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1.
J Biol Chem ; 300(6): 107374, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38762180

RESUMO

The pre-integration steps of the HIV-1 viral cycle are some of the most valuable targets of recent therapeutic innovations. HIV-1 integrase (IN) displays multiple functions, thanks to its considerable conformational flexibility. Recently, such flexible proteins have been characterized by their ability to form biomolecular condensates as a result of Liquid-Liquid-Phase-Separation (LLPS), allowing them to evolve in a restricted microenvironment within cells called membrane-less organelles (MLO). The LLPS context constitutes a more physiological approach to study the integration of molecular mechanisms performed by intasomes (complexes containing viral DNA, IN, and its cellular cofactor LEDGF/p75). We investigated here if such complexes can form LLPS in vitro and if IN enzymatic activities were affected by this LLPS environment. We observed that the LLPS formed by IN-LEDGF/p75 functional complexes modulate the in vitro IN activities. While the 3'-processing of viral DNA ends was drastically reduced inside LLPS, viral DNA strand transfer was strongly enhanced. These two catalytic IN activities appear thus tightly regulated by the environment encountered by intasomes.


Assuntos
Integrase de HIV , HIV-1 , Integração Viral , Integrase de HIV/metabolismo , Integrase de HIV/química , Integrase de HIV/genética , HIV-1/metabolismo , HIV-1/fisiologia , Humanos , DNA Viral/metabolismo , DNA Viral/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/química
2.
Plant J ; 118(1): 124-140, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38113339

RESUMO

As in many other organisms, tRNA-derived RNAs (tDRs) exist in plants and likely have multiple functions. We previously showed that tDRs are present in Arabidopsis under normal growth conditions, and that the ones originating from alanine tRNAs are the most abundant in leaves. We also showed that tDRs Ala of 20 nt produced from mature tRNAAla (AGC) can block in vitro protein translation. Here, we report that first, these tDRs Ala (AGC) can be found within peculiar foci in the cell that are neither P-bodies nor stress granules and, second, that they assemble into intermolecular RNA G-quadruplex (rG4) structures. Such tDR Ala rG4 structures can specifically interact with an Arabidopsis DEA(D/H) RNA helicase, the DExH1 protein, and unwind them. The rG4-DExH1 protein interaction relies on a glycine-arginine domain with RGG/RG/GR/GRR motifs present at the N-terminal extremity of the protein. Mutations on the four guanine residues located at the 5' extremity of the tDR Ala abolish its rG4 structure assembly, association with the DExH1 protein, and foci formation, but they do not prevent protein translation inhibition in vitro. Our data suggest that the sequestration of tDRs Ala into rG4 complexes might represent a way to modulate accessible and functional tDRs for translation inhibition within the plant cell via the activity of a specific RNA helicase, DExH1.


Assuntos
Arabidopsis , Quadruplex G , Arabidopsis/genética , RNA Helicases/genética , RNA , RNA de Transferência
3.
J Biol Chem ; 299(8): 104940, 2023 08.
Artigo em Inglês | MEDLINE | ID: mdl-37343702

RESUMO

Ostreolysin A6 (OlyA6) is an oyster mushroom-derived membrane-binding protein that, upon recruitment of its partner protein, pleurotolysin B, forms a cytolytic membrane pore complex. OlyA6 itself is not cytolytic but has been reported to exhibit pro-apoptotic activities in cell culture. Here we report the formation dynamics and the structure of OlyA6 assembly on a lipid membrane containing an OlyA6 high-affinity receptor, ceramide phosphoethanolamine, and cholesterol. High-speed atomic force microscopy revealed the reorganization of OlyA6 dimers from initial random surface coverage to 2D protein crystals composed of hexameric OlyA6 repeat units. Crystal growth took place predominantly in the longitudinal direction by the association of OlyA6 dimers, forming a hexameric unit cell. Molecular-level examination of the OlyA6 crystal elucidated the arrangement of dimers within the unit cell and the structure of the dimer that recruits pleurotolysin B for pore formation.


Assuntos
Proteínas Fúngicas , Proteínas Hemolisinas , Modelos Moleculares , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/ultraestrutura , Proteínas Hemolisinas/química , Proteínas Hemolisinas/metabolismo , Proteínas Hemolisinas/ultraestrutura , Proteínas de Membrana , Cristalização , Microscopia de Força Atômica , Multimerização Proteica , Estrutura Terciária de Proteína
4.
J Virol ; 97(9): e0004023, 2023 09 28.
Artigo em Inglês | MEDLINE | ID: mdl-37695057

RESUMO

The human immunodeficiency virus-1 (HIV-1) nucleocapsid protein (NCp7) is a nucleic acid chaperone protein with two highly conserved zinc fingers. To exert its key roles in the viral cycle, NCp7 interacts with several host proteins. Among them, the human NoL12 protein (hNoL12) was previously identified in genome wide screens as a potential partner of NCp7. hNoL12 is a highly conserved 25 kDa nucleolar RNA-binding protein implicated in the 5'end processing of ribosomal RNA in the nucleolus and thus in the assembly and maturation of ribosomes. In this work, we confirmed the NCp7/hNoL12 interaction in cells by Förster resonance energy transfer visualized by Fluorescence Lifetime Imaging Microscopy and co-immunoprecipitation. The interaction between NCp7 and hNoL12 was found to strongly depend on their both binding to RNA, as shown by the loss of interaction when the cell lysates were pretreated with RNase. Deletion mutants of hNoL12 were tested for their co-immunoprecipitation with NCp7, leading to the identification of the exonuclease domain of hNoL12 as the binding domain for NCp7. Finally, the interaction with hNoL12 was found to be specific of the mature NCp7 and to require NCp7 basic residues. IMPORTANCE HIV-1 mature nucleocapsid (NCp7) results from the maturation of the Gag precursor in the viral particle and is thus mostly abundant in the first phase of the infection which ends with the genomic viral DNA integration in the cell genome. Most if not all the nucleocapsid partners identified so far are not specific of the mature form. We described here the specific interaction in the nucleolus between NCp7 and the human nucleolar protein 12, a protein implicated in ribosomal RNA maturation and DNA damage response. This interaction takes place in the cell nucleolus, a subcellular compartment where NCp7 accumulates. The absence of binding between hNoL12 and Gag makes hNoL12 one of the few known specific cellular partners of NCp7.


Assuntos
HIV-1 , Proteínas Nucleares , Proteínas do Nucleocapsídeo , Proteínas de Ligação a RNA , Produtos do Gene gag do Vírus da Imunodeficiência Humana , Humanos , Proteínas do Capsídeo/química , Proteínas do Capsídeo/metabolismo , Produtos do Gene gag do Vírus da Imunodeficiência Humana/química , Produtos do Gene gag do Vírus da Imunodeficiência Humana/metabolismo , HIV-1/genética , HIV-1/metabolismo , Proteínas Nucleares/metabolismo , Proteínas do Nucleocapsídeo/química , Proteínas do Nucleocapsídeo/metabolismo , RNA Ribossômico/metabolismo , Proteínas de Ligação a RNA/metabolismo , Dedos de Zinco , Transferência Ressonante de Energia de Fluorescência , Ligação Proteica , Imunoprecipitação
5.
Chemphyschem ; 25(13): e202400101, 2024 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-38563617

RESUMO

Spectrally-resolved single-molecule localization microscopy (srSMLM) has emerged as a powerful tool for exploring the spectral properties of single emitters in localization microscopy. By simultaneously capturing the spatial positions and spectroscopic signatures of individual fluorescent molecules, srSMLM opens up the possibility of investigating an additional dimension in super-resolution imaging. However, appropriate and dedicated tools are required to fully capitalize on the spectral dimension. Here, we propose the application of the spectral phasor analysis as an effective method for summarizing and analyzing the spectral information obtained from srSMLM experiments. The spectral phasor condenses the complete spectrum of a single emitter into a two-dimensional space, preserving key spectral characteristics for single-molecule spectral exploration. We demonstrate the effectiveness of spectral phasor in efficiently classifying single Nile Red fluorescence emissions from largely overlapping cyanine fluorescence signals in dual-color PAINT experiments. Additionally, we employed spectral phasor with srSMLM to reveal subtle alterations occurring in the membrane of Gram-positive Enterococcus hirae in response to gramicidin exposure, a membrane-perturbing antibiotic treatment. Spectral phasor provides a robust, model-free analytic tool for the detailed analysis of the spectral component of srSMLM, enhancing the capabilities of multi-color spectrally-resolved single-molecule imaging.


Assuntos
Microscopia de Fluorescência , Imagem Individual de Molécula , Imagem Individual de Molécula/métodos , Microscopia de Fluorescência/métodos , Corantes Fluorescentes/química , Gramicidina/química , Oxazinas/química
6.
Cell Mol Life Sci ; 80(6): 167, 2023 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-37249637

RESUMO

Monosialoganglioside GM3 is the simplest ganglioside involved in various cellular signaling. Cell surface distribution of GM3 is thought to be crucial for the function of GM3, but little is known about the cell surface GM3 distribution. It was shown that anti-GM3 monoclonal antibody binds to GM3 in sparse but not in confluent melanoma cells. Our model membrane study evidenced that monoclonal anti-GM3 antibodies showed stronger binding when GM3 was in less fluid membrane environment. Studies using fluorescent GM3 analogs suggested that GM3 was clustered in less fluid membrane. Moreover, fluorescent lifetime measurement showed that cell surface of high density melanoma cells is more fluid than that of low density cells. Lipidomics and fatty acid supplementation experiment suggested that monounsaturated fatty acid-containing phosphatidylcholine contributed to the cell density-dependent membrane fluidity. Our results indicate that anti-GM3 antibody senses GM3 clustering and the number and/or size of GM3 cluster differ between sparse and confluent melanoma cells.


Assuntos
Gangliosídeo G(M3) , Melanoma , Humanos , Gangliosídeo G(M3)/metabolismo , Membrana Celular/metabolismo , Anticorpos Monoclonais , Melanoma/metabolismo , Contagem de Células
7.
Int J Mol Sci ; 25(11)2024 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-38892200

RESUMO

The pyoverdine siderophore is produced by Pseudomonas aeruginosa to access iron. Its synthesis involves the complex coordination of four nonribosomal peptide synthetases (NRPSs), which are responsible for assembling the pyoverdine peptide backbone. The precise cellular organization of these NRPSs and their mechanisms of interaction remain unclear. Here, we used a combination of several single-molecule microscopy techniques to elucidate the spatial arrangement of NRPSs within pyoverdine-producing cells. Our findings reveal that PvdL differs from the three other NRPSs in terms of localization and mobility patterns. PvdL is predominantly located in the inner membrane, while the others also explore the cytoplasmic compartment. Leveraging the power of multicolor single-molecule localization, we further reveal co-localization between PvdL and the other NRPSs, suggesting a pivotal role for PvdL in orchestrating the intricate biosynthetic pathway. Our observations strongly indicates that PvdL serves as a central orchestrator in the assembly of NRPSs involved in pyoverdine biosynthesis, assuming a critical regulatory function.


Assuntos
Oligopeptídeos , Peptídeo Sintases , Pseudomonas aeruginosa , Pseudomonas aeruginosa/metabolismo , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/enzimologia , Oligopeptídeos/biossíntese , Oligopeptídeos/metabolismo , Peptídeo Sintases/metabolismo , Peptídeo Sintases/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Sideróforos/biossíntese , Sideróforos/metabolismo
8.
J Biol Chem ; 298(8): 102208, 2022 08.
Artigo em Inglês | MEDLINE | ID: mdl-35772496

RESUMO

Antibiotic resistance via epigenetic methylation of ribosomal RNA is one of the most prevalent strategies adopted by multidrug resistant pathogens. The erythromycin-resistance methyltransferase (Erm) methylates rRNA at the conserved A2058 position and imparts resistance to macrolides such as erythromycin. However, the precise mechanism adopted by Erm methyltransferases for locating the target base within a complicated rRNA scaffold remains unclear. Here, we show that a conserved RNA architecture, including specific bulge sites, present more than 15 Å from the reaction center, is key to methylation at the pathogenic site. Using a set of RNA sequences site-specifically labeled by fluorescent nucleotide surrogates, we show that base flipping is a prerequisite for effective methylation and that distal bases assist in the recognition and flipping at the reaction center. The Erm-RNA complex model revealed that intrinsically flipped-out bases in the RNA serve as a putative anchor point for the Erm. Molecular dynamic simulation studies demonstrated the RNA undergoes a substantial change in conformation to facilitate an effective protein-rRNA handshake. This study highlights the importance of unique architectural features exploited by RNA to impart fidelity to RNA methyltransferases via enabling allosteric crosstalk. Moreover, the distal trigger sites identified here serve as attractive hotspots for the development of combination drug therapy aimed at reversing resistance.


Assuntos
Metiltransferases , RNA Ribossômico , Antibacterianos/farmacologia , Farmacorresistência Bacteriana , Resistência Microbiana a Medicamentos/genética , Eritromicina/farmacologia , Metiltransferases/metabolismo , RNA , RNA Ribossômico/genética , RNA Ribossômico/metabolismo
9.
Small ; 19(33): e2300728, 2023 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-37093225

RESUMO

Spectrally-resolved single molecule localization microscopy (srSMLM) is a recent technique enriching single molecule localization microscopy with the simultaneous recording of spectra of the single emitters. srSMLM resolution is limited by the number of photons collected per emitters. Sharing a photon budget to record the localization and the spectroscopic information results in a loss of spatial and spectral resolution-or forces the sacrifice of one at the expense of the other. Here, srUnet-a deep-learning Unet-based image processing routine trained to increase the spectral and spatial signals to compensate for the resolution loss inherent in additionally recording the spectral component is reported. Both localization and spectral precision are improved by srUnet-particularly for the low-emitting species. srUnet increases the fraction of localization whose signal can be both spatially and spectrally characterized. It preserves spectral shifts and the linearity of the dispersion of light. It strongly facilitates wavelength assignment in multicolor experiments. srUnet is a simple post-processing add-on boosting srSMLM performance close to conventional SMLM with the potential to turn srSMLM into the new standard for multicolor single molecule imaging.

10.
Hepatology ; 76(4): 1164-1179, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-35388524

RESUMO

BACKGROUND AND AIMS: Numerous HCV entry factors have been identified, and yet information regarding their spatiotemporal dynamics is still limited. Specifically, one of the main entry factors of HCV is occludin (OCLN), a protein clustered at tight junctions (TJs), away from the HCV landing site. Thus, whether HCV particles slide toward TJs or, conversely, OCLN is recruited away from TJs remain debated. APPROACH AND RESULTS: Here, we generated CRISPR/CRISPR-associated protein 9 edited Huh7.5.1 cells expressing endogenous levels of enhanced green fluorescent protein/OCLN and showed that incoming HCV particles recruit OCLN outside TJs, independently of claudin 1 (CLDN1) expression, another important HCV entry factor located at TJs. Using ex vivo organotypic culture of hepatic slices obtained from human liver explants, a physiologically relevant model that preserves the overall tissue architecture, we confirmed that HCV associates with OCLN away from TJs. Furthermore, we showed, by live cell imaging, that increased OCLN recruitment beneath HCV particles correlated with lower HCV motility. To decipher the mechanism underlying virus slow-down upon OCLN recruitment, we performed CRISPR knockout (KO) of CLDN1, an HCV entry factor proposed to act upstream of OCLN. Although CLDN1 KO potently inhibits HCV infection, OCLN kept accumulating underneath the particle, indicating that OCLN recruitment is CLDN1 independent. Moreover, inhibition of the phosphorylation of Ezrin, a protein involved in HCV entry that links receptors to the actin cytoskeleton, increased OCLN accumulation and correlated with more efficient HCV internalization. CONCLUSIONS: Together, our data provide robust evidence that HCV particles interact with OCLN away from TJs and shed mechanistic insights regarding the manipulation of transmembrane receptor localization by extracellular virus particles.


Assuntos
Hepatite C , Junções Íntimas , Proteína 9 Associada à CRISPR/metabolismo , Claudina-1/genética , Hepacivirus/fisiologia , Hepatite C/metabolismo , Hepatócitos/metabolismo , Humanos , Ocludina , Vírion , Internalização do Vírus
11.
Bioorg Chem ; 137: 106616, 2023 08.
Artigo em Inglês | MEDLINE | ID: mdl-37247564

RESUMO

Ubiquitin-like containing PHD and RING finger domain 1 (UHRF1) is a nuclear multi-domain protein overexpressed in numerous human cancer types. We previously disclosed the anthraquinone derivative UM63 that inhibits UHRF1-SRA domain base-flipping activity, although having DNA intercalating properties. Herein, based on the UM63 structure, new UHRF1-SRA inhibitors were identified through a multidisciplinary approach, combining molecular modelling, biophysical assays, molecular and cell biology experiments. We identified AMSA2 and MPB7, that inhibit UHRF1-SRA mediated base flipping at low micromolar concentrations, but do not intercalate into DNA, which is a key advantage over UM63. These molecules prevent UHRF1/DNMT1 interaction at replication forks and decrease the overall DNA methylation in cells. Moreover, both compounds specifically induce cell death in numerous cancer cell lines, displaying marginal effect on non-cancer cells, as they preferentially affect cells with high level of UHRF1. Overall, these two compounds are promising leads for the development of anti-cancer drugs targeting UHRF1.


Assuntos
Proteínas Estimuladoras de Ligação a CCAAT , Neoplasias , Humanos , Proteínas Estimuladoras de Ligação a CCAAT/química , Proteínas Estimuladoras de Ligação a CCAAT/genética , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Metilação de DNA , DNA/química , Modelos Moleculares , Neoplasias/genética
12.
Nucleic Acids Res ; 49(8): 4550-4563, 2021 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-33872352

RESUMO

The nucleocapsid (NC) protein plays key roles in Human Immunodeficiency Virus 1 (HIV-1) replication, notably by condensing and protecting the viral RNA genome and by chaperoning its reverse transcription into double-stranded DNA (dsDNA). Recent findings suggest that integration of viral dsDNA into the host genome, and hence productive infection, is linked to a small subpopulation of viral complexes where reverse transcription was completed within the intact capsid. Therefore, the synthesized dsDNA has to be tightly compacted, most likely by NC, to prevent breaking of the capsid in these complexes. To investigate NC's ability to compact viral dsDNA, we here characterize the compaction of single dsDNA molecules under unsaturated NC binding conditions using nanofluidic channels. Compaction is shown to result from accumulation of NC at one or few compaction sites, which leads to small dsDNA condensates. NC preferentially initiates compaction at flexible regions along the dsDNA, such as AT-rich regions and DNA ends. Upon further NC binding, these condensates develop into a globular state containing the whole dsDNA molecule. These findings support NC's role in viral dsDNA compaction within the mature HIV-1 capsid and suggest a possible scenario for the gradual dsDNA decondensation upon capsid uncoating and NC loss.


Assuntos
DNA Viral/química , Proteínas de Ligação a DNA/química , DNA/química , HIV-1/química , Proteínas do Nucleocapsídeo/química , Nucleocapsídeo/química , Sequência de Aminoácidos , DNA/síntese química , Proteínas de Ligação a DNA/genética , HIV-1/genética , Conformação de Ácido Nucleico , Nucleocapsídeo/metabolismo , Proteínas do Nucleocapsídeo/síntese química , RNA Viral/química , Software , Dedos de Zinco/genética
13.
Nucleic Acids Res ; 49(12): e72, 2021 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-33872373

RESUMO

The sensitivity of FRET-based sensing is usually limited by the spectral overlaps of the FRET donor and acceptor, which generate a poor signal-to-noise ratio. To overcome this limitation, a quenched donor presenting a large Stokes shift can be combined with a bright acceptor to perform Dark Resonance Energy Transfer (DRET). The consequent fluorogenic response from the acceptor considerably improves the signal-to-noise ratio. To date, DRET has mainly relied on a donor that is covalently bound to the acceptor. In this context, our aim was to develop the first intermolecular DRET pair for specific sensing of nucleic acid sequences. To this end, we designed DFK, a push-pull probe based on a fluorenyl π-platform that is strongly quenched in water. DFK was incorporated into a series of oligonucleotides and used as a DRET donor with Cy5-labeled complementary sequences. In line with our expectations, excitation of the dark donor in the double-labeled duplex switched on the far-red Cy5 emission and remained free of cross-excitation. The DRET mechanism was supported by time-resolved fluorescence measurements. This concept was then applied with binary probes, which confirmed the distance dependence of DRET as well as its potency in detecting sequences of interest with low background noise.


Assuntos
Corantes Fluorescentes/química , Espectrometria de Fluorescência/métodos , Transferência Ressonante de Energia de Fluorescência , Corantes Fluorescentes/síntese química , Oligonucleotídeos/química
14.
Nucleic Acids Res ; 49(19): e111, 2021 11 08.
Artigo em Inglês | MEDLINE | ID: mdl-34450653

RESUMO

Interconversions between nucleic acid structures play an important role in transcriptional and translational regulation and also in repair and recombination. These interconversions are frequently promoted by nucleic acid chaperone proteins. To monitor their kinetics, Förster resonance energy transfer (FRET) is widely exploited using ensemble fluorescence intensity measurements in pre-steady-state stopped-flow experiments. Such experiments only provide a weighted average of the emission of all species in solution and consume large quantities of materials. Herein, we lift these limitations by combining time-resolved fluorescence (TRF) with droplet microfluidics (DmF). We validate the innovative TRF-DmF approach by investigating the well characterized annealing of the HIV-1 (+)/(-) Primer Binding Sequences (PBS) promoted by a HIV-1 nucleocapsid peptide. Upon rapid mixing of the FRET-labelled (-)PBS with its complementary (+)PBS sequence inside microdroplets, the TRF-DmF set-up enables resolving the time evolution of sub-populations of reacting species and reveals an early intermediate with a ∼50 ps donor fluorescence lifetime never identified so far. TRF-DmF also favorably compares with single molecule experiments, as it offers an accurate control of concentrations with no upper limit, no need to graft one partner on a surface and no photobleaching issues.


Assuntos
Primers do DNA/química , HIV-1/química , Chaperonas Moleculares/química , Proteínas do Nucleocapsídeo/química , Peptídeos/química , Albumina Sérica Humana/química , Pareamento de Bases , Primers do DNA/metabolismo , Fluoresceínas/química , Fluorescência , Transferência Ressonante de Energia de Fluorescência , HIV-1/metabolismo , Humanos , Cinética , Técnicas Analíticas Microfluídicas , Chaperonas Moleculares/metabolismo , Conformação de Ácido Nucleico , Proteínas do Nucleocapsídeo/metabolismo , Peptídeos/metabolismo , Albumina Sérica Humana/metabolismo , p-Dimetilaminoazobenzeno/análogos & derivados , p-Dimetilaminoazobenzeno/química
15.
Molecules ; 28(16)2023 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-37630248

RESUMO

Cancer is one of the leading causes of death worldwide, and its incidence and mortality are increasing each year. Improved therapeutic strategies against cancer have progressed, but remain insufficient to invert this trend. Along with several other risk factors, abnormal genetic and epigenetic regulations play a critical role in the initiation of cellular transformation, as well as tumorigenesis. The epigenetic regulator UHRF1 (ubiquitin-like, containing PHD and RING finger domains 1) is a multidomain protein with oncogenic abilities overexpressed in most cancers. Through the coordination of its multiple domains and other epigenetic key players, UHRF1 regulates DNA methylation and histone modifications. This well-coordinated dialogue leads to the silencing of tumor-suppressor genes (TSGs) and facilitates tumor cells' resistance toward anticancer drugs, ultimately promoting apoptosis escape and uncontrolled proliferation. Several studies have shown that the downregulation of UHRF1 with natural compounds in tumor cells induces the reactivation of various TSGs, inhibits cell growth, and promotes apoptosis. In this review, we discuss the underlying mechanisms and the potential of various natural and synthetic compounds that can inhibit/minimize UHRF1's oncogenic activities and/or its expression.


Assuntos
Carcinogênese , Transformação Celular Neoplásica , Humanos , Apoptose , Ciclo Celular , Epigênese Genética , Proteínas Estimuladoras de Ligação a CCAAT/genética , Ubiquitina-Proteína Ligases/genética
16.
Q Rev Biophys ; 53: e1, 2020 01 09.
Artigo em Inglês | MEDLINE | ID: mdl-31915092

RESUMO

The bio-synthesis of pyoverdine (PVD) in Pseudomonas aeruginosa involves multiple enzymatic steps including the action of non-ribosomal peptide synthetases (NRPSs). One hallmark of NRPS is their ability to make usage of non-proteinogenic amino-acids synthesized by co-expressed accessory enzymes. It is generally proposed that different enzymes of a secondary metabolic pathway assemble into large supra-molecular complexes. However, evidence for the assembly of sequential enzymes in the cellular context is sparse. Here, we used in cellulo single-molecule tracking and Förster resonance energy transfer measured by fluorescence lifetime microscopy (FRET-FLIM) to explore the spatial partitioning of the ornithine hydroxylase PvdA and its interactions with NRPS. We found PvdA was mostly diffusing bound to large complexes in the cytoplasm with a small exchangeable trapped fraction. FRET-FLIM clearly showed that PvdA is physically interacting with PvdJ, PvdI, PvdL, and PvdD, the four NRPS involved in the PVD pathway in Pseudomonas aeruginosa PAO1. The binding modes of PvdA were strikingly different according to the NRPS it is interacting with, suggesting that PvdA binding sites have co-evolved with the enzymatic active sites of NRPS. Our data provide evidence for strongly organized multi-enzymatic complexes responsible for the bio-synthesis of PVD and illustrate how binding sites have evolved to finely control the co-localization of sequential enzymes and promote metabolic pathway efficiency.


Assuntos
Oxigenases de Função Mista/metabolismo , Oligopeptídeos/química , Pseudomonas aeruginosa/metabolismo , Imagem Individual de Molécula/métodos , Análise por Conglomerados , Citoplasma/metabolismo , Escherichia coli/metabolismo , Transferência Ressonante de Energia de Fluorescência , Proteínas de Fluorescência Verde/metabolismo , Microscopia de Fluorescência/métodos , Mutação , Oligopeptídeos/metabolismo , Ligação Proteica
17.
J Am Chem Soc ; 144(29): 13356-13365, 2022 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-35771602

RESUMO

We have prepared a hetero-tetrametallic assembly consisting of three ytterbium ions coordinated to a central [Ru(bpm)3]2+ (bpm = 2,2'-bipyrimidine) motif. Irradiation into the absorption band of the peripheral ytterbium ions at 980 nm engenders emission of the 3MLCT state of the central [Ru(bpm)3]2+ core at 636 nm, which represents the first example of f → d molecular upconversion (UC). Time-resolved measurements reveal a slow rise of the UC emission, which was modeled with a mathematical treatment of the observed kinetics according to a cooperative photosensitization mechanism using a virtual Yb centered doubly excited state followed by energy transfer to the Ru centered 1MLCT state.


Assuntos
Itérbio , Transferência de Energia , Íons
18.
Nucleic Acids Res ; 48(17): 9969-9985, 2020 09 25.
Artigo em Inglês | MEDLINE | ID: mdl-32974652

RESUMO

Retinoic acid receptors (RARs) as a functional heterodimer with retinoid X receptors (RXRs), bind a diverse series of RA-response elements (RAREs) in regulated genes. Among them, the non-canonical DR0 elements are bound by RXR-RAR with comparable affinities to DR5 elements but DR0 elements do not act transcriptionally as independent RAREs. In this work, we present structural insights for the recognition of DR5 and DR0 elements by RXR-RAR heterodimer using x-ray crystallography, small angle x-ray scattering, and hydrogen/deuterium exchange coupled to mass spectrometry. We solved the crystal structures of RXR-RAR DNA-binding domain in complex with the Rarb2 DR5 and RXR-RXR DNA-binding domain in complex with Hoxb13 DR0. While cooperative binding was observed on DR5, the two molecules bound non-cooperatively on DR0 on opposite sides of the DNA. In addition, our data unveil the structural organization and dynamics of the multi-domain RXR-RAR DNA complexes providing evidence for DNA-dependent allosteric communication between domains. Differential binding modes between DR0 and DR5 were observed leading to differences in conformation and structural dynamics of the multi-domain RXR-RAR DNA complexes. These results reveal that the topological organization of the RAR binding element confer regulatory information by modulating the overall topology and structural dynamics of the RXR-RAR heterodimers.


Assuntos
Sítio Alostérico , Elementos de Resposta , Receptores X de Retinoides/química , Regulação Alostérica , DNA/química , DNA/metabolismo , Proteínas de Homeodomínio/genética , Humanos , Simulação de Acoplamento Molecular , Ligação Proteica , Receptores X de Retinoides/metabolismo
19.
Nucleic Acids Res ; 48(16): 9218-9234, 2020 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-32797159

RESUMO

The HIV-1 Gag protein playing a key role in HIV-1 viral assembly has recently been shown to interact through its nucleocapsid domain with the ribosomal protein L7 (RPL7) that acts as a cellular co-factor promoting Gag's nucleic acid (NA) chaperone activity. To further understand how the two proteins act together, we examined their mechanism individually and in concert to promote the annealing between dTAR, the DNA version of the viral transactivation element and its complementary cTAR sequence, taken as model HIV-1 sequences. Gag alone or complexed with RPL7 was found to act as a NA chaperone that destabilizes cTAR stem-loop and promotes its annealing with dTAR through the stem ends via a two-step pathway. In contrast, RPL7 alone acts as a NA annealer that through its NA aggregating properties promotes cTAR/dTAR annealing via two parallel pathways. Remarkably, in contrast to the isolated proteins, their complex promoted efficiently the annealing of cTAR with highly stable dTAR mutants. This was confirmed by the RPL7-promoted boost of the physiologically relevant Gag-chaperoned annealing of (+)PBS RNA to the highly stable tRNALys3 primer, favoring the notion that Gag recruits RPL7 to overcome major roadblocks in viral assembly.


Assuntos
Infecções por HIV/genética , HIV-1/genética , Proteínas Ribossômicas/genética , Produtos do Gene gag do Vírus da Imunodeficiência Humana/genética , Sequência de Aminoácidos/genética , Infecções por HIV/virologia , HIV-1/patogenicidade , Interações Hospedeiro-Patógeno/genética , Humanos , Chaperonas Moleculares/genética , Conformação de Ácido Nucleico , Ácidos Nucleicos/genética , RNA Viral/genética , Montagem de Vírus/genética
20.
Chem Soc Rev ; 50(12): 7062-7107, 2021 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-33956014

RESUMO

Fluorescent nucleoside analogues (FNAs) are structurally diverse mimics of the natural essentially non-fluorescent nucleosides which have found numerous applications in probing the structure and dynamics of nucleic acids as well as their interactions with various biomolecules. In order to minimize disturbance in the labelled nucleic acid sequences, the FNA chromophoric groups should resemble the natural nucleobases in size and hydrogen-bonding patterns. Isomorphic and expanded FNAs are the two groups that best meet the criteria of non-perturbing fluorescent labels for DNA and RNA. Significant progress has been made over the past decades in understanding the fundamental photophysics that governs the spectroscopic and environmentally sensitive properties of these FNAs. Herein, we review recent advances in the spectroscopic and computational studies of selected isomorphic and expanded FNAs. We also show how this information can be used as a rational basis to design new FNAs, select appropriate sequences for optimal spectroscopic response and interpret fluorescence data in FNA applications.


Assuntos
Fluorescência , Corantes Fluorescentes/química , Nucleosídeos/química , Processos Fotoquímicos
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