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1.
Bioinformatics ; 2019 Oct 04.
Artigo em Inglês | MEDLINE | ID: mdl-31584626

RESUMO

MOTIVATION: Leucine-aspartic acid (LD) motifs are short linear interaction motifs (SLiMs) that link paxillin family proteins to factors controlling cell adhesion, motility and survival. The existence and importance of LD motifs beyond the paxillin family is poorly understood. RESULTS: To enable a proteome-wide assessment of LD motifs, we developed an active-learning based framework (LDmotif finder; LDMF) that iteratively integrates computational predictions with experimental validation. Our analysis of the human proteome revealed a dozen new proteins containing LD motifs. We found that LD motif signalling evolved in unicellular eukaryotes more than 800 Myr ago, with paxillin and vinculin as core constituents, and nuclear export signal (NES) as a likely source of de novo LD motifs. We show that LD motif proteins form a functionally homogenous group, all being involved in cell morphogenesis and adhesion. This functional focus is recapitulated in cells by GFP-fused LD motifs, suggesting that it is intrinsic to the LD motif sequence, possibly through their effect on binding partners. Our approach elucidated the origin and dynamic adaptations of an ancestral SLiM, and can serve as a guide for the identification of other SLiMs for which only few representatives are known. AVAILABILITY: LDMF is freely available online at www.cbrc.kaust.edu.sa/ldmf; Source code is available at https://github.com/tanviralambd/LD/. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

2.
J Inorg Biochem ; 198: 110716, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31153112

RESUMO

Human serum albumin (HSA) is a monomeric, globular, multi-carrier and the most abundant protein in the blood. HSA displays multiple ligand binding sites with extraordinary binding capacity for a wide range of ions and molecules. For decades, HSA's ability to bind to various ligands has led many scientists to study its physiological properties and protein structure; indeed, a better understanding of HSA-ligand interactions in human blood, at the atomic level, will likely foster the development of more potent, and overall more performant, diagnostic and therapeutic tools against serious human disorders such as diabetes, cardiovascular disorders, and cancer. Here, we present a concise overview of the current knowledge of HSA's structural characteristics, and its coordination chemistry with transition metal ions, within the scope and limitations of current techniques and biophysical methods to reach atomic resolution in solution and in blood serum. We also highlight the overwhelming need of a detailed atomistic understanding of HSA dynamic structures and interactions that are transient, weak, multi-site and multi-step, and allosterically affected by each other. Considering the fact that HSA is a current clinical tool for drug delivery systems and a potential contender as molecular cargo and nano-vehicle used in biophysical, clinical and industrial fields, we underline the emerging need for novel approaches to target the dynamic functional coordination chemistry of the human blood serum albumin in solution, at the atomic level.

4.
Am J Hum Genet ; 104(3): 542-552, 2019 Mar 07.
Artigo em Inglês | MEDLINE | ID: mdl-30827498

RESUMO

Polyglutamine expansions in the transcriptional co-repressor Atrophin-1, encoded by ATN1, cause the neurodegenerative condition dentatorubral-pallidoluysian atrophy (DRPLA) via a proposed novel toxic gain of function. We present detailed phenotypic information on eight unrelated individuals who have de novo missense and insertion variants within a conserved 16-amino-acid "HX repeat" motif of ATN1. Each of the affected individuals has severe cognitive impairment and hypotonia, a recognizable facial gestalt, and variable congenital anomalies. However, they lack the progressive symptoms typical of DRPLA neurodegeneration. To distinguish this subset of affected individuals from the DRPLA diagnosis, we suggest using the term CHEDDA (congenital hypotonia, epilepsy, developmental delay, digit abnormalities) to classify the condition. CHEDDA-related variants alter the particular structural features of the HX repeat motif, suggesting that CHEDDA results from perturbation of the structural and functional integrity of the HX repeat. We found several non-homologous human genes containing similar motifs of eight to 10 HX repeat sequences, including RERE, where disruptive variants in this motif have also been linked to a separate condition that causes neurocognitive and congenital anomalies. These findings suggest that perturbation of the HX motif might explain other Mendelian human conditions.

5.
Nucleic Acids Res ; 47(5): 2666-2680, 2019 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-30597093

RESUMO

As an environment-dependent pleiotropic gene regulator in Gram-negative bacteria, the H-NS protein is crucial for adaptation and toxicity control of human pathogens such as Salmonella, Vibrio cholerae or enterohaemorrhagic Escherichia coli. Changes in temperature affect the capacity of H-NS to form multimers that condense DNA and restrict gene expression. However, the molecular mechanism through which H-NS senses temperature and other physiochemical parameters remains unclear and controversial. Combining structural, biophysical and computational analyses, we show that human body temperature promotes unfolding of the central dimerization domain, breaking up H-NS multimers. This unfolding event enables an autoinhibitory compact H-NS conformation that blocks DNA binding. Our integrative approach provides the molecular basis for H-NS-mediated environment-sensing and may open new avenues for the control of pathogenic multi-drug resistant bacteria.


Assuntos
Proteínas de Bactérias/química , DNA Bacteriano/genética , Proteínas de Ligação a DNA/química , Desdobramento de Proteína , Proteínas de Bactérias/genética , DNA Bacteriano/química , Proteínas de Ligação a DNA/genética , Escherichia coli Êntero-Hemorrágica/genética , Escherichia coli Êntero-Hemorrágica/patogenicidade , Interação Gene-Ambiente , Humanos , Domínios Proteicos , Multimerização Proteica/genética , Salmonella/genética , Salmonella/patogenicidade , Temperatura Ambiente , Vibrio cholerae/genética , Vibrio cholerae/patogenicidade
6.
J Inorg Biochem ; 191: 69-76, 2018 Nov 10.
Artigo em Inglês | MEDLINE | ID: mdl-30468944

RESUMO

Islet Amyloid Polypeptide (IAPP), also known as amylin, is a 37-amino-acid peptide hormone that is secreted by pancreatic islet ß-cells. Amylin is complementary to insulin in regulating and maintaining blood glucose levels in the human body. The misfolding and aggregation of amylin is primarily associated with type 2 diabetes mellitus, which is classified as an amyloid disease. Recently, the interactions between amylin and specific metal ions, e.g., copper(II), zinc(II), and iron(II), were found to impact its performance and aggregation processes. Therefore, the focus in this review will be on how the chemistry and structural properties of amylin are affected by these interactions. In addition, the impact of amylin and other amyloidogenic peptides interacting with metal ions on the cell membranes is discussed. In particular, recent studies on the interactions of amylin with copper, zinc, iron, nickel, gold, ruthenium, and vanadium are discussed.

7.
Sci Rep ; 8(1): 10462, 2018 Jul 11.
Artigo em Inglês | MEDLINE | ID: mdl-29993003

RESUMO

Pathological levels of oxidative stress (OS) have been implicated in many diseases including diabetes mellitus, neurodegenerative diseases, inflammatory diseases, atherosclerosis, and cancer. Studies of oxidative stress are however complicated by the low concentration of oxidation products. To resolve this problem, we tested a new derivative of aminoadipic semialdehyde (Fmoc-Aea-OH) in the solid-phase synthesis of carbonylated peptides. We prepared a series of peptides with free and acetylated N-terminal amino groups using the Fmoc-Aea-OH reagent. LC-MS, ESI-MS, and MS/MS spectra confirmed the sequences of the modified peptides, although the LC-MS and ESI-MS spectra were dominated by signals corresponding to dehydration products. NMR studies of acetylated products revealed that the dominant product formed in this reaction contains a 1,2,3,4-tetrahydropyridine-2-carboxylic acid residue. Another side reaction in this system was the cleavage of the amide bond between the Aea residue and the amino acid moiety preceding it resulting in the formation of a side product with a six-membered ring at the N-terminus (2,3,4,5-tetrahydropyridine-2-carboxylic acid residue). We found that, depending on the peptide sequence, one of those side products is predominant. Our work suggests new methods for the solid-state synthesis of peptides containing unnatural amino acids.

8.
FEBS J ; 285(11): 1988-2003, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29619777

RESUMO

The inflammatory chemokine CCL5, which binds the chemokine receptor CCR5 in a two-step mechanism so as to activate signaling pathways in hematopoetic cells, plays an important role in immune surveillance, inflammation, and development as well as in several immune system pathologies. The recently published crystal structure of CCR5 bound to a high-affinity variant of CCL5 lacks the N-terminal segment of the receptor that is post-translationally sulfated and is known to be important for high-affinity binding. Here, we report the NMR solution structure of monomeric CCL5 bound to a synthetic doubly sulfated peptide corresponding to the missing first 27 residues of CCR5. Our structures show that two sulfated tyrosine residues, sY10 and sY14, as well as the unsulfated Y15 form a network of strong interactions with a groove on a surface of CCL5 that is formed from evolutionarily conserved basic and hydrophobic amino acids. We then use our NMR structures, in combination with available crystal data, to create an atomic model of full-length wild-type CCR5:CCL5. Our findings reveal the structural determinants involved in the recognition of CCL5 by the CCR5 N terminus. These findings, together with existing structural data, provide a complete structural framework with which to understand the specificity of receptor:chemokine interactions. DATABASE: Structural data are available in the PDB under the accession number 6FGP.

9.
J Biomol NMR ; 70(4): 219-228, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29594733

RESUMO

Simple and convenient method of protein dynamics evaluation from the insufficient experimental 15N relaxation data is presented basing on the ratios, products, and differences of longitudinal and transverse 15N relaxation rates obtained at a single magnetic field. Firstly, the proposed approach allows evaluating overall tumbling correlation time (nanosecond time scale). Next, local parameters of the model-free approach characterizing local mobility of backbone amide N-H vectors on two different time scales, S2 and R ex , can be elucidated. The generalized order parameter, S2, describes motions on the time scale faster than the overall tumbling correlation time (pico- to nanoseconds), while the chemical exchange term, R ex , identifies processes slower than the overall tumbling correlation time (micro- to milliseconds). Advantages and disadvantages of different methods of data handling are thoroughly discussed.


Assuntos
Simulação de Dinâmica Molecular , Ressonância Magnética Nuclear Biomolecular/métodos , Proteínas/química , Movimento (Física) , Isótopos de Nitrogênio , Fatores de Tempo
10.
Angew Chem Int Ed Engl ; 57(12): 3246-3250, 2018 Mar 12.
Artigo em Inglês | MEDLINE | ID: mdl-29314492

RESUMO

The microtubule-associated protein Tau promotes the polymerization of tubulin and modulates the function of microtubules. As a consequence of the dynamic nature of the Tau-tubulin interaction, the structural basis of this complex has remained largely elusive. By using NMR methods optimized for ligand-receptor interactions in combination with site-directed mutagenesis we demonstrate that the flanking domain downstream of the four microtubule-binding repeats of Tau binds competitively to a site on the α-tubulin surface. The binding process is complex, involves partial coupling of different interacting regions, and is modulated by phosphorylation at Y394 and S396. This study strengthens the hypothesis of an intimate relationship between Tau phosphorylation and tubulin binding and highlights the power of the INPHARMA NMR method to characterize the interaction of peptides derived from intrinsically disordered proteins with their molecular partners.

11.
Nat Commun ; 8: 14893, 2017 03 30.
Artigo em Inglês | MEDLINE | ID: mdl-28358007

RESUMO

Cholesterol is an important regulator of membrane protein function. However, the exact mechanisms involved in this process are still not fully understood. Here we study how the tertiary and quaternary structure of the mitochondrial translocator protein TSPO, which binds cholesterol with nanomolar affinity, is affected by this sterol. Residue-specific analysis of TSPO by solid-state NMR spectroscopy reveals a dynamic monomer-dimer equilibrium of TSPO in the membrane. Binding of cholesterol to TSPO's cholesterol-recognition motif leads to structural changes across the protein that shifts the dynamic equilibrium towards the translocator monomer. Consistent with an allosteric mechanism, a mutation within the oligomerization interface perturbs transmembrane regions located up to 35 Å away from the interface, reaching TSPO's cholesterol-binding motif. The lower structural stability of the intervening transmembrane regions provides a mechanistic basis for signal transmission. Our study thus reveals an allosteric signal pathway that connects membrane protein tertiary and quaternary structure with cholesterol binding.


Assuntos
Colesterol/metabolismo , Mitocôndrias/metabolismo , Receptores de GABA/química , Receptores de GABA/metabolismo , Regulação Alostérica , Sequência de Aminoácidos , Animais , Membrana Celular/metabolismo , Bicamadas Lipídicas/metabolismo , Espectroscopia de Ressonância Magnética , Camundongos , Multimerização Proteica , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Transdução de Sinais
12.
Nat Commun ; 7: 13343, 2016 11 09.
Artigo em Inglês | MEDLINE | ID: mdl-27827373

RESUMO

BMI1 is a core component of the polycomb repressive complex 1 (PRC1) and emerging data support a role of BMI1 in cancer. The central domain of BMI1 is involved in protein-protein interactions and is essential for its oncogenic activity. Here, we present the structure of BMI1 bound to the polyhomeotic protein PHC2 illustrating that the central domain of BMI1 adopts an ubiquitin-like (UBL) fold and binds PHC2 in a ß-hairpin conformation. Unexpectedly, we find that the UBL domain is involved in homo-oligomerization of BMI1. We demonstrate that both the interaction of BMI1 with polyhomeotic proteins and homo-oligomerization via UBL domain are necessary for H2A ubiquitination activity of PRC1 and for clonogenic potential of U2OS cells. Here, we also emphasize need for joint application of NMR spectroscopy and X-ray crystallography to determine the overall structure of the BMI1-PHC2 complex.


Assuntos
Histonas/metabolismo , Complexo Repressor Polycomb 1/metabolismo , Complexo Repressor Polycomb 2/metabolismo , Multimerização Proteica , Linhagem Celular Tumoral , Cristalografia por Raios X , Humanos , Espectroscopia de Ressonância Magnética , Complexo Repressor Polycomb 1/química , Domínios Proteicos , Estrutura Terciária de Proteína , Ubiquitinação
13.
Angew Chem Int Ed Engl ; 55(35): 10518-21, 2016 08 22.
Artigo em Inglês | MEDLINE | ID: mdl-27461260

RESUMO

(15) N spin-relaxation rates are demonstrated to provide critical information about the long-range structure and internal motions of membrane proteins. Combined with an improved calculation method, the relaxation-rate-derived structure of the 283-residue human voltage-dependent anion channel revealed an anisotropically shaped barrel with a rigidly attached N-terminal helix. Our study thus establishes an NMR spectroscopic approach to determine the structure and dynamics of mammalian membrane proteins at high accuracy and resolution.


Assuntos
Proteínas de Membrana/química , Simulação de Dinâmica Molecular , Ressonância Magnética Nuclear Biomolecular , Humanos , Conformação Proteica
14.
Pharmacol Rep ; 68(2): 502-5, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26922560

RESUMO

BACKGROUND: 3-Bromopyruvic acid (3-BP), a glycolytic inhibitor and a promising anticancer compound, induces oxidative stress and depletes cells of glutathione (GSH). The causes of GSH loss remain unclear. The aim of this study was to ascertain whether 3-BP forms a conjugate with glutathione. METHODS: GSH was incubated with various amounts of 3-BP and the extent of reaction was titrated with (1)H NMR and (1)H-(1)H NMR. The reaction outcome was identified by MS/MS. Intracellular formation of the conjugate was assessed in cells treated with 3-BP and 3-BP((13)C) and analyzed using the targeted LC-MS/MS method in negative ionization MRM mode. RESULTS: 3-BP was found to react with GSH in a 1:1 ratio forming an S-conjugate. The same conjugate was formed intracellularly in erythrocytes and MCF-7 cells. CONCLUSIONS: 3-BP reacts with GSH in the absence of cells and intracellularly. This reaction appears to be the main cause of GSH loss in 3-BP treated cells.


Assuntos
Antineoplásicos/farmacologia , Glutationa/metabolismo , Piruvatos/farmacologia , Linhagem Celular Tumoral , Cromatografia Líquida/métodos , Eritrócitos/efeitos dos fármacos , Eritrócitos/metabolismo , Humanos , Células MCF-7 , Espectroscopia de Ressonância Magnética/métodos , Estresse Oxidativo/efeitos dos fármacos , Espectrometria de Massas em Tandem/métodos
15.
Biomol NMR Assign ; 10(1): 79-83, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26364056

RESUMO

The integral polytopic membrane protein TSPO is the target for numerous endogenous and synthetic ligands. However, the affinity of many ligands is influenced by a common polymorphism in TSPO, in which an alanine at position 147 is replaced by threonine, thereby complicating the use of several radioligands for clinical diagnosis. In contrast, the best-characterized TSPO ligand (R)-PK11195 binds with similar affinity to both variants of mitochondrial TSPO (wild-type and A147T variant). Here we report the (1)H, (13)C, (15)N backbone and side-chain resonance assignment of the A147T polymorph of TSPO from Mus Musculus in complex with (R)-PK11195 in DPC detergent micelles. More than 90 % of all resonances were sequence-specifically assigned, demonstrating the ability to obtain high-quality spectral data for both the backbone and the side-chains of medically relevant integral membrane proteins.


Assuntos
Ressonância Magnética Nuclear Biomolecular , Polimorfismo de Nucleotídeo Único/genética , Compostos Radiofarmacêuticos/metabolismo , Receptores de GABA/química , Receptores de GABA/genética , Animais , Ligantes , Camundongos , Estrutura Secundária de Proteína
16.
Biochem Soc Trans ; 43(4): 566-71, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-26551694

RESUMO

The 3D structure of the 18-kDa transmembrane (TM) protein TSPO (translocator protein)/PBR (peripheral benzodiazepine receptor), which contains a binding site for benzodiazepines, is important to better understand its function and regulation by endogenous and synthetic ligands. We have recently determined the structure of mammalian TSPO/PBR in complex with the diagnostic ligand PK11195 [1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinolinecarboxamide; Jaremko et al. (2014) Science 343: , 1363-1366], providing for the first time atomic-level insight into the conformation of this protein, which is up-regulated in various pathological conditions including Alzheimer's disease and Parkinson's disease. Here, we review the studies which have probed the structural properties of mammalian TSPO/PBR as well as the homologues bacterial tryptophan-rich sensory proteins (TspOs) over the years and provide detailed insight into the 3D structure of mouse TSPO (mTSPO)/PBR in complex with PK11195.


Assuntos
Proteínas de Bactérias/química , Isoquinolinas/farmacologia , Mamíferos/metabolismo , Receptores de GABA/química , Animais , Proteínas de Bactérias/metabolismo , Sítios de Ligação/efeitos dos fármacos , Humanos , Camundongos , Modelos Moleculares , Estrutura Secundária de Proteína , Receptores de GABA/metabolismo
17.
Biometals ; 28(6): 975-86, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26407665

RESUMO

Hydrogen peroxide is an important regulator of protein tyrosine phosphatase activity via reversible oxidation. However, the role of iron in this reaction has not been yet elucidated. Here we compare the influence of hydrogen peroxide and the ferrous iron (reagent for Fenton reaction) on the enzymatic activity of recombinant CD45, LAR, PTP1B phosphatases and cellular CD45 in Jurkat cells. The obtained results show that ferrous iron (II) is potent inhibitor of CD45, LAR and PTP1B, but the inhibitory effect is concentration dependent. We found that the higher concentrations of ferrous iron (II) increase the inactivation of CD45, LAR and PTP1B phosphatase caused by hydrogen peroxide, but the addition of the physiological concentration (500 nM) of ferrous iron (II) has even a slightly preventive effect on the phosphatase activity against hydrogen peroxide.


Assuntos
Compostos Ferrosos/farmacologia , Peróxido de Hidrogênio/farmacologia , Antígenos Comuns de Leucócito/genética , Proteína Tirosina Fosfatase não Receptora Tipo 1/genética , Proteínas Tirosina Fosfatases Classe 2 Semelhantes a Receptores/genética , Relação Dose-Resposta a Droga , Interações de Medicamentos , Regulação da Expressão Gênica , Humanos , Células Jurkat , Antígenos Comuns de Leucócito/antagonistas & inibidores , Antígenos Comuns de Leucócito/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 1/antagonistas & inibidores , Proteína Tirosina Fosfatase não Receptora Tipo 1/metabolismo , Proteínas Tirosina Fosfatases Classe 2 Semelhantes a Receptores/antagonistas & inibidores , Proteínas Tirosina Fosfatases Classe 2 Semelhantes a Receptores/metabolismo , Transdução de Sinais
18.
Chemistry ; 21(46): 16555-63, 2015 Nov 09.
Artigo em Inglês | MEDLINE | ID: mdl-26394723

RESUMO

The translocator protein (TSPO) is an integral membrane protein that interacts with a wide variety of endogenous ligands, such as cholesterol and porphyrins, and is also the target for several small molecules with substantial in vivo efficacy. When complexed with the TSPO-specific radioligand (R)-PK11195, TSPO folds into a rigid five-helix bundle. However, little is known about the structure and dynamics of TSPO in the absence of high-affinity ligands. By means of NMR spectroscopy, we show that TSPO exchanges between multiple conformations in the absence of (R)-PK11195. Extensive motions on time scales from pico- to microseconds occur all along the primary sequence of the protein, leading to a loss of stable tertiary interactions and local unfolding of the helical structure in the vicinity of the ligand-binding site. The flexible nature of TSPO highlights the importance of conformational plasticity in integral membrane proteins.


Assuntos
Isoquinolinas/química , Isoquinolinas/farmacologia , Proteínas de Membrana/química , Sítios de Ligação , Ligantes , Espectroscopia de Ressonância Magnética , Proteínas de Membrana/metabolismo , Conformação Proteica
19.
J Phys Chem B ; 119(36): 11978-87, 2015 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-26301699

RESUMO

Nuclear magnetic relaxation provides a powerful method giving insight into molecular motions at atomic resolution on a broad time scale. Dynamics of biological macromolecules has been widely exploited by measuring (15)N and (13)C relaxation data. Interpretation of these data relies almost exclusively on the use of the model-free approach (MFA) and its extended version (EMFA) which requires no particular physical model of motion and a small number of parameters. It is shown that EMFA is often unable to cope with three different time scales and fails to describe slow internal motions properly. In contrast to EMFA, genuine MFA with two time scales can reproduce internal motions slower than the overall tumbling. It is also shown that MFA and simplified EMFA are equivalent with respect to the values of the N-H bond length and chemical shift anisotropy. Therefore, the vast majority of (15)N relaxation data for proteins can be satisfactorily interpreted solely with MFA.


Assuntos
Modelos Teóricos , Proteínas/química , Espectroscopia de Ressonância Magnética , Estatística como Assunto
20.
Angew Chem Int Ed Engl ; 54(35): 10347-51, 2015 Aug 24.
Artigo em Inglês | MEDLINE | ID: mdl-26094605

RESUMO

Microtubules are regulated by microtubule-associated proteins. However, little is known about the structure of microtubule-associated proteins in complex with microtubules. Herein we show that the microtubule-associated protein Tau, which is intrinsically disordered in solution, locally folds into a stable structure upon binding to microtubules. While Tau is highly flexible in solution and adopts a ß-sheet structure in amyloid fibrils, in complex with microtubules the conserved hexapeptides at the beginning of the Tau repeats two and three convert into a hairpin conformation. Thus, binding to microtubules stabilizes a unique conformation in Tau.


Assuntos
Amiloide/química , Microtúbulos/química , Dobramento de Proteína , Proteínas tau/química , Humanos , Espectroscopia de Ressonância Magnética , Conformação Proteica
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