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1.
Cell Rep ; 43(1): 113639, 2024 01 23.
Artigo em Inglês | MEDLINE | ID: mdl-38175753

RESUMO

The nuclear cap-binding complex (CBC) coordinates co-transcriptional maturation, transport, or degradation of nascent RNA polymerase II (Pol II) transcripts. CBC with its partner ARS2 forms mutually exclusive complexes with diverse "effectors" that promote either productive or destructive outcomes. Combining AlphaFold predictions with structural and biochemical validation, we show how effectors NCBP3, NELF-E, ARS2, PHAX, and ZC3H18 form competing binary complexes with CBC and how PHAX, NCBP3, ZC3H18, and other effectors compete for binding to ARS2. In ternary CBC-ARS2 complexes with PHAX, NCBP3, or ZC3H18, ARS2 is responsible for the initial effector recruitment but inhibits their direct binding to the CBC. We show that in vivo ZC3H18 binding to both CBC and ARS2 is required for nuclear RNA degradation. We propose that recruitment of PHAX to CBC-ARS2 can lead, with appropriate cues, to competitive displacement of ARS2 and ZC3H18 from the CBC, thus promoting a productive rather than a degradative RNA fate.


Assuntos
Complexo Proteico Nuclear de Ligação ao Cap , RNA , Ligação Competitiva , Complexo Proteico Nuclear de Ligação ao Cap/química , RNA/genética , RNA Polimerase II/metabolismo , RNA Nuclear
2.
Life Sci Alliance ; 6(11)2023 11.
Artigo em Inglês | MEDLINE | ID: mdl-37673444

RESUMO

RIPK2 is an essential adaptor for NOD signalling and its kinase domain is a drug target for NOD-related diseases, such as inflammatory bowel disease. However, recent work indicates that the phosphorylation activity of RIPK2 is dispensable for signalling and that inhibitors of both RIPK2 activity and RIPK2 ubiquitination prevent the essential interaction between RIPK2 and the BIR2 domain of XIAP, the key RIPK2 ubiquitin E3 ligase. Moreover, XIAP BIR2 antagonists also block this interaction. To reveal the molecular mechanisms involved, we combined native mass spectrometry, NMR, and cryo-electron microscopy to determine the structure of the RIPK2 kinase BIR2 domain complex and validated the interface with in cellulo assays. The structure shows that BIR2 binds across the RIPK2 kinase antiparallel dimer and provides an explanation for both inhibitory mechanisms. It also highlights why phosphorylation of the kinase activation loop is dispensable for signalling while revealing the structural role of RIPK2-K209 residue in the RIPK2-XIAP BIR2 interaction. Our results clarify the features of the RIPK2 conformation essential for its role as a scaffold protein for ubiquitination.


Assuntos
Bioensaio , Ubiquitina-Proteína Ligases , Ubiquitina-Proteína Ligases/genética , Microscopia Crioeletrônica , Fosforilação , Ubiquitinação
3.
Science ; 381(6663): 1217-1225, 2023 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-37708276

RESUMO

The mitogen-activated protein kinase (MAPK) p38α is a central component of signaling in inflammation and the immune response and is, therefore, an important drug target. Little is known about the molecular mechanism of its activation by double phosphorylation from MAPK kinases (MAP2Ks), because of the challenge of trapping a transient and dynamic heterokinase complex. We applied a multidisciplinary approach to generate a structural model of p38α in complex with its MAP2K, MKK6, and to understand the activation mechanism. Integrating cryo-electron microscopy with molecular dynamics simulations, hydrogen-deuterium exchange mass spectrometry, and experiments in cells, we demonstrate a dynamic, multistep phosphorylation mechanism, identify catalytically relevant interactions, and show that MAP2K-disordered amino termini determine pathway specificity. Our work captures a fundamental step of cell signaling: a kinase phosphorylating its downstream target kinase.


Assuntos
MAP Quinase Quinase 2 , MAP Quinase Quinase 6 , Proteína Quinase 14 Ativada por Mitógeno , Microscopia Crioeletrônica , Ativação Enzimática , MAP Quinase Quinase 2/química , MAP Quinase Quinase 6/química , Proteína Quinase 14 Ativada por Mitógeno/química , Fosforilação , Especificidade por Substrato , Conformação Proteica
4.
PLoS One ; 18(4): e0265297, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37068110

RESUMO

BACKGROUND: Caregivers of people with Multiple Sclerosis are required to provide ongoing assistance especially during the advanced stages of the disease. They have to manage interventions and assume responsibilities which significantly impact both their personal quality of life and family's dynamics. OBJECTIVE: A qualitative phenomenological study was carried out to understand the experience of burden in caregivers and their resources to manage it. The study also explores how healthcare services involved in the Multiple Sclerosis Clinical Pathway respond to the needs of well-being of patients and family members. METHODS: 17 caregivers were involved in focus groups and in semi-structured individual interviews. RESULTS: Fatigue is experienced by all respondents and it starts when physical disabilities increase or when people become aware of them. Many caregivers declare that they refer to intrinsic (love towards their relatives, patience and dedication) or extrinsic (family members, hobbies) resources to cope with the burden of assistance. Patient associations and the Multiple Sclerosis Clinical Pathway play a significant role in supporting caregivers. CONCLUSIONS: Fatigue, loneliness, and isolation are experienced by caregivers and strongly affect their quality of life and health status. The study highlights caregivers' need to reconcile working times with care times, to give more space to self-care and to have moments to share their experiences with someone else. These needs should be at the core of health policies in order to avoid physical and emotional breakdowns which could lead to the rupture of the relational balance on which home care is based.


Assuntos
Cuidadores , Esclerose Múltipla , Humanos , Cuidadores/psicologia , Qualidade de Vida , Estresse Psicológico/psicologia , Família/psicologia , Pesquisa Qualitativa
5.
Chemistry ; 25(36): 8484-8488, 2019 Jun 26.
Artigo em Inglês | MEDLINE | ID: mdl-31038818

RESUMO

Cell signaling by small G proteins uses an ON to OFF signal based on conformational changes following the hydrolysis of GTP to GDP and release of dihydrogen phosphate (Pi ). The catalytic mechanism of GTP hydrolysis by RhoA is strongly accelerated by a GAP protein and is now well defined, but timing of inorganic phosphate release and signal change remains unresolved. We have generated a quaternary complex for RhoA-GAP-GDP-Pi . Its 1.75 Šcrystal structure shows geometry for ionic and hydrogen bond coordination of GDP and Pi in an intermediate state. It enables the selection of a QM core for DFT exploration of a 20 H-bonded network. This identifies serial locations of the two mobile protons from the original nucleophilic water molecule, showing how they move in three rational steps to form a stable quaternary complex. It also suggests how two additional proton transfer steps can facilitate Pi release.


Assuntos
Teoria da Densidade Funcional , GTP Fosfo-Hidrolases/química , Guanosina Difosfato/química , Guanosina Trifosfato/química , Sítios de Ligação , Biocatálise , Domínio Catalítico , Cristalografia por Raios X , GTP Fosfo-Hidrolases/metabolismo , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Ligação de Hidrogênio , Hidrólise , Simulação de Dinâmica Molecular , Fosfatos/química , Prótons
6.
Nat Commun ; 9(1): 4043, 2018 10 02.
Artigo em Inglês | MEDLINE | ID: mdl-30279485

RESUMO

Activation of the innate immune pattern recognition receptor NOD2 by the bacterial muramyl-dipeptide peptidoglycan fragment triggers recruitment of the downstream adaptor kinase RIP2, eventually leading to NF-κB activation and proinflammatory cytokine production. Here we show that full-length RIP2 can form long filaments mediated by its caspase recruitment domain (CARD), in common with other innate immune adaptor proteins. We further show that the NOD2 tandem CARDs bind to one end of the RIP2 CARD filament, suggesting a mechanism for polar filament nucleation by activated NOD2. We combine X-ray crystallography, solid-state NMR and high-resolution cryo-electron microscopy to determine the atomic structure of the helical RIP2 CARD filament, which reveals the intermolecular interactions that stabilize the assembly. Using structure-guided mutagenesis, we demonstrate the importance of RIP2 polymerization for the activation of NF-κB signalling by NOD2. Our results could be of use to develop new pharmacological strategies to treat inflammatory diseases characterised by aberrant NOD2 signalling.


Assuntos
NF-kappa B/metabolismo , Proteína Adaptadora de Sinalização NOD2/metabolismo , Proteína Serina-Treonina Quinase 2 de Interação com Receptor/metabolismo , Domínio de Ativação e Recrutamento de Caspases , Células HEK293 , Humanos , Conformação Proteica , Proteína Serina-Treonina Quinase 2 de Interação com Receptor/genética
7.
PLoS One ; 12(5): e0177161, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28545134

RESUMO

Innate immune receptors NOD1 and NOD2 are activated by bacterial peptidoglycans leading to recruitment of adaptor kinase RIP2, which, upon phosphorylation and ubiquitination, becomes a scaffold for downstream effectors. The kinase domain (RIP2K) is a pharmaceutical target for inflammatory diseases caused by aberrant NOD2-RIP2 signalling. Although structures of active RIP2K in complex with inhibitors have been reported, the mechanism of RIP2K activation remains to be elucidated. Here we analyse RIP2K activation by combining crystal structures of the active and inactive states with mass spectrometric characterization of their phosphorylation profiles. The active state has Helix αC inwardly displaced and the phosphorylated Activation Segment (AS) disordered, whilst in the inactive state Helix αC is outwardly displaced and packed against the helical, non-phosphorylated AS. Biophysical measurements show that the active state is a stable dimer whilst the inactive kinase is in a monomer-dimer equilibrium, consistent with the observed structural differences at the dimer interface. We conclude that RIP2 kinase auto-phosphorylation is intimately coupled to dimerization, similar to the case of BRAF. Our results will help drug design efforts targeting RIP2 as a potential treatment for NOD2-RIP2 related inflammatory diseases.


Assuntos
Proteína Serina-Treonina Quinase 2 de Interação com Receptor/química , Proteína Serina-Treonina Quinase 2 de Interação com Receptor/metabolismo , Animais , Cristalografia por Raios X , Ativação Enzimática , Humanos , Ligação de Hidrogênio , Mutação , Fosforilação , Conformação Proteica , Multimerização Proteica , Estabilidade Proteica , Proteína Serina-Treonina Quinase 2 de Interação com Receptor/genética , Ultracentrifugação
8.
Angew Chem Int Ed Engl ; 56(33): 9732-9735, 2017 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-28498638

RESUMO

We report X-ray crystallographic and 19 F NMR studies of the G-protein RhoA complexed with MgF3- , GDP, and RhoGAP, which has the mutation Arg85'Ala. When combined with DFT calculations, these data permit the identification of changes in transition state (TS) properties. The X-ray data show how Tyr34 maintains solvent exclusion and the core H-bond network in the active site by relocating to replace the missing Arg85' sidechain. The 19 F NMR data show deshielding effects that indicate the main function of Arg85' is electronic polarization of the transferring phosphoryl group, primarily mediated by H-bonding to O3G and thence to PG . DFT calculations identify electron-density redistribution and pinpoint why the TS for guanosine 5'-triphosphate (GTP) hydrolysis is higher in energy when RhoA is complexed with RhoGAPArg85'Ala relative to wild-type (WT) RhoGAP. This study demonstrates that 19 F NMR measurements, in combination with X-ray crystallography and DFT calculations, can reliably dissect the response of small GTPases to site-specific modifications.


Assuntos
Teoria da Densidade Funcional , GTP Fosfo-Hidrolases/genética , Cristalografia por Raios X , Flúor/química , GTP Fosfo-Hidrolases/química , GTP Fosfo-Hidrolases/metabolismo , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Mutação
9.
Structure ; 25(1): 16-26, 2017 01 03.
Artigo em Inglês | MEDLINE | ID: mdl-27889209

RESUMO

The causative agent of toxoplasmosis, the intracellular parasite Toxoplasma gondii, delivers a protein, GRA24, into the cells it infects that interacts with the mitogen-activated protein (MAP) kinase p38α (MAPK14), leading to activation and nuclear translocation of the host kinase and a subsequent inflammatory response that controls the progress of the parasite. The purification of a recombinant complex of GRA24 and human p38α has allowed the molecular basis of this activation to be determined. GRA24 is shown to be intrinsically disordered, binding two kinases that act independently, and is the only factor required to bypass the canonical mitogen-activated protein kinase activation pathway. An adapted kinase interaction motif (KIM) forms a highly stable complex that competes with cytoplasmic regulatory partners. In addition, the recombinant complex forms a powerful in vitro tool to evaluate the specificity and effectiveness of p38α inhibitors that have advanced to clinical trials, as it provides a hitherto unavailable stable and highly active form of p38α.


Assuntos
Proteína Quinase 14 Ativada por Mitógeno/química , Proteína Quinase 14 Ativada por Mitógeno/metabolismo , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo , Sítios de Ligação , Núcleo Celular/metabolismo , Humanos , Proteínas Intrinsicamente Desordenadas/química , Proteínas Intrinsicamente Desordenadas/metabolismo , Sistema de Sinalização das MAP Quinases , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Domínios Proteicos , Multimerização Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo
10.
Proc Natl Acad Sci U S A ; 111(34): 12384-9, 2014 Aug 26.
Artigo em Inglês | MEDLINE | ID: mdl-25104750

RESUMO

ß-Phosphoglucomutase (ßPGM) catalyzes isomerization of ß-D-glucose 1-phosphate (ßG1P) into D-glucose 6-phosphate (G6P) via sequential phosphoryl transfer steps using a ß-D-glucose 1,6-bisphosphate (ßG16BP) intermediate. Synthetic fluoromethylenephosphonate and methylenephosphonate analogs of ßG1P deliver novel step 1 transition state analog (TSA) complexes for ßPGM, incorporating trifluoromagnesate and tetrafluoroaluminate surrogates of the phosphoryl group. Within an invariant protein conformation, the ß-D-glucopyranose ring in the ßG1P TSA complexes (step 1) is flipped over and shifted relative to the G6P TSA complexes (step 2). Its equatorial hydroxyl groups are hydrogen-bonded directly to the enzyme rather than indirectly via water molecules as in step 2. The (C)O-P bond orientation for binding the phosphate in the inert phosphate site differs by ∼ 30° between steps 1 and 2. By contrast, the orientations for the axial O-Mg-O alignment for the TSA of the phosphoryl group in the catalytic site differ by only ∼ 5°, and the atoms representing the five phosphorus-bonded oxygens in the two transition states (TSs) are virtually superimposable. The conformation of ßG16BP in step 1 does not fit into the same invariant active site for step 2 by simple positional interchange of the phosphates: the TS alignment is achieved by conformational change of the hexose rather than the protein.


Assuntos
Hexoses/química , Hexoses/metabolismo , Organofosfonatos/química , Organofosfonatos/metabolismo , Fosfoglucomutase/química , Fosfoglucomutase/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Catálise , Cristalografia por Raios X , Flúor/química , Glucose-6-Fosfato/química , Glucose-6-Fosfato/metabolismo , Glucofosfatos/química , Glucofosfatos/metabolismo , Isomerismo , Cinética , Lactococcus lactis/enzimologia , Magnésio/química , Modelos Moleculares , Estrutura Molecular , Ressonância Magnética Nuclear Biomolecular , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Eletricidade Estática , Termodinâmica
11.
Acta Crystallogr D Biol Crystallogr ; 67(Pt 10): 902-6, 2011 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-21931222

RESUMO

Over the last 20 years cryocrystallography has revolutionized the field of macromolecular crystallography, greatly reducing radiation damage and allowing the collection of complete data sets at synchrotron sources. However, in order to cool crystals to 100 K cryoprotective agents must usually be added to prevent the formation of crystalline ice, which disrupts the macromolecular crystal lattice and often results in a degradation of diffraction quality. This process can involve the extensive testing of solution compositions and soaking protocols to find suitable conditions that maintain diffraction quality. In this study, it is demonstrated that when some crystals of macromolecules are mounted in the complete absence of surrounding liquid no crystalline ice is formed and the diffraction resolution, merging R factors and mosaic spread values are comparable to those of crystals cryocooled in the presence of a cryoprotectant. This potentially removes one of the most onerous manual steps in the structure-solution pipeline and could alleviate some of the foreseen difficulties in the automation of crystal mounting.


Assuntos
Crioprotetores/química , Cristalografia/métodos , Proteínas/química , Substâncias Macromoleculares/química , Fosfoglicerato Quinase/química , Fosfotransferases (Fosfomutases)/química , Tripsina/química , Proteína rhoA de Ligação ao GTP/química
12.
J Struct Biol ; 175(2): 236-43, 2011 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-21385612

RESUMO

The increase in the number of large multi-component complexes and membrane protein crystal structures determined over the last few years can be ascribed to a number of factors such as better protein expression and purification systems, the emergence of high-throughput crystallization techniques and the advent of 3rd generation synchrotron sources. However, many systems tend to produce crystals that can be extremely heterogeneous in their diffraction properties. This prevents, in many cases, the collection of diffraction data of sufficient quality to yield useful biological or phase information. Techniques that can increase the diffraction quality of macromolecular crystals can therefore be essential in the successful conclusion of these challenging projects. No technique is universal but encouraging results have been recently achieved by carrying out the controlled dehydration of crystals of biological macromolecules. A new device that delivers a stream of air with a precisely controlled relative humidity to the complicated sample environment found at modern synchrotron beamlines has been conceived at the EMBL Grenoble and developed by the EMBL and the ESRF as part of the SPINE2 complexes project, a European Commission funded protein structure initiative. The device, the HC1b, has been available for three years at the ESRF macromolecular crystallography beamlines and many systems have benefitted from on-line controlled dehydration. Here we describe a standard dehydration experiment, highlight some successful cases and discuss the different possible uses of the device.


Assuntos
Cristalografia por Raios X/instrumentação , Dessecação/instrumentação , Complexos Multiproteicos/química , Transição de Fase , Amiloide/química , Temperatura Baixa , Cristalografia por Raios X/métodos , Dessecação/métodos , Humanos , Fosfoglicerato Quinase/química , Complexo de Proteína do Fotossistema I/química , Síncrotrons/instrumentação
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