Design, Synthesis and Biological Evaluation of Peptidomimetic Prenyl Transferase Inhibitors

De enzymen protein:farnesyl transferase (PFT) en protein:geranylgeranyl transferase-1 (PGGT-1) zijn verantwoordelijk voor de post-translationele modificatie van kleine G-eiwitten noodzakelijk voor hun binding aan celmembranen. Sinds het begin van de jaren __80 is bekend dat het remmen van PFT voorkomt dat bepaalde gemuteerde G-eiwitten die kanker veroorzaken volwassen worden. Naast anti-kanker therapie_n, zijn er de laatste jaren steeds meer duidelijke aanwijzingen dat PFT en PGGT-1 remmers voor andere therapeutische toepassingen gebruikt kunnen. Hierbij gaat het om bestrijding van osteoporose (botontkalking), atherosclerose en restenose (aderverkalking), angiogenese (de vorming van nieuwe bloedvaten), malaria en multiple sclerose. Mogelijk zal dit jaar de PFT remmer Tipifarnib__ (Johnson & Johnson) goedgekeurd worden voor de behandeling van Acute Myelo_de Leukemie. Het proefschrift beschrijft enkele synthetische methoden voor het ontwikkelen van potente PFT en PGGT-1 remmers (in samenwerking met Gaubius Laboratorium, TNO Quality of Life). Zo is voor PFT een potente en selectieve PFT remmer ontwikkeld waarvan de werking in cellen ook is aangetoond. Naast de bio-organische synthetische methoden, is een __proteomics__ (onderzoek naar eiwitten en hun functie in de cel) methode ontwikkeld dat het mogelijk moet maken om tot nu toe onbekende PFT en PGGT-1 substraten te isoleren en identificeren.|The prenyl transferases protein:farnesyl transferase (PFT) and protein:geranylgeranyl transferase-1 (PGGT-1) are responsible for the post-translational modification of small Gproteins. This post-translational modification comprises the attachment of an isoprenoid (farnesyl or gerangeranyl) to the C-terminus of the G-protein and is essential for a proper cellular membrane localization and function. Since the beginning of the 80__s it is known that inhibition of PFT prevents the maturation of certain mutated (small) G-proteins involved in tumorogenesis. Besides anti-cancer therapies, it is becoming more and more obvious that PFT and/or PGGT-1 inhibitors also hold promise for the treatment of osteoporosis (bone resorption), atherosclerosis and restenosis (thickening of the inner lining of an artery), angiogenesis (formation of new bloodvessels), malaria and multiple sclerosis. At the moment the PFT inhibitor Tipifarnib_ (Johnson & Johnson) has been submitted for approval by the FDA for the treatment of Acute Myeloid Leukemia (AML). The Thesis by Farid El Oualid presents several synthethic approaches toward the development of potent PFT and/or PGGT-1 inhibitors (in cooperation with the Gaubius Laboratory, TNO Quality of Life). For PFT a potent and very selective inhibitor was found. Besides being active against pure PFT, the inhibitor was also shown to be active in cultured cells. In addition to the bio-organic synthethic methods, a __proteomics__ strategy was developed which should make it possible to isolate and identify unknown PFT (and PGGT-1) substratesThe work described in this Thesis was conducted at the division of Bio-Organic Synthesis (BIOSYN) of the Leiden Institute of Chemistry (LIC, Leiden University) and was financed by the Netherlands Technology Foundation (STW, project GC 790.35.356) and Netherlands Organization for Scientific Research (NWO).UBL - phd migration 201

Les erreurs d’orthographe grammaticale et d’orthographe lexicale des lycéens de Tanger en contexte de la compréhension de l’écrit

Les représentations négatives que se font la plupart des lycéens Tangérois de l’écrit sont décourageantes. Pourtant, l’écrit  constitue  la  norme pour l’évaluation des connaissances acquises. Nous avons remarqué que peu de lycéens réussissent parfaitement leurs écrits. La majorité d’entre eux commettent beaucoup d’erreurs, celles qui relèvent de l’orthographe lexicale et grammaticale  sont les plus fréquentes. Cette situation angoissante nous incite à nous interroger : pourquoi  les élèves commettent-ils ces erreurs  malgré les longues d’années d’apprentissage du français? Quel dispositif devons-nous adopter au tant qu’enseignants de langue  pour aider les élèves en difficultés. Nous essayons par le biais   de ce travail  de soumettre  une réponse envisageable à ces interrogations. Cette étude, est  en effet, consacrée à présenter  les résultats d’une recherche que nous avons menée au sein des lycées de la région Tanger –Asila

Metabolic control of BRISC–SHMT2 assembly regulates immune signalling

Serine hydroxymethyltransferase 2 (SHMT2) regulates one-carbon transfer reactions that are essential for amino acid and nucleotide metabolism, and uses pyridoxal-5′-phosphate (PLP) as a cofactor. Apo SHMT2 exists as a dimer with unknown functions, whereas PLP binding stabilizes the active tetrameric state. SHMT2 also promotes inflammatory cytokine signalling by interacting with the deubiquitylating BRCC36 isopeptidase complex (BRISC), although it is unclear whether this function relates to metabolism. Here we present the cryo-electron microscopy structure of the human BRISC–SHMT2 complex at a resolution of 3.8 Å. BRISC is a U-shaped dimer of four subunits, and SHMT2 sterically blocks the BRCC36 active site and inhibits deubiquitylase activity. Only the inactive SHMT2 dimer—and not the active PLP-bound tetramer—binds and inhibits BRISC. Mutations in BRISC that disrupt SHMT2 binding impair type I interferon signalling in response to inflammatory stimuli. Intracellular levels of PLP regulate the interaction between BRISC and SHMT2, as well as inflammatory cytokine responses. These data reveal a mechanism in which metabolites regulate deubiquitylase activity and inflammatory signalling

Strategy for development of site-specific ubiquitin antibodies

Protein ubiquitination is a key post-translational modification regulating a wide range of biological processes. Ubiquitination involves the covalent attachment of the small protein ubiquitin to a lysine of a protein substrate. In addition to its well-established role in protein degradation, protein ubiquitination plays a role in protein-protein interactions, DNA repair, transcriptional regulation, and other cellular functions. Understanding the mechanisms and functional relevance of ubiquitin as a signaling system requires the generation of antibodies or alternative reagents that specifically detect ubiquitin in a site-specific manner. However, in contrast to other post-translational modifications such as acetylation, phosphorylation, and methylation, the instability and size of ubiquitin-76 amino acids-complicate the preparation of suitable antigens and the generation antibodies detecting such site-specific modifications. As a result, the field of ubiquitin research has limited access to specific antibodies. This severely hampers progress in understanding the regulation and function of site-specific ubiquitination in many areas of biology, specifically in epigenetics and cancer. Therefore, there is a high demand for antibodies recognizing site-specific ubiquitin modifications. Here we describe a strategy for the development of site-specific ubiquitin antibodies. Based on a recently developed antibody against site-specific ubiquitination of histone H2B, we provide detailed protocols for chemical synthesis methods for antigen preparation and discuss considerations for screening and quality control experiments.Chemical Immunolog

Polyubiquitin-Photoactivatable Crosslinking Reagents for Mapping Ubiquitin Interactome Identify Rpn1 as a Proteasome Ubiquitin-Associating Subunit

Chemical Immunolog

Screening of DUB activity and specificity by MALDI-TOF mass spectrometry

Deubiquitylases (DUBs) are key regulators of the ubiquitin system which cleave ubiquitin moieties from proteins and polyubiquitin chains. Several DUBs have been implicated in various diseases and are attractive drug targets. We have developed a sensitive and fast assay to quantify in vitro DUB enzyme activity using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Unlike other current assays, this method uses unmodified substrates, such as diubiquitin topoisomers. By analyzing 42 human DUBs against all diubiquitin topoisomers we provide an extensive characterization of DUB activity and specificity. Our results confirm the high specificity of many members of the OTU and JAMM DUB families and highlight that all USPs tested display low linkage selectivity. We also demonstrate that this assay can be deployed to assess the potency and specificity of DUB inhibitors by profiling 11 compounds against a panel of 32 DUBs

Olfactory Stem Cells, a New Cellular Model for Studying Molecular Mechanisms Underlying Familial Dysautonomia

International audienceBackground: Familial dysautonomia (FD) is a hereditary neuropathy caused by mutations in the IKBKAP gene, the most common of which results in variable tissue-specific mRNA splicing with skipping of exon 20. Defective splicing is especially severe in nervous tissue, leading to incomplete development and progressive degeneration of sensory and autonomic neurons. The specificity of neuron loss in FD is poorly understood due to the lack of an appropriate model system. To better understand and modelize the molecular mechanisms of IKBKAP mRNA splicing, we collected human olfactory ecto-mesenchymal stem cells (hOE-MSC) from FD patients. hOE-MSCs have a pluripotent ability to differentiate into various cell lineages, including neurons and glial cells.Methodology/Principal Findings: We confirmed IKBKAP mRNA alternative splicing in FD hOE-MSCs and identified 2 novel spliced isoforms also present in control cells. We observed a significant lower expression of both IKBKAP transcript and IKAP/hELP1 protein in FD cells resulting from the degradation of the transcript isoform skipping exon 20. We localized IKAP/hELP1 in different cell compartments, including the nucleus, which supports multiple roles for that protein. We also investigated cellular pathways altered in FD, at the genome-wide level, and confirmed that cell migration and cytoskeleton reorganization were among the processes altered in FD. Indeed, FD hOE-MSCs exhibit impaired migration compared to control cells. Moreover, we showed that kinetin improved exon 20 inclusion and restores a normal level of IKAP/hELP1 in FD hOE-MSCs. Furthermore, we were able to modify the IKBKAP splicing ratio in FD hOE-MSCs, increasing or reducing the WT (exon 20 inclusion):MU (exon 20 skipping) ratio respectively, either by producing free-floating spheres, or by inducing cells into neural differentiation.Conclusions/Significance: hOE-MSCs isolated from FD patients represent a new approach for modeling FD to better understand genetic expression and possible therapeutic approaches. This model could also be applied to other neurological genetic diseases

Molecular basis of Lys11-polyubiquitin specificity in the deubiquitinase Cezanne

The post-translational modification of proteins with polyubiquitin regulates virtually all aspects of cell biology. Eight distinct chain linkage types in polyubiquitin co-exist and are independently regulated in cells. This ‘ubiquitin code’ determines the fate of the modified protein1. Deubiquitinating enzymes of the Ovarian Tumour (OTU) family regulate cellular signalling by targeting distinct linkage types within polyubiquitin2, and understanding their mechanisms of linkage specificity gives fundamental insights into the ubiquitin system. We here reveal how the deubiquitinase Cezanne/OTUD7B specifically targets Lys11-linked polyubiquitin. Crystal structures of Cezanne alone and in complex with mono- and Lys11-linked diubiquitin, in combination with hydrogen-deuterium exchange mass spectrometry, enable reconstruction of the enzymatic cycle in exquisite detail. An intricate mechanism of ubiquitin-assisted conformational changes activate the enzyme, and while all chain types interact with the enzymatic S1 site, only Lys11-linked chains can bind productively across the active site and stimulate catalytic turnover. Our work highlights the fascinating plasticity of deubiquitinases, and indicates that new conformational states can occur when a true substrate, such as diubiquitin, is bound at the active site

Structural insights into the catalysis and regulation of E3 ubiquitin ligases

Covalent attachment (conjugation) of one or more ubiquitin molecules to protein substrates governs numerous eukaryotic cellular processes, including apoptosis, cell division and immune responses. Ubiquitylation was originally associated with protein degradation, but it is now clear that ubiquitylation also mediates processes such as protein–protein interactions and cell signalling depending on the type of ubiquitin conjugation. Ubiquitin ligases (E3s) catalyse the final step of ubiquitin conjugation by transferring ubiquitin from ubiquitin-conjugating enzymes (E2s) to substrates. In humans, more than 600 E3s contribute to determining the fates of thousands of substrates; hence, E3s need to be tightly regulated to ensure accurate substrate ubiquitylation. Recent findings illustrate how E3s function on a structural level and how they coordinate with E2s and substrates to meticulously conjugate ubiquitin. Insights regarding the mechanisms of E3 regulation, including structural aspects of their autoinhibition and activation are also emerging

Impact of opioid-free analgesia on pain severity and patient satisfaction after discharge from surgery: multispecialty, prospective cohort study in 25 countries

Background: Balancing opioid stewardship and the need for adequate analgesia following discharge after surgery is challenging. This study aimed to compare the outcomes for patients discharged with opioid versus opioid-free analgesia after common surgical procedures.Methods: This international, multicentre, prospective cohort study collected data from patients undergoing common acute and elective general surgical, urological, gynaecological, and orthopaedic procedures. The primary outcomes were patient-reported time in severe pain measured on a numerical analogue scale from 0 to 100% and patient-reported satisfaction with pain relief during the first week following discharge. Data were collected by in-hospital chart review and patient telephone interview 1 week after discharge.Results: The study recruited 4273 patients from 144 centres in 25 countries; 1311 patients (30.7%) were prescribed opioid analgesia at discharge. Patients reported being in severe pain for 10 (i.q.r. 1-30)% of the first week after discharge and rated satisfaction with analgesia as 90 (i.q.r. 80-100) of 100. After adjustment for confounders, opioid analgesia on discharge was independently associated with increased pain severity (risk ratio 1.52, 95% c.i. 1.31 to 1.76; P < 0.001) and re-presentation to healthcare providers owing to side-effects of medication (OR 2.38, 95% c.i. 1.36 to 4.17; P = 0.004), but not with satisfaction with analgesia (beta coefficient 0.92, 95% c.i. -1.52 to 3.36; P = 0.468) compared with opioid-free analgesia. Although opioid prescribing varied greatly between high-income and low- and middle-income countries, patient-reported outcomes did not.Conclusion: Opioid analgesia prescription on surgical discharge is associated with a higher risk of re-presentation owing to side-effects of medication and increased patient-reported pain, but not with changes in patient-reported satisfaction. Opioid-free discharge analgesia should be adopted routinely