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1.
Cell Death Differ ; 31(2): 217-238, 2024 02.
Artigo em Inglês | MEDLINE | ID: mdl-38238520

RESUMO

Selective removal of dysfunctional mitochondria via autophagy is crucial for the maintenance of cellular homeostasis. This event is initiated by the translocation of the E3 ubiquitin ligase Parkin to damaged mitochondria, and it requires the Serine/Threonine-protein kinase PINK1. In a coordinated set of events, PINK1 operates upstream of Parkin in a linear pathway that leads to the phosphorylation of Parkin, Ubiquitin, and Parkin mitochondrial substrates, to promote ubiquitination of outer mitochondrial membrane proteins. Ubiquitin-decorated mitochondria are selectively recruiting autophagy receptors, which are required to terminate the organelle via autophagy. In this work, we show a previously uncharacterized molecular pathway that correlates the activation of the Ca2+-dependent phosphatase Calcineurin to Parkin translocation and Parkin-dependent mitophagy. Calcineurin downregulation or genetic inhibition prevents Parkin translocation to CCCP-treated mitochondria and impairs stress-induced mitophagy, whereas Calcineurin activation promotes Parkin mitochondrial recruitment and basal mitophagy. Calcineurin interacts with Parkin, and promotes Parkin translocation in the absence of PINK1, but requires PINK1 expression to execute mitophagy in MEF cells. Genetic activation of Calcineurin in vivo boosts basal mitophagy in neurons and corrects locomotor dysfunction and mitochondrial respiratory defects of a Drosophila model of impaired mitochondrial functions. Our study identifies Calcineurin as a novel key player in the regulation of Parkin translocation and mitophagy.


Assuntos
Calcineurina , Proteínas de Drosophila , Animais , Calcineurina/metabolismo , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Mitofagia/genética , Mitocôndrias/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina/metabolismo , Drosophila/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo
2.
Haematologica ; 108(8): 2130-2145, 2023 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-36794499

RESUMO

Sustained ANKRD26 expression associated with germline ANKRD26 mutations causes thrombocytopenia 2 (THC2), an inherited platelet disorder associated with a predisposition to leukemia. Some patients also present with erythrocytosis and/or leukocytosis. Using multiple human-relevant in vitro models (cell lines, primary patients' cells and patient-derived induced pluripotent stem cells) we demonstrate for the first time that ANKRD26 is expressed during the early steps of erythroid, megakaryocyte and granulocyte differentiation, and is necessary for progenitor cell proliferation. As differentiation progresses, ANKRD26 expression is progressively silenced, to complete the cellular maturation of the three myeloid lineages. In primary cells, abnormal ANKRD26 expression in committed progenitors directly affects the proliferation/differentiation balance for the three cell types. We show that ANKRD26 interacts with and crucially modulates the activity of MPL, EPOR and G-CSFR, three homodimeric type I cytokine receptors that regulate blood cell production. Higher than normal levels of ANKRD26 prevent the receptor internalization that leads to increased signaling and cytokine hypersensitivity. These findings afford evidence how ANKRD26 overexpression or the absence of its silencing during differentiation is responsible for myeloid blood cell abnormalities in patients with THC2.


Assuntos
Leucemia , Receptores de Citocinas , Humanos , Citocinas , Hematopoese , Leucemia/patologia , Diferenciação Celular , Peptídeos e Proteínas de Sinalização Intercelular
3.
Blood ; 138(17): 1603-1614, 2021 10 28.
Artigo em Inglês | MEDLINE | ID: mdl-34115825

RESUMO

EZH2, the enzymatic component of PRC2, has been identified as a key factor in hematopoiesis. EZH2 loss-of-function mutations have been found in myeloproliferative neoplasms, particularly in myelofibrosis, but the precise function of EZH2 in megakaryopoiesis is not fully delineated. Here, we show that EZH2 inhibition by small molecules and short hairpin RNA induces megakaryocyte (MK) commitment by accelerating lineage marker acquisition without change in proliferation. Later in differentiation, EZH2 inhibition blocks proliferation and polyploidization and decreases proplatelet formation. EZH2 inhibitors similarly reduce MK polyploidization and proplatelet formation in vitro and platelet levels in vivo in a JAK2V617F background. In transcriptome profiling, the defect in proplatelet formation was associated with an aberrant actin cytoskeleton regulation pathway, whereas polyploidization was associated with an inhibition of expression of genes involved in DNA replication and repair and an upregulation of cyclin-dependent kinase inhibitors, particularly CDKN1A and CDKN2D. The knockdown of CDKN1A and to a lesser extent CDKN2D could partially rescue the percentage of polyploid MKs. Moreover, H3K27me3 and EZH2 chromatin immunoprecipitation assays revealed that CDKN1A is a direct EZH2 target and CDKN2D expression is not directly regulated by EZH2, suggesting that EZH2 controls MK polyploidization directly through CDKN1A and indirectly through CDKN2D.


Assuntos
Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Megacariócitos/citologia , Trombopoese , Animais , Plaquetas/citologia , Plaquetas/metabolismo , Células Cultivadas , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Humanos , Megacariócitos/metabolismo , Camundongos , Interferência de RNA , Transcriptoma
4.
Blood ; 138(6): 480-485, 2021 08 12.
Artigo em Inglês | MEDLINE | ID: mdl-34010413

RESUMO

Congenital amegakaryocytic thrombocytopenia (CAMT) is a severe inherited thrombocytopenia due to loss-of-function mutations affecting the thrombopoietin (TPO) receptor, MPL. Here, we report a new homozygous MPL variant responsible for CAMT in 1 consanguineous family. The propositus and her sister presented with severe thrombocytopenia associated with mild anemia. Next-generation sequencing revealed the presence of a homozygous MPLR464G mutation resulting in a weak cell-surface expression of the receptor in platelets. In cell lines, we observed a defect in MPLR464G maturation associated with its retention in the endoplasmic reticulum. The low cell-surface expression of MPLR464G induced very limited signaling with TPO stimulation, leading to survival and reduced proliferation of cells. Overexpression of a myeloproliferative neoplasm-associated calreticulin (CALR) mutant did not rescue trafficking of MPLR464G to the cell surface and did not induce constitutive signaling. However, it unexpectedly restored a normal response to eltrombopag (ELT), but not to TPO. This effect was only partially mimicked by the purified recombinant CALR mutant protein. Finally, the endogenous CALR mutant was able to restore the megakaryocyte differentiation of patient CD34+ cells carrying MPLR464G in response to ELT.


Assuntos
Benzoatos/farmacologia , Calreticulina , Síndrome Congênita de Insuficiência da Medula Óssea , Hidrazinas/farmacologia , Mutação de Sentido Incorreto , Pirazóis/farmacologia , Receptores de Trombopoetina , Trombocitopenia , Adulto , Substituição de Aminoácidos , Calreticulina/genética , Calreticulina/metabolismo , Criança , Pré-Escolar , Síndrome Congênita de Insuficiência da Medula Óssea/tratamento farmacológico , Síndrome Congênita de Insuficiência da Medula Óssea/genética , Síndrome Congênita de Insuficiência da Medula Óssea/metabolismo , Síndrome Congênita de Insuficiência da Medula Óssea/patologia , Feminino , Células HEK293 , Homozigoto , Humanos , Lactente , Masculino , Receptores de Trombopoetina/genética , Receptores de Trombopoetina/metabolismo , Trombocitopenia/tratamento farmacológico , Trombocitopenia/genética , Trombocitopenia/metabolismo , Trombocitopenia/patologia
5.
Small GTPases ; 12(5-6): 399-415, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33570449

RESUMO

Megakaryocytes (MKs) are the bone marrow (BM) cells that generate blood platelets by a process that requires: i) polyploidization responsible for the increased MK size and ii) cytoplasmic organization leading to extension of long pseudopods, called proplatelets, through the endothelial barrier to allow platelet release into blood. Low level of localized RHOA activation prevents actomyosin accumulation at the cleavage furrow and participates in MK polyploidization. In the platelet production, RHOA and CDC42 play opposite, but complementary roles. RHOA inhibits both proplatelet formation and MK exit from BM, whereas CDC42 drives the development of the demarcation membranes and MK migration in BM. Moreover, the RhoA or Cdc42 MK specific knock-out in mice and the genetic alterations in their down-stream effectors in human induce a thrombocytopenia demonstrating their key roles in platelet production. A better knowledge of Rho-GTPase signalling is thus necessary to develop therapies for diseases associated with platelet production defects.Abbreviations: AKT: Protein Kinase BARHGEF2: Rho/Rac Guanine Nucleotide Exchange Factor 2ARP2/3: Actin related protein 2/3BM: Bone marrowCDC42: Cell division control protein 42 homologCFU-MK: Colony-forming-unit megakaryocyteCIP4: Cdc42-interacting protein 4mDIA: DiaphanousDIAPH1; Protein diaphanous homolog 1ECT2: Epithelial Cell Transforming Sequence 2FLNA: Filamin AGAP: GTPase-activating proteins or GTPase-accelerating proteinsGDI: GDP Dissociation InhibitorGEF: Guanine nucleotide exchange factorHDAC: Histone deacetylaseLIMK: LIM KinaseMAL: Megakaryoblastic leukaemiaMARCKS: Myristoylated alanine-rich C-kinase substrateMKL: Megakaryoblastic leukaemiaMLC: Myosin light chainMRTF: Myocardin Related Transcription FactorOTT: One-Twenty Two ProteinPACSIN2: Protein Kinase C And Casein Kinase Substrate In Neurons 2PAK: P21-Activated KinasePDK: Pyruvate Dehydrogenase kinasePI3K: Phosphoinositide 3-kinasePKC: Protein kinase CPTPRJ: Protein tyrosine phosphatase receptor type JRAC: Ras-related C3 botulinum toxin substrate 1RBM15: RNA Binding Motif Protein 15RHO: Ras homologousROCK: Rho-associated protein kinaseSCAR: Suppressor of cAMP receptorSRF: Serum response factorSRC: SarcTAZ: Transcriptional coactivator with PDZ motifTUBB1: Tubulin ß1VEGF: Vascular endothelial growth factorWAS: Wiskott Aldrich syndromeWASP: Wiskott Aldrich syndrome proteinWAVE: WASP-family verprolin-homologous proteinWIP: WASP-interacting proteinYAP: Yes-associated protein.


Assuntos
Plaquetas/fisiologia , Citoesqueleto/fisiologia , Megacariócitos/fisiologia , Proteínas rho de Ligação ao GTP/metabolismo , Animais , Plaquetas/enzimologia , Citoesqueleto/enzimologia , Humanos , Megacariócitos/enzimologia , Transdução de Sinais
6.
Stem Cell Res ; 49: 102060, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33142254

RESUMO

The derivation of human pluripotent stem cell (iPSC) lines by in vitro reprogramming of somatic cells revolutionized research: iPSCs have been used for disease modeling, drug screening and regenerative medicine for many disorders, especially when combined with cutting-edge genome editing technologies. In hematology, malignant transformation is often a multi-step process, that starts with either germline or acquired genetic alteration, followed by progressive acquisition of mutations combined with the selection of one or more pre-existing clones. iPSCs are an excellent model to study the cooperation between different genetic alterations and to test relevant therapeutic drugs. In this review, we will describe the use of iPSCs for pathophysiological studies and drug testing in inherited and acquired hematological malignancies.


Assuntos
Neoplasias Hematológicas , Células-Tronco Pluripotentes Induzidas , Células-Tronco Pluripotentes , Desenvolvimento de Medicamentos , Edição de Genes , Neoplasias Hematológicas/genética , Humanos
7.
Cell Calcium ; 87: 102186, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32120195

RESUMO

The outer mitochondrial membrane protein VDAC interacts with the ER protein IP3R via chaperone Grp75 to form a molecular complex that couples mitochondria to the ER and contributes to functional mitochondria-ER contacts (MERCs), essential for efficient calcium (Ca2+) transfer. A new study by Liu et al. identifies the PD protein DJ-1 as a component of the IP3R-Grp75-VDAC complex. DJ-1 ablation impairs mitochondria-ER association and Ca2+ crosstalk, and impacts the stability of the trio.


Assuntos
Retículo Endoplasmático/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Proteínas de Membrana/metabolismo , Mitocôndrias/metabolismo , Proteína Desglicase DJ-1/metabolismo , Humanos , Ligação Proteica , Canal de Ânion 1 Dependente de Voltagem/metabolismo
8.
Pharmacol Res ; 138: 43-56, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30219582

RESUMO

Parkin, an E3 ubiquitin ligase and a Parkinson's disease (PD) related gene, translocates to impaired mitochondria and drives their elimination via autophagy, a process known as mitophagy. Mitochondrial pro-fusion protein Mitofusins (Mfn1 and Mfn2) were found to be a target for Parkin mediated ubiquitination. Mfns are transmembrane GTPase embedded in the outer membrane of mitochondria, which are required on adjacent mitochondria to mediate fusion. In mammals, Mfn2 also forms complexes that are capable of tethering mitochondria to endoplasmic reticulum (ER), a structural feature essential for mitochondrial energy metabolism, calcium (Ca2+) transfer between the organelles and Ca2+ dependent cell death. Despite its fundamental physiological role, the molecular mechanisms that control ER-mitochondria cross talk are obscure. Ubiquitination has recently emerged as a powerful tool to modulate protein function, via regulation of protein subcellular localization and protein ability to interact with other proteins. Ubiquitination is also a reversible mechanism, which can be actively controlled by opposing ubiquitination-deubiquitination events. In this work we found that in Parkin deficient cells and parkin mutant human fibroblasts, the tether between ER and mitochondria is decreased. We identified the site of Parkin dependent ubiquitination and showed that the non-ubiquitinatable Mfn2 mutant fails to restore ER-mitochondria physical and functional interaction. Finally, we took advantage of an established in vivo model of PD to demonstrate that manipulation of ER-mitochondria tethering by expressing an ER-mitochondria synthetic linker is sufficient to rescue the locomotor deficit associated to an in vivo Drosophila model of PD.


Assuntos
Retículo Endoplasmático/fisiologia , GTP Fosfo-Hidrolases/fisiologia , Mitocôndrias/fisiologia , Proteínas Mitocondriais/fisiologia , Doença de Parkinson/fisiopatologia , Ubiquitina-Proteína Ligases/fisiologia , Animais , Drosophila , Feminino , Fibroblastos/metabolismo , Células HEK293 , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Interferência de RNA , RNA Interferente Pequeno/genética , Ubiquitinação
9.
Biol Direct ; 12(1): 6, 2017 02 21.
Artigo em Inglês | MEDLINE | ID: mdl-28222786

RESUMO

Mutations in the gene encoding for the E3 ubiquitin ligase Parkin are associated to a rare form of familiar autosomal recessive Parkinsonism. Despite decades of research on the Parkin protein, whose structure has been recently solved, little is known about the specific signalling pathways that lead to Parkin activation. Parkin activity spans from mitochondria quality control to tumor suppression and stress protection; it is thus tempting to hypothesize that the broad impact of Parkin on cellular physiology might be the result of different post translational modifications that can be controlled by balanced opposing events. Sequence alignment of Parkin from different species indicates high homology between domains across Parkin orthologs and identifies highly conserved amino acid residues that, if modified, impinge on Parkin functions. In this review, we summarize findings on post translational modifications that have been shown to affect Parkin activity and stability. REVIEWERS: This article was reviewed by Prof. Dr. Konstanze F. Winklhofer and by Prof. Thomas Simmen. Both reviewers have been nominated by Professor Luca Pellegrini.


Assuntos
Processamento de Proteína Pós-Traducional , Ubiquitina-Proteína Ligases/metabolismo , Animais , Humanos , Camundongos , Modelos Moleculares , Fosforilação , Estabilidade Proteica , Alinhamento de Sequência , Transdução de Sinais , Ubiquitina-Proteína Ligases/química , Ubiquitinação
10.
JIMD Rep ; 28: 119-126, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26589310

RESUMO

Hyperornithinemia-hyperammonemia-homocitrullinuria (HHH) syndrome is an autosomal recessive metabolic disorder usually presenting in the neonatal period with intermittent episodes of hyperammonemia, psychomotor delay, and progressive encephalopathy. Adult cases usually evolve into frank spastic paraparesis. The syndrome is caused by mutations in SLC25A15/ORNT1 encoding the mitochondrial ornithine transporter; a second ornithine transporter, ORNT2 of unknown function, is also present in most placental mammals. ORNT2 is believed to originate from an ancient retro-transposition event. In yeast Saccharomyces cerevisiae the major function of the transporter (encoded by Arg11) is to shuttle ornithine from the mitochondrial matrix to the cytosol. Its inactivation abolishes growth in the absence of arginine.In this work, we used functional complementation in S. cerevisiae to characterize the function of human ORNT2 and to test the pathogenicity of ORNT1 mutations found in HHH patients. Notably, we found that human ORNT1 but not ORNT2 complements the deletion of the yeast gene, despite their high level of homology. However, we identified some key residues in ORNT2, which may recover its functional competence when replaced with the corresponding residues of ORNT1, suggesting that roles of the two transporters are different. Moreover, we used this system to test a series of missense mutations of ORNT1 identified in patients with HHH syndrome. All mutations had a detrimental effect on the functionality of the human gene, without however clear genotype-phenotype correlations. Our data support yeast as a simple and effective model to validate missense mutations occurring in patients with HHH.

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