Proteaszóma szubsztrátok felismerési mechanizmusának genetikai analízise = Genetic analysis of the protein delivery mechanism for proteasomal degradation

P elemre épülő klasszikus és molekuláris genetikai technikákkal mutációkat izoláltunk, és/vagy transzgenikus RNS-interferencia vonalakat állítottunk elő az ecetmuslica ubikvitin-proteaszóma rendszerét (UPR) kódoló génekben. A mutánsok részletes genetikai, biokémiai és molekuláris analíziséből új ismereteket szereztünk az UPR egyes komponenseinek szerepéről a sejtciklus szabályozásában. Azonosítottuk és jellemeztük a Drosophila anafázis-promoting komplex (APC) katalitikus moduljának összetételét és szerepét. Azonosítottuk egy új ubikvitin konjugáló enzim, az Ube2S Drosophila ortológját, és kimutattuk, hogy stressz-indukált ubikvitinációban van szerepe. Kimutattuk, hogy a p54, Dsk2 és Rad23 poliubikvitin-kötő receptorok funkcionális interakciót mutatnak egymás között. Átfogó bioinformatikai analízissel azonosítottunk 40 dezubikvitináló enzimet kódoló gént Drosophilában, majd meghatároztuk mutánsaik egyedfejlődési és citológiai fenotípusait. Kimutattuk, hogy a DmUsb5 dezubikvitináló enzimnek szerepe van az ubikvitin homeosztázis fenntartásában és az apoptózis szabályozásában. Végezetül demonstráltuk, hogy az APC ciklikus aktivitást mutat endomitózis során is, ahol a ciklinek ubikvitin-függő degradációjával lehetővé teszi a replikációs komplex ismételt összeszerelődését. | P element induced mutants and transgenic RNA-interference lines affecting genes of the ubiquitin-proteasome system (UPS) were established and characterized. Valuable new information about their function in cell cycle regulation was gained following detailed genetic, biochemical and molecular analyses of these mutants. Through genetic and physical interaction studies, we identified and characterized components of the catalytic module of the anaphase promoting complex (APC). The Drosophila orthologue of a new ubiquitin conjugating enzyme, DmUbe2S was identified and demonstrated, that it functions in stress related ubiquitylation. We have shown that the p54, Dsk2 and Rad23 polyubiquitin binding receptors functionally interact with each other. Through comprehensive bioinformatic analyses, forty Drosophila genes coding for deubiquitylating enzymes were identified and characterized preliminarily. It was established that the DmUsb5 deubiquitylating enzyme has a role in maintaining intracellular ubiquitin homeostasis and in regulating apoptosis. Finally, we have demonstrated that the APC shows cyclical activity during endomitózis, in which it promotes cell cycle progression by lowering cyclin concentration in order to facilitate the relicensing of replication origins

A fehérjelebontás szerepének tanulmányozása a kromoszóma szegregáció és a mitózis szabályozásában = Determining the role of protein degradation in the regulation of chromosome segregation and mitosis

Az sejtciklus szabályozásában alapvető szerepet játszó komplex, az APC genetikai és molekuláris biológiai analízisét végeztük el elsőként egy soksejtű eukarióta modell organizmusban, az ecetmuslicában. Azonosítottuk a Drosophila APC komplexet alkotó tíz alegységet kódoló géneket. Ezek közül, P elemre épülő technikákkal, nyolc gén mutáns alléljait és transzgénikus RNSi vonalait sikerült előállítani és jellemezni. Egy kivételével valamennyi alegység funkciója nélkülözhetetlennek bizonyult az állatok egyedfejlődése szempontjából, és kiesésük jellegzetes mitótikus fenotípusok kialakulását eredményezte. Az egyes mutánsok esetében egymáshoz hasonló, de jelentősen eltérő fenotípus-jegyeket is kimutattunk. Ezekből arra következtettünk, hogy a különböző alegységeknek eltérő szerepe van a komplex funkciójának kialakításában. Genetikai és fizikai interakciókat is kimutattunk az egyes alegységek között, amelyek jó egyezést mutatnak az időközben megjelent szerkezetvizsgálati eredményekkel. Kimutattuk, hogy a két protein modifikációs rendszer, az ubiquitinálás és a sumoilálás funkcionálisan összefügg egymással. Kimutattuk, hogy a csak soksejtű eukariótákban található APC7 alegység egy hét TPR (tetratrico-peptide repeat) motívumot hordozó fehérje, amelynek funkciója, egyedüliként, nem eszenciális a teljes komplex funkciója szempontjából. Végezetül kidolgoztunk egy kísérleti rendszert a proteindegradáció követésére mind in vivo, mind pedig in vitro. | In this project we have accomplished the genetic and molecular biological analysis of the anaphase promoting complex, or APC, in a multicellular model organism, the fruit fly, Drosophila melanogaster. First, we identified all genes coding for ten subunits of the Drosophila APC. Mutant alleles and transgenic RNA interference lines corresponding to eight subunit genes were isolated and characterised. The function of all but one subunit proved to be essential for development, and their loss resulted in characteristic mitotic phenotypes. The significant differences in their mitotic phenotypes indicate that the subunits have distinct roles and they contribute differently to the functions of the whole complex. In addition to this, we have demonstrated genetic and physical interactions among subunits. These data are in good agreement with the physico-chemical data emerging from structural studies of the APC. We have shown that the two protein modification system, the ubiquitination and sumoilation are functionally related to each other. We have also shown that the APC7 subunit, found only in multicellular eukaryotes, contains seven TPR (tetratrico-peptide-repeat) repeats, and it is the only subunit whose function is not essential for the functioning of the whole complex. In the course of this project we established an experimental system to monitor protein degradation both in vivo and in vitro

Ubiquitylation of Drosophila p54/Rpn10/S5a regulates its interaction with the UBA-UBL polyubiquitin receptors.

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.This work was supported by the Institute of Biochemistry (Biological Research Centre of the Hungarian Academy of Sciences)

Deletion of proteasomal subunit S5a/Rpn10/p54 causes lethality, multiple mitotic defects and overexpression of proteasomal genes in Drosophila melanogaster

The regulatory complex of the 26S proteasome is responsible for the selective recognition and binding of multiubiquitinated proteins. It was earlier shown that the subunit S5a/Rpn10/p54 of the regulatory complex is the only cellular protein capable of binding multiubiquitin chains in an in vitro overlay assay. The role of this subunit in substrate selection, however, is a subject of debate, following the observation that its deletion in Saccharomyces cerevisiae is not lethal and instead causes only a mild phenotype. To study the function of this subunit in higher eukaryotes, a mutant Drosophila strain was constructed by deleting the single copy gene encoding subunit S5a/Rpn10/p54. This deletion caused larval-pupal polyphasic lethality, multiple mitotic defects, the accumulation of higher multimers of ubiquitinated proteins and a huge accumulation of defective 26S proteasome particles. Deletion of the subunit S5a/Rpn10/p54 does not destabilise the regulatory complex and does not disturb the assembly of the regulatory complex and the catalytic core. The pupal lethality is a consequence of the depletion of the maternally provided 26S proteasome during the larval stages and a sudden increase in the proteasomal activity demands during the first few hours of pupal development. The huge accumulation of the fully assembled 26S proteasome in the deletion mutant and the lack of free subunits or partially assembled particles indicate that there is a highly coordinated accumulation of all the subunits of the 26S proteasome. This suggests that in higher eukaryotes, as with yeast, a feedback circuit coordinately regulates the expression of the proteasomal genes, and this adjusts the actual proteasome concentration in the cells according to the temporal and/or spatial proteolytic demand

A Drosophila 26S proteasoma molekuláris jellemzése = Molecular characterization of the Drosophila 26S proteasome

Módszert dolgoztunk ki Drosophila melanogaster multiubiquitinált fehérjéinak tisztítására és tömegspektrometriás azonosítására. A Drosophila 26S proteaszóma multiubiquitin receptor alegységének háromszorosan tag-gelt változatát állítottuk elő, ez a módosított fehérje megtartotta multiubiquitinált fehérje kötő képességét. Ezt a fehérjét használva affinitás kromatográfiás rendszerben sikerült szelektíven megkötni a multiubiquitinált fehérjéket és megszabadulni a bakteriális és Drosophila eredetű szennyező fehérjéktől. A módszer lehetőséget teremt a Drosophila multiubiquitinált proteómjának feltérképezésére, e proteom fejlődés- és szövetspecifikus változásainak jellemzésére. A Drosophila 26S proteaszóma multiubiquitin receptor alegységének extraproteaszómális szerepét vizsgálva megállapítottuk, hogy a receptor alegység C-terminális felének ektópikus expressziója transzgénikus állatokban lárvális letalitást okoz, és a receptor fehérje ubiquitinálódik. Ez az ubiquitináció azonban nem degradációs szignál, mert a transzgén terméke egy hosszú élettartamú, stabil fehérje. Az ubiquitinációnak feltehetően szerepe van a receptor fehérje extraproteaszómális funkciójában, és ennek a funkciónak megzavarása letális. Molekulárisan jellemeztük a 26S proteaszóma egyik ATPase alegységének szerkezetét és szerepét az alegységre mutáns Drosophila törzsben. | We have established a method for the purification and mass spectrometric identification of multiubiquitinated proteins in Drosophila melanogaster. Triple-tagged version of the ubiquitin receptor subunit of the 26S proteasome has been constructed, this modified version of the subunit retained its multiubiquitin-binding property. This modified subunit was used as an affinity matrix for the purification of multiubiquitinated proteins. Consecutive affinity purifications on tag-specific columns allowed the elimination of contaminating bacterial and Drosophila proteins. This method opens the way for the establishment of Drosophila multiubiquitinated proteome, and the analysis of the developmental and tissue specific variations of this proteome. Analyzing the extraproteasomal function of the ubiquitin receptor subunit of the Drosophila 26S proteasome we have shown that the ectopic expression of the C-terminal half of the subunit in transgenic Drosophila results in larval lethality, and the transgenic protein becomes ubiquitinated. This ubiquitination, however, is not a degradation signal, because the transgenic protein is a stable, long-lived protein. The ubiquitination of this subunit is probably connected with its extraproteasomal function, and the disturbance of this function is lethal. Using a mutant Drosophila stock we presented a detailed molecular characterization of an ATPase subunit of the 26S proteasome

Role of the deubiquitylating enzyme DmUsp5 in coupling ubiquitin equilibrium to development and apoptosis in Drosophila melanogaster.

Protein ubiquitylation is a dynamic process that affects the function and stability of proteins and controls essential cellular processes ranging from cell proliferation to cell death. This process is regulated through the balanced action of E3 ubiquitin ligases and deubiquitylating enzymes (DUB) which conjugate ubiquitins to, and remove them from target proteins, respectively. Our genetic analysis has revealed that the deubiquitylating enzyme DmUsp5 is required for maintenance of the ubiquitin equilibrium, cell survival and normal development in Drosophila. Loss of the DmUsp5 function leads to late larval lethality accompanied by the induction of apoptosis. Detailed analyses at a cellular level demonstrated that DmUsp5 mutants carry multiple abnormalities, including a drop in the free monoubiquitin level, the excessive accumulation of free polyubiquitins, polyubiquitylated proteins and subunits of the 26S proteasome. A shortage in free ubiquitins results in the induction of a ubiquitin stress response previously described only in the unicellular budding yeast. It is characterized by the induction of the proteasome-associated deubiquitylase DmUsp14 and sensitivity to cycloheximide. Removal of DmUsp5 also activates the pro-apoptotic machinery thereby resulting in widespread apoptosis, indicative of an anti-apoptotic role of DmUsp5. Collectively, the pleiotropic effects of a loss of DmUsp5 function can be explained in terms of the existence of a limited pool of free monoubiquitins which makes the ubiquitin-dependent processes mutually interdependent

Intimate relationship between the genes of two transcriptional coactivators, ADA2a and PIMT, of Drosophila

PIMT, a transcriptional coactivator which interacts with and enhances nuclear receptor coactivator PRIP function, was identified recently in mammalian cells and suggested to function as a link between two major multiprotein complexes anchored by CBP/p300 and PBP. Here we describe that the gene of the Drosophila homologue of PIMT, designated as Dtl, is closely associated and has an overlapping promoter with a gene encoding another transcriptional coactivator, ADA2a, which in turn participates in GCN5 HAT-containing complexes. Ada2a also produces an RNA polII subunit, RPB4, via alternative splicing; consequently, an overlapping regulatory region serves for the production of three proteins, each involved in transcription. By studying expression of reporter gene fusions in tissue culture cells and transgenic animals we have demonstrated that the regulatory regions of Ada2a/Rpb4 and Dtl overlap and the Dtl promoter is partly within the Ada2a/Rpb4 coding region. The shared regulatory region contains a DRE element, binding site of DREF, the protein factor involved in the regulation of a number of genes which play a role in DNA replication and cell proliferation. Despite the perfectly symmetrical DRE, DREF seems to have a more decisive role in Ada2a/Rpb4 transcription than in the transcription of Dtl

lemmingA encodes the Apc11 subunit of the APC/C in Drosophila melanogaster that forms a ternary complex with the E2-C type ubiquitin conjugating enzyme, Vihar and Morula/Apc2

<p>Abstract</p> <p>Background</p> <p>Ubiquitin-dependent protein degradation is a critical step in key cell cycle events, such as metaphase-anaphase transition and mitotic exit. The anaphase promoting complex/cyclosome (APC/C) plays a pivotal role in these transitions by recognizing and marking regulatory proteins for proteasomal degradation. Its overall structure and function has been elucidated mostly in yeasts and mammalian cell lines. The APC/C is, however, a multisubunit assembly with at least 13 subunits and their function and interaction within the complex is still relatively uncharacterized, particularly in metazoan systems. Here, <it>lemming </it>(<it>lmg</it>) mutants were used to study the APC/C subunit, Apc11, and its interaction partners in <it>Drosophila melanogaster</it>.</p> <p>Results</p> <p>The <it>lmg </it>gene was initially identified through a pharate adult lethal P element insertion mutation expressing developmental abnormalities and widespread apoptosis in larval imaginal discs and pupal abdominal histoblasts. Larval neuroblasts were observed to arrest mitosis in a metaphase-like state with highly condensed, scattered chromosomes and frequent polyploidy. These neuroblasts contain high levels of both cyclin A and cyclin B. The <it>lmg </it>gene was cloned by virtue of the <it>lmg⁰³⁴²⁴</it>P element insertion which is located in the 5' untranslated region. The <it>lemming </it>locus is transcribed to give a 2.0 kb mRNA that contains two ORFs, <it>lmgA </it>and <it>lmgB</it>. The <it>lmgA </it>ORF codes for a putative protein with more than 80% sequence homology to the APC11 subunit of the human APC/C. The 85 amino acid protein also contains a RING-finger motif characteristic of known APC11 subunits. The <it>lmgA </it>ORF alone was sufficient to rescue the lethal and mitotic phenotypes of the <it>lmg¹³⁸</it>null allele and to complement the temperature sensitive lethal phenotype of the <it>APC11-myc9 </it>budding yeast mutant. The LmgA protein interacts with Mr/Apc2, and they together form a binding site for Vihar, the E2-C type ubiquitin conjugating enzyme. Despite being conserved among <it>Drosophila </it>species, the LmgB protein is not required for viability or fertility.</p> <p>Conclusions</p> <p>Our work provides insight into the subunit structure of the <it>Drosophila </it>APC/C with implications for its function. Based on the presented data, we suggest that the Lmg/Apc11 subunit recruits the E2-C type ubiquitin conjugating enzyme, Vihar, to the APC/C together with Mr/Apc2 by forming a ternary complex.</p