10 research outputs found
Direct m6A recognition by IMP1 underlays an alternative model of target selection for non-canonical methyl-readers
m6A methylation provides an essential layer of regulation in organismal development, and is aberrant in a range of cancers and neuro-pathologies. The information encoded by m6A methylation is integrated into existing RNA regulatory networks by RNA binding proteins that recognise methylated sites, the m6A readers. m6A readers include a well-characterised class of dedicated proteins, the YTH proteins, as well as a broader group of multi-functional regulators where recognition of m6A is only partially understood. Molecular insight in this recognition is essential to build a mechanistic understanding of global m6A regulation. In this study, we show that the reader IMP1 recognises the m6A using a dedicated hydrophobic platform that assembles on the methyl moiety, creating a stable high-affinity interaction. This recognition is conserved across evolution and independent from the underlying sequence context but is layered upon the strong sequence specificity of IMP1 for GGAC RNA. This leads us to propose a concept for m6A regulation where methylation plays a context-dependent role in the recognition of selected IMP1 targets that is dependent on the cellular concentration of available IMP1, differing from that observed for the YTH proteins
Regolazione delle Cellule Staminali Mesenchimali: effetti dell'omeogene Otx1 su proliferazione e commitment
Le Cellule Staminali Mesenchimali (Mesenchymal Stem Cells – MSCs) sono cellule pluripotenti capaci di differenziare in elementi maturi di lineages mesodermici, quali osteociti, adipociti e condrociti. Le MSCs risiedono principalmente nel midollo osseo, dove sono state isolate per la prima volta, ma possono trovarsi in quasi tutti i tessuti adulti. La loro capacità di secernere molecole trofiche e immunoregolatorie e la loro possibilità di espansione e differenziazione ex vivo, hanno generato forte interesse per le MSCs quali potenziali strumenti terapeutici nella medicina rigenerativa e nell’ingegneria tissutale. Tuttavia, per l’applicazione clinica delle MSCs, è necessario comprendere a fondo i meccanismi molecolari che ne regolano il self-renewal e la differenziazione nei diversi lineages. Nel laboratorio dove ho svolto il mio lavoro di tesi, è stato ipotizzato un ruolo chiave nella regolazione delle MSCs da parte degli omeogeni, così chiamati in quanto contengono una sequenza altamente conservata di 183 nucleotidi, l’omeobox, che codifica l’omeodominio. Quest’ultimo presenta struttura helix-turn-helix, che permette il legame al DNA e che quindi consente di classificare le omeoproteine come fattori di trascrizione. L’omeogene Otx1, appartenente alla sotto-famiglia degli omeogeni Otx, caratterizzati da un dominio bicoid, è omologo nei Vertebrati del gene Orthodenticle di Drosophila e gioca un ruolo fondamentale nella morfogenesi del sistema nervoso e nello sviluppo degli organi di senso. Studi effettuati precedentemente nel nostro laboratorio hanno dimostrato che Otx1, durante la vita adulta, è coinvolto nella regolazione delle cellule staminali ematopoietiche (Hematopoietic Stem Cells – HSCs) e contribuisce alla produzione dei precursori della linea eritroide. Nella regolazione dell’attività delle HSCs, è sempre più evidente il ruolo chiave della nicchia, composta da cellule differenziate di alcuni lineages mesenchimali, in particolare gli osteoblasti, e dalle MSCs. Poiché abbiamo rilevato che l’inattivazione in vivo di Otx1 provoca una notevole fragilità ossea, è verosimile ipotizzare che l’omeogene possa essere coinvolto anche nella regolazione delle MSCs, in particolare nella loro differenziazione osteogenica. A tal proposito, sono stati precedentemente condotti studi di espressione a livello sia trascrizionale che proteico in MSCs primarie murine ed in pre-osteoblasti della linea cellulare MC3T3-E1, una linea clonale osteogenica non tumorale generata a partire dalle ossa della base cranica del topo. Essa rappresenta la finestra temporale idonea per studiare i fenomeni che stanno alla base dell’osteogenesi, dallo stadio di pre-osteoblasti, fino alla differenziazione terminale. Tali studi hanno mostrato un aumento di espressione sia dell’mRNA sia della proteina di Otx1 nel corso del processo differenziativo, che riproduce fedelmente il pattern di espressione di Runx2, master gene dell’osteogenesi. Inoltre, è stato dimostrato che l’espressione di Runx2 e di ALP, ecto-enzima che catalizza la produzione dell’idrossiapatite di calcio, il mineralizzante della matrice ossea, è fortemente ridotta durante la differenziazione osteogenica di MSCs Otx1-/-. Il mio lavoro di tesi si è focalizzato sullo stu-dio della funzione di Otx1 nella regolazione delle mMSCs, analizzando gli effetti della perdita e del guadagno di funzione del gene. Il confronto di mMSCs wt e Otx1-/- allo stadio indifferenziato ha evidenziato che l’inattivazione dell’omeogene determina: 1) un netto aumento della sopravvivenza/proliferazione delle MSCs, anche a passaggi in coltura in cui le cellule wt smettono di dividersi; 2) una riduzione significativa della senescenza cellulare; 3) un decremento dell’espressione di geni del checkpoint G1 → S, quali p53, p21WAF1, p16INK4a e p19ARF/INK4d. Successivi esperimenti di trasfezione transiente con vettore contenente Otx1 hanno evidenziato effetti diametralmente opposti sull’espressione dei geni del checkpoint G1 → S, suggerendo un’azione diretta o indiretta di Otx1 su questi regolatori del ciclo cellulare. Risultati preliminari ottenuti con Saggi di Immunoprecipitazione della Cromatina sembrano indicare che l’omeoproteina sia capace di legare specificamente una sequenza consensus TAATCT/C sul promotore di p53 nelle mMSCs allo stadio indifferenziato, e su quello di p21 in mMSCs indotte in senso osteogenico per 6 giorni. Questi dati supportano l’ipotesi che Otx1 sia coinvolto nel controllo dell’equilibrio fra proliferazione e senescenza e prospettano un modello dinamico in cui l’omeoproteina cambia i suoi targets d’azione, a seconda dello stadio differenziativo delle cellule in cui viene espresso. Inoltre ho analizzato l’espressione di una serie di geni implicati nell’osteogenesi in mMSCs indifferenziate, in seguito ad inattivazione e sovra-espressione di Otx1, e nelle MC3T3-E1, dopo trasfezione transiente del gene. I risultati hanno mostrato un effetto solo sull’attività trascrizionale di geni delle fasi precoci dell’osteogenesi, a livello delle mMSCs, mentre non influenzano le fasi tardive del processo. Tali risultati, rafforzati anche da dati preliminari di ChIP, suggeriscono che l’azione di Otx1 sia relegata prevalentemente alle fasi del commitment osteogenico. Questa ipotesi è avvalorata anche dall’osservazione che le MSCs Otx1-/- mostrano una capacità osteogenica ridotta, ma una differenziazione adipogenica aumentata rispetto alle cellule wt, sia in assenza che in presenza di induzione. Questo dato, sebbene debba essere confermato da analisi più approfondite, prospetta uno scenario in cui Otx1 può agire da “interruttore molecolare” (molecular switch) che “accende” il destino osteogenico e “spegne” quello adipogenico. In conclusione, Otx1 è coinvolto nel controllo della sopravvivenza/proliferazione e differenziazione delle MSCs e nella vita adulta gioca un ruolo di importante regolatore di cellule staminali e progenitori di diversi tessuti
Expression, purification, and characterisation of human soluble Epoxide Hydrolase (hsEH), and of its functional C-terminal domain
Isothermal Titration Calorimetry Enables Rapid Characterization of Enzyme Kinetics and Inhibition for the Human Soluble Epoxide Hydrolase
Isothermal titration calorimetry (ITC) is conventionally used to acquire thermodynamic data for biological interactions. In recent years, ITC has emerged as a powerful tool to characterise enzyme kinetics. In this study, we have adapted a single-injection method (SIM) to study the kinetics of human soluble epoxide hydrolase (hsEH), an enzyme involved in cardiovascular homeostasis, hypertension, nociception and insulin sensitivity through the metabolism of epoxy-fatty acids (EpFAs). In the SIM method, the rate of reaction is determined by monitoring the thermal power while the substrate is being depleted, overcoming the need for synthetic substrates, and reducing postreaction processing. Our results show that ITC enables the detailed, rapid and reproducible characterisation of the hsEH-mediated hydrolysis of several natural EpFA substrates. Furthermore, we have applied a variant of the single-injection ITC method for the detailed description of enzyme inhibition, proving the power of this approach in the rapid screening and discovery of new hsEH inhibitors using the enzyme’s physiological substrates. The methods described herein will enable further studies on EpFAs metabolism and biology, as well as drug discovery investigations to identify and characterise hsEH inhibitors. This also promises to provide a general approach for the characterisation of lipid catalysis, given the challenges that lipid metabolism studies pose to traditional spectroscopic techniques
Allosteric Regulation of the Soluble Epoxide Hydrolase by Nitro Fatty Acids:a Combined Experimental and Computational Approach: Allosteric regulation of hsEH by nitro fatty acids
The human soluble epoxide hydrolase (hsEH) is a key regulator of epoxy fatty acid (EpFA) metabolism. Inhibition of sEH can maintain endogenous levels of beneficial EpFAs and reduce the levels of their corresponding diol products, thus ameliorating a variety of pathological conditions including cardiovascular, central nervous system and metabolic diseases. The quest for orthosteric drugs that bind directly to the catalytic crevice of hsEH has been prolonged and sustained over the past decades, but the disappointing outcome of clinical trials to date warrants alternative pharmacological approaches. Previously, we have shown that hsEH can be allosterically inhibited by the endogenous electrophilic lipid 15-deoxy-Δ12,14-Prostaglandin-J2, via covalent adduction to two cysteines, C423 and C522. In this study, we explore the properties and behaviour of three electrophilic lipids belonging to the class of the nitro fatty acids, namely 9- and 10-nitrooleate and 10-nitrolinoleate. Biochemical and biophysical investigations revealed that, in addition to C423 and C522, nitro fatty acids can covalently bind to additional nucleophilic residues in hsEH C-terminal domain (CTD), two of which predicted in this study to be latent allosteric sites. Systematic mapping of the protein mutational space and evaluation of possible propagation pathways delineated selected residues, both in the allosteric patches and in other regions of the enzyme, envisaged to play a role in allosteric signalling. The responses elicited by the ligands on the covalent adduction sites supports future fragment-based design studies of new allosteric effectors for hsEH with increased efficacy and selectivity.</p
Affinity-enhanced RNA-binding domains as tools to understand RNA recognition
13 p.-4 fig.-1 graph. abst.Understanding how the RNA-binding domains of a protein regulator are used to recognize its RNA targets is a key problem in RNA biology, but RNA-binding domains with very low affinity do not perform well in the methods currently available to characterize protein-RNA interactions. Here, we propose to use conservative mutations that enhance the affinity of RNA-binding domains to overcome this limitation. As a proof of principle, we have designed and validated an affinity-enhanced K-homology (KH) domain mutant of the fragile X syndrome protein FMRP, a key regulator of neuronal development, and used this mutant to determine the domain’s sequence preference and to explain FMRP recognition of specific RNA motifs in the cell. Our results validate our concept and our nuclear magnetic resonance (NMR)-based workflow. While effective mutant design requires an understanding of the underlying principles of RNA recognition by the relevant domain type, we expect the method will be used effectively in many RNA-binding domains.This work was supported by the UK Medical Research Council (MC_PC_13051, U117574558, and MR/S000305/1) and the UK BBRSC research grant S014438/1. It was also supported by University College London and by the Francis Crick Institute, which receives its core funding from Cancer Research UK (CC2029), the UK Medical Research Council (CC2029), and the Wellcome Trust (CC2029). NMR spectra were recorded at the MRC Biomedical NMR Facility Francis Crick Institute UK, which is funded by Cancer Research UK (CC1078), the UK Medical Research Council (CC1078), and the Wellcome Trust (CC1078) and at UCL NMR facilities.Peer reviewe
Development of a specific and potent IGF2BP1 inhibitor: A promising therapeutic agent for IGF2BP1-expressing cancers
IGF2BP1 is a protein that controls the stability, localization, and translation of various mRNA targets. Poor clinical outcomes in numerous cancer types have been associated with its overexpression. As it has been demonstrated to impede tumor growth and metastasis in animal models, inhibiting IGF2BP1 function is a promising strategy for combating cancer. A lead chemical, 7773, which specifically decreased IGF2BP1 RNA binding and cellular activities, was previously identified in a high-throughput screen for effective IGF2BP1 inhibitors. Additional optimization of 7773 described in this manuscript led to the discovery of six compounds that performed equally well or better than 7773. In cell lines with high levels of endogenous IGF2BP1, one of 7773 derivatives, AVJ16, was found to be most efficient at preventing cell migration. Further, AVJ16 was found to be IGF2BP1-specific because it had no effect on cell lines that expressed little or no IGF2BP1 protein. The direct binding of AVJ16 to IGF2BP1 was validated by binding tests, with a 12-fold increase in binding efficiency over the lead compound. AVJ16 was shown to bind to a hydrophobic region at the protein's KH34 di-domain interface between the KH3 and KH4 domains. Overall, the findings imply that AVJ16 is a potent and specific inhibitor of IGF2BP1 activity
Direct m6A recognition by IMP1 underlays an alternative model of target selection for non-canonical methyl-readers
PDXK mutations cause polyneuropathy responsive to pyridoxal 5'-phosphate supplementation
Objective: To identify disease-causing variants in autosomal recessive axonal polyneuropathy with optic atrophy and provide targeted replacement therapy. Methods: We performed genome-wide sequencing, homozygosity mapping, and segregation analysis for novel disease-causing gene discovery. We used circular dichroism to show secondary structure changes and isothermal titration calorimetry to investigate the impact of variants on adenosine triphosphate (ATP) binding. Pathogenicity was further supported by enzymatic assays and mass spectroscopy on recombinant protein, patient-derived fibroblasts, plasma, and erythrocytes. Response to supplementation was measured with clinical validated rating scales, electrophysiology, and biochemical quantification. Results: We identified biallelic mutations in PDXK in 5 individuals from 2 unrelated families with primary axonal polyneuropathy and optic atrophy. The natural history of this disorder suggests that untreated, affected individuals become wheelchair-bound and blind. We identified conformational rearrangement in the mutant enzyme around the ATP-binding pocket. Low PDXK ATP binding resulted in decreased erythrocyte PDXK activity and low pyridoxal 5'-phosphate (PLP) concentrations. We rescued the clinical and biochemical profile with PLP supplementation in 1 family, improvement in power, pain, and fatigue contributing to patients regaining their ability to walk independently during the first year of PLP normalization. Interpretation: We show that mutations in PDXK cause autosomal recessive axonal peripheral polyneuropathy leading to disease via reduced PDXK enzymatic activity and low PLP. We show that the biochemical profile can be rescued with PLP supplementation associated with clinical improvement. As B6 is a cofactor in diverse essential biological pathways, our findings may have direct implications for neuropathies of unknown etiology characterized by reduced PLP levels.ANN NEUROL 2019;86:225-24