disfunzione mitocondriale e controllo di hif vhl 2018...

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Le prolil idrossilasi (PHD) hanno ruolo chiave e sono finemente regolate

Fe2+, which binds the proline substrate and the oxygen molecule, undergoes oxidation Ascorbate /glutathione maintains iron in the active site of PHDs in the reduced (ferrous) state.

g Emerging novel functions of the oxygen-sensing prolyl hydroxylase domain enzymes Brian W. Won

Prolyl hydroxylase domain enzyme (PHD) activityPHDs are α-ketoglutarate/2-oxoglutarate (2-OG)-dependent hydroxylase and the cofactors oxygen and iron to hydroxylate substrates

HIF-1α

2-oxaloglutarate

Inhibitory factors of PHD include the metabolic intermediates succinate and fumarate

reactive oxygen species (ROS) can disrupt oxygen interaction with PHDs. PCBP1 delivers Fe2+ to PHDs and iron to ferritin for intracellular iron storage

g Emerging novel functions of the oxygen-sensing prolyl hydroxylase domain enzymes Brian W. Won

Regulation of prolyl hydroxylase domain enzyme (PHD) activity

2-oxoglutarate

Concentrations of oxygen in tissues -range 10–30 μM-

below the Km for oxygen of the hydroxylases

Concentrations of oxygen is limiting for enzyme activity over the entire physiological range.

How to model Mitochondrial disease (1)

Isha H. Jain et al. Science 2016;352:54-61Published by AAAS

act as competitive antagonists of 2-oxoglutarate, a cofactor that accepts one oxygen from molecular dioxygen to become succinate as the second oxygen forms trans-4-hydroxyproline

….PHD inhibition


Immunoblot

Fig. 3 FG-4592 causes normoxic stabilization of HIF1α and rewires energy metabolism.


± FG-4592 under normoxia (21% O2) or hypoxia (1% O2) FG-4592 administration stabilizes HIF1α even during normoxia.

Immunoblot

FG-4592 treatment activates the HIF response in zebrafish embryos and alleviates death caused by Respiratoty Chain inhibition.

Isha H. Jain et al. Science 2016;352:54-61

FG-4592 treatment activates the HIF response in zebrafish embryos


HIF-responsive promoter -EGFP embryos

FG-4592 treatment activates the HIF response in zebrafish embryos


Known HIF targets glut1 and ldha1 are overexpressed in 96 hpf zebrafish embryos treated with FG-4592 for 6 hours

19 | 13Tymoczko et al., PRINCIPI DI BIOCHIMICA, Zanichelli editore S.p.A. Copyright © 2010

La fosforilazione ossidativa nel mitocondrio

Respiratoty Chain


Published by AAAS

Respiratoty Chain inhibition



RC inhibition by 2.5 nM antimycin in 4 days post fertilization (dpf) embryos results in significant death within the first 24 hours of treatmentCoexposure of antimycin with FG-4592 (2.5 μM) doubles embryo survival, whereas FG-4592 alone has no impact. Exposure to FG-4592 rescues antimycin-induced zebrafish embryonic death.

Antimycin=AntiRespiratoty Chain inhibition

Retinopathy of prematurity (ROP) causes 100,000 new cases of childhood blindness each year. ROP is initiated by oxygen supplementation necessary to prevent neonatal death.

hypoxia-inducible factor (HIF) stabilization via HIF prolyl hydroxylase inhibition using the isoquinolone Roxadustat

Roxadustat directs retinal HIF stabilization

Roxadustat rescued the HIF-1 knockout mouse from retinal oxygen toxicity

This provides a rationale for protecting the severely premature infant from oxygen toxicity.

Effect of Roxadustat on neural retina apoptosis.

George Hoppe et al. PNAS 2016;113:E2516-E2525

©2016 by National Academy of Sciences

FG-4592

Quantification of active caspase 3-positive cells demonstrates statistically significant reduction in apoptosis in the inner nuclear layer of animals treated with Roxadustat (RXD)

outer nuclear layer (ONL), inner nuclear layer (INL), and ganglion cell layer (GCL).

HIF e Metabolismo (Mitocondrio)

HIFa Control of Cell Metabolismfatty acid storage

Glucose consumptionand glycolysis


act as competitive antagonists of 2-oxoglutarate, a cofactor that accepts one oxygen from molecular dioxygen to become succinate as the second oxygen forms trans-4-hydroxyproline

….PHD inhibition


Published by AAAS

+Respiratoty Chain inhibition …..

Mutations in mitochondrial disease

The Journal of Pathology2 NOV 2016 DOI: 10.1002/path.4809http://onlinelibrary.wiley.com/doi/10.1002/path.4809/full#path4809-fig-0001

bold= genes with mutations



RC inhibition by 2.5 nM antimycin in 4 days post fertilization (dpf) embryos results in significant death within the first 24 hours of treatmentCoexposure of antimycin with FG-4592 (2.5 μM) doubles embryo survival, whereas FG-4592 alone has no impact. Exposure to FG-4592 rescues antimycin-induced zebrafish embryonic death.

Antimycin=AntiRespiratoty Chain inhibition

Programming the CRISPR (clustered regularly interspaced short palindromic repeats)–associated nuclease Cas9 to modify

specific genomic loci

Doudna et al., 2014

Lentiviral delivery of Cas9 and sgRNA provides efficient depletion of target genes

Ophir Shalem et al. Science 2014;343:84-87Published by AAAS

programming the CRISPR (clustered regularly interspaced short palindromic repeats)–associated nuclease Cas9 to modify specific genomic loci

synthetic single-guide RNA (sgRNA) targeted to specific coding regions of genes

Lentiviral expression vector for Cas9 and sgRNA (lentiCRISPR)

.


synthetic single-guide RNA (sgRNA) targeted to specific coding regions of genes

ability to simultaneously deliver Cas9 and sgRNA through a single vector enables application to any cell type of interest

puro, puromycin selection marker; psi+, psi packaging signal; RRE, rev response element; cPPT, central polypurine tract; EFS, elongation factor-1α short promoter; P2A, 2A self-cleaving peptide; WPRE, posttranscriptional regulatory element

Genome-scale Cas9-mediated knockout screen during states of mitochondrial dysfunction.

Published by AAAS

Mitochondrial disease was modeled with the addition of the complex III inhibitor antimycin (moderate disease) addition of antimycin and removal of pyruvate (severe disease).

Genome-scale Cas9-mediated knockout screen during states of mitochondrial dysfunction.


cells were infected with the genome-scale Cas9-mediated knockout library

3 conditions U untreated, M moderate disease, S severe disease Samples were taken at a pretreatment time point and after 3 weeks of selection

genome-scale negative selection screening


significantly depleted gene sets

18,080 genes in the human genome with an average coverage of 3 to 4 sgRNAs per gene

Genome-scale Cas9-mediated knockout screen identifies VHL inhibition as protective during states of mitochondrial dysfunction.

enrichment of sgRNAs in severe disease S relative to pretreatment conditions U

Genetic (CAS9) or small-molecule activation of the HIF response is protective against multiple forms of RC inhibition,


FG-4592 acts as competitive antagonists of 2-oxoglutarate, a cofactor that accepts one oxygen from molecular dioxygen to become succinate as the second oxygen forms trans-4-hydroxyproline

vhl KO activates the HIF response in zebrafish embryos and alleviates death caused by RC

inhibition.


Anti = Respiratory Chain inhibition

The Function of VHL and pVHL Protein

• Helps to regulate and destroy the alpha subunit of hypoxia-inducible factor or HIF-1

• HIF-1 is a transcription factor that has a myriad of target genes

• Products are involved in angiogenesis, erythropoiesis, energy metabolism, glucose transport

Normal VHL Function

Patients with a germline mutation in von Hippel-Lindau (VHL)

develop renal cell cancers and hypervascular tumors of the brain, adrenal glands, and pancreas as well as

erythrocytosis

These phenotypes are driven by aberrant expression of HIF2α, which induces expression of genes involved in cell proliferation, angiogenesis, and red blood cell production

Inactivation of vhl in zebrafish (vhl–/– ) led to constitutive activation of HIF2α

von Hippel-Lindau (VHL)

Mutations in mitochondrial disease

The Journal of Pathology2 NOV 2016 DOI: 10.1002/path.4809http://onlinelibrary.wiley.com/doi/10.1002/path.4809/full#path4809-fig-0001

bold= genes with mutations

Chronic hypoxia extends life span and alleviates disease in a mouse model of Leigh syndrome (KO) whereas chronic hyperoxia exacerbates disease.


Published by AAAS

mouse model of Leigh syndrome(KO) Ndufs4 -/- NADH:ubiquinone oxidoreductase subunit S4

Fig. 5 Chronic hypoxia extends life span and alleviates disease in a mouse model of Leigh syndrome (KO) whereas chronic hyperoxia exacerbates disease.


Published by AAAS

mouse model of Leigh syndrome(KO) Ndufs4 -/- NADH:ubiquinone oxidoreductase subunit S4

Breathing 11% O2 in late-stage neurological disease reverses pathological inflammation in the brains of Ndufs4 KO mice.

Michele Ferrari et al. PNAS 2017;114:E4241-E4250©2017 by National Academy of Sciences

KO mice breathing 21% O2 up to 55 d and then breathing 11% O2 to 160 d.

Genome-scale Cas9-mediated knockout screen and states of mitochondrial dysfunction.

enrichment of sgRNAs in severe disease S relative to pretreatment conditions U

Sin3A= global transcription regulators that provide a platform for chromatin-modifying activities

Within 24 h of reoxygenation, the hypoxia-induced transcription returned to basal levels and the nucleosome structure was reassembled in the hypoxia-inducible form.

Mutazioni nella Pathway oxygen sensing

Policitemia di Chuvash

- Policitemia autosomica recessiva trovata in Russia

Ang et al. Nature Genetics 2002

Sequenziamento gene von Hippel Lindau (VHL) C/T transition, Arg/Trp200 (Pazienti omozigoti)

Disruption of oxygen homeostasis underlies congenital Chuvash polycythemia

Sonny O. Ang

Nature genetics 2002, volume 32 no. 4 pp 614 - 621

Western blot, 5 pazienti + 5 controlli

- Livelli di proteina VHL normali in mutato e Wt

- Livelli di HIF1α maggiori nei soggetti affetti

20% O2:

Disruption of oxygen homeostasis underlies congenital Chuvash polycythemia

Sonny O. Ang

Nature genetics 2002, volume 32 no. 4 pp 614 - 621

V= controllo (Wild type)

H= eterozigote

P1= paziente (omozigote)

La forma ubiquitinizzata è meno presente nelle cellule del paziente

Mutazione Arg200Trp:

- Ridotta ubiquitinizzazione di HIF1α

- Aumentata espressione del gene Epo policitemia

pVHL is the substrate-recognizing component of a multiprotein E3 ubiquitin ligase complex containing elongins C and B, Cullin 2, and the RING-H2 finger protein Rbx-1

(B) Under low oxygen tension HIFa associates with HIFb. The heterodimer binds to a core consensus sequence at the promoters of HIF-responsive genes, and upon binding to the coactivators p300/CBP and PKM2, initiates transcription.The interaction between HIFa and p300 may be regulated by a variety of factors that to influence the transcriptional activity.

(PHD, prolyl-hydroxylase domain-containing enzyme; SIRT, sirtuin; FIH, factor inhibiting HIF; CBP, Creb-binding protein; OH, hydroxyl group; STAT3, signal transducer and activator of transcription 3; ub, ubiquitin moiety; EloB/C, elongins B and C; Cul2, cullin 2; pVHL, von Hippel-Lindau protein; ROS, reactive oxygen species; CITED2/4, CBP/p300 interacting transactivator; PKM2, pyruvatekinase isoform M2; hnRNPs, heterogeneous nuclear ribonucleoproteins).

disfunzione mitocondriale e controllo di hif vhl 2018...

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