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1.
Nat Commun ; 11(1): 5210, 2020 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-33060578

RESUMO

Human insulinomas are rare, benign, slowly proliferating, insulin-producing beta cell tumors that provide a molecular "recipe" or "roadmap" for pathways that control human beta cell regeneration. An earlier study revealed abnormal methylation in the imprinted p15.5-p15.4 region of chromosome 11, known to be abnormally methylated in another disorder of expanded beta cell mass and function: the focal variant of congenital hyperinsulinism. Here, we compare deep DNA methylome sequencing on 19 human insulinomas, and five sets of normal beta cells. We find a remarkably consistent, abnormal methylation pattern in insulinomas. The findings suggest that abnormal insulin (INS) promoter methylation and altered transcription factor expression create alternative drivers of INS expression, replacing canonical PDX1-driven beta cell specification with a pathological, looping, distal enhancer-based form of transcriptional regulation. Finally, NFaT transcription factors, rather than the canonical PDX1 enhancer complex, are predicted to drive INS transactivation.


Assuntos
Regulação Neoplásica da Expressão Gênica , Insulina/genética , Insulina/metabolismo , Insulinoma/genética , Insulinoma/metabolismo , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Adulto , Idoso , Sítios de Ligação , Biologia Computacional , Metilação de DNA , Feminino , Proteínas de Homeodomínio/metabolismo , Humanos , Células Secretoras de Insulina/metabolismo , Masculino , Doenças Metabólicas/genética , Doenças Metabólicas/metabolismo , Pessoa de Meia-Idade , Regiões Promotoras Genéticas , Transativadores/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Adulto Jovem
2.
J Comput Aided Mol Des ; 34(12): 1237-1259, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33034007

RESUMO

Computational protein-ligand docking is well-known to be prone to inaccuracies in input receptor structures, and it is challenging to obtain good docking results with computationally predicted receptor structures (e.g. through homology modeling). Here we introduce a fragment-based docking method and test if it reduces requirements on the accuracy of an input receptor structures relative to non-fragment docking approaches. In this method, small rigid fragments are docked first using AutoDock Vina to generate a large number of favorably docked poses spanning the receptor binding pocket. Then a graph theory maximum clique algorithm is applied to find combined sets of docked poses of different fragment types onto which the complete ligand can be properly aligned. On the basis of these alignments, possible binding poses of complete ligand are determined. This docking method is first tested for bound docking on a series of Cytochrome P450 (CYP450) enzyme-substrate complexes, in which experimentally determined receptor structures are used. For all complexes tested, ligand poses of less than 1 Å root mean square deviations (RMSD) from the actual binding positions can be recovered. Then the method is tested for unbound docking with modeled receptor structures for a number of protein-ligand complexes from different families including the very recent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) protease. For all complexes, poses with RMSD less than 3 Å from actual binding positions can be recovered. Our results suggest that for docking with approximately modeled receptor structures, fragment-based methods can be more effective than common complete ligand docking approaches.


Assuntos
Betacoronavirus/enzimologia , Infecções por Coronavirus/tratamento farmacológico , Cisteína Endopeptidases/efeitos dos fármacos , Simulação de Acoplamento Molecular , Pandemias , Pneumonia Viral/tratamento farmacológico , Proteínas não Estruturais Virais/efeitos dos fármacos , ATPases Associadas a Diversas Atividades Celulares/química , ATPases Associadas a Diversas Atividades Celulares/metabolismo , Cisteína Endopeptidases/química , Cisteína Endopeptidases/metabolismo , Sistema Enzimático do Citocromo P-450/química , Sistema Enzimático do Citocromo P-450/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Humanos , Ligantes , Modelos Químicos , Modelos Moleculares , Chaperonas Moleculares/química , Chaperonas Moleculares/metabolismo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Ligação Proteica , Conformação Proteica , Receptores Acoplados a Proteínas-G/química , Receptores Acoplados a Proteínas-G/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/metabolismo
3.
Nat Commun ; 11(1): 5425, 2020 10 27.
Artigo em Inglês | MEDLINE | ID: mdl-33110077

RESUMO

Transcription factors are key protein effectors in the regulation of gene transcription, and in many cases their activity is regulated via a complex network of protein-protein interactions (PPI). The chemical modulation of transcription factor activity is a long-standing goal in drug discovery but hampered by the difficulties associated with the targeting of PPIs, in particular when extended and flat protein interfaces are involved. Peptidomimetics have been applied to inhibit PPIs, however with variable success, as for certain interfaces the mimicry of a single secondary structure element is insufficient to obtain high binding affinities. Here, we describe the design and characterization of a stabilized protein tertiary structure that acts as an inhibitor of the interaction between the transcription factor TEAD and its co-repressor VGL4, both playing a central role in the Hippo signalling pathway. Modification of the inhibitor with a cell-penetrating entity yielded a cell-permeable proteomimetic that activates cell proliferation via regulation of the Hippo pathway, highlighting the potential of protein tertiary structure mimetics as an emerging class of PPI modulators.


Assuntos
Peptidomiméticos , Fatores de Transcrição/química , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Humanos , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
4.
Nat Commun ; 11(1): 4578, 2020 09 14.
Artigo em Inglês | MEDLINE | ID: mdl-32929090

RESUMO

Thalidomide and its derivatives exert not only therapeutic effects as immunomodulatory drugs (IMiDs) but also adverse effects such as teratogenicity, which are due in part to different C2H2 zinc-finger (ZF) transcription factors, IKZF1 (or IKZF3) and SALL4, respectively. Here, we report the structural bases for the SALL4-specific proteasomal degradation induced by 5-hydroxythalidomide, a primary thalidomide metabolite generated by the enzymatic activity of cytochrome P450 isozymes, through the interaction with cereblon (CRBN). The crystal structure of the metabolite-mediated human SALL4-CRBN complex and mutagenesis studies elucidate the complex formation enhanced by the interaction between CRBN and an additional hydroxy group of (S)-5-hydroxythalidomide and the variation in the second residue of ß-hairpin structure that underlies the C2H2 ZF-type neo-morphic substrate (neosubstrate) selectivity of 5-hydroxythalidomide. These findings deepen our understanding of the pharmaceutical action of IMiDs and provide structural evidence that the glue-type E3 ligase modulators cause altered neosubstrate specificities through their metabolism.


Assuntos
Fatores Imunológicos/química , Fatores Imunológicos/farmacologia , Talidomida/análogos & derivados , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Sequência de Aminoácidos , Células HEK293 , Humanos , Ligação Proteica/efeitos dos fármacos , Domínios Proteicos , Estereoisomerismo , Homologia Estrutural de Proteína , Especificidade por Substrato , Talidomida/química , Talidomida/farmacologia , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo
5.
Proc Natl Acad Sci U S A ; 117(38): 23548-23556, 2020 09 22.
Artigo em Inglês | MEDLINE | ID: mdl-32887801

RESUMO

Mitochondrial complex II, also known as succinate dehydrogenase (SDH), is an integral-membrane heterotetramer (SDHABCD) that links two essential energy-producing processes, the tricarboxylic acid (TCA) cycle and oxidative phosphorylation. A significant amount of information is available on the structure and function of mature complex II from a range of organisms. However, there is a gap in our understanding of how the enzyme assembles into a functional complex, and disease-associated complex II insufficiency may result from incorrect function of the mature enzyme or from assembly defects. Here, we investigate the assembly of human complex II by combining a biochemical reconstructionist approach with structural studies. We report an X-ray structure of human SDHA and its dedicated assembly factor SDHAF2. Importantly, we also identify a small molecule dicarboxylate that acts as an essential cofactor in this process and works in synergy with SDHAF2 to properly orient the flavin and capping domains of SDHA. This reorganizes the active site, which is located at the interface of these domains, and adjusts the pKa of SDHAR451 so that covalent attachment of the flavin adenine dinucleotide (FAD) cofactor is supported. We analyze the impact of disease-associated SDHA mutations on assembly and identify four distinct conformational forms of the complex II flavoprotein that we assign to roles in assembly and catalysis.


Assuntos
Ácidos Dicarboxílicos/metabolismo , Complexo II de Transporte de Elétrons , Flavinas/metabolismo , Proteínas Mitocondriais , Ácidos Dicarboxílicos/química , Complexo II de Transporte de Elétrons/química , Complexo II de Transporte de Elétrons/metabolismo , Flavinas/química , Humanos , Proteínas Mitocondriais/química , Proteínas Mitocondriais/metabolismo , Modelos Moleculares , Dobramento de Proteína , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo
6.
Nat Struct Mol Biol ; 27(9): 836-845, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32747783

RESUMO

Interactions between chromatin-associated proteins and the histone landscape play major roles in dictating genome topology and gene expression. Cancer-specific fusion oncoproteins, which display unique chromatin localization patterns, often lack classical DNA-binding domains, presenting challenges in identifying mechanisms governing their site-specific chromatin targeting and function. Here we identify a minimal region of the human SS18-SSX fusion oncoprotein (the hallmark driver of synovial sarcoma) that mediates a direct interaction between the mSWI/SNF complex and the nucleosome acidic patch. This binding results in altered mSWI/SNF composition and nucleosome engagement, driving cancer-specific mSWI/SNF complex targeting and gene expression. Furthermore, the C-terminal region of SSX confers preferential affinity to repressed, H2AK119Ub-marked nucleosomes, underlying the selective targeting to polycomb-marked genomic regions and synovial sarcoma-specific dependency on PRC1 function. Together, our results describe a functional interplay between a key nucleosome binding hub and a histone modification that underlies the disease-specific recruitment of a major chromatin remodeling complex.


Assuntos
Proteínas Cromossômicas não Histona/metabolismo , Histonas/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas de Fusão Oncogênica/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Repressoras/metabolismo , Sarcoma Sinovial/metabolismo , Fatores de Transcrição/metabolismo , Ubiquitinas/metabolismo , Linhagem Celular Tumoral , Proteínas Cromossômicas não Histona/química , Células HEK293 , Humanos , Modelos Moleculares , Proteínas de Neoplasias/química , Nucleossomos/metabolismo , Nucleossomos/patologia , Proteínas de Fusão Oncogênica/química , Conformação Proteica , Proteínas Proto-Oncogênicas/química , Proteínas Repressoras/química , Sarcoma Sinovial/patologia , Fatores de Transcrição/química , Ubiquitinação
7.
Plant Mol Biol ; 104(4-5): 483-498, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32813232

RESUMO

The phytohormone ethylene is widely involved in many developmental processes and is a crucial regulator of defense responses against biotic and abiotic stresses in plants. Ethylene-responsive element binding protein, a member of the APETALA2/ethylene response factor (AP2/ERF) superfamily, is a transcription factor that regulates stress-responsive genes by recognizing a specific cis-acting element of target DNA. A previous study showed only the NMR structure of the AP2/ERF domain of AtERF100 in complex with a GCC box DNA motif. In this report, we determined the crystal structure of AtERF96 in complex with a GCC box at atomic resolution. We analyzed the binding residues of the conserved AP2/ERF domain in the DNA recognition sequence. In addition to the AP2/ERF domain, an N-terminal α-helix of AtERF96 participates in DNA interaction in the flanking region. We also demonstrated the structure of AtERF96 EDLL motif, a unique conserved motif in the group IX of AP2/ERF family, might involve in the transactivation of defense-related genes. Our study establishes the structural basis of the AtERF96 transcription factor in complex with the GCC box, as well as the DNA binding mechanisms of the N-terminal α-helix and AP2/ERF domain.


Assuntos
Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Arabidopsis/química , Arabidopsis/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Sítios de Ligação , Cristalografia por Raios X , DNA de Plantas/metabolismo , Modelos Moleculares , Mutação , Conformação Proteica , Protoplastos , Fatores de Transcrição/genética
8.
Nucleic Acids Res ; 48(17): 9943-9958, 2020 09 25.
Artigo em Inglês | MEDLINE | ID: mdl-32821917

RESUMO

Nucleotide excision repair (NER) is an essential pathway to remove bulky lesions affecting one strand of DNA. Defects in components of this repair system are at the ground of genetic diseases such as xeroderma pigmentosum (XP) and Cockayne syndrome (CS). The XP complementation group G (XPG) endonuclease cleaves the damaged DNA strand on the 3' side of the lesion coordinated with DNA re-synthesis. Here, we determined crystal structures of the XPG nuclease domain in the absence and presence of DNA. The overall fold exhibits similarities to other flap endonucleases but XPG harbors a dynamic helical arch that is uniquely oriented and defines a gateway. DNA binding through a helix-2-turn-helix motif, assisted by one flanking α-helix on each side, shows high plasticity, which is likely relevant for DNA scanning. A positively-charged canyon defined by the hydrophobic wedge and ß-pin motifs provides an additional DNA-binding surface. Mutational analysis identifies helical arch residues that play critical roles in XPG function. A model for XPG participation in NER is proposed. Our structures and biochemical data represent a valuable tool to understand the atomic ground of XP and CS, and constitute a starting point for potential therapeutic applications.


Assuntos
Reparo do DNA , Proteínas de Ligação a DNA/química , Endonucleases/química , Proteínas Nucleares/química , Fatores de Transcrição/química , Sítios de Ligação , Cristalografia por Raios X , DNA/química , DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Endonucleases/metabolismo , Humanos , Simulação de Acoplamento Molecular , Proteínas Nucleares/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Fatores de Transcrição/metabolismo
9.
Plant Mol Biol ; 104(3): 309-325, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32833148

RESUMO

KEY MESSAGE: FtMYB18 plays a role in the repression of anthocyanins and proanthocyanidins accumulation by strongly down-regulating the CHS and DFR genes in Tartary buckwheat, and the C5 motif plays an important role in this process. Anthocyanins and proanthocyanidins (PAs) are important flavonoids in Tartary buckwheat (Fagopyrum tataricum Gaertn.), which provides various vibrant color and stronge abiotic stress resistance. Their synthesis is generally regulated by MYB transcription factors at transcription level. However, the negative regulations of MYB and their effects on flavonol metabolism are poorly understood. A SG4-like MYB subfamily TF, FtMYB18, containing C5 motif was identified from Tartary buckwheat. The expression of FtMYB18 was not only showed a negative correlation with anthocyanins and PAs content but also strongly respond to MeJA and ABA. As far as the transgenic lines with FtMYB18 overexpression, anthocyanins and PAs accumulations were decreased through down-regulating expression levels of NtCHS and NtDFR in tobacco, AtDFR and AtTT12 in Arabidopsis, FtCHS, FtDFR and FtANS in Tartary buckwheat hairy roots, respectively. However, FtMYB18 showed no effect on the FLS gene expression and the metabolites content in flavonol synthesis branch. The further molecular interaction analysis indicated FtMYB18 could mediate the inhibition of anthocyanins and PAs synthesis by forming MBW transcriptional complex with FtTT8 and FtTTG1, or MYB-JAZ complex with FtJAZ1/-3/-4/-7. Importantly, in FtMYB18 mutant lines with C5 motif deletion (FtMYB18-C), both of anthocyanins and PAs accumulations had recovered to the similar level as that in wild type, which was attributed to the weakened MBW complex activity or the deficient molecular interaction between FtMYB18ΔC5 with FtJAZ3/-4. The results showed that FtMYB18 could suppress anthocyanins and PAs synthesis at transcription level through the specific interaction of C5 motif with other proteins in Tartary buckwheat.


Assuntos
Antocianinas/biossíntese , Fagopyrum/metabolismo , Proteínas de Plantas/metabolismo , Proantocianidinas/biossíntese , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Arabidopsis , Fagopyrum/genética , Flavonoides/metabolismo , Regulação da Expressão Gênica de Plantas , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas , Estresse Fisiológico , Tabaco/genética , Fatores de Transcrição/química
10.
Nature ; 585(7824): 256-260, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32848244

RESUMO

Temperature controls plant growth and development, and climate change has already altered the phenology of wild plants and crops1. However, the mechanisms by which plants sense temperature are not well understood. The evening complex is a major signalling hub and a core component of the plant circadian clock2,3. The evening complex acts as a temperature-responsive transcriptional repressor, providing rhythmicity and temperature responsiveness to growth through unknown mechanisms2,4-6. The evening complex consists of EARLY FLOWERING 3 (ELF3)4,7, a large scaffold protein and key component of temperature sensing; ELF4, a small α-helical protein; and LUX ARRYTHMO (LUX), a DNA-binding protein required to recruit the evening complex to transcriptional targets. ELF3 contains a polyglutamine (polyQ) repeat8-10, embedded within a predicted prion domain (PrD). Here we find that the length of the polyQ repeat correlates with thermal responsiveness. We show that ELF3 proteins in plants from hotter climates, with no detectable PrD, are active at high temperatures, and lack thermal responsiveness. The temperature sensitivity of ELF3 is also modulated by the levels of ELF4, indicating that ELF4 can stabilize the function of ELF3. In both Arabidopsis and a heterologous system, ELF3 fused with green fluorescent protein forms speckles within minutes in response to higher temperatures, in a PrD-dependent manner. A purified fragment encompassing the ELF3 PrD reversibly forms liquid droplets in response to increasing temperatures in vitro, indicating that these properties reflect a direct biophysical response conferred by the PrD. The ability of temperature to rapidly shift ELF3 between active and inactive states via phase transition represents a previously unknown thermosensory mechanism.


Assuntos
Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas Priônicas/química , Temperatura , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Aclimatação/fisiologia , Arabidopsis/química , Temperatura Alta , Modelos Moleculares , Peptídeos/metabolismo , Transição de Fase , Domínios Proteicos , Proteínas Repressoras/química , Proteínas Repressoras/metabolismo
11.
Proc Natl Acad Sci U S A ; 117(33): 20292-20297, 2020 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-32747529

RESUMO

Nuclear Factor of Activated T cells 5 (NFAT5) is a transcription factor (TF) that mediates protection from adverse effects of hypertonicity by increasing transcription of genes, including those that lead to cellular accumulation of protective organic osmolytes. NFAT5 has three intrinsically ordered (ID) activation domains (ADs). Using the NFAT5 N-terminal domain (NTD), which contains AD1, as a model, we demonstrate by biophysical methods that the NTD senses osmolytes and hypertonicity, resulting in stabilization of its ID regions. In the presence of sufficient NaCl or osmolytes, trehalose and sorbitol, the NFAT5 NTD undergoes a disorder-to-order shift, adopting higher average secondary and tertiary structure. Thus, NFAT5 is activated by the stress that it protects against. In its salt and/or osmolyte-induced more ordered conformation, the NTD interacts with several proteins, including HMGI-C, which is known to protect against apoptosis. These findings raise the possibility that the increased intracellular ionic strength and elevated osmolytes caused by hypertonicity activate and stabilize NFAT5.


Assuntos
Proteínas Intrinsicamente Desordenadas/química , Fatores de Transcrição/química , Escherichia coli/metabolismo , Pressão Osmótica , Ligação Proteica , Dobramento de Proteína , Cloreto de Sódio , Sorbitol , Fatores de Transcrição/metabolismo , Trealose
12.
Plant Mol Biol ; 104(4-5): 429-450, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32808190

RESUMO

KEY MESSAGE: OsWRKY36 represses plant height and grain size by inhibiting gibberellin signaling. Plant height and grain size are important agronomic traits affecting yield in cereals, including rice. Gibberellins (GAs) are plant hormones that promote plant growth and developmental processions such as stem elongation and grain size. WRKYs are transcription factors that regulate stress tolerance and plant development including height and grain size. However, the relationship between GA signaling and WRKY genes is still poorly understood. Here, we characterized a small grain and semi-dwarf 3 (sgsd3) mutant in rice cv. Hwayoung (WT). A T-DNA insertion in the 5'-UTR of OsWRKY36 induced overexpression of OsWRKY36 in the sgsd3 mutant, likely leading to the mutant phenotype. This was confirmed by the finding that overexpression of OsWRKY36 caused a similar small grain and semi-dwarf phenotype to the sgsd3 mutant whereas knock down and knock out caused larger grain phenotypes. The sgsd3 mutant was also hyposensitive to GA and accumulated higher mRNA and protein levels of SLR1 (a GA signaling DELLA-like inhibitor) compared with the WT. Further assays showed that OsWRKY36 enhanced SLR1 transcription by directly binding to its promoter. In addition, we found that OsWRKY36 can protect SLR1 from GA-mediated degradation. We thus identified a new GA signaling repressor OsWRKY36 that represses GA signaling through stabilizing the expression of SLR1.


Assuntos
Oryza/crescimento & desenvolvimento , Oryza/genética , Proteínas de Plantas/genética , Fatores de Transcrição/genética , Regiões 5' não Traduzidas , DNA Bacteriano , Regulação da Expressão Gênica de Plantas , Giberelinas/metabolismo , Mutação , Oryza/citologia , Fenótipo , Células Vegetais , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Caules de Planta/citologia , Caules de Planta/genética , Regiões Promotoras Genéticas , Estabilidade Proteica , Interferência de RNA , Sementes/genética , Sementes/crescimento & desenvolvimento , Transdução de Sinais , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo
13.
Nat Struct Mol Biol ; 27(8): 735-742, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32632277

RESUMO

The cytosolic iron-sulfur (Fe-S) assembly (CIA) pathway is required for the insertion of Fe-S clusters into cytosolic and nuclear client proteins, including many DNA replication and repair factors. The molecular mechanisms of client protein recognition and Fe-S cluster transfer remain unknown. Here, we report crystal structures of the CIA targeting complex (CTC), revealing that its CIAO2B subunit is centrally located and bridges CIAO1 and the client adaptor protein MMS19. Cryo-EM reconstructions of human CTC bound either to the DNA replication factor primase or to the DNA helicase DNA2, combined with biochemical, biophysical and yeast complementation assays, reveal an evolutionarily conserved, bipartite client recognition mode facilitated by CIAO1 and the structural flexibility of the MMS19 subunit. Unexpectedly, the primase Fe-S cluster is located ~70 Å away from the CTC reactive cysteine, implicating conformational dynamics of the CTC or additional maturation factors in the mechanism of Fe-S cluster transfer.


Assuntos
Proteínas com Ferro-Enxofre/metabolismo , Metalochaperonas/metabolismo , Metaloproteínas/metabolismo , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Animais , Linhagem Celular , Cristalografia por Raios X , Drosophila , Células HEK293 , Humanos , Proteínas com Ferro-Enxofre/química , Metalochaperonas/química , Metaloproteínas/química , Camundongos , Modelos Moleculares , Proteínas Nucleares/química , Ligação Proteica , Conformação Proteica , Fatores de Transcrição/química
14.
Nat Commun ; 11(1): 3398, 2020 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-32636384

RESUMO

SWI/SNF remodelers play a key role in regulating chromatin architecture and gene expression. Here, we report the cryo-EM structure of the Saccharomyces cerevisiae Swi/Snf complex in a nucleosome-free state. The structure consists of a stable triangular base module and a flexible Arp module. The conserved subunits Swi1 and Swi3 form the backbone of the complex and closely interact with other components. Snf6, which is specific for yeast Swi/Snf complex, stabilizes the binding of the ATPase-containing subunit Snf2 to the base module. Comparison of the yeast Swi/Snf and RSC complexes reveals conserved structural features that govern the assembly and function of these two subfamilies of chromatin remodelers. Our findings complement those from recent structures of the yeast and human chromatin remodelers and provide further insights into the assembly and function of the SWI/SNF remodelers.


Assuntos
Adenosina Trifosfatases/química , Cromatina/química , Proteínas Cromossômicas não Histona/química , Proteínas Nucleares/química , Proteínas Repressoras/química , Proteínas de Saccharomyces cerevisiae/química , Fatores de Transcrição/química , Microscopia Crioeletrônica , Proteínas de Ligação a DNA/química , Humanos , Nucleossomos , Ligação Proteica , Domínios Proteicos , Saccharomyces cerevisiae/química
15.
Gene ; 757: 144935, 2020 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-32653482

RESUMO

MYB transcription factors (TFs) play important roles in the plant's response to abiotic stress. In this study, we cloned a novel MYB TF gene from Vaccinium corymbosum (blueberry) using rapid amplification of cDNA ends (RACE). The cDNA contained a 798-bp open reading frame that encodes a 265-amino acid protein. VcMYB4a possessed a C2/EAR-repressor motif domain and phylogenetic analysis showed that it clustered into a subgroup 4 with six Arabidopsis thaliana MYBs. Quantitative RT-PCR analysis demonstrated that VcMYB4a expression was downregulated by salt, drought, and cold treatment, but was induced by freezing and heat. Overexpression of VcMYB4a in blueberry callus enhanced sensitivity to salt, drought, cold, freezing, and heat stress. These results indicate that VcMYB4a may be an important repressor of abiotic stress in blueberry.


Assuntos
Mirtilos Azuis (Planta)/genética , Resposta ao Choque Térmico , Proteínas de Plantas/genética , Tolerância ao Sal , Termotolerância , Fatores de Transcrição/genética , Mirtilos Azuis (Planta)/metabolismo , Secas , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Domínios Proteicos , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo
16.
Nat Commun ; 11(1): 3703, 2020 07 24.
Artigo em Inglês | MEDLINE | ID: mdl-32710080

RESUMO

Mycobacterium tuberculosis is a pathogen with a unique cell envelope including very long fatty acids, implicated in bacterial resistance and host immune modulation. FasR is a TetR-like transcriptional activator that plays a central role in sensing mycobacterial long-chain fatty acids and regulating lipid biosynthesis. Here we disclose crystal structures of M. tuberculosis FasR in complex with acyl effector ligands and with DNA, uncovering its molecular sensory and switching mechanisms. A long tunnel traverses the entire effector-binding domain, enabling long fatty acyl effectors to bind. Only when the tunnel is entirely occupied, the protein dimer adopts a rigid configuration with its DNA-binding domains in an open state, leading to DNA dissociation. The protein-folding hydrophobic core connects the two domains, and is completed into a continuous spine when the effector binds. Such a transmission spine is conserved in a large number of TetR-like regulators, offering insight into effector-triggered allosteric functional control.


Assuntos
Acil Coenzima A/química , Proteínas de Bactérias/química , Proteínas de Ligação a DNA/química , Mycobacterium tuberculosis/metabolismo , Fatores de Transcrição/química , Acil Coenzima A/metabolismo , Sítio Alostérico , Proteínas de Bactérias/metabolismo , Parede Celular/metabolismo , Cristalografia por Raios X , DNA Bacteriano/química , Proteínas de Ligação a DNA/metabolismo , Ácidos Graxos/metabolismo , Ligantes , Modelos Moleculares , Conformação Proteica , Fatores de Transcrição/metabolismo
17.
Nat Commun ; 11(1): 3740, 2020 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-32719356

RESUMO

Mfd couples transcription to nucleotide excision repair, and acts on RNA polymerases when elongation is impeded. Depending on impediment severity, this action results in either transcription termination or elongation rescue, which rely on ATP-dependent Mfd translocation on DNA. Due to its role in antibiotic resistance, Mfd is also emerging as a prime target for developing anti-evolution drugs. Here we report the structure of DNA-bound Mfd, which reveals large DNA-induced structural changes that are linked to the active site via ATPase motif VI. These changes relieve autoinhibitory contacts between the N- and C-termini and unmask UvrA recognition determinants. We also demonstrate that translocation relies on a threonine in motif Ic, widely conserved in translocases, and a family-specific histidine near motif IVa, reminiscent of the "arginine clamp" of RNA helicases. Thus, Mfd employs a mode of DNA recognition that at its core is common to ss/ds translocases that act on DNA or RNA.


Assuntos
Proteínas de Bactérias/metabolismo , Reparo do DNA , DNA/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Genética , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Sítios de Ligação , DNA/química , DNA/ultraestrutura , Escherichia coli/metabolismo , Modelos Moleculares , Ligação Proteica , Domínios Proteicos , RNA Helicases/metabolismo , Fatores de Transcrição/química
18.
SAR QSAR Environ Res ; 31(7): 547-570, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32657160

RESUMO

It is well known that bromodomain-containing protein 4 (BRD4) has been thought as a promising target utilized for treating various human diseases, such as inflammatory disorders, malignant tumours, acute myelogenous leukaemia (AML), bone diseases, etc. For this study, molecular dynamics (MD) simulations, binding free energy calculations, and principal component analysis (PCA) were integrated together to uncover binding modes of inhibitors 8P9, 8PU, and 8PX to BRD4(1). The results obtained from binding free energy calculations show that van der Waals interactions act as the main regulator in bindings of inhibitors to BRD4(1). The information stemming from PCA reveals that inhibitor associations extremely affect conformational changes, internal dynamics, and movement patterns of BRD4(1). Residue-based free energy decomposition method was wielded to unveil contributions of independent residues to inhibitor bindings and the data signify that hydrogen bonding interactions and hydrophobic interactions are decisive factors affecting bindings of inhibitors to BRD4(1). Meanwhile, eight residues Trp81, Pro82, Val87, Leu92, Leu94, Cys136, Asn140, and Ile146 are recognized as the common hot interaction spots of three inhibitors with BRD4(1). The results from this work are expected to provide a meaningfully theoretical guidance for design and development of effective inhibitors inhibiting of the activity of BRD4.


Assuntos
Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/química , Simulação de Dinâmica Molecular , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/química , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Análise de Componente Principal , Ligação Proteica
19.
Proc Natl Acad Sci U S A ; 117(31): 18858-18868, 2020 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-32694206

RESUMO

Buried seedlings undergo dramatic developmental transitions when they emerge from soil into sunlight. As central transcription factors suppressing light responses, PHYTOCHROME-INTERACTING FACTORs (PIFs) and ETHYLENE-INSENSITIVE 3 (EIN3) actively function in darkness and must be promptly repressed upon light to initiate deetiolation. Microproteins are evolutionarily conserved small single-domain proteins that act as posttranslational regulators in eukaryotes. Although hundreds to thousands of microproteins are predicted to exist in plants, their target molecules, biological roles, and mechanisms of action remain largely unknown. Here, we show that two microproteins, miP1a and miP1b (miP1a/b), are robustly stimulated in the dark-to-light transition. miP1a/b are primarily expressed in cotyledons and hypocotyl, exhibiting tissue-specific patterns similar to those of PIFs and EIN3 We demonstrate that PIFs and EIN3 assemble functional oligomers by self-interaction, while miP1a/b directly interact with and disrupt the oligomerization of PIFs and EIN3 by forming nonfunctional protein complexes. As a result, the DNA binding capacity and transcriptional activity of PIFs and EIN3 are predominantly suppressed. These biochemical findings are further supported by genetic evidence. miP1a/b positively regulate photomorphogenic development, and constitutively expressing miP1a/b rescues the delayed apical hook unfolding and cotyledon development of plants overexpressing PIFs and EIN3 Our study reveals that microproteins provide a temporal and negative control of the master transcription factors' oligomerization to achieve timely developmental transitions upon environmental changes.


Assuntos
Proteínas de Arabidopsis , Proteínas de Ligação a DNA , Desenvolvimento Vegetal/efeitos da radiação , Transdução de Sinais/efeitos da radiação , Fatores de Transcrição , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica de Plantas/efeitos da radiação , Luz , Especificidade de Órgãos , Multimerização Proteica/efeitos da radiação , Fatores de Transcrição/química , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
20.
Science ; 368(6498): 1449-1454, 2020 06 26.
Artigo em Inglês | MEDLINE | ID: mdl-32587015

RESUMO

Gene regulation is chiefly determined at the level of individual linear chromatin molecules, yet our current understanding of cis-regulatory architectures derives from fragmented sampling of large numbers of disparate molecules. We developed an approach for precisely stenciling the structure of individual chromatin fibers onto their composite DNA templates using nonspecific DNA N6-adenine methyltransferases. Single-molecule long-read sequencing of chromatin stencils enabled nucleotide-resolution readout of the primary architecture of multikilobase chromatin fibers (Fiber-seq). Fiber-seq exposed widespread plasticity in the linear organization of individual chromatin fibers and illuminated principles guiding regulatory DNA actuation, the coordinated actuation of neighboring regulatory elements, single-molecule nucleosome positioning, and single-molecule transcription factor occupancy. Our approach and results open new vistas on the primary architecture of gene regulation.


Assuntos
Montagem e Desmontagem da Cromatina , Cromatina/química , Regulação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Imagem Individual de Molécula/métodos , Animais , DNA/química , DNA/genética , Drosophila melanogaster , Humanos , Células K562 , Nucleossomos/química , Regiões Promotoras Genéticas , DNA Metiltransferases Sítio Específica (Adenina-Específica)/química , Fatores de Transcrição/química
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