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1.
Nature ; 574(7777): 278-282, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31578520

RESUMO

In eukaryotes, accurate chromosome segregation in mitosis and meiosis maintains genome stability and prevents aneuploidy. Kinetochores are large protein complexes that, by assembling onto specialized Cenp-A nucleosomes1,2, function to connect centromeric chromatin to microtubules of the mitotic spindle3,4. Whereas the centromeres of vertebrate chromosomes comprise millions of DNA base pairs and attach to multiple microtubules, the simple point centromeres of budding yeast are connected to individual microtubules5,6. All 16 budding yeast chromosomes assemble complete kinetochores using a single Cenp-A nucleosome (Cenp-ANuc), each of which is perfectly centred on its cognate centromere7-9. The inner and outer kinetochore modules are responsible for interacting with centromeric chromatin and microtubules, respectively. Here we describe the cryo-electron microscopy structure of the Saccharomyces cerevisiae inner kinetochore module, the constitutive centromere associated network (CCAN) complex, assembled onto a Cenp-A nucleosome (CCAN-Cenp-ANuc). The structure explains the interdependency of the constituent subcomplexes of CCAN and shows how the Y-shaped opening of CCAN accommodates Cenp-ANuc to enable specific CCAN subunits to contact the nucleosomal DNA and histone subunits. Interactions with the unwrapped DNA duplex at the two termini of Cenp-ANuc are mediated predominantly by a DNA-binding groove in the Cenp-L-Cenp-N subcomplex. Disruption of these interactions impairs assembly of CCAN onto Cenp-ANuc. Our data indicate a mechanism of Cenp-A nucleosome recognition by CCAN and how CCAN acts as a platform for assembly of the outer kinetochore to link centromeres to the mitotic spindle for chromosome segregation.


Assuntos
Proteína Centromérica A/metabolismo , Cinetocoros/química , Cinetocoros/metabolismo , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Nucleossomos/química , Nucleossomos/metabolismo , Proteína Centromérica A/química , Proteína Centromérica A/ultraestrutura , Microscopia Crioeletrônica , DNA/química , DNA/metabolismo , DNA/ultraestrutura , Cinetocoros/ultraestrutura , Modelos Moleculares , Complexos Multiproteicos/ultraestrutura , Nucleossomos/ultraestrutura , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/ultraestrutura
2.
BMC Bioinformatics ; 20(1): 464, 2019 Sep 09.
Artigo em Inglês | MEDLINE | ID: mdl-31500562

RESUMO

BACKGROUND: The function of oligomeric proteins is inherently linked to their quaternary structure. In the absence of high-resolution data, low-resolution information in the form of spatial restraints can significantly contribute to the precision and accuracy of structural models obtained using computational approaches. To obtain such restraints, chemical cross-linking coupled with mass spectrometry (XL-MS) is commonly used. However, the use of XL-MS in the modeling of protein complexes comprised of identical subunits (homo-oligomers) is often hindered by the inherent ambiguity of intra- and inter-subunit connection assignment. RESULTS: We present a comprehensive evaluation of (1) different methods for inter-residue distance calculations, and (2) different approaches for the scoring of spatial restraints. Our results show that using Solvent Accessible Surface distances (SASDs) instead of Euclidean distances (EUCs) greatly reduces the assignation ambiguity and delivers better modeling precision. Furthermore, ambiguous connections should be considered as inter-subunit only when the intra-subunit alternative exceeds the distance threshold. Modeling performance can also be improved if symmetry, characteristic for most homo-oligomers, is explicitly defined in the scoring function. CONCLUSIONS: Our findings provide guidelines for proper evaluation of chemical cross-linking-based spatial restraints in modeling homo-oligomeric protein complexes, which could facilitate structural characterization of this important group of proteins.


Assuntos
Reagentes para Ligações Cruzadas/química , Modelos Moleculares , Complexos Multiproteicos/química , Multimerização Proteica , Simulação de Acoplamento Molecular , Subunidades Proteicas/química , Solventes
3.
Nucleic Acids Res ; 47(18): 9696-9707, 2019 10 10.
Artigo em Inglês | MEDLINE | ID: mdl-31400115

RESUMO

Ubiquitous Structural Maintenance of Chromosomes (SMC) complexes use a proteinaceous ring-shaped architecture to organize and individualize chromosomes, thereby facilitating chromosome segregation. They utilize cycles of adenosine triphosphate (ATP) binding and hydrolysis to transport themselves rapidly with respect to DNA, a process requiring protein conformational changes and multiple DNA contact sites. By analysing changes in the architecture and stoichiometry of the Escherichia coli SMC complex, MukBEF, as a function of nucleotide binding to MukB and subsequent ATP hydrolysis, we demonstrate directly the formation of dimer of MukBEF dimer complexes, dependent on dimeric MukF kleisin. Using truncated and full length MukB, in combination with MukEF, we show that engagement of the MukB ATPase heads on nucleotide binding directs the formation of dimers of heads-engaged dimer complexes. Complex formation requires functional interactions between the C- and N-terminal domains of MukF with the MukB head and neck, respectively, and MukE, which organizes the complexes by stabilizing binding of MukB heads to MukF. In the absence of head engagement, a MukF dimer bound by MukE forms complexes containing only a dimer of MukB. Finally, we demonstrate that cells expressing MukBEF complexes in which MukF is monomeric are Muk-, with the complexes failing to associate with chromosomes.


Assuntos
Proteínas Cromossômicas não Histona/química , Proteínas de Escherichia coli/genética , Proteínas Repressoras/genética , Proteínas Cromossômicas não Histona/genética , Cromossomos/química , Cromossomos/genética , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Escherichia coli/química , Escherichia coli/genética , Proteínas de Escherichia coli/química , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Ligação Proteica , Proteínas Repressoras/química
4.
Genes Dev ; 33(15-16): 1031-1047, 2019 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-31196865

RESUMO

Aneuploidy, a condition characterized by chromosome gains and losses, causes reduced fitness and numerous cellular stresses, including increased protein aggregation. Here, we identify protein complex stoichiometry imbalances as a major cause of protein aggregation in aneuploid cells. Subunits of protein complexes encoded on excess chromosomes aggregate in aneuploid cells, which is suppressed when expression of other subunits is coordinately altered. We further show that excess subunits are either degraded or aggregate and that protein aggregation is nearly as effective as protein degradation at lowering levels of excess proteins. Our study explains why proteotoxic stress is a universal feature of the aneuploid state and reveals protein aggregation as a form of dosage compensation to cope with disproportionate expression of protein complex subunits.


Assuntos
Aneuploidia , Citosol/metabolismo , Compensação de Dosagem (Genética)/fisiologia , Agregados Proteicos/genética , Humanos , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Agregação Patológica de Proteínas , Subunidades Proteicas/metabolismo , Proteólise , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
5.
Int J Mol Sci ; 20(12)2019 Jun 23.
Artigo em Inglês | MEDLINE | ID: mdl-31234561

RESUMO

The channeling of metabolites is an essential step of metabolic regulation in all living organisms. Multifunctional enzymes with defined domains for metabolite compartmentalization are rare, but in many cases, larger assemblies forming multimeric protein complexes operate in defined metabolic shunts. In Arabidopsis thaliana, a multimeric complex was discovered that contains a 13-lipoxygenase and allene oxide synthase (AOS) as well as allene oxide cyclase. All three plant enzymes are localized in chloroplasts, contributing to the biosynthesis of jasmonic acid (JA). JA and its derivatives act as ubiquitous plant defense regulators in responses to both biotic and abiotic stresses. AOS belongs to the superfamily of cytochrome P450 enzymes and is named CYP74A. Another CYP450 in chloroplasts, hydroperoxide lyase (HPL, CYP74B), competes with AOS for the common substrate. The products of the HPL reaction are green leaf volatiles that are involved in the deterrence of insect pests. Both enzymes represent non-canonical CYP450 family members, as they do not depend on O2 and NADPH-dependent CYP450 reductase activities. AOS and HPL activities are crucial for plants to respond to different biotic foes. In this mini-review, we aim to summarize how plants make use of the LOX2-AOS-AOC2 complex in chloroplasts to boost JA biosynthesis over volatile production and how this situation may change in plant communities during mass ingestion by insect pests.


Assuntos
Aldeído Liases/metabolismo , Arabidopsis/fisiologia , Sistema Enzimático do Citocromo P-450/metabolismo , Resistência à Doença , Oxirredutases Intramoleculares/metabolismo , Aldeído Liases/química , Aldeído Liases/genética , Sequência de Aminoácidos , Cloroplastos/metabolismo , Ciclopentanos/metabolismo , Sistema Enzimático do Citocromo P-450/química , Sistema Enzimático do Citocromo P-450/genética , Resistência à Doença/genética , Oxirredutases Intramoleculares/química , Oxirredutases Intramoleculares/genética , Redes e Vias Metabólicas , Modelos Moleculares , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Oxilipinas/metabolismo , Desenvolvimento Vegetal/genética , Ligação Proteica , Relação Estrutura-Atividade
6.
Nat Cell Biol ; 21(5): 614-626, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31036939

RESUMO

Cell growth is controlled by a lysosomal signalling complex containing Rag small GTPases and mammalian target of rapamycin complex 1 (mTORC1) kinase. Here, we carried out a microscopy-based genome-wide human short interfering RNA screen and discovered a lysosome-localized G protein-coupled receptor (GPCR)-like protein, GPR137B, that interacts with Rag GTPases, increases Rag localization and activity, and thereby regulates mTORC1 translocation and activity. High GPR137B expression can recruit and activate mTORC1 in the absence of amino acids. Furthermore, GPR137B also regulates the dissociation of activated Rag from lysosomes, suggesting that GPR137B controls a cycle of Rag activation and dissociation from lysosomes. GPR137B-knockout cells exhibited defective autophagy and an expanded lysosome compartment, similar to Rag-knockout cells. Like zebrafish RagA mutants, GPR137B-mutant zebrafish had upregulated TFEB target gene expression and an expanded lysosome compartment in microglia. Thus, GPR137B is a GPCR-like lysosomal regulatory protein that controls dynamic Rag and mTORC1 localization and activity as well as lysosome morphology.


Assuntos
Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Genoma Humano/genética , Proteínas Monoméricas de Ligação ao GTP/genética , Receptores Acoplados a Proteínas-G/genética , Animais , Autofagia/genética , Regulação da Expressão Gênica/genética , Humanos , Lisossomos/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Microglia/metabolismo , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , RNA Interferente Pequeno/genética , Receptores Acoplados a Proteínas-G/antagonistas & inibidores , Peixe-Zebra/genética , Peixe-Zebra/crescimento & desenvolvimento
7.
Nature ; 568(7751): 259-263, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30944473

RESUMO

The genetic compensation response (GCR) has recently been proposed as a possible explanation for the phenotypic discrepancies between gene-knockout and gene-knockdown1,2; however, the underlying molecular mechanism of the GCR remains uncharacterized. Here, using zebrafish knockdown and knockout models of the capn3a and nid1a genes, we show that mRNA bearing a premature termination codon (PTC) promptly triggers a GCR that involves Upf3a and components of the COMPASS complex. Unlike capn3a-knockdown embryos, which have small livers, and nid1a-knockdown embryos, which have short body lengths2, capn3a-null and nid1a-null mutants appear normal. These phenotypic differences have been attributed to the upregulation of other genes in the same families. By analysing six uniquely designed transgenes, we demonstrate that the GCR is dependent on both the presence of a PTC and the nucleotide sequence of the transgene mRNA, which is homologous to the compensatory endogenous genes. We show that upf3a (a member of the nonsense-mediated mRNA decay pathway) and components of the COMPASS complex including wdr5 function in GCR. Furthermore, we demonstrate that the GCR is accompanied by an enhancement of histone H3 Lys4 trimethylation (H3K4me3) at the transcription start site regions of the compensatory genes. These findings provide a potential mechanistic basis for the GCR, and may help lead to the development of therapeutic strategies that treat missense mutations associated with genetic disorders by either creating a PTC in the mutated gene or introducing a transgene containing a PTC to trigger a GCR.


Assuntos
Códon sem Sentido/genética , Teste de Complementação Genética , Complexos Multiproteicos/metabolismo , RNA Mensageiro/genética , Peixe-Zebra/genética , Animais , Proteínas de Ligação ao Cálcio/deficiência , Proteínas de Ligação ao Cálcio/genética , Deleção de Genes , Células HCT116 , Histonas/metabolismo , Humanos , Complexos Multiproteicos/química , Degradação do RNAm Mediada por Códon sem Sentido , Organismos Geneticamente Modificados , Proteínas de Peixe-Zebra/deficiência , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
8.
Phys Chem Chem Phys ; 21(18): 9265-9276, 2019 May 08.
Artigo em Inglês | MEDLINE | ID: mdl-31016301

RESUMO

As applications in mass spectrometry continue to expand into the field of structural biology, there have been an increasing number of studies on noncovalent biological assemblies. Ensuring that protein complexes maintain native-like conformations and architectures during the transition from solution to the gas phase is a key aim. Probing composition and arrangement of subunits of multi-charged complexes via tandem mass spectrometry (MS/MS) may lead to protein unfolding and the redistribution of charges on the constituent subunits, leading to asymmetric charge partitioning and ejection of a high-charged monomer. Additionally, the overall dissociation efficiency of many ion activation methods is often suppressed for low charge states, hindering the effectiveness of MS/MS for complexes that have low charge density. Ultraviolet photodissociation (UVPD) of proteins using 193 nm photons is a high-energy alternative to collisional activation and demonstrates little to no charge state dependence. Here the symmetry of charge partitioning upon UVPD is evaluated for an array of multimeric protein complexes as a function of initial charge state. The results demonstrate that high laser energies (3 mJ) for UVPD induces more symmetric charge partitioning and ejection of low-charged, presumably compact monomers than higher-energy collisional dissociation (HCD).


Assuntos
Complexos Multiproteicos/química , Complexos Multiproteicos/efeitos da radiação , Conformação Proteica/efeitos da radiação , Raios Ultravioleta , Espectrometria de Massas em Tandem
9.
Biochim Biophys Acta Rev Cancer ; 1872(1): 11-23, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31034924

RESUMO

The ubiquitous family of AP-1 dimeric transcription complexes is involved in virtually all cellular and physiological functions. It is paramount for cells to reprogram gene expression in response to cues of many sorts and is involved in many tumorigenic processes. How AP-1 controls gene transcription has largely remained elusive till recently. The advent of the "omics" technologies permitting genome-wide studies of transcription factors has however changed and improved our view of AP-1 mechanistical actions. If these studies confirm that AP-1 can sometimes act as a local transcriptional switch operating in the vicinity of transcription start sites (TSS), they strikingly indicate that AP-1 principally operates as a remote command binding to distal enhancers, placing chromatin architecture dynamics at the heart of its transcriptional actions. They also unveil novel constraints operating on AP-1, as well as novel mechanisms used to regulate gene expression via transcription-pioneering-, chromatin-remodeling- and chromatin accessibility maintenance effects.


Assuntos
Complexos Multiproteicos/genética , Fator de Transcrição AP-1/genética , Transcrição Genética , Ativação Transcricional/genética , Sítios de Ligação/genética , Núcleo Celular/genética , Montagem e Desmontagem da Cromatina/genética , Humanos , Complexos Multiproteicos/química , Fator de Transcrição AP-1/química , Sítio de Iniciação de Transcrição
10.
Mol Cell ; 74(4): 701-712.e9, 2019 05 16.
Artigo em Inglês | MEDLINE | ID: mdl-30948266

RESUMO

Alternative 3' untranslated regions (3' UTRs) are widespread, but their functional roles are largely unknown. We investigated the function of the long BIRC3 3' UTR, which is upregulated in leukemia. The 3' UTR does not regulate BIRC3 protein localization or abundance but is required for CXCR4-mediated B cell migration. We established an experimental pipeline to study the mechanism of regulation and used mass spectrometry to identify BIRC3 protein interactors. In addition to 3'-UTR-independent interactors involved in known BIRC3 functions, we detected interactors that bind only to BIRC3 protein encoded from the mRNA with the long 3' UTR. They regulate several functions, including CXCR4 trafficking. We further identified RNA-binding proteins differentially bound to the alternative 3' UTRs and found that cooperative binding of Staufen and HuR mediates 3'-UTR-dependent complex formation. We show that the long 3' UTR is required for the formation of specific protein complexes that enable additional functions of BIRC3 protein beyond its 3'-UTR-independent functions.


Assuntos
Proteína 3 com Repetições IAP de Baculovírus/genética , Leucemia/genética , Complexos Multiproteicos/genética , Receptores CXCR4/genética , Regiões 3' não Traduzidas/genética , Linfócitos B/metabolismo , Linfócitos B/patologia , Proteína 3 com Repetições IAP de Baculovírus/química , Movimento Celular/genética , Proteínas do Citoesqueleto/genética , Proteína Semelhante a ELAV 1/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Leucemia/patologia , Complexos Multiproteicos/química , Transporte Proteico , RNA Mensageiro/genética , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética
11.
Molecules ; 24(7)2019 Apr 11.
Artigo em Inglês | MEDLINE | ID: mdl-30979095

RESUMO

Many nucleic acid-protein structures have been resolved, though quantitative structure-activity relationship remains unclear in many cases. Thrombin complexes with G-quadruplex aptamers are striking examples of a lack of any correlation between affinity, interface organization, and other common parameters. Here, we tested the hypothesis that affinity of the aptamer-protein complex is determined with the capacity of the interface to dissipate energy of binding. Description and detailed analysis of 63 nucleic acid-protein structures discriminated peculiarities of high-affinity nucleic acid-protein complexes. The size of the amino acid sidechain in the interface was demonstrated to be the most significant parameter that correlates with affinity of aptamers. This observation could be explained in terms of need of efficient energy transfer from interacting residues. Application of energy dissipation theory provided an illustrative tool for estimation of efficiency of aptamer-protein complexes. These results are of great importance for a design of efficient aptamers.


Assuntos
Aptâmeros de Nucleotídeos/química , Quadruplex G , Ácidos Nucleicos/química , Proteínas/química , Fenômenos Biofísicos , Transferência de Energia , Fenômenos Mecânicos , Complexos Multiproteicos/química , Conformação de Ácido Nucleico , Ligação Proteica , Conformação Proteica , Relação Estrutura-Atividade , Trombina/química
12.
Nature ; 567(7749): 486-490, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30894744

RESUMO

In Gram-negative bacteria, lipopolysaccharide is essential for outer membrane formation and antibiotic resistance. The seven lipopolysaccharide transport (Lpt) proteins A-G move lipopolysaccharide from the inner to the outer membrane. The ATP-binding cassette transporter LptB2FG, which tightly associates with LptC, extracts lipopolysaccharide out of the inner membrane. The mechanism of the LptB2FG-LptC complex (LptB2FGC) and the role of LptC in lipopolysaccharide transport are poorly understood. Here we characterize the structures of LptB2FG and LptB2FGC in nucleotide-free and vanadate-trapped states, using single-particle cryo-electron microscopy. These structures resolve the bound lipopolysaccharide, reveal transporter-lipopolysaccharide interactions with side-chain details and uncover how the capture and extrusion of lipopolysaccharide are coupled to conformational rearrangements of LptB2FGC. LptC inserts its transmembrane helix between the two transmembrane domains of LptB2FG, which represents a previously unknown regulatory mechanism for ATP-binding cassette transporters. Our results suggest a role for LptC in achieving efficient lipopolysaccharide transport, by coordinating the action of LptB2FG in the inner membrane and Lpt protein interactions in the periplasm.


Assuntos
Microscopia Crioeletrônica , Proteínas de Escherichia coli/metabolismo , Escherichia coli/química , Escherichia coli/metabolismo , Lipopolissacarídeos/química , Lipopolissacarídeos/metabolismo , Proteínas de Membrana/metabolismo , Complexos Multiproteicos/química , Complexos Multiproteicos/ultraestrutura , Escherichia coli/ultraestrutura , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/ultraestrutura , Proteínas de Membrana/química , Proteínas de Membrana/ultraestrutura , Modelos Moleculares , Complexos Multiproteicos/metabolismo , Ligação Proteica/efeitos dos fármacos , Domínios Proteicos/efeitos dos fármacos , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Relação Estrutura-Atividade , Vanadatos/química , Vanadatos/metabolismo , Vanadatos/farmacologia
13.
Nature ; 567(7749): 550-553, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30894747

RESUMO

Gram-negative bacteria are surrounded by an inner cytoplasmic membrane and by an outer membrane, which serves as a protective barrier to limit entry of many antibiotics. The distinctive properties of the outer membrane are due to the presence of lipopolysaccharide1. This large glycolipid, which contains numerous sugars, is made in the cytoplasm; a complex of proteins forms a membrane-to-membrane bridge that mediates transport of lipopolysaccharide from the inner membrane to the cell surface1. The inner-membrane components of the protein bridge comprise an ATP-binding cassette transporter that powers transport, but how this transporter ensures unidirectional lipopolysaccharide movement across the bridge to the outer membrane is unknown2. Here we describe two crystal structures of a five-component inner-membrane complex that contains all the proteins required to extract lipopolysaccharide from the membrane and pass it to the protein bridge. Analysis of these structures, combined with biochemical and genetic experiments, identifies the path of lipopolysaccharide entry into the cavity of the transporter and up to the bridge. We also identify a protein gate that must open to allow movement of substrate from the cavity onto the bridge. Lipopolysaccharide entry into the cavity is ATP-independent, but ATP is required for lipopolysaccharide movement past the gate and onto the bridge. Our findings explain how the inner-membrane transport complex controls efficient unidirectional transport of lipopolysaccharide against its concentration gradient.


Assuntos
Transportadores de Cassetes de Ligação de ATP/química , Proteínas de Bactérias/química , Membrana Celular/metabolismo , Lipopolissacarídeos/metabolismo , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Vibrio cholerae/química , Transportadores de Cassetes de Ligação de ATP/metabolismo , Trifosfato de Adenosina/metabolismo , Proteínas de Bactérias/metabolismo , Transporte Biológico , Cristalografia por Raios X , Escherichia coli , Proteínas de Escherichia coli/química , Klebsiella pneumoniae , Lipopolissacarídeos/química , Proteínas de Membrana/química , Modelos Moleculares , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Pseudomonas aeruginosa , Vibrio cholerae/citologia , Vibrio cholerae/metabolismo
14.
Nucleic Acids Res ; 47(9): 4462-4475, 2019 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-30864669

RESUMO

The general transcription factor P-TEFb, a master regulator of RNA polymerase (Pol) II elongation, phosphorylates the C-terminal domain (CTD) of Pol II and negative elongation factors to release Pol II from promoter-proximal pausing. We show here that P-TEFb surprisingly inhibits the myoblast differentiation into myotubes, and that P-TEFb and its two positive complexes are eliminated in this process. In contrast, DYRK1A, another CTD kinase known to control transcription of a subset of genes important for development and tissue homeostasis, is found to activate transcription of key myogenic genes. We show that active DYRK1A exists in a complex with the WD40-repeat protein DCAF7 that stabilizes and tethers DYRK1A to Pol II, so that DYRK1A-DCAF7 can co-migrate with and phosphorylate Pol II along the myogenic gene loci. Thus, DCAF7 modulates the kinase signaling output of DYRK1A on Pol II to stimulate myogenic transcription after active P-TEFb function is shut off.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Desenvolvimento Muscular/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Tirosina Quinases/genética , Transcrição Genética , Animais , Diferenciação Celular/genética , Ciclina T/genética , Quinase 9 Dependente de Ciclina/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Humanos , Camundongos , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Fibras Musculares Esqueléticas/metabolismo , Mioblastos/metabolismo , Proteínas Nucleares/genética , RNA Polimerase II/química , RNA Polimerase II/genética , Canais de Translocação SEC/genética , Fatores de Transcrição/genética
15.
Int J Mol Sci ; 20(6)2019 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-30875920

RESUMO

The present study gives an overview of the binding energetics of the homologous heterodimers of cruzipain-chagasin based on the binding energy (ΔGb) prediction obtained with FoldX. This analysis involves a total of 70 homologous models of the cruzipain-chagasin complex which were constructed by homology from the combinatory variation of nine papain-like cysteine peptidase structures and seven cysteine protease inhibitor structures (as chagasin-like and cystatin-like inhibitors). Only 32 systems have been evaluated experimentally, ΔGbexperimental values previously reported. Therefore, the result of the multiple analysis in terms of the thermodynamic parameters, are shown as relative energy |ΔΔG| = |ΔGbfrom FoldX - ΔGbexperimental|. Nine models were identified that recorded |ΔΔG| < 1.3, five models to 2.8 > |ΔΔG| > 1.3 and the other 18 models, values of |ΔΔG| > 2.8. The energetic analysis of the contribution of ΔH and ΔS to ΔGb to the 14-molecular model presents a ΔGb mostly ΔH-driven at neutral pH and at an ionic strength (I) of 0.15 M. The dependence of ΔGb(I,pH) at 298 K to the cruzipain-chagasin complex predicts a linear dependence of ΔGb(I). The computational protocol allowed the identification and prediction of thermodynamics binding energy parameters for cruzipain-chagasin-like heterodimers.


Assuntos
Cisteína Endopeptidases/metabolismo , Complexos Multiproteicos/química , Proteínas de Protozoários/metabolismo , Trypanosoma cruzi/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Simulação por Computador , Cisteína Endopeptidases/química , Humanos , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Proteínas de Protozoários/química , Homologia Estrutural de Proteína
16.
Adv Mater ; 31(17): e1807521, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30866118

RESUMO

The development of extracellular matrix mimetics that imitate niche stem cell microenvironments and support cell growth for technological applications is intensely pursued. Specifically, mimetics are sought that can enact control over the self-renewal and directed differentiation of human pluripotent stem cells (hPSCs) for clinical use. Despite considerable progress in the field, a major impediment to the clinical translation of hPSCs is the difficulty and high cost of large-scale cell production under xeno-free culture conditions using current matrices. Here, a bioactive, recombinant, protein-based polymer, termed ZTFn , is presented that closely mimics human plasma fibronectin and serves as an economical, xeno-free, biodegradable, and functionally adaptable cell substrate. The ZTFn substrate supports with high performance the propagation and long-term self-renewal of human embryonic stem cells while preserving their pluripotency. The ZTFn polymer can, therefore, be proposed as an efficient and affordable replacement for fibronectin in clinical grade cell culturing. Further, it can be postulated that the ZT polymer has significant engineering potential for further orthogonal functionalization in complex cell applications.


Assuntos
Autorrenovação Celular/efeitos dos fármacos , Células-Tronco Embrionárias/metabolismo , Matriz Extracelular/química , Fibronectinas/química , Complexos Multiproteicos/química , Sequência de Aminoácidos , Materiais Biomiméticos/química , Técnicas de Cultura de Células/métodos , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Reagentes para Ligações Cruzadas/química , Humanos , Polímeros/química , Conformação Proteica
17.
Mol Cell ; 74(2): 245-253.e6, 2019 04 18.
Artigo em Inglês | MEDLINE | ID: mdl-30826165

RESUMO

Transcription factors (TFs) control gene expression by binding DNA recognition sites in genomic regulatory regions. Although most forkhead TFs recognize a canonical forkhead (FKH) motif, RYAAAYA, some forkheads recognize a completely different (FHL) motif, GACGC. Bispecific forkhead proteins recognize both motifs, but the molecular basis for bispecific DNA recognition is not understood. We present co-crystal structures of the FoxN3 DNA binding domain bound to the FKH and FHL sites, respectively. FoxN3 adopts a similar conformation to recognize both motifs, making contacts with different DNA bases using the same amino acids. However, the DNA structure is different in the two complexes. These structures reveal how a single TF binds two unrelated DNA sequences and the importance of DNA shape in the mechanism of bispecific recognition.


Assuntos
Proteínas de Ciclo Celular/química , Proteínas de Ligação a DNA/química , DNA/química , Conformação de Ácido Nucleico , Proteínas Repressoras/química , Sequência de Aminoácidos/genética , Sequência de Bases/genética , Sítios de Ligação/genética , Proteínas de Ciclo Celular/genética , Cristalografia por Raios X , DNA/genética , Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica/genética , Humanos , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Motivos de Nucleotídeos/genética , Sequências Reguladoras de Ácido Nucleico/genética , Proteínas Repressoras/genética
18.
Mol Cell ; 74(2): 347-362.e6, 2019 04 18.
Artigo em Inglês | MEDLINE | ID: mdl-30853401

RESUMO

Selective autophagy recycles damaged organelles and clears intracellular pathogens to prevent their aberrant accumulation. How ULK1 kinase is targeted and activated during selective autophagic events remains to be elucidated. In this study, we used chemically inducible dimerization (CID) assays in tandem with CRISPR KO lines to systematically analyze the molecular basis of selective autophagosome biogenesis. We demonstrate that ectopic placement of NDP52 on mitochondria or peroxisomes is sufficient to initiate selective autophagy by focally localizing and activating the ULK1 complex. The capability of NDP52 to induce mitophagy is dependent on its interaction with the FIP200/ULK1 complex, which is facilitated by TBK1. Ectopically tethering ULK1 to cargo bypasses the requirement for autophagy receptors and TBK1. Focal activation of ULK1 occurs independently of AMPK and mTOR. Our findings provide a parsimonious model of selective autophagy, which highlights the coordination of ULK1 complex localization by autophagy receptors and TBK1 as principal drivers of targeted autophagosome biogenesis.


Assuntos
Proteína Homóloga à Proteína-1 Relacionada à Autofagia/genética , Autofagia/genética , Proteínas Nucleares/genética , Proteínas Serina-Treonina Quinases/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Células HeLa , Humanos , Mitocôndrias/química , Mitocôndrias/genética , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Peroxissomos/química , Peroxissomos/genética , Fosforilação , Proteínas Quinases/genética , Multimerização Proteica , Proteínas Tirosina Quinases/química , Proteínas Tirosina Quinases/genética , Transdução de Sinais/genética , Serina-Treonina Quinases TOR/genética
19.
Mol Cell ; 74(2): 320-329.e6, 2019 04 18.
Artigo em Inglês | MEDLINE | ID: mdl-30853402

RESUMO

Xenophagy, a selective autophagy pathway that protects the cytosol against bacterial invasion, relies on cargo receptors that juxtapose bacteria and phagophore membranes. Whether phagophores are recruited from a constitutive pool or are generated de novo at prospective cargo remains unknown. Phagophore formation in situ would require recruitment of the upstream autophagy machinery to prospective cargo. Here, we show that, essential for anti-bacterial autophagy, the cargo receptor NDP52 forms a trimeric complex with FIP200 and SINTBAD/NAP1, which are subunits of the autophagy-initiating ULK and the TBK1 kinase complex, respectively. FIP200 and SINTBAD/NAP1 are each recruited independently to bacteria via NDP52, as revealed by selective point mutations in their respective binding sites, but only in their combined presence does xenophagy proceed. Such recruitment of the upstream autophagy machinery by NDP52 reveals how detection of cargo-associated "eat me" signals, induction of autophagy, and juxtaposition of cargo and phagophores are integrated in higher eukaryotes.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Autofagia/genética , Proteínas Nucleares/genética , Proteínas Tirosina Quinases/genética , Proteínas Adaptadoras de Transdução de Sinal/química , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/genética , Sítios de Ligação/genética , Citoplasma/microbiologia , Citosol/microbiologia , Humanos , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Proteínas Nucleares/química , Mutação Puntual/genética , Ligação Proteica/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Tirosina Quinases/química , Salmonella typhimurium/genética , Salmonella typhimurium/patogenicidade
20.
J Biol Chem ; 294(16): 6416-6429, 2019 04 19.
Artigo em Inglês | MEDLINE | ID: mdl-30819806

RESUMO

BTB-Kelch proteins are substrate-specific adaptors for cullin-3 (Cul3) RING-box-based E3 ubiquitin ligases, mediating protein ubiquitylation for subsequent proteasomal degradation. Vaccinia virus encodes three BTB-Kelch proteins: A55, C2, and F3. Viruses lacking A55 or C2 have altered cytopathic effects in cultured cells and altered pathology in vivo Previous studies have shown that the ectromelia virus orthologue of A55 interacts with Cul3 in cells. We report that the N-terminal BTB-BACK (BB) domain of A55 binds directly to the Cul3 N-terminal domain (Cul3-NTD), forming a 2:2 complex in solution. We solved the structure of an A55BB/Cul3-NTD complex from anisotropic crystals diffracting to 2.3/3.7 Å resolution in the best/worst direction, revealing that the overall interaction and binding interface closely resemble the structures of cellular BTB/Cul3-NTD complexes, despite low sequence identity between A55 and cellular BTB domains. Surprisingly, despite this structural similarity, the affinity of Cul3-NTD for A55BB was stronger than for cellular BTB proteins. Glutamate substitution of the A55 residue Ile-48, adjacent to the canonical φX(D/E) Cul3-binding motif, reduced affinity of A55BB for Cul3-NTD by at least 2 orders of magnitude. Moreover, Ile-48 and the φX(D/E) motif are conserved in A55 orthologues from other poxviruses, but not in the vaccinia virus proteins C2 or F3. The high-affinity interaction between A55BB and Cul3-NTD suggests that, in addition to directing the Cul3-RING E3 ligase complex to degrade cellular/viral target proteins that are normally unaffected, A55 may also sequester Cul3 from cellular adaptor proteins, thereby protecting substrates of these cellular adaptors from ubiquitylation and degradation.


Assuntos
Proteínas Culina/química , Complexos Multiproteicos/química , Vírus Vaccinia/química , Proteínas Virais/química , Substituição de Aminoácidos , Proteínas Culina/genética , Proteínas Culina/metabolismo , Células HEK293 , Humanos , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Mutação de Sentido Incorreto , Domínios Proteicos , Estrutura Quaternária de Proteína , Proteólise , Ubiquitinação/genética , Vaccinia/genética , Vaccinia/metabolismo , Vírus Vaccinia/genética , Vírus Vaccinia/metabolismo , Proteínas Virais/genética
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