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1.
Cell ; 177(4): 881-895.e17, 2019 05 02.
Artigo em Inglês | MEDLINE | ID: mdl-31051106

RESUMO

Non-alcoholic fatty liver is the most common liver disease worldwide. Here, we show that the mitochondrial protein mitofusin 2 (Mfn2) protects against liver disease. Reduced Mfn2 expression was detected in liver biopsies from patients with non-alcoholic steatohepatitis (NASH). Moreover, reduced Mfn2 levels were detected in mouse models of steatosis or NASH, and its re-expression in a NASH mouse model ameliorated the disease. Liver-specific ablation of Mfn2 in mice provoked inflammation, triglyceride accumulation, fibrosis, and liver cancer. We demonstrate that Mfn2 binds phosphatidylserine (PS) and can specifically extract PS into membrane domains, favoring PS transfer to mitochondria and mitochondrial phosphatidylethanolamine (PE) synthesis. Consequently, hepatic Mfn2 deficiency reduces PS transfer and phospholipid synthesis, leading to endoplasmic reticulum (ER) stress and the development of a NASH-like phenotype and liver cancer. Ablation of Mfn2 in liver reveals that disruption of ER-mitochondrial PS transfer is a new mechanism involved in the development of liver disease.


Assuntos
GTP Fosfo-Hidrolases/metabolismo , Proteínas Mitocondriais/metabolismo , Hepatopatia Gordurosa não Alcoólica/metabolismo , Fosfatidilserinas/metabolismo , Animais , Modelos Animais de Doenças , Retículo Endoplasmático/metabolismo , Estresse do Retículo Endoplasmático/fisiologia , Hepatócitos/metabolismo , Hepatócitos/patologia , Humanos , Inflamação/metabolismo , Fígado/patologia , Hepatopatias/etiologia , Hepatopatias/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/metabolismo , Cultura Primária de Células , Transporte Proteico/fisiologia , Transdução de Sinais , Triglicerídeos/metabolismo
2.
Proc Natl Acad Sci U S A ; 118(29)2021 07 20.
Artigo em Inglês | MEDLINE | ID: mdl-34272277

RESUMO

Cell survival in response to stress is determined by the coordination of various signaling pathways. The kinase p38α is activated by many stresses, but the intensity and duration of the signal depends on the stimuli. How different p38α-activation dynamics may impact cell life/death decisions is unclear. Here, we show that the p38α-signaling output in response to stress is modulated by the expression levels of the downstream kinase MK2. We demonstrate that p38α forms a complex with MK2 in nonstimulated mammalian cells. Upon pathway activation, p38α phosphorylates MK2, the complex dissociates, and MK2 is degraded. Interestingly, transient p38α activation allows MK2 reexpression, reassembly of the p38α-MK2 complex, and cell survival. In contrast, sustained p38α activation induced by severe stress interferes with p38α-MK2 interaction, resulting in irreversible MK2 loss and cell death. MK2 degradation is mediated by the E3 ubiquitin ligase MDM2, and we identify four lysine residues in MK2 that are directly ubiquitinated by MDM2. Expression of an MK2 mutant that cannot be ubiquitinated by MDM2 enhances the survival of stressed cells. Our results indicate that MK2 reexpression and binding to p38α is critical for cell viability in response to stress and illustrate how particular p38α-activation patterns induced by different signals shape the stress-induced cell fate.


Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , Estresse Fisiológico , Animais , Diferenciação Celular , Linhagem Celular , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Camundongos , Proteína Quinase 14 Ativada por Mitógeno/genética , Proteína Quinase 14 Ativada por Mitógeno/metabolismo , Ligação Proteica , Proteínas Serina-Treonina Quinases/genética , Proteólise , Proteínas Proto-Oncogênicas c-mdm2/genética , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Ubiquitinação
3.
Acc Chem Res ; 53(3): 654-661, 2020 03 17.
Artigo em Inglês | MEDLINE | ID: mdl-32134250

RESUMO

This Account highlights recent advances and discusses major challenges in investigations of cryptic (hidden) binding sites by molecular simulations. Cryptic binding sites are not visible in protein targets crystallized without a ligand and only become visible crystallographically upon binding events. These sites have been shown to be druggable and might provide a rare opportunity to target difficult proteins. However, due to their hidden nature, they are difficult to find through experimental screening. Computational methods based on atomistic molecular simulations remain one of the best approaches to identify and characterize cryptic binding sites. However, not all methods are equally efficient. Some are more apt at quickly probing protein dynamics but do not provide thermodynamic or druggability information, while others that are able to provide such data are demanding in terms of time and resources. Here, we review the recent contributions of mixed-solvent simulations, metadynamics, Markov state models, and other enhanced sampling methods to the field of cryptic site identification and characterization. We discuss how these methods were able to provide precious information on the nature of the site opening mechanisms, to predict previously unknown sites which were used to design new ligands, and to compute the free energy landscapes and kinetics associated with the opening of the sites and the binding of the ligands. We highlight the potential and the importance of such predictions in drug discovery, especially for difficult ("undruggable") targets. We also discuss the major challenges in the field and their possible solutions.


Assuntos
Simulação de Dinâmica Molecular , Sítios de Ligação , Descoberta de Drogas , Ligantes , Cadeias de Markov , Solventes/química
4.
Biophys J ; 106(3): 677-86, 2014 Feb 04.
Artigo em Inglês | MEDLINE | ID: mdl-24507608

RESUMO

Protein crystallization is frequently induced by the addition of various precipitants, which directly affect protein solubility. In addition to organic cosolvents and long-chain polymers, salts belong to the most widely used precipitants in protein crystallography. However, despite such widespread usage, their mode of action at the atomistic level is still largely unknown. Here, we perform extensive molecular dynamics simulations of the villin headpiece crystal unit cell to examine its stability at different concentrations of sodium sulfate. We show that the inclusion of ions in crystal solvent at high concentration can prevent large rearrangements of the asymmetric units and a loss of symmetry of the unit cell without significantly affecting protein dynamics. Of importance, a similar result can be achieved by neutralizing several specific charged residues suggesting that they may play an active role in crystal destabilization due to unfavorable electrostatic interactions. Our results provide a microscopic picture behind salt-induced stabilization of a protein crystal and further suggest that adequate modeling of realistic crystallization conditions may be necessary for successful molecular dynamics simulations of protein crystals.


Assuntos
Proteínas de Drosophila/química , Proteínas dos Microfilamentos/química , Simulação de Dinâmica Molecular , Sódio/química , Eletricidade Estática , Sequência de Aminoácidos , Aminoácidos/química , Animais , Cristalografia por Raios X/métodos , Drosophila/química , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Solventes/química
5.
Biophys J ; 98(5): 861-71, 2010 Mar 03.
Artigo em Inglês | MEDLINE | ID: mdl-20197040

RESUMO

Root mean-square deviation (RMSD) after roto-translational least-squares fitting is a measure of global structural similarity of macromolecules used commonly. On the other hand, experimental x-ray B-factors are used frequently to study local structural heterogeneity and dynamics in macromolecules by providing direct information about root mean-square fluctuations (RMSF) that can also be calculated from molecular dynamics simulations. We provide a mathematical derivation showing that, given a set of conservative assumptions, a root mean-square ensemble-average of an all-against-all distribution of pairwise RMSD for a single molecular species, (1/2), is directly related to average B-factors () and (1/2). We show this relationship and explore its limits of validity on a heterogeneous ensemble of structures taken from molecular dynamics simulations of villin headpiece generated using distributed-computing techniques and the Folding@Home cluster. Our results provide a basis for quantifying global structural diversity of macromolecules in crystals directly from x-ray experiments, and we show this on a large set of structures taken from the Protein Data Bank. In particular, we show that the ensemble-average pairwise backbone RMSD for a microscopic ensemble underlying a typical protein x-ray structure is approximately 1.1 A, under the assumption that the principal contribution to experimental B-factors is conformational variability.


Assuntos
Proteínas dos Microfilamentos/química , Cristalografia por Raios X , Proteínas dos Microfilamentos/metabolismo , Conformação Proteica , Dobramento de Proteína , Estrutura Terciária de Proteína
6.
J Phys Chem Lett ; 10(8): 1928-1934, 2019 Apr 18.
Artigo em Inglês | MEDLINE | ID: mdl-30933516

RESUMO

Whether recent updates and new releases of atomistic force fields can model the structural and dynamical properties of proteins containing both folded and partially disordered domains is still unclear. To address this fundamental question, we tested eight recently released force fields against our set of nuclear magnetic resonance (NMR) observables for a complex and medically relevant system, the major factor VIII binding region on the von Willebrand factor. This biomedically important region comprises both a folded and a partially structured domain. By using an enhanced sampling technique (temperature replica-exchange molecular dynamics simulations), we find that some force fields indeed rise to the challenge and capture the structural and dynamical features of the NMR ensemble and, therefore, are the appropriate choice for simulations of proteins with partially structured domains. What is more, we show that only such force fields can qualitatively capture the effects of a pathogenic mutation on the structural ensemble.

7.
Structure ; 27(1): 78-89.e3, 2019 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-30393051

RESUMO

Nerve growth factor (NGF) is an important neurotrophic factor involved in the regulation of cell differentiation and survival of target neurons. Expressed as a proNGF precursor, NGF is matured by furin-mediated protease cleavage. Increasing evidence suggests that NGF and proNGF have distinct functional roles. While the structure of mature NGF is available, little is known about that of the pro-domain because of its dynamical structural features. We exploited an ad hoc hybrid strategy based on nuclear magnetic resonance and modeling validated by small-angle X-ray scattering to gain novel insights on the pro-domain, both in isolation and in the context of proNGF. We show that the isolated pro-domain is intrinsically unstructured but forms transient intramolecular contacts with mature NGF and has per se the ability to induce growth cone collapse, indicating functional independence. Our data represent an important step toward the structural and functional characterization of the properties of proNGF.


Assuntos
Fator de Crescimento Neural/química , Precursores de Proteínas/química , Animais , Células Cultivadas , Cones de Crescimento/metabolismo , Espectroscopia de Ressonância Magnética , Camundongos , Simulação de Dinâmica Molecular , Fator de Crescimento Neural/metabolismo , Domínios Proteicos , Precursores de Proteínas/metabolismo , Processamento de Proteína Pós-Traducional , Proteólise , Espalhamento a Baixo Ângulo , Difração de Raios X
8.
Nat Commun ; 9(1): 4325, 2018 10 18.
Artigo em Inglês | MEDLINE | ID: mdl-30337523

RESUMO

Our current understanding of epidermal growth factor receptor (EGFR) autoinhibition is based on X-ray structural data of monomer and dimer receptor fragments and does not explain how mutations achieve ligand-independent phosphorylation. Using a repertoire of imaging technologies and simulations we reveal an extracellular head-to-head interaction through which ligand-free receptor polymer chains of various lengths assemble. The architecture of the head-to-head interaction prevents kinase-mediated dimerisation. The latter, afforded by mutation or intracellular treatments, splits the autoinhibited head-to-head polymers to form stalk-to-stalk flexible non-extended dimers structurally coupled across the plasma membrane to active asymmetric tyrosine kinase dimers, and extended dimers coupled to inactive symmetric kinase dimers. Contrary to the previously proposed main autoinhibitory function of the inactive symmetric kinase dimer, our data suggest that only dysregulated species bear populations of symmetric and asymmetric kinase dimers that coexist in equilibrium at the plasma membrane under the modulation of the C-terminal domain.


Assuntos
Receptores ErbB/antagonistas & inibidores , Receptores ErbB/química , Multimerização Proteica , Animais , Células CHO , Membrana Celular/metabolismo , Cricetinae , Cricetulus , Matriz Extracelular/metabolismo , Corantes Fluorescentes/química , Corantes Fluorescentes/metabolismo , Ligantes , Modelos Biológicos , Modelos Moleculares , Fotodegradação , Polímeros/química , Domínios Proteicos , Proteínas Quinases/química , Proteínas Quinases/metabolismo
9.
Elife ; 62017 04 26.
Artigo em Inglês | MEDLINE | ID: mdl-28445123

RESUMO

p38α is a Ser/Thr protein kinase involved in a variety of cellular processes and pathological conditions, which makes it a promising pharmacological target. Although the activity of the enzyme is highly regulated, its molecular mechanism of activation remains largely unexplained, even after decades of research. By using state-of-the-art molecular dynamics simulations, we decipher the key elements of the complex molecular mechanism refined by evolution to allow for a fine tuning of p38α kinase activity. Our study describes for the first time the molecular effects of different regulators of the enzymatic activity, and provides an integrative picture of the activation mechanism that explains the seemingly contradictory X-ray and NMR data.


Assuntos
Ativação Enzimática , Simulação de Dinâmica Molecular , Proteínas Quinases p38 Ativadas por Mitógeno/química , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Ligação Proteica , Conformação Proteica
10.
Nat Commun ; 5: 3220, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24504120

RESUMO

Biomolecular X-ray structures typically provide a static, time- and ensemble-averaged view of molecular ensembles in crystals. In the absence of rigid-body motions and lattice defects, B-factors are thought to accurately reflect the structural heterogeneity of such ensembles. In order to study the effects of averaging on B-factors, we employ molecular dynamics simulations to controllably manipulate microscopic heterogeneity of a crystal containing 216 copies of villin headpiece. Using average structure factors derived from simulation, we analyse how well this heterogeneity is captured by high-resolution molecular-replacement-based model refinement. We find that both isotropic and anisotropic refined B-factors often significantly deviate from their actual values known from simulation: even at high 1.0 Å resolution and Rfree of 5.9%, B-factors of some well-resolved atoms underestimate their actual values even sixfold. Our results suggest that conformational averaging and inadequate treatment of correlated motion considerably influence estimation of microscopic heterogeneity via B-factors, and invite caution in their interpretation.


Assuntos
Proteínas dos Microfilamentos/química , Cristalização , Simulação de Dinâmica Molecular
11.
J Chem Theory Comput ; 8(10): 3820-9, 2012 Oct 09.
Artigo em Inglês | MEDLINE | ID: mdl-26593023

RESUMO

We study the contribution of linear, pairwise atom-positional correlations (covariances) to absolute and relative conformational entropy as calculated by quasi-harmonic analysis of molecular dynamics (MD) trajectories (SQH and ΔSQH). By analyzing a total of 25 µs of MD simulations of ubiquitin and six of its binding partners in bound and unbound states, and 2.4 µs of simulations of eight different proteins in phosphorylated and unphosphorylated states, we show that ΔSQH represents a remarkably constant fraction of a quasi-harmonic entropy change obtained if one ignores the contribution of covariance terms and uses mass-weighted atom-positional variances only (ΔSVAR). In other words, the relative contribution of linear correlations to conformational entropy change for different proteins and in different biomolecular processes appears to be largely constant. Based on this, we establish an empirical relationship between relative quasi-harmonic conformational entropy and changes in crystallographic B-factors induced by different processes, and we use it to estimate conformational-entropic contribution to the free energy of binding for a large set of protein complexes based on their X-ray structures. Our results suggest a simple way for relating other types of dynamical observables with conformational entropy in the absence of information on correlated motions, such as in the case of NMR order parameters.

12.
J Mol Biol ; 411(1): 286-97, 2011 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-21645520

RESUMO

Atomic positions obtained by X-ray crystallography are time and space averages over many molecules in the crystal. Importantly, interatomic distances, calculated between such average positions and frequently used in structural and mechanistic analyses, can be substantially different from the more appropriate time-average and ensemble-average interatomic distances. Using crystallographic B-factors, one can deduce corrections, which have so far been applied exclusively to small molecules, to obtain correct average distances as a function of the type of atomic motion. Here, using 4774 high-quality protein X-ray structures, we study the significance of such corrections for different types of atomic motion. Importantly, we show that for distances shorter than 5 Å, corrections greater than 0.5 Å may apply, especially for noncorrelated or anticorrelated motion. For example, 14% of the studied structures have at least one pair of atoms with a correction of ≥0.5 Å in the case of noncorrelated motion. Using molecular dynamics simulations of villin headpiece, ubiquitin, and SH3 domain unit cells, we demonstrate that the majority of average interatomic distances in these proteins agree with noncorrelated corrections, suggesting that such deviations may be truly relevant. Importantly, we demonstrate that the corrections do not significantly affect stereochemistry and the overall quality of final refined X-ray structures, but can provide marked improvements in starting unrefined models obtained from low-resolution X-ray data. Finally, we illustrate the potential mechanistic and biological significance of the calculated corrections for KcsA ion channel and show that they provide indirect evidence that motions in its selectivity filter are highly correlated.


Assuntos
Cristalografia por Raios X/métodos , Simulação de Dinâmica Molecular , Conformação Proteica , Proteínas/química , Modelos Moleculares
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