported by the observation that, while transgenic p54 can fully rescue the lethal phenotype of the Delta p54 null mutation, its derivative from which the cluster of conserved lysines is deleted shifts the lethality from the early pupa to pharate adult stage but cannot rescue the Delta p54 mutation, suggesting that ubiquitylated extraproteasomal p54 has an essential role in the pupa adult transition.}, year = {2012}, eissn = {1520-4995}, pages = {2461-2470}, orcid-numbers = {Lipinszki, Zoltán/0000-0002-2067-0832; Kovács, Levente/0000-0002-3226-3740} } @article{MTMT:1921997, title = {Overexpression of Dsk2/dUbqln results in severe developmental defects and lethality in Drosophila melanogaster that can be rescued by overexpression of the p54/Rpn10/S5a proteasomal subunit.}, url = {https://m2.mtmt.hu/api/publication/1921997}, author = {Lipinszki, Zoltán and Pál, Margit and Nagy, Olga Mária and Deák, Péter and Hunyadi-Gulyás Éva, Csilla and Udvardy, Andor}, doi = {10.1111/j.1742-4658.2011.08383.x}, journal-iso = {FEBS J}, journal = {FEBS JOURNAL}, volume = {278}, unique-id = {1921997}, issn = {1742-464X}, abstract = {Polyubiquitin receptors execute the targeting of polyubiquitylated proteins to the 26S proteasome. In vitro studies indicate that disturbance of the physiological balance among different receptor proteins impairs the proteasomal degradation of polyubiquitylated proteins. To study the physiological consequences of shifting the in vivo equilibrium between the p54/Rpn10 proteasomal and the Dsk2/dUbqln extraproteasomal polyubiquitin receptors, transgenic Drosophila lines were constructed in which the overexpression or RNA interference-mediated silencing of these receptors can be induced. Flies overexpressing Flag-p54 were viable and fertile, without any detectable morphological abnormalities, although detectable accumulation of polyubiquitylated proteins demonstrated a certain level of proteolytic disturbance. Flag-p54 was assembled into the 26S proteasome and could fully complement the lethal phenotype of a p54 null mutant Drosophila line. The overexpression of Dsk2 caused severe morphological abnormalities in the late pupal stages, leading to pharate adult lethality, accompanied by a huge accumulation of highly polyubiquitylated proteins. The lethal phenotype of Dsk2 overexpression could be rescued in a double transgenic line coexpressing Flag-Dsk2 and Flag-p54. Although the double transgenic line was viable and fertile, it did not restore the proteolytic defects; the accumulation of the highly polyubiquitylated proteins was even more severe in the double transgenic line. Significant differences were found in the Dsk2-26S proteasome interaction in Drosophila melanogaster as compared with Saccharomyces cerevisiae. In yeast, Dsk2 can interact only with DeltaRpn10 proteasomes and not with the wild-type one. In Drosophila, Dsk2 does not interact with Deltap54 proteasomes, but the interaction can be fully restored by complementing the Deltap54 deletion with Flag-p54. Structured digital abstract * Dsk2 physically interacts with p54 by pull down (View Interaction: 1, 2) * p54 binds to Dsk2 by pull down (View interaction).}, keywords = {Animals; ARTICLE; Saccharomyces cerevisiae; Saccharomyces cerevisiae; priority journal; controlled study; nonhuman; developmental biology; Cell Cycle Proteins; RNA Interference; Animals, Genetically Modified; Drosophila melanogaster; Drosophila melanogaster; Drosophila melanogaster; unclassified drug; protein protein interaction; Proteasome Endopeptidase Complex; proteasome; 26S PROTEASOME; 26S PROTEASOME; Carrier Proteins; transgenic animal; gene overexpression; lethality; protein degradation; Drosophila Proteins; Ubiquitination; larval development; Pupa; Ubiquitylation; Dsk2/dUbqln; p54/Rpn10/S5a; polyubiquitin receptors; Dsk2 protein; polyubiquitin; polyubiquitin; protein p54; pupa (life cycle stage)}, year = {2011}, eissn = {1742-4658}, pages = {4833-4844}, orcid-numbers = {Lipinszki, Zoltán/0000-0002-2067-0832; Nagy, Olga Mária/0000-0003-1471-5165} } @article{MTMT:1920783, title = {Developmental-stage-specific regulation of the polyubiquitin receptors in drosophila melanogaster}, url = {https://m2.mtmt.hu/api/publication/1920783}, author = {Lipinszki, Zoltán and Kiss, Petra and Pál, Margit and Deák, Péter and Szabó, Áron and Hunyadi-Gulyás Éva, Csilla and Klement, Éva and Medzihradszky F., Katalin and Udvardy, Andor}, doi = {10.1242/jcs.049049}, journal-iso = {J CELL SCI}, journal = {JOURNAL OF CELL SCIENCE}, volume = {122}, unique-id = {1920783}, issn = {0021-9533}, abstract = {Recognition of polyubiquitylated substrates by the proteasome is a highly regulated process that requires polyubiquitin receptors. We show here that the concentrations of the proteasomal and extraproteasomal polyubiquitin receptors change in a developmentally regulated fashion. The stoichiometry of the proteasomal p54/Rpn10 polyubiquitin receptor subunit, relative to that of other regulatory particle (RP) subunits falls suddenly at the end of embryogenesis, remains low throughout the larval stages, starts to increase again in the late third instar larvae and remains high in the pupae, adults and embryos. A similar developmentally regulated fluctuation was observed in the concentrations of the Rad23 and Dsk2 extraproteasomal polyubiquitin receptors. Depletion of the polyubiquitin receptors at the end of embryogenesis is due to the emergence of a developmentally regulated selective proteolytic activity. To follow the fate of subunit p54/Rpn10 in vivo, transgenic Drosophila melanogaster lines encoding the N-terminal half (NTH), the C-terminal half (CTH) or the full-length p54/Rpn10 subunit were established in the inducible Gal4-UAS system. The daughterless-Gal4-driven whole-body expression of the full-length subunit or its NTH did not produce any detectable phenotypic changes, and the transgenic products were incorporated into the 26S proteasome. The transgene-encoded CTH was not incorporated into the 26S proteasome, caused third instar larval lethality and was found to be multi-ubiquitylated. This modification, however, did not appear to be a degradation signal because the half-life of the CTH was over 48 hours. Accumulation of the CTH disturbed the developmentally regulated changes in subunit composition of the RP and the emergence of the selective proteolytic activity responsible for the depletion of the polyubiquitin receptors. Build-up of subunit p54/Rpn10 in the RP had already started in 84-hour-old larvae and reached the full complement characteristic of the non-larval developmental stages at the middle of the third instar larval stage, just before these larvae perished. Similar shifts were observed in the concentrations of the Rad23 and Dsk2 polyubiquitin receptors. The postsynthetic modification of CTH might be essential for this developmental regulation, or it might regulate an essential extraproteasomal function(s) of subunit p54/Rpn10 that is disturbed by the expression of an excess of CTH.}, keywords = {Animals; Female; PHENOTYPE; ARTICLE; priority journal; controlled study; nonhuman; developmental stage; regulatory mechanism; Gene Expression Regulation, Developmental; Gene Expression; Drosophila melanogaster; Drosophila melanogaster; Drosophila melanogaster; down regulation; 26S PROTEASOME; gene function; Drosophila Proteins; embryo development; Ubiquitination; polyubiquitin receptors; polyubiquitin; polyubiquitin; protein p54; pupa (life cycle stage); Multiubiquitylation; Regulatory particle; larval stage}, year = {2009}, eissn = {1477-9137}, pages = {3083-3092}, orcid-numbers = {Lipinszki, Zoltán/0000-0002-2067-0832} } @article{MTMT:1247741, title = {A novel 2D electrophoresis technique for the identification of intrinsically unstructured proteins.}, url = {https://m2.mtmt.hu/api/publication/1247741}, author = {Csizmók, Veronika and Szőllősi, E and Friedrich, Péter and Tompa, Péter}, doi = {10.1074/mcp.M500181-MCP200}, journal-iso = {MOL CELL PROTEOMICS}, journal = {MOLECULAR & CELLULAR PROTEOMICS}, volume = {5}, unique-id = {1247741}, issn = {1535-9476}, abstract = {Intrinsically unstructured proteins (IUPs) lack a well defined three-dimensional structure under physiological conditions. They constitute a significant fraction of various proteomes, but only a handful of them have so far been identified. Here we report the development of a two-dimensional electrophoresis technique for their de novo recognition and characterization. This technique consists of the combination of native and 8 m urea electrophoresis of heat-treated proteins where IUPs are expected to run into the diagonal, whereas globular proteins either precipitate upon heat treatment or unfold and run off the diagonal in the second dimension. This behavior was born out by a collection of 10 known IUPs and four globular proteins. By running Escherichia coli and Saccharomyces cerevisiae extracts, several novel IUPs were also identified by mass spectrometric analysis of spots at or near the diagonal. By comparing this novel method to several other techniques, such as the PONDR(R) predictor, hydrophobicity-net charge plot, CD analysis, and gel filtration chromatography, it was shown to provide dependable global assessment of disorder even in dubious cases. Overall the reproducibility and ease of performance of this technique may promote the proteomic scale recognition and characterization of protein disorder.}, year = {2006}, eissn = {1535-9484}, pages = {265-273} } @article{MTMT:1907322, title = {Mapping the ubiquitin-binding domains in the p54 regulatory complex subunit of the Drosophila 26S protease.}, url = {https://m2.mtmt.hu/api/publication/1907322}, author = {Haracska, Lajos and Udvardy, Andor}, doi = {10.1016/S0014-5793(97)00808-9}, journal-iso = {FEBS LETT}, journal = {FEBS LETTERS}, volume = {412}, unique-id = {1907322}, issn = {0014-5793}, year = {1997}, eissn = {1873-3468}, pages = {331-336} }