Loss-of-function genetic screens are a powerful approach to identify the genes involved in biological processes. For nearly a century, forward genetic screens in model organisms have provided enormous insight into many cellular processes. However, the difficulty in generating and recovering bi-allelic mutations in diploid cells severely hindered the performance of forward genetic screens in mammalian cells. The development of a retroviral gene-trap vector to mutagenise the human near-haploid KBM7 cell line transformed forward genetic screens in human cells. The re-purposing of the microbial CRISPR/Cas9 system now offers an effective method to generate gene knockouts in diploid cells. Here, I performed a head-to-head comparison of retroviral gene-trap mutagenesis screens and genome-wide CRISPR knockout screens in KBM7 cells. The two screening approaches were equally effective at identifying genes required for the endoplasmic reticulum (ER)-associated degradation of MHC class I molecules. The ER-resident enzyme HMG-CoA reductase (HMGCR) catalyses the rate-limiting step in the cholesterol biosynthesis pathway and is targeted therapeutically by statins. To maintain cholesterol homeostasis, the expression of HMGCR is tightly regulated by sterols transcriptionally and post-translationally. Sterols induce the association of HMGCR with Insig proteins, which recruit E3 ubiquitin ligase complexes to mediate degradation of HMGCR by the ubiquitin proteasome system. However, the identity of the E3 ligase(s) responsible for HMGCR ubiquitination is controversial. Here, I use a series of genome-wide CRISPR knockout screens using a fluorescently-tagged HMGCR exogenous reporter and an endogenous HMGCR knock-in as an unbiased approach to identify the E3 ligases and any additional components required for HMGCR degradation. The CRISPR screens identified a role for the poorly characterised ERAD E3 ligase RNF145. I found RNF145 to be functionally redundant with gp78, an E3 ligase previously implicated in HMGCR degradation, and the loss of both E3 ligases was required to significantly inhibit the sterol-induced degradation and ubiquitination of HMGCR. A focused E3 ligase CRISPR screen revealed that the combined loss of gp78, RNF145 and Hrd1 was required to completely block the sterol-induced degradation of HMGCR. I present a model to account for this apparent complexity.

The ubiquitin proteasome system targets proteins to the proteasome where they are degraded. Substrate recognition and processing prior to degradation take place at the 19S regulatory particle of the proteasome. A polyubiquitin chain, linked through isopeptide bonds formed between the C-terminal G76 and K48, is the signal responsible for delivery to the proteasome. Because chains linked via any of the seven lysine residues of ubiquitin exist in vivo and encode signals unrelated to protein degradation it is crucial for cells to avoid crosstalk between these different pathways. Several ubiquitin receptors related to proteasomal degradation have been identified but the selectivity between the different ubiquitin chains has not been assessed quantitatively while avoiding artefacts attributed to GST-dimerisation. By employing isothermal titration calorimetry, analytical ultracentrifugation and nuclear magnetic resonance, discrimination between K48- and K63-linked diubiquitin was established for the S. pombe proteasomal receptor Rpn10 and the shuttle protein Rhp23. The same methods allowed us to propose a discriminatory model for Rpn10. The crystal structures of the 19S regulatory particle subunits Rpn101-193 and Rpn121-224 have been determined and possible protein-protein interaction sites were identified by surface conservation and electrostatics analysis. Rpn12 surface residues were identified that had a negative effect on Rpn10-binding. This interaction was studied by surface plasmon resonance, fluorescence anisotropy and nuclear magnetic resonance. These experiments revealed a binding site on Rpn10 that is exclusively occupied by either ubiquitin or Rpn12 and for the first time demonstrated the interaction of a ubiquitin interacting motif with a protein other than ubiquitin.

Inactivating EGF signalling is key to modulating cell growth and avoiding cancer. To do this, the EGF receptor is ubiquitinated, internalized and sorted to lysosome for degradation. This latter process is coordinated by the endosomal sorting complex required for transport (ESCRT) machinery, a multi-complex protein machinery divided into four groups: ESCRT-0, I, II, III. ESCRTs recognise ubiquitinated cargoes and sort them from the limiting membrane of intermediate vesicles of maturing endosomes. In mammalian cells, the ESCRT machinery is also involved in other membrane related events, such as cytokinesis and viral budding. Certain ESCRTs, such as ESCRT-0, seem to be specifically required for cargo sorting to lysosomes, whereas other downstream ESCRTs, such as ESCRT-I, are required for all the cellular processes where the ESCRT machinery is involved. The existence of multiple variants of ESCRT-I components may suggest that ESCRT-I itself exists in different variants, each specific for a different membrane-based event. A bioinformatic study suggested Ubiquitin Associated Protein1 (UBAP1)as a novel variant of the ESCRT-I component MVB12. Moreover, a preliminary Y2H study identified UBAP1 as a potential binding partner of the ESCRT machinery regulator, HDPTP. This study aims to characterise UBAP1 as a variant MVB12 and a novel member of ESCRT-I. The results show that loss of UBAP1 impairs EGFR trafficking to lysosomes and causes the accumulation of ubiquitinated proteins on aberrant vacuolar structures. In cells, UBAP1 is incorporated in a complex with the ESCRT-I members TSG101, VPS28 and VPS37A. Importantly, UBAP1 uses three tandem ubiquitin associated (UBA) domains to bind ubiquitin and this activity is key for UBAP1 to function in cells. UBAP1 binds HDPTP via a peptide motif located about 100 aa. proximal to the tandem UBA domains. Altogether, the data shown in this thesis suggest that UBAP1 represents a subunit of an endosome-specific ESCRT-I complex, whose function may be coordinated by the ESCRT machinery regulator HDPTP.

Doctorat en Sciences médicales / info:eu-repo/semantics/nonPublished

The expression of the Drosophila melanogaster morphogen Hedgehog (Hh) plays a key role in co-ordinating proliferation and differentiation during animal development. Tight spatial and temporal regulation of Hh expression is essential for its correct function in these essential processes. Both mis-expression of its mammalian orthologue Sonic Hedgehog (Shh) and aberrant stimulation of the associated signalling pathway occur in a wide range of human tumours. Although there is extensive knowledge of the signal transduction pathway that is activated in a Hh-stimulated cell, very little is known about pathways governing the expression of the morphogen itself. The Drosophila tumour suppressor protein Hyperplastic Discs (Hyd), an E3 ubiquitin ligase, negatively regulates hedgehog (hh) expression and Hh pathway activity by independent mechanisms in the developing Drosophila eye. Genetically generated hyd mutant clones in the eye mis-express hh and the transcriptional activator of Hh target genes, Cubitus interruptus (Ci), and cause overgrowth of the surrounding wildtype tissue. However, the underlying molecular mechanism(s) by which Hyd regulates these morphogen regulatory pathways is not known. Hyd may be involved in ubiquitylating target proteins in these pathways, which could have degradative or non-degradative outcomes. In order to elucidate Hyd’s molecular role in potential morphogen regulatory pathways, I applied a proteomics-based approach to identify novel Hyd binding partners and ubiquitylated substrates. Tandem affinity purification in combination with mass spectrometry was used to purify and identify Hyd and its complexed binding partners from Drosophila cells. Binding and ubiquitylation assays were subsequently used to verify and characterize the interactions. In addition, a biased, literature-guided approach was applied to identify likely Hyd binding partners based on their involvement in morphogen signalling and conservation across species. Finally, to assess the functional consequences of a newly identified interaction, I used a Drosophila in vivo model to determine whether the novel binding partner was capable of modifying the hyd mutant phenotype. For this purpose, the Mosaic Analysis with a Repressible Cell Marker (MARCM) technique was used to generate hyd mutant clones in the developing larval eye, which were expressing transgenes resulting in either the over-expression or RNAi-mediated knockdown of the gene of interest. My results indicate that Hyd is involved in regulating both Hh and Wg morphogen signalling in the Drosophila eye, and that the molecular mechanism of action may, at least in part, involve the protein kinase Shaggy (Sgg). Hyd interacts with the Hh and Wg transcriptional activator proteins Ci and Armadillo, respectively, as well as the Sgg kinase. Sgg is a negative regulator of both the Hh and Wg pathways, and acts to direct the proteolytic processing or degradation of the transcriptional effectors of these morphogen pathways. Sgg and its mammalian orthologue GSK3β were ubiquitylated in vitro, and GSK3β ubiquitylation was negatively regulated by the mammalian homologue of Hyd, EDD. Knockdown of sgg in eye disc cells mutant for hyd resulted in a dramatic rescue of the overgrowth phenotype. Loss of hyd in clones located in the anterior region of the eye disc resulted in low levels of the full-length Hh transcriptional activator protein Ci. This effect was reversed completely as a result of sgg knockdown. Furthermore, loss of hyd in eye disc clones resulted in elevated Hh and Wg morphogen expression. Mis-expression of hh in hyd mutant clones was significantly reduced upon over-expression of a constitutively active Sgg kinase. Hence sgg appears to genetically act downstream of hyd to regulate hh gene expression and Ci expression. In summary my results identify Sgg as a novel regulator of hh gene expression, whose activity may be regulated by ubiquitylation, and which may be acting downstream of Hyd in a ubiquitin-regulated manner to control both hh gene expression and Hh pathway activity in the developing Drosophila eye. Hyd may also regulate Hh pathway activity by directly interacting with Ci and affecting its activity. The results also indicate that Hyd may be a master regulator of both Hh and Wg morphogen signalling during Drosophila development.

Protein phosphatase 2A (PP2A), a ubiquitous and pleiotropic regulator of intracellular signaling, is composed of a core dimer (A scaffolding and C catalytic subunits) bound to a variable (B) regulatory subunit of either the B, B' or B" families. Further genetic expansion and alternative splicing within each B subunit family affords the enzyme tremendous functional heterogeneity as PP2A contributes dozens of heterotrimers with varying subcellular locations and cellular substrates dictated by the variable B subunit. B'β is a brain-specific PP2A regulatory subunit that mediates dephosphorylation of Ca2+/calmodulin-dependent protein kinase II and tyrosine hydroxylase. Unbiased proteomic screens for B'β interactors identified Cullin3 (Cul3), a scaffolding component of E3 ubiquitin ligase complexes, and the previously uncharacterized Kelch-like 15 (KLHL15). KLHL15 is one of more than 40 Kelch-like proteins, many of which have been defined as adaptors for the recruitment of substrates to Cul3-based E3 ubiquitin ligases. KLHL15/Cul3 specifically targets B'β to promote protein turnover via ubiquitination and proteasomal degradation. Comparison of KLHL15 and B'β expression profiles suggest that the E3 ligase adaptor contributes to selective expression of the PP2A/B'β holoenzyme in the brain. Mapping of KLHL15 residues critical for Cul3 binding and protein dimerization indicate two distinct and independent functions of KLHL15's N-terminal BTB domain while similar analysis of the C-terminal kelch domain identifies a B'β-specific binding core. While B' regulatory subunit association with the AC dimer is mediated by a highly conserved inner core of roughly 400 amino acids, the divergent N-terminus of B'β is found to be both necessary and sufficient for KLHL15-mediated degradation, with Tyr52 having an obligatory role, underlying the selective association of KLHL15 with the B'β regulatory subunit only. KLHL15 can interact with not only the monomeric version of B'β but also the more stable, PP2A/B'β heterotrimer. However, proteasomal targeting is reserved for the B subunit only. The loss of B'β promotes an exchange of B subunits and a reciprocal upregulation of alternative B subunit-containing heterotrimers. That is, excess KLHL15 may not only downregulate B'β-guided PP2A dephosphorylation activity, but moreover, may upregulate PP2A activity dictated by alternative B subunits. Taken together, these data suggest regulatory subunit-specific ubiquitination and proteasomal degradation as a novel mechanism for controlling total cellular PP2A activity.

<p>The modification of proteins by the covalent attachment of ubiquitin is a natural process that crucially regulates a wide range of eukaryotic signaling outcomes. This process has been understood as the linking of the C-terminus of ubiquitin to the lysine residue of a target protein via an isopeptide linkage, catalyzed by the coordinated effort by E1, E2, and E3 enzymes. Importantly, ubiquitination has only been observed to be a eukaryotic phenomenon. In recent years though, intracellular bacteria, including human pathogens, have been observed to possess ubiquitin-interacting proteins in their genomes. These proteins serve to subdue and manipulate their hosts’ ubiquitin signaling for their own benefit. While some of these proteins act within the eukaryotic context, more recent findings reveal the existence of prokaryotic enzymes that catalyze ubiquitination using mechanisms never before seen in nature. These remarkable processes utilize different cofactors and target different amino acid residues of both ubiquitin as well as substrate protein. The findings reported in this Thesis involve structural and biochemical studies on two new ubiquitinating proteins, the only two proteins known to catalyze ubiquitination outside of the canonical pathway. Both proteins are present in the genome of the intracellular human pathogen <i>Legionella pneumophila</i>: the SidE family, which catalyzes ubiquitination via a mechanism combining ADP-ribosylation and phosphodiesterase activities, and MavC, which utilizes a mechanism reminiscent of transglutaminases. Key insights provided in this document include the discovery that SidE enzymes can modify multiple ubiquitin moieties within a ubiquitin chain, and that modified ubiquitin chains are resistant to hydrolytic cleavage from many deubiquitinating enyzmes. Also, the development of a robust, continuous assay for SidE-catalyzed ubiquitination using a synthetic substrate is described. The catalytic action of MavC, which differs from both canonical E1/E2/E3 ubiquitination and SidE ubiquitination is also here elucidated. The crystal structure of MavC in complex with its ubiquitinated product is presented and provides an atomic view into the basis of substrate recognition. These findings bring to light a new dimension of host-pathogen interactions, where pathogenic ubiquitinating enzymes have appeared to arise from convergent evolution. The regulation of these pathogenic enzymes by other effectors is also discussed, as well as biochemical studies of these regulators. Further, these findings describe possible new drug discovery strategies, as well as possible techniques for discovering similar enzymes in organisms besides <i>Legionella</i>.</p>

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22738号 / 医博第4656号 / 新制||医||1046(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 竹内 理, 教授 中川 一路, 教授 生田 宏一 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Background: Heart attack or myocardial infarction (MI) is a major cause of death worldwide. MI is generally attributed to the detrimental effects of myocardial ischemia/reperfusion (I/R) injury. I/R injury induces cell death and reduces heart function. To compensate, the heart remodels with an associated increase in cell death, fibrosis, and hypertrophy, which can further compromise heart function. Ubiquitin (UB) is an evolutionarily conserved protein. Our lab has shown that pre-I/R injury treatment with exogenous UB preserves heart function and reduces fibrosis 3-days post-I/R in mice. A major objective of this study is to analyze the long-term cardioprotective potential of UB post-I/R injury. Here the UB treatment was continued until 28 days post-I/R to include the entire remodeling period. To enhance the clinical applicability, UB treatment was started at the time of reperfusion. Methods: C57BL/6 mice (aged ~3 months) underwent myocardial I/R surgery. Mice were anesthetized and the left anterior descending coronary artery (LAD) was ligated for 45 minutes. The ligature was then removed for reperfusion. Mice were treated with UB (1µg/g body weight; intraperitoneal (IP) injection) or saline at the time of reperfusion; followed by 3-days of saline or UB IP treatment twice per day. The mice were then implanted with micro-osmotic pumps containing UB (1 μg·g−1·h−1) or saline to continue treatment 28-days post I/R. Mice were sacrificed at 28-days post I/R injury. Sham animals underwent the same surgery without LAD ligation. Heart functional parameters (percent ejection fraction and fractional shortening) were analyzed by echocardiography in a time-dependent manner (3, 7, 14 and 28 days post-I/R). Extracted hearts were embedded in paraffin. Heart sections (5µm) were stained with Mason’s Trichrome to measure fibrosis, TUNEL to measure apoptosis, and fluorescein-conjugated wheat germ agglutinin to measure hypertrophy. Index of fibrosis was quantified as a percentage of total left ventricular area, apoptosis was quantified as a percentage of the total number of nuclei, and hypertrophy was quantified by measuring the myocyte cross-sectional area. Major findings: 1) I/R+saline exhibited a significant decrease in the functional parameters of the heart at 3, 7, 14 and 28 days post-I/R vs sham (n=4-12). No significant decrease in heart function observed between I/R+UB vs sham, and heart function was significantly lower in I/R+saline compared to UB+I/R (n=7-12); 2) I/R surgery significantly increased fibrosis in the myocardium of I/R+saline vs sham. No significant difference was observed between UB+I/R and sham, and fibrosis was significantly lower in UB+I/R vs I/R+saline (n=4-6); 3) Apoptosis was significantly higher in I/R+saline vs sham (p4) Myocyte hypertrophy was significantly higher in I/R+saline vs sham (pConclusion: Long-term UB treatment has the potential to preserve heart function with effects on myocardial fibrosis, myocyte apoptosis, and hypertrophy following myocardial I/R injury.

The PhD thesis is focused on the effect of porcine sperm cell extracellular ubiquitination on early embryonic development up to the blastocyst stage after ICSI. In addition, it also presents a potential improvement of the technique of in vitro fertilization using oocyte incubation with ion channels regulators. To address these aims, we established an entirely novel methodology for sperm cell sorting using flow cytometry and subsequent cryopreservation. We determined the conditions for successful sperm cell sorting based on extracellular ubiquitination rate providing highly specific selection as well as sufficient numbers of viable sperms for fertilization using the ICSI method. Concerning the following cryopreservation, established methods were optimized to enable freezing of a minimal sperm cell suspension volume with low cell numbers. The performed experiments showed a direct relationship between the rate of extracellular ubiquitination and the capability of sperms to give rise to a properly developing embryo. Highly ubiquitinated sperm cells were less successful regarding the embryonic development to the blastocyst stage if compared with the lowly ubiquitinated group (6,2 % vs. 16,7 %, P<0,001). Interestingly, the rate of extracellular ubiquitination showed no effect on the pronuclear formation...