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1.
Biochem Soc Trans ; 50(1): 473-485, 2022 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-35129615

RESUMO

Due to its fundamental role in all eukaryotic cells, a deeper understanding of the molecular mechanisms underlying ubiquitination is of central importance. Being responsible for chain specificity and substrate recognition, E3 ligases are the selective elements of the ubiquitination process. In this review, we discuss different cellular pathways regulated by one of the first identified E3 ligase, NEDD4, focusing on its pathophysiological role, its known targets and modulators. In addition, we highlight small molecule inhibitors that act on NEDD4 and discuss new strategies to effectively target this E3 enzyme.


Assuntos
Complexos Endossomais de Distribuição Requeridos para Transporte , Ubiquitina , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Ubiquitina-Proteína Ligases Nedd4/metabolismo , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação
2.
Cell Rep ; 42(8): 113001, 2023 08 29.
Artigo em Inglês | MEDLINE | ID: mdl-37590133

RESUMO

Tissue fluidification and collective motility are pivotal in regulating embryonic morphogenesis, wound healing, and tumor metastasis. These processes frequently require that each cell constituent of a tissue coordinates its migration activity and directed motion through the oriented extension of lamellipodium cell protrusions, promoted by RAC1 activity. While the upstream RAC1 regulators in individual migratory cells or leader cells during invasion or wound healing are well characterized, how RAC1 is controlled in follower cells remains unknown. Here, we identify a MYO6-DOCK7 axis essential for spatially restricting RAC1 activity in a planar polarized fashion in model tissue monolayers. The MYO6-DOCK7 axis specifically controls the extension of cryptic lamellipodia required to drive tissue fluidification and cooperative-mode motion in otherwise solid and static carcinoma cell collectives.


Assuntos
Mama , Pseudópodes , Cicatrização , Movimento (Física)
3.
bioRxiv ; 2023 Jan 23.
Artigo em Inglês | MEDLINE | ID: mdl-36747801

RESUMO

Tissue fluidification and collective motility are pivotal in regulating embryonic morphogenesis, wound healing and tumor metastasis. These processes frequently require that each cell constituent of a tissue coordinates its migration activity and directed motion through the oriented extension of lamellipodia cell protrusions, promoted by RAC1 activity. While the upstream RAC1 regulators in individual migratory cells or leader cells during invasion or wound healing are well characterized, how RAC1 is controlled in follower cells remains unknown. Here, we identify a novel MYO6-DOCK7 axis that is critical for spatially restriction of RAC1 activity in a planar polarized fashion in model tissue monolayers. The MYO6-DOCK7 axis specifically controls the extension of cryptic lamellipodia required to drive tissue fluidification and cooperative mode motion in otherwise solid and static carcinoma cell collectives. Highlights: Collective motion of jammed epithelia requires myosin VI activityThe MYO6-DOCK7 axis is critical to restrict the activity of RAC1 in a planar polarized fashionMYO6-DOCK7-RAC1 activation ensures long-range coordination of movements by promoting orientation and persistence of cryptic lamellipodiaMyosin VI overexpression is exploited by infiltrating breast cancer cells.

4.
Cell Rep Med ; 4(11): 101266, 2023 11 21.
Artigo em Inglês | MEDLINE | ID: mdl-37944530

RESUMO

The spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has fueled the COVID-19 pandemic with its enduring medical and socioeconomic challenges because of subsequent waves and long-term consequences of great concern. Here, we chart the molecular basis of COVID-19 pathogenesis by analyzing patients' immune responses at single-cell resolution across disease course and severity. This approach confirms cell subpopulation-specific dysregulation in COVID-19 across disease course and severity and identifies a severity-associated activation of the receptor for advanced glycation endproducts (RAGE) pathway in monocytes. In vitro THP1-based experiments indicate that monocytes bind the SARS-CoV-2 S1-receptor binding domain (RBD) via RAGE, pointing to RAGE-Spike interaction enabling monocyte infection. Thus, our results demonstrate that RAGE is a functional receptor of SARS-CoV-2 contributing to COVID-19 severity.


Assuntos
COVID-19 , Humanos , Monócitos , Pandemias , Receptor para Produtos Finais de Glicação Avançada/genética , SARS-CoV-2
5.
Front Physiol ; 10: 370, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31001145

RESUMO

Ubiquitination plays a pivotal role in several cellular processes and is critical for protein degradation and signaling. E3 ubiquitin ligases are the matchmakers in the ubiquitination cascade, responsible for substrate recognition. In order to achieve selectivity and specificity on their substrates, HECT E3 enzymes are tightly regulated and exert their function in a spatially and temporally controlled fashion in the cells. These characteristics made HECT E3s intriguing targets in drug discovery in the context of cancer biology.

6.
Methods Enzymol ; 619: 293-318, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30910026

RESUMO

In this chapter, we describe techniques to detect ubiquitination events occurring in vivo. We focus on methodologies capable of preserving and detecting target protein ubiquitination in physiological conditions, without overexpressing a tagged version of ubiquitin. We provide detailed protocols for mammalian and Drosophila melanogaster systems using linkage-specific antibodies against ubiquitin. We analyze immunoblotting and immunofluorescence approaches as well as an ELISA-based quantitative approach, which allows comparing more samples and conditions. Finally, we describe the use of in vitro ubiquitination assays with purified proteins that allows a direct analysis of the molecular mechanisms behind specific E3 activity.


Assuntos
Ensaio de Imunoadsorção Enzimática/métodos , Imunofluorescência/métodos , Ubiquitinação , Animais , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Células HeLa , Humanos , Immunoblotting/métodos , Poliubiquitina/metabolismo , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo
7.
Biomolecules ; 9(7)2019 07 17.
Artigo em Inglês | MEDLINE | ID: mdl-31319543

RESUMO

Ubiquitin binding domains (UBDs) are modular elements that bind non-covalently to ubiquitin and act as downstream effectors and amplifiers of the ubiquitination signal. With few exceptions, UBDs recognize the hydrophobic path centered on Ile44, including residues Leu8, Ile44, His68, and Val70. A variety of different orientations, which can be attributed to specific contacts between each UBD and surface residues surrounding the hydrophobic patch, specify how each class of UBD specifically contacts ubiquitin. Here, we describe the structural model of a novel ubiquitin-binding domain that we identified in NEDD4 binding protein 1 (N4BP1). By performing protein sequence analysis, mutagenesis, and nuclear magnetic resonance (NMR) spectroscopy of the 15N isotopically labeled protein, we demonstrate that a Phe-Pro motif in N4BP1 recognizes the canonical hydrophobic patch of ubiquitin. This recognition mode resembles the molecular mechanism evolved in the coupling of ubiquitin conjugation to endoplasmic-reticulum (ER) degradation (CUE) domain family, where an invariant proline, usually following a phenylalanine, is required for ubiquitin binding. Interestingly, this novel UBD, which is not evolutionary related to CUE domains, shares a 40% identity and 47% similarity with cullin binding domain associating with NEDD8 (CUBAN), a protein module that also recognizes the ubiquitin-like NEDD8. Based on these features, we dubbed the region spanning the C-terminal 50 residues of N4BP1 the CoCUN domain, for Cousin of CUBAN. By performing circular dichroism and 15N NMR chemical shift perturbation of N4BP1 in complex with ubiquitin, we demonstrate that the CoCUN domain lacks the NEDD8 binding properties observed in CUBAN. We also show that, in addition to mediating the interaction with ubiquitin and ubiquitinated substrates, both CUBAN and CoCUN are poly-ubiquitinated in cells. The structural and the functional characterization of this novel UBD can contribute to a deeper understanding of the molecular mechanisms governing N4BP1 function, providing at the same time a valuable tool for clarifying how the discrimination between ubiquitin and the highly related NEDD8 is achieved.


Assuntos
Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Ubiquitina/metabolismo , Sequência de Aminoácidos , Biologia Computacional , Humanos , Espectroscopia de Ressonância Magnética , Ligação Proteica , Estrutura Terciária de Proteína , Ubiquitinação/fisiologia
9.
Nat Commun ; 10(1): 4974, 2019 10 31.
Artigo em Inglês | MEDLINE | ID: mdl-31672988

RESUMO

Clathrin light chains (CLCa and CLCb) are major constituents of clathrin-coated vesicles. Unique functions for these evolutionary conserved paralogs remain elusive, and their role in clathrin-mediated endocytosis in mammalian cells is debated. Here, we find and structurally characterize a direct and selective interaction between CLCa and the long isoform of the actin motor protein myosin VI, which is expressed exclusively in highly polarized tissues. Using genetically-reconstituted Caco-2 cysts as proxy for polarized epithelia, we provide evidence for coordinated action of myosin VI and CLCa at the apical surface where these proteins are essential for fission of clathrin-coated pits. We further find that myosin VI and Huntingtin-interacting protein 1-related protein (Hip1R) are mutually exclusive interactors with CLCa, and suggest a model for the sequential function of myosin VI and Hip1R in actin-mediated clathrin-coated vesicle budding.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Cadeias Leves de Clatrina/metabolismo , Vesículas Revestidas por Clatrina/metabolismo , Invaginações Revestidas da Membrana Celular/metabolismo , Proteínas dos Microfilamentos/metabolismo , Cadeias Pesadas de Miosina/metabolismo , Actinas/metabolismo , Células CACO-2 , Técnicas de Cultura de Células , Cadeias Leves de Clatrina/ultraestrutura , Cistos , Endocitose , Humanos , Espectroscopia de Ressonância Magnética , Cadeias Pesadas de Miosina/ultraestrutura , Ligação Proteica , Conformação Proteica , Isoformas de Proteínas
10.
Elife ; 52016 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-26744780

RESUMO

The neuronal DNA-/RNA-binding protein Pur-alpha is a transcription regulator and core factor for mRNA localization. Pur-alpha-deficient mice die after birth with pleiotropic neuronal defects. Here, we report the crystal structure of the DNA-/RNA-binding domain of Pur-alpha in complex with ssDNA. It reveals base-specific recognition and offers a molecular explanation for the effect of point mutations in the 5q31.3 microdeletion syndrome. Consistent with the crystal structure, biochemical and NMR data indicate that Pur-alpha binds DNA and RNA in the same way, suggesting binding modes for tri- and hexanucleotide-repeat RNAs in two neurodegenerative RNAopathies. Additionally, structure-based in vitro experiments resolved the molecular mechanism of Pur-alpha's unwindase activity. Complementing in vivo analyses in Drosophila demonstrated the importance of a highly conserved phenylalanine for Pur-alpha's unwinding and neuroprotective function. By uncovering the molecular mechanisms of nucleic-acid binding, this study contributes to understanding the cellular role of Pur-alpha and its implications in neurodegenerative diseases.


Assuntos
DNA de Cadeia Simples/química , DNA de Cadeia Simples/metabolismo , Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Conformação de Ácido Nucleico , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Animais , Cristalografia por Raios X , Drosophila , Deleção de Genes , Teste de Complementação Genética , Modelos Moleculares , Ligação Proteica , Conformação Proteica
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