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1.
EBioMedicine ; 87: 104390, 2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36584595

RESUMO

BACKGROUND: The COVID-19 pandemic is an infectious disease caused by SARS-CoV-2. The first step of SARS-CoV-2 infection is the recognition of angiotensin-converting enzyme 2 (ACE2) receptors by the receptor-binding domain (RBD) of the viral Spike (S) glycoprotein. Although the molecular and structural bases of the SARS-CoV-2-RBD/hACE2 interaction have been thoroughly investigated in vitro, the relationship between hACE2 expression and in vivo infection is less understood. METHODS: Here, we developed an efficient SARS-CoV-2-RBD binding assay suitable for super resolution microscopy and simultaneous hACE2 immunodetection and mapped the correlation between hACE2 receptor abundance and SARS-CoV-2-RBD binding, both in vitro and in human lung biopsies. Next, we explored the specific proteome of SARS-CoV-2-RBD/hACE2 through a comparative mass spectrometry approach. FINDINGS: We found that only a minority of hACE2 positive spots are actually SARS-CoV-2-RBD binding sites, and that the relationship between SARS-CoV-2-RBD binding and hACE2 presence is variable, suggesting the existence of additional factors. Indeed, we found several interactors that are involved in receptor localization and viral entry and characterized one of them: SLC1A5, an amino acid transporter. High-resolution receptor-binding studies showed that co-expression of membrane-bound SLC1A5 with hACE2 predicted SARS-CoV-2 binding and entry better than hACE2 expression alone. SLC1A5 depletion reduces SARS-CoV-2 binding and entry. Notably, the Omicron variant is more efficient in binding hACE2 sites, but equally sensitive to SLC1A5 downregulation. INTERPRETATION: We propose a method for mapping functional SARS-CoV-2 receptors in vivo. We confirm the existence of hACE2 co-factors that may contribute to differential sensitivity of cells to infection. FUNDING: This work was supported by an unrestricted grant from "Fondazione Romeo ed Enrica Invernizzi" to Stefano Biffo and by AIRC under MFAG 2021 - ID. 26178 project - P.I. Manfrini Nicola.


Assuntos
COVID-19 , SARS-CoV-2 , Humanos , SARS-CoV-2/metabolismo , Internalização do Vírus , Pandemias , Receptores Virais/química , Receptores Virais/metabolismo , Ligação Proteica , Pulmão/metabolismo , Antígenos de Histocompatibilidade Menor/metabolismo , Sistema ASC de Transporte de Aminoácidos/metabolismo
2.
iScience ; 25(5): 104239, 2022 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-35434540

RESUMO

Developing strategies against the SARS-CoV-2 is currently a main research subject. SARS-CoV-2 infects host cells by binding to human ACE2 receptors. Both, virus and ACE2, are highly glycosylated, and exploiting glycans of the SARS-CoV-2 envelope as binding sites for ACE2 represents a virus strategy for attacking the human host. We report here that a family of mannose-binding synthetic carbohydrate-binding agents (CBAs) inhibits SARS-CoV-2 infection, showing broad neutralizing activity vs. several variants of the spike protein. Preliminary tests indicated that the investigated CBAs interact with the spike protein rather than with ACE2. For a lead compound (IDS060), which has been selected among others for its lack of cytotoxicity, evidence of binding to the RBD of the spike protein has been found by NMR experiments, while competitive binding assays in the presence of IDS060 showed inhibition of binding of RBD to hACE2, although neutralizing activity was also observed with variants showing reduced or depleted binding.

3.
Cell Rep Methods ; 2(12): 100367, 2022 12 19.
Artigo em Inglês | MEDLINE | ID: mdl-36590694

RESUMO

Stem cell engineering of striatal medium spiny neurons (MSNs) is a promising strategy to understand diseases affecting the striatum and for cell-replacement therapies in different neurological diseases. Protocols to generate cells from human pluripotent stem cells (PSCs) are scarce and how well they recapitulate the endogenous fetal cells remains poorly understood. We have developed a protocol that modulates cell seeding density and exposure to specific morphogens that generates authentic and functional D1- and D2-MSNs with a high degree of reproducibility in 25 days of differentiation. Single-cell RNA sequencing (scRNA-seq) shows that our cells can mimic the cell-fate acquisition steps observed in vivo in terms of cell type composition, gene expression, and signaling pathways. Finally, by modulating the midkine pathway we show that we can increase the yield of MSNs. We expect that this protocol will help decode pathogenesis factors in striatal diseases and eventually facilitate cell-replacement therapies for Huntington's disease (HD).


Assuntos
Neurônios Espinhosos Médios , Células-Tronco Pluripotentes , Humanos , Reprodutibilidade dos Testes , Neurogênese , Corpo Estriado , Células-Tronco Pluripotentes/metabolismo
4.
Nat Genet ; 52(12): 1397-1411, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33169020

RESUMO

The genetic elements required to tune gene expression are partitioned in active and repressive nuclear condensates. Chromatin compartments include transcriptional clusters whose dynamic establishment and functioning depend on multivalent interactions occurring among transcription factors, cofactors and basal transcriptional machinery. However, how chromatin players contribute to the assembly of transcriptional condensates is poorly understood. By interrogating the effect of KMT2D (also known as MLL4) haploinsufficiency in Kabuki syndrome, we found that mixed lineage leukemia 4 (MLL4) contributes to the assembly of transcriptional condensates through liquid-liquid phase separation. MLL4 loss of function impaired Polycomb-dependent chromatin compartmentalization, altering the nuclear architecture. By releasing the nuclear mechanical stress through inhibition of the mechanosensor ATR, we re-established the mechanosignaling of mesenchymal stem cells and their commitment towards chondrocytes both in vitro and in vivo. This study supports the notion that, in Kabuki syndrome, the haploinsufficiency of MLL4 causes an altered functional partitioning of chromatin, which determines the architecture and mechanical properties of the nucleus.


Assuntos
Anormalidades Múltiplas/genética , Núcleo Celular/fisiologia , Cromatina/metabolismo , Face/anormalidades , Haploinsuficiência/genética , Doenças Hematológicas/genética , Histona-Lisina N-Metiltransferase/genética , Doenças Vestibulares/genética , Células 3T3 , Animais , Linhagem Celular , Linhagem da Célula/genética , Condrócitos/citologia , Condrogênese/genética , Regulação da Expressão Gênica/genética , Células HEK293 , Humanos , Mecanotransdução Celular/fisiologia , Células-Tronco Mesenquimais/citologia , Camundongos , Osteócitos/citologia , Osteogênese/genética , Proteínas do Grupo Polycomb/genética , Estresse Mecânico
5.
Neuron ; 104(2): 271-289.e13, 2019 10 23.
Artigo em Inglês | MEDLINE | ID: mdl-31515109

RESUMO

Mutations in one SETD5 allele are genetic causes of intellectual disability and autistic spectrum disorders. However, the mechanisms by which SETD5 regulates brain development and function remain largely elusive. Herein, we found that Setd5 haploinsufficiency impairs the proliferative dynamics of neural progenitors and synaptic wiring of neurons, ultimately resulting in behavioral deficits in mice. Mechanistically, Setd5 inactivation in neural stem cells, zebrafish, and mice equally affects genome-wide levels of H3K36me3 on active gene bodies. Notably, we demonstrated that SETD5 directly deposits H3K36me3, which is essential to allow on-time RNA elongation dynamics. Hence, Setd5 gene loss leads to abnormal transcription, with impaired RNA maturation causing detrimental effects on gene integrity and splicing. These findings identify SETD5 as a fundamental epigenetic enzyme controlling the transcriptional landscape in neural progenitors and their derivatives and illuminate the molecular events that connect epigenetic defects with neuronal dysfunctions at the basis of related human diseases.


Assuntos
Encéfalo/embriologia , Cromatina/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/genética , Código das Histonas/genética , Metiltransferases/genética , Proteínas de Peixe-Zebra/fisiologia , Animais , Comportamento Animal , Encéfalo/metabolismo , Sequenciamento de Cromatina por Imunoprecipitação , Cognição , Epigênese Genética , Histona Metiltransferases/genética , Metiltransferases/fisiologia , Camundongos , Mutação , Células-Tronco Neurais/metabolismo , Splicing de RNA/genética , RNA-Seq , Comportamento Social , Elongação da Transcrição Genética , Peixe-Zebra , Proteínas de Peixe-Zebra/genética
6.
Nat Commun ; 9(1): 3921, 2018 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-30237396

RESUMO

The original version of this Article contained an error in the spelling of the author Miriam Gaggianesi, which was incorrectly given as Miriam Giaggianesi. Furthermore, the affiliation details for Gabriella Gaudioso, Valentina Vaira, and Silvano Bosari incorrectly omitted 'Division of Pathology, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milan, 20122, Italy'. Finally, the affiliation details for Alice Turdo, Miriam Gaggianesi, Aurora Chinnici and Elisa Lipari were incorrectly given as 'Dipartimento di Biotecnologie Mediche e Medicina Legale Sezione di Biochimica Medica, Facoltà di Medicina e Chirurgia, Policlinico "P.Giaccone", Università di Palermo, Palermo, 90127, Italy'. The correct affiliation is 'Department of Surgical, Oncological and Stomatological Sciences, University of Palermo, Palermo, 90127, Italy'. These errors have now been corrected in both the PDF and HTML versions of the Article.

7.
Nat Commun ; 9(1): 1024, 2018 03 09.
Artigo em Inglês | MEDLINE | ID: mdl-29523784

RESUMO

Breast cancer consists of highly heterogeneous tumors, whose cell of origin and driver oncogenes are difficult to be uniquely defined. Here we report that MYC acts as tumor reprogramming factor in mammary epithelial cells by inducing an alternative epigenetic program, which triggers loss of cell identity and activation of oncogenic pathways. Overexpression of MYC induces transcriptional repression of lineage-specifying transcription factors, causing decommissioning of luminal-specific enhancers. MYC-driven dedifferentiation supports the onset of a stem cell-like state by inducing the activation of de novo enhancers, which drive the transcriptional activation of oncogenic pathways. Furthermore, we demonstrate that the MYC-driven epigenetic reprogramming favors the formation and maintenance of tumor-initiating cells endowed with metastatic capacity. This study supports the notion that MYC-driven tumor initiation relies on cell reprogramming, which is mediated by the activation of MYC-dependent oncogenic enhancers, thus establishing a therapeutic rational for treating basal-like breast cancers.


Assuntos
Neoplasias da Mama/metabolismo , Epigênese Genética , Células-Tronco Neoplásicas/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/fisiopatologia , Carcinogênese , Linhagem Celular Tumoral , Reprogramação Celular , Elementos Facilitadores Genéticos , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Camundongos SCID , Células-Tronco Neoplásicas/citologia
8.
Nat Commun ; 7: 11903, 2016 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-27301576

RESUMO

Stem cell identity depends on the integration of extrinsic and intrinsic signals, which directly influence the maintenance of their epigenetic state. Although Myc transcription factors play a major role in stem cell self-renewal and pluripotency, their integration with signalling pathways and epigenetic regulators remains poorly defined. We addressed this point by profiling the gene expression and epigenetic pattern in ESCs whose growth depends on conditional Myc activity. Here we show that Myc potentiates the Wnt/ß-catenin signalling pathway, which cooperates with the transcriptional regulatory network in sustaining ESC self-renewal. Myc activation results in the transcriptional repression of Wnt antagonists through the direct recruitment of PRC2 on these targets. The consequent potentiation of the autocrine Wnt/ß-catenin signalling induces the transcriptional activation of the endogenous Myc family members, which in turn activates a Myc-driven self-reinforcing circuit. Thus, our data unravel a Myc-dependent self-propagating epigenetic memory in the maintenance of ESC self-renewal capacity.


Assuntos
Redes Reguladoras de Genes/genética , Células-Tronco Embrionárias Murinas/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Animais , Autorrenovação Celular/efeitos dos fármacos , Epigênese Genética/efeitos dos fármacos , Retroalimentação Fisiológica/efeitos dos fármacos , Redes Reguladoras de Genes/efeitos dos fármacos , Fator Inibidor de Leucemia/farmacologia , Camundongos , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/efeitos dos fármacos , Proteínas do Grupo Polycomb/metabolismo , Transcrição Gênica/efeitos dos fármacos , Via de Sinalização Wnt/efeitos dos fármacos , Via de Sinalização Wnt/genética
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