Papers by Zoltan Lipinszki
Journal of Proteome Research, 2010
A chemical derivatization approach has been developed for the enrichment of O-GlcNAc modified pro... more A chemical derivatization approach has been developed for the enrichment of O-GlcNAc modified proteins. The procedure is based on the isolation technique used for N-glycoproteins with appropriate modifications because of the differences in the two types of glycosylation: a prolonged periodate oxidation is followed by hydrazide resin capture, on-resin proteolytic digestion and release of the modified peptides by hydroxylamine. This enrichment strategy offers a fringe benefit in mass spectrometry analysis. Upon collisional activation the presence of the open carbohydrate ring leads to characteristic fragmentation facilitating both glycopeptide identification and site assignment. The enrichment protocol was applied to the Drosophila proteasome complex previously described as O-GlcNAc modified. The O-GlcNAc modification was located on proteasome interacting proteins, deubiquitinating enzyme Faf (CG1945) and a ubiquitin-like domain containing protein (CG7546). Three other proteins were also found GlcNAc modified, a HSP70 homologue (CG2918), scribbled (CG5462) and the 205 kDa microtubule-associated protein (CG1483). Interestingly, in the HSP70 homologue the GlcNAc modification is attached to an asparagine residue of a N-glycosylation motif. . Supporting Information Available. MALDI-TOF mass spectra of the periodate oxidized O-GlcNAc peptide standard; base peak chromatogram of an enriched fraction of the proteasome; extracted ion chromatogram and full-scan mass spectrum of a selected glycopeptide (m/z 745.85); CID and ETD spectra of the oxime or BEMAD derivatives of O-GlcNAc modified peptides. This material is available free of charge via the Internet at
Molecular Genetics and Genomics, 2007
The function and the molecular properties of the Rpt1/p48B ATPase subunit of the regulatory parti... more The function and the molecular properties of the Rpt1/p48B ATPase subunit of the regulatory particle of the Drosophila melanogaster 26S proteasome have been studied by analyzing three mutant Drosophila stocks in which P-element insertions occurred in the 5Ј-non-translated region of the Rpt1/p48B gene. These P-element insertions resulted in larval lethality during the second instar larval phase. Since the Rpt1/p48B gene resides within a long intron of an annotated, but uncharacterized Drosophila gene (CG17985), the second instar larval lethality may be a consequence of a combined damage to two independent genes. To analyze the phenotypic eVect of the mutations aVecting the Rpt1/p48B gene alone, imprecise P-element excision mutants were selected. One of them, the pupal lethal P1 mutation, is a hypomorphic allele of the Rpt1/ p48B gene, in which the displacement of two essential regulatory sequences of the gene occurred due to the insertion of a 32 bp residual P-element sequence. This mutation caused a 30-fold drop in the cellular concentration of the Rpt1/p48B mRNA. The decline in the cellular Rpt1/p48B protein concentration induced serious damage in the assembly of the 26S proteasomes, the accumulation of multiubiquitinated proteins, a change in the phosphorylation pattern of the subunit and depletion of this ATPase protein from the chromatin.
FEBS Journal, 2011
Overexpression of Dsk2 ⁄dUbqln results in severe developmental defects and lethality in Drosophil... more 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
Biochemistry, 2012
Analysis of the in vivo ubiquitylation of the p54/Rpn10 polyubiquitin receptor subunit of the Dro... more 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 Δp54 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 Δp54 mutation, suggesting that ubiquitylated extraproteasomal p54 has an essential role in the pupa−adult transition.
Biochemical Journal, 2005
In the presence of Zn 2+ , the Drosophila 26 S proteasome disassembles into RP (regulatory partic... more In the presence of Zn 2+ , the Drosophila 26 S proteasome disassembles into RP (regulatory particle) and CP (catalytic particle), this process being accompanied by the dissociation of subunit Rpn10/p54, the ubiquitin receptor subunit of the proteasome. The dissociation of Rpn10/p54 induces extensive rearrangements within the lid subcomplex of the RP, while the structure of the ATPase ring of the base subcomplex seems to be maintained. As a consequence of the dissociation of the RP, the peptidase activity of the 26 S proteasome is lost. The Zn 2+ -induced structural and functional changes are fully reversible; removal of Zn 2+ is followed by reassociation of subunit Rpn10/p54 to the RP, reassembly of the 26 S proteasome and resumption of the peptidase activity. After the Zn 2+ -induced dissociation, Rpn10/p54 interacts with a set of non-proteasomal proteins. Hsp82 (heatshock protein 82) has been identified by MS as the main Rpn10/p54-interacting protein, suggesting its role in the reas-sembly of the 26 S proteasome after Zn 2+ removal. The physiological relevance of another Rpn10/p54-interacting protein, the Smt3 SUMO (small ubiquitin-related modifier-1)-activating enzyme, detected by chemical cross-linking, has been confirmed by yeast two-hybrid analysis. Besides the Smt3 SUMO-activating enzyme, the Ubc9 SUMO-conjugating enzyme also exhibited in vivo interaction with the 5 -half of Rpn10/p54 in yeast cells. The mechanism of 26 S proteasome disassembly after ATP depletion is clearly different from that induced by Zn 2+ . Rpn10/p54 is permanently RP-bound during the ATP-dependent assemblydisassembly cycle, but during the Zn 2+ cycle it reversibly shuttles between the RP-bound and free states.
Journal of Cell Science, 2009
Centromeric binding and activity of Protein Phosphatase 4
Nature Communications, 2015
The cell division cycle requires tight coupling between protein phosphorylation and dephosphoryla... more The cell division cycle requires tight coupling between protein phosphorylation and dephosphorylation. However, understanding the cell cycle roles of multimeric protein phosphatases has been limited by the lack of knowledge of how their diverse regulatory subunits target highly conserved catalytic subunits to their sites of action. Phosphoprotein phosphatase 4 (PP4) has been recently shown to participate in the regulation of cell cycle progression. We now find that the EVH1 domain of the regulatory subunit 3 of Drosophila PP4, Falafel (Flfl), directly interacts with the centromeric protein C (CENP-C). Unlike other EVH1 domains that interact with proline-rich ligands, the crystal structure of the Flfl amino-terminal EVH1 domain bound to a CENP-C peptide reveals a new target-recognition mode for the phosphatase subunit. We also show that binding of Flfl to CENP-C is required to bring PP4 activity to centromeres to maintain CENP-C and attached core kinetochore proteins at chromosomes during mitosis.
Nature Communications, 2015
The cell division cycle requires tight coupling between protein phosphorylation and dephosphoryla... more The cell division cycle requires tight coupling between protein phosphorylation and dephosphorylation. However, understanding the cell cycle roles of multimeric protein phosphatases has been limited by the lack of knowledge of how their diverse regulatory subunits target highly conserved catalytic subunits to their sites of action. Phosphoprotein phosphatase 4 (PP4) has been recently shown to participate in the regulation of cell cycle progression. We now find that the EVH1 domain of the regulatory subunit 3 of Drosophila PP4, Falafel (Flfl), directly interacts with the centromeric protein C (CENP-C). Unlike other EVH1 domains that interact with proline-rich ligands, the crystal structure of the Flfl amino-terminal EVH1 domain bound to a CENP-C peptide reveals a new target-recognition mode for the phosphatase subunit. We also show that binding of Flfl to CENP-C is required to bring PP4 activity to centromeres to maintain CENP-C and attached core kinetochore proteins at chromosomes during mitosis.
Plk4 Phosphorylates Ana2 to Trigger Sas6 Recruitment and Procentriole Formation
Current Biology, 2014
Centrioles are 9-fold symmetrical structures at the core of centrosomes and base of cilia whose d... more Centrioles are 9-fold symmetrical structures at the core of centrosomes and base of cilia whose dysfunction has been linked to a wide range of inherited diseases and cancer. Their duplication is regulated by a protein kinase of conserved structure, the C. elegans ZYG-1 or its Polo-like kinase 4 (Plk4) counterpart in other organisms. Although Plk4's centriolar partners and mechanisms that regulate its stability are known, its crucial substrates for centriole duplication have never been identified. Here we show that Drosophila Plk4 phosphorylates four conserved serines in the STAN motif of the core centriole protein Ana2 to enable it to bind and recruit its Sas6 partner. Ana2 and Sas6 normally load onto both mother and daughter centrioles immediately after their disengagement toward the end of mitosis to seed procentriole formation. Nonphosphorylatable Ana2 still localizes to the centriole but can no longer recruit Sas6 and centriole duplication fails. Thus, following centriole disengagement, recruitment of Ana2 and its phosphorylation by Plk4 are the earliest known events in centriole duplication to recruit Sas6 and thereby establish the architecture of the new procentriole engaged with its parent.
The ability to identify protein interactions is key to elucidating the molecular mechanisms of ce... more The ability to identify protein interactions is key to elucidating the molecular mechanisms of cellular processes, including mitosis and cell cycle regulation. Drosophila melanogaster , as a model system, provides powerful tools to study cell division using genetics, microscopy, and RNAi. Drosophila early embryos are highly enriched in mitotic protein complexes as their nuclei undergo 13 rounds of rapid, synchronous mitotic nuclear divisions in a syncytium during the fi rst 2 h of development. Here, we describe simple methods for the affi nity purifi cation of protein complexes from transgenic fl y embryos via protein A-and green fl uorescent protein-tags fused to bait proteins of interest. This in vivo proteomics approach has allowed the identifi cation of several known and novel mitotic protein interactions using mass spectrometry, and it expands the use of the Drosophila model in modern molecular biology.
FEBS Journal, 2011
Overexpression of Dsk2 ⁄dUbqln results in severe developmental defects and lethality in Drosophil... more 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
Molecular Genetics and Genomics, 2007
The function and the molecular properties of the Rpt1/p48B ATPase subunit of the regulatory parti... more The function and the molecular properties of the Rpt1/p48B ATPase subunit of the regulatory particle of the Drosophila melanogaster 26S proteasome have been studied by analyzing three mutant Drosophila stocks in which P-element insertions occurred in the 5Ј-non-translated region of the Rpt1/p48B gene. These P-element insertions resulted in larval lethality during the second instar larval phase. Since the Rpt1/p48B gene resides within a long intron of an annotated, but uncharacterized Drosophila gene (CG17985), the second instar larval lethality may be a consequence of a combined damage to two independent genes. To analyze the phenotypic eVect of the mutations aVecting the Rpt1/p48B gene alone, imprecise P-element excision mutants were selected. One of them, the pupal lethal P1 mutation, is a hypomorphic allele of the Rpt1/ p48B gene, in which the displacement of two essential regulatory sequences of the gene occurred due to the insertion of a 32 bp residual P-element sequence. This mutation caused a 30-fold drop in the cellular concentration of the Rpt1/p48B mRNA. The decline in the cellular Rpt1/p48B protein concentration induced serious damage in the assembly of the 26S proteasomes, the accumulation of multiubiquitinated proteins, a change in the phosphorylation pattern of the subunit and depletion of this ATPase protein from the chromatin.
Journal of Proteome Research, 2010
A chemical derivatization approach has been developed for the enrichment of O-GlcNAc modified pro... more A chemical derivatization approach has been developed for the enrichment of O-GlcNAc modified proteins. The procedure is based on the isolation technique used for N-glycoproteins with appropriate modifications because of the differences in the two types of glycosylation: a prolonged periodate oxidation is followed by hydrazide resin capture, on-resin proteolytic digestion and release of the modified peptides by hydroxylamine. This enrichment strategy offers a fringe benefit in mass spectrometry analysis. Upon collisional activation the presence of the open carbohydrate ring leads to characteristic fragmentation facilitating both glycopeptide identification and site assignment. The enrichment protocol was applied to the Drosophila proteasome complex previously described as O-GlcNAc modified. The O-GlcNAc modification was located on proteasome interacting proteins, deubiquitinating enzyme Faf (CG1945) and a ubiquitin-like domain containing protein (CG7546). Three other proteins were also found GlcNAc modified, a HSP70 homologue (CG2918), scribbled (CG5462) and the 205 kDa microtubule-associated protein (CG1483). Interestingly, in the HSP70 homologue the GlcNAc modification is attached to an asparagine residue of a N-glycosylation motif. . Supporting Information Available. MALDI-TOF mass spectra of the periodate oxidized O-GlcNAc peptide standard; base peak chromatogram of an enriched fraction of the proteasome; extracted ion chromatogram and full-scan mass spectrum of a selected glycopeptide (m/z 745.85); CID and ETD spectra of the oxime or BEMAD derivatives of O-GlcNAc modified peptides. This material is available free of charge via the Internet at
Journal of Cell Science, 2009
Biochemistry, 2012
Analysis of the in vivo ubiquitylation of the p54/Rpn10 polyubiquitin receptor subunit of the Dro... more 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 Δp54 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 Δp54 mutation, suggesting that ubiquitylated extraproteasomal p54 has an essential role in the pupa−adult transition.
Biochemical Journal, 2005
In the presence of Zn 2+ , the Drosophila 26 S proteasome disassembles into RP (regulatory partic... more In the presence of Zn 2+ , the Drosophila 26 S proteasome disassembles into RP (regulatory particle) and CP (catalytic particle), this process being accompanied by the dissociation of subunit Rpn10/p54, the ubiquitin receptor subunit of the proteasome. The dissociation of Rpn10/p54 induces extensive rearrangements within the lid subcomplex of the RP, while the structure of the ATPase ring of the base subcomplex seems to be maintained. As a consequence of the dissociation of the RP, the peptidase activity of the 26 S proteasome is lost. The Zn 2+ -induced structural and functional changes are fully reversible; removal of Zn 2+ is followed by reassociation of subunit Rpn10/p54 to the RP, reassembly of the 26 S proteasome and resumption of the peptidase activity. After the Zn 2+ -induced dissociation, Rpn10/p54 interacts with a set of non-proteasomal proteins. Hsp82 (heatshock protein 82) has been identified by MS as the main Rpn10/p54-interacting protein, suggesting its role in the reas-sembly of the 26 S proteasome after Zn 2+ removal. The physiological relevance of another Rpn10/p54-interacting protein, the Smt3 SUMO (small ubiquitin-related modifier-1)-activating enzyme, detected by chemical cross-linking, has been confirmed by yeast two-hybrid analysis. Besides the Smt3 SUMO-activating enzyme, the Ubc9 SUMO-conjugating enzyme also exhibited in vivo interaction with the 5 -half of Rpn10/p54 in yeast cells. The mechanism of 26 S proteasome disassembly after ATP depletion is clearly different from that induced by Zn 2+ . Rpn10/p54 is permanently RP-bound during the ATP-dependent assemblydisassembly cycle, but during the Zn 2+ cycle it reversibly shuttles between the RP-bound and free states.
Biochemical Journal, 2013
The concentrations of the Drosophila proteasomal and extraproteasomal polyubiquitin receptors flu... more The concentrations of the Drosophila proteasomal and extraproteasomal polyubiquitin receptors fluctuate in a developmentally regulated fashion. This fluctuation is generated by a previously unidentified proteolytic activity. In the present paper, we describe the purification, identification and characterization of this protease (endoproteinase I). Its expression increases sharply at the L1-L2 larval stages, remains high until the second half of the L3 stage, then declines dramatically. This sharp decrease coincides precisely with the increase of polyubiquitin receptor concentrations in late L3 larvae, which suggests a tight developmental co-regulation. RNAi-induced down-regulation of endoproteinase I results in pupal lethality. Interestingly, we found a cross-talk between the 26S proteasome and this larval protease: transgenic overexpression of the in vivo target of endoproteinase I, the C-terminal half of the proteasomal polyubiquitin receptor subunit p54/Rpn10 results in transcriptional down-regulation of endoproteinase I and consequently a lower level of proteolytic elimination of the polyubiquitin receptors. Another larval protease, Jonah65A-IV, which degrades only unfolded proteins and exhibits similar cross-talk with the proteasome has also been purified and characterized. It may prevent the accumulation of polyubiquitylated proteins in larvae contrary to the low polyubiquitin receptor concentration.
Background: The identification of interaction networks between proteins and complexes holds the p... more Background: The identification of interaction networks between proteins and complexes holds the promise of offering novel insights into the molecular mechanisms that regulate many biological processes. With increasing volumes of such datasets, especially in model organisms such as Drosophila melanogaster, there exists a pressing need for specialised tools, which can seamlessly collect, integrate and analyse these data. Here we describe a database coupled with a mining tool for protein-protein interactions (DAPPER), developed as a rich resource for studying multi-protein complexes in Drosophila melanogaster.
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Papers by Zoltan Lipinszki