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Disulfide bridges in peptides and proteins play an essential role in maintaining their conformation, structural integrity, and consequently function. Despite ongoing efforts, it is still not possible to detect disulfide bonds and the connectivity of multiply bridged peptides directly through a simple and sufficiently validated protein sequencing or peptide mapping method. Partial or complete reduction and chemical cysteine modification are required as initial steps, followed by the application of a proper detection method. Edman degradation (ED) has been used for primary sequence determination but is largely neglected since the establishment of mass spectrometry (MS)-based protein sequencing. Here, we evaluated and thoroughly characterized the phenyl thiohydantoin (PTH) cysteine derivatives PTH-S-methyl cysteine and PTH-S-carbamidomethyl cysteine as bioanalytical standards for cysteine detection and quantification as well as for the elucidation of the disulfide connectivity in peptides by ED. Validation of the established derivatives was performed according to the guidelines of the International Committee of Harmonization on bioanalytical method validation, and their analytical properties were confirmed as reference standards. A series of model peptides was sequenced to test the usability of the PTH-Cys-derivatives as standards, whereas the native disulfide-bonded peptides CCAP-vil, µ-conotoxin KIIIA, and human insulin were used as case studies to determine their disulfide bond connectivity completely independent of MS analysis.
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Cisteína , Disulfuros , Humanos , Cisteína/química , Disulfuros/química , Péptidos/química , Proteínas , Secuencia de AminoácidosRESUMEN
Ultraviolet (UV) B irradiation induces protein modification, especially the conformational rearrangement of proteins, and is therefore promising as a non-thermal and non-chemical functionalization technique. Nevertheless, UVB irradiation introduces radicals and oxidizes side chains resulting in the loss of food quality. Thus, assessing the UVB irradiation-based functionalization of ß-lactoglobulin (BLG) versus its oxidative degradation is of interest. UVB irradiation of up to 8 h was successfully applied to loosen the rigid folding of BLG and increase its flexibility. Thereby, the cysteine at position 121 and hydrophobic regions became surface-exposed as indicated by the increase in accessible thiol groups and increased surface hydrophobicity. Furthermore, we demonstrated the cleavage of the "outer" disulfide bond C66-C160 by LC-MS/MS after tryptic digestion of BLG. The 2-h-irradiated BLG showed adequate conformational rearrangement for protein functionalization while being minimally oxidized.
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Lactoglobulinas , Espectrometría de Masas en Tándem , Lactoglobulinas/química , Cromatografía Liquida , Rayos Ultravioleta , Estrés OxidativoRESUMEN
Heme is a double-edged sword. On the one hand, it has a pivotal role as a prosthetic group of hemoproteins in many biological processes ranging from oxygen transport and storage to miRNA processing. On the other hand, heme can transiently associate with proteins, thereby regulating biochemical pathways. During hemolysis, excess heme, which is released into the plasma, can bind to proteins and regulate their activity and function. The role of heme in these processes is under-investigated, with one problem being the lack of knowledge concerning recognition mechanisms for the initial association of heme with the target protein and the formation of the resulting complex. A specific heme-binding sequence motif is a prerequisite for such complex formation. Although numerous short signature sequences indicating a particular protein function are known, a comprehensive analysis of the heme-binding motifs (HBMs) which have been identified in proteins, concerning specific patterns and structural peculiarities, is missing. In this report, we focus on the evaluation of known mammalian heme-regulated proteins concerning specific recognition and structural patterns in their HBMs. The Cys-Pro dipeptide motifs are particularly emphasized because of their more frequent occurrence. This analysis presents a comparative insight into the sequence and structural anomalies observed during transient heme binding, and consequently, in the regulation of the relevant protein.
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Hemoproteínas , Animales , Proteínas de Unión al Hemo/metabolismo , Fenómenos Biofísicos , Hemoproteínas/genética , Hemoproteínas/metabolismo , Hemo/metabolismo , Unión Proteica , Mamíferos/metabolismoRESUMEN
The modulatory interactions between neurotensin (NT) and the dopaminergic neurotransmitter system in the brain suggest that NT may be associated with the progression of Parkinson's disease (PD). NT exerts its neurophysiological effects by interactions with the human NT receptors type 1 (hNTS1) and 2 (hNTS2). Therefore, both receptor subtypes are promising targets for the development of novel NT-based analogs for the treatment of PD. In this study, we used a virtually guided molecular modeling approach to predict the activity of NT(8-13) analogs by investigating the docking models of ligands designed for binding to the human NTS1 and NTS2 receptors. The importance of the residues at positions 8 and/or 9 for hNTS1 and hNTS2 receptor binding affinity was experimentally confirmed by radioligand binding assays. Further in vitro ADME profiling and in vivo studies revealed that, compared to the parent peptide NT(8-13), compound 10 exhibited improved stability and BBB permeability combined with a significant enhancement of the motor function and memory in a mouse model of PD. The herein reported NTS1/NTS2 dual-specific NT(8-13) analogs represent an attractive tool for the development of therapeutic strategies against PD and potentially other CNS disorders.
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Neurotensina , Enfermedad de Parkinson , Animales , Humanos , Ratones , Dopamina , Ligandos , Neurotensina/farmacología , Neurotensina/metabolismo , Enfermedad de Parkinson/tratamiento farmacológico , Unión Proteica , Receptores de Neurotensina/metabolismoRESUMEN
Structural and functional integrities of formulated proteins are key characteristics that provide a better understanding of influencing factors and their adjustment during formulation development. Here, the procedures commonly used for protein analysis were applied and optimized to obtain a higher degree of accuracy, reproducibility, and reliability for the analysis of lysozyme extracts from hot-melt extrudates (HME). The extrudates were prepared with polyethylene glycol 20 000. The test lysozyme HMEs were subjected to extraction procedures and analytical methods following the International Council of Harmonization guidelines for testing the active protein ingredient Q 1 A (R2) in its pure and formulated form. Therefore, reversed-phase high-pressure liquid chromatography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, matrix-assisted laser desorption ionization mass spectrometry, and fluorescence-based activity measurements were applied to study lysozyme stability and function after formulation. Long-term accelerated stability studies were performed for the pure and formulated protein. Our findings revealed a high degree of stability for lysozyme toward different temperatures and storage times, confirming that HME is a suitable formulation alternative that preserves lysozyme's properties and stability. The presented methods and workflow are recommended to be exploited for further protein drugs to assess usability and compatibility concerning different pharmaceutical applications.
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Fe(III) heme is known to possess low catalytic activity when exposed to hydrogen peroxide and a reducing substrate. Efficient non-covalently linked Fe(III) heme-peptide complexes may represent suitable alternatives as a new group of green catalysts. Here, we evaluated a set of heme-peptide complexes by determination of their peroxidase-like activity and the kinetics of the catalytic conversion in both, the soluble and the immobilized state. We show the impact of peptide length on binding of the peptides to Fe(III) heme and the catalytic activity. Immobilization of the peptide onto a polymer support maintains the catalytic performance of the Fe(III) heme-peptide complex. This study thus opens up a new perspective with regard to the development of heterogeneous biocatalysts with a peroxidase-like activity.
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Compuestos Férricos , Hemo , Hemo/química , Compuestos Férricos/química , Catálisis , Péptidos , Peroxidasas/química , Peroxidasas/metabolismoRESUMEN
Gα proteins as part of heterotrimeric G proteins are molecular switches essential for G protein-coupled receptor- mediated intracellular signaling. The role of the Gα subunits has been examined for decades with various guanine nucleotides to elucidate the activation mechanism and Gα protein-dependent signal transduction. Several approaches describe fluorescent ligands mimicking the GTP function, yet lack the efficient estimation of the proteins' GTP binding activity and the fraction of active protein. Herein, we report the development of a reliable fluorescence anisotropy-based method to determine the affinity of ligands at the GTP-binding site and to quantify the fraction of active Gαi1 protein. An advanced bacterial expression protocol was applied to produce active human Gαi1 protein, whose GTP binding capability was determined with novel fluorescently labeled guanine nucleotides acting as high-affinity Gαi1 binders compared to the commonly used BODIPY FL GTPγS. This study thus contributes a new method for future investigations of the characterization of Gαi and other Gα protein subunits, exploring their corresponding signal transduction systems and potential for biomedical applications.
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Nucleótidos de Guanina , Proteínas de Unión al GTP Heterotriméricas , Polarización de Fluorescencia , Nucleótidos de Guanina/metabolismo , Guanosina Trifosfato/metabolismo , Proteínas de Unión al GTP Heterotriméricas/metabolismo , Humanos , Ligandos , Unión Proteica , Subunidades de Proteína/metabolismo , Receptores Acoplados a Proteínas G/metabolismoRESUMEN
Heme, an iron-protoporphyrin IX complex, is a cofactor bound to various hemoproteins and supports a broad range of functions, such as electron transfer, oxygen transport, signal transduction, and drug metabolism. In recent years, there has been a growing recognition of heme as a non-genomic modulator of ion channel functions. Here, we show that intracellular free heme and hemin modulate human ether à go-go (hEAG1, Kv10.1) voltage-gated potassium channels. Application of hemin to the intracellular side potently inhibits Kv10.1 channels with an IC50 of about 4 nM under ambient and 63 nM under reducing conditions in a weakly voltage-dependent manner, favoring inhibition at resting potential. Functional studies on channel mutants and biochemical analysis of synthetic and recombinant channel fragments identified a heme-binding motif CxHx8H in the C-linker region of the Kv10.1 C terminus, with cysteine 541 and histidines 543 and 552 being important for hemin binding. Binding of hemin to the C linker may induce a conformational constraint that interferes with channel gating. Our results demonstrate that heme and hemin are endogenous modulators of Kv10.1 channels and could be exploited to modulate Kv10.1-mediated cellular functions.
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Canales de Potasio Éter-A-Go-Go , Hemina , Humanos , Potenciales de la MembranaRESUMEN
Chemical probes that specifically modulate the activity of heterotrimeric G proteins provide excellent tools for investigating G protein-mediated cell signaling. Herein, we report a family of selective peptidyl Gαi/s modulators derived from peptide library screening and optimization. Conjugation to a cell-penetrating peptide rendered the peptides cell-permeable and biologically active in cell-based assays. The peptides exhibit potent guanine-nucleotide exchange modulator-like activity toward Gαi and Gαs. Molecular docking and dynamic simulations revealed the molecular basis of the protein-ligand interactions and their effects on GDP binding. This study demonstrates the feasibility of developing direct Gαi/s modulators and provides a novel chemical probe for investigating cell signaling through GPCRs/G proteins.
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Proteínas de Unión al GTP Heterotriméricas , Nucleótidos , Proteínas de Unión al GTP Heterotriméricas/metabolismo , Proteínas de Unión al GTP Heterotriméricas/farmacología , Simulación del Acoplamiento Molecular , Nucleótidos/metabolismo , Péptidos/química , Transducción de SeñalRESUMEN
Significance: The excessive production of reactive oxygen species (ROS) has been linked to neurodegenerative diseases (NDs), and, therefore, many scientific works were published on the impact of ROS on the development of prevalent NDs, such as Alzheimer's disease (AD) and Parkinson's disease (PD). Since quantitative and qualitative bibliometric analyses in this research area have not yet been done, the aim of this work is to explore the scientific literature implying ROS in NDs and to identify the major contributors, mainstream research themes, and topics on the rise. Recent Advances: Overall, 22,885 publications were identified and analyzed within the Web of Science (WoS) Core Collection electronic database (Clarivate Analytics, Philadelphia, PA). Most of the manuscripts were published in the 21st century. The publications were mainly related to the WoS categories Neurosciences and Biochemistry molecular biology. The United States is the major contributor, harboring the most productive authors and institutions. China, South Korea, and India have emerged as upcoming major contributors in the 2010s. Two most productive journals were Journal of Neurochemistry and Free Radical Biology and Medicine. Critical Issues: AD, PD, and amyotrophic lateral sclerosis were much more investigated than multiple sclerosis and Huntington's disease. Vitamin E and curcumin were frequently mentioned as potential antioxidant therapeutics, but their efficacy in treating NDs requires more clinical studies, since the existing evidence was mainly from in vitro experiments and in vivo animal studies. Future Directions: Mitochondrial dysfunction, autophagy, and nuclear factor erythroid 2-related factor 2 were among the author keywords with rising prevalence. Further research in these directions should advance our understanding of the mechanism and treatment of NDs. Antioxid. Redox Signal. 34, 402-420.
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Susceptibilidad a Enfermedades , Enfermedades Neurodegenerativas/etiología , Enfermedades Neurodegenerativas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Animales , Humanos , Medicina en la Literatura , Enfermedades Neurodegenerativas/diagnóstico , Enfermedades Neurodegenerativas/terapia , Oxidación-ReducciónRESUMEN
De Novo metalloprotein design assesses the relationship between metal active site architecture and catalytic reactivity. Herein, we use an α-helical scaffold to control the iron coordination geometry when a heme cofactor is allowed to bind to either histidine or cysteine ligands, within a single artificial protein. Consequently, we uncovered a reversible pH-induced switch of the heme axial ligation within this simplified scaffold. Characterization of the specific heme coordination modes was done by using UV/Vis and Electron Paramagnetic Resonance spectroscopies. The penta- or hexa-coordinate thiolate heme (9≤pH≤11) and the penta-coordinate imidazole heme (6≤pH≤8.5) reproduces well the heme ligation in chloroperoxidases or cyt P450 monooxygenases and peroxidases, respectively. The stability of heme coordination upon ferric/ferrous redox cycling is a crucial property of the construct. At basic pHs, the thiolate mini-heme protein can catalyze O2 reduction when adsorbed onto a pyrolytic graphite electrode.
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Cisteína/metabolismo , Hemo/metabolismo , Histidina/metabolismo , Metaloproteínas/metabolismo , Secuencia de Aminoácidos , Catálisis , Cisteína/química , Espectroscopía de Resonancia por Spin del Electrón , Hemo/química , Histidina/química , Concentración de Iones de Hidrógeno , Hierro/química , Metaloproteínas/química , Oxidación-Reducción , Oxígeno/química , Péptidos/química , Péptidos/metabolismo , Conformación Proteica en Hélice alfaRESUMEN
Many research institutions, clinical diagnostic laboratories, and blood banks are desperately searching for a possibility to identify and quantify heme in different physiological and pathological settings as well as various research applications. The reasons for this are the toxicity of the heme and the fact that it acts as a hemolytic and pro-inflammatory molecule. Heme only exerts these severe and undesired effects when it is not incorporated in hemoproteins. Upon release from the hemoproteins, it enters a biologically available state (labile heme), in which it is loosely associated with proteins, lipids, nucleic acids, or other molecules. While the current methods and procedures for quantitative determination of heme have been used for many years in different settings, their value is limited by the challenging chemical properties of heme. A major cause of inadequate quantification is the separation of labile and permanently bound heme and its high aggregation potential. Thus, none of the current methods are utilized as a generally applicable, standardized approach. The aim of this Feature is to describe and summarize the most common and frequently used chemical, analytical, and biochemical methods for the quantitative determination of heme. Based on this overview, the most promising approaches for future solutions to heme quantification are highlighted.
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Cromatografía/métodos , Electroforesis Capilar/métodos , Pruebas de Enzimas/métodos , Hemo/química , Hemoproteínas/química , Humanos , Estructura Molecular , Análisis EspectralRESUMEN
BACKGROUND & MOTIVATION: Peptides and proteins can interact with heme through His, Tyr, or Cys in heme-regulatory motifs (HRMs). The Cys-Pro dipeptide is a well investigated HRM, but for His and Tyr such a distinct motif is currently unknown. In addition, many heme-peptide complexes, such as heme-amyloid ß, can display a peroxidase-like activity, albeit there is little understanding of how the local primary and secondary coordination environment influences catalytic activity. We thus systematically evaluated a series of His- and Tyr-based peptides to identify sequence features for high-affinity heme binding and their impact on the catalytic activity of heme. METHODS: We employed solid-phase peptide synthesis to produce 58 nonapeptides, which were investigated by UV/vis, resonance Raman, and 2D NMR spectroscopy. A chromogenic assay was used to determine the catalytic activity of the heme-peptide complexes. RESULTS: Heme-binding affinity and binding mode were found to be dependent on the coordinating amino acid and spacer length between multiple potential coordination sites in a motif. In particular, HXH and HXXXH motifs showed strong heme binding. Analysis of the peroxidase-like activity revealed that some of these peptides and also HXXXY motifs enhance the catalytic activity of heme significantly. CONCLUSIONS: We identify HXH, HXXXH, and HXXXY as potential new HRMs with functional properties. Several peptides displayed a strikingly high peroxidase-like activity. GENERAL SIGNIFICANCE: The identification of HRMs allows to discover yet unknown heme-regulated proteins, and consequently, enhances our current understanding of pathologies involving labile heme.
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Hemo , Hemoproteínas , Péptidos beta-Amiloides , Hemo/metabolismo , Hemoproteínas/metabolismo , Espectroscopía de Resonancia Magnética , PeroxidasasRESUMEN
Cytokines of the interleukin (IL)-1 family regulate immune and inflammatory responses. The recently discovered IL-36 family members are involved in psoriasis, rheumatoid arthritis, and pulmonary diseases. Here, we show that IL-36α interacts with heme thereby contributing to its regulation. Based on in-depth spectroscopic analyses, we describe two heme-binding sites in IL-36α that associate with heme in a pentacoordinated fashion. Solution NMR analysis reveals structural features of IL-36α and its complex with heme. Structural investigation of a truncated IL-36α supports the notion that the N-terminus is necessary for association with its cognate receptor. Consistent with our structural studies, IL-36-mediated signal transduction was negatively regulated by heme in synovial fibroblast-like synoviocytes from rheumatoid arthritis patients. Taken together, our results provide a structural framework for heme-binding proteins and add IL-1 cytokines to the group of potentially heme-regulated proteins.
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Hemo/metabolismo , Interleucina-1/metabolismo , Artritis Reumatoide/metabolismo , Artritis Reumatoide/patología , Células Cultivadas , Citocinas/agonistas , Citocinas/química , Citocinas/metabolismo , Fibroblastos/metabolismo , Fibroblastos/patología , Humanos , Mediadores de Inflamación/agonistas , Mediadores de Inflamación/química , Mediadores de Inflamación/metabolismo , Interleucina-1/agonistas , Interleucina-1/química , Modelos Moleculares , Simulación de Dinámica Molecular , Unión Proteica , Conformación Proteica , Psoriasis/metabolismo , Psoriasis/patología , Relación Estructura-Actividad , Membrana Sinovial/metabolismo , Membrana Sinovial/patologíaRESUMEN
Deviant levels of available heme and related molecules can result from pathological situations such as impaired heme biosynthesis or increased hemolysis as a consequence of vascular trauma or bacterial infections. Heme-related biological processes are affected by these situations, and it is essential to fully understand the underlying mechanisms. While heme has long been known as an important prosthetic group of various proteins, its function as a regulatory and signaling molecule is poorly understood. Diseases such as porphyria are caused by impaired heme metabolism, and heme itself might be used as a drug in order to downregulate its own biosynthesis. In addition, heme-driven side effects and symptoms emerging from heme-related pathological conditions are not fully comprehended and thus impede adequate medical treatment. Several heme-regulated proteins have been identified in the past decades, however, the molecular basis of transient heme-protein interactions remains to be explored. Herein, we summarize the results of an in-depth analysis of heme binding to proteins, which revealed specific binding modes and affinities depending on the amino acid sequence. Evaluating the binding behavior of a plethora of heme-peptide complexes resulted in the implementation of a prediction tool (SeqD-HBM) for heme-binding motifs, which eventually led and will perspectively lead to the identification and verification of so far unknown heme-regulated proteins. This systematic approach resulted in a broader picture of the alternative functions of heme as a regulator of proteins. However, knowledge on heme regulation of proteins is still a bottomless barrel that leaves much scope for future research and development.
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Hemo/genética , Hemoproteínas/genética , Complejos Multiproteicos/genética , Péptidos/genética , Secuencia de Aminoácidos , Bases de Datos Genéticas , Hemo/metabolismo , Hemoproteínas/metabolismo , Humanos , Complejos Multiproteicos/química , Péptidos/química , Unión Proteica/genéticaRESUMEN
Direct targeting of intracellular Gα subunits of Gâ protein-coupled receptors by chemical tools is a challenging task in current pharmacological studies and in the development of novel therapeutic approaches. In this study we analyzed novel FR900359-based analogs from natural sources, synthetic cyclic peptides, as well as all so-far known Gq α inhibitors in a comprehensive study to devise a strategy for the elucidation of characteristics that determine interactions with and inhibition of Gq in the specific FR/YM-binding pocket. Using 2D NMR spectroscopy and molecular docking we identified unique features in the macrocyclic structures responsible for binding to the target protein correlating with inhibitory activity. While all novel compounds were devoid of effects on Gi and Gs proteins, no inhibitor surpassed the biological activity of FR. This raises the question of whether depsipeptides such as FR already represent valuable chemical tools for specific inhibition of Gq and, at the same time, are suitable natural lead structures for the development of novel compounds to target Gα subunits other than Gq .
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Depsipéptidos/farmacología , Inhibidores Enzimáticos/farmacología , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/antagonistas & inhibidores , Animales , Sitios de Unión , Células CHO , Bovinos , Cricetulus , Depsipéptidos/síntesis química , Depsipéptidos/química , Depsipéptidos/metabolismo , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/química , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/metabolismo , Células HEK293 , Humanos , Espectroscopía de Resonancia Magnética , Ratones , Simulación del Acoplamiento Molecular , Estructura Molecular , Unión Proteica , Ratas , Relación Estructura-ActividadRESUMEN
Aqueous two-phase systems (ATPS) occur by the mixture of two polymers or a polymer and an inorganic salt in water. It was shown that not only polymers but also ionic liquids in combination with inorganic cosmotrophic salts are able to build ATPS. Suitable for the formation of ionic liquid-based ATPS systems are hydrophilic water miscible ionic liquids. To understand the driving force for amino acid and peptide distribution in IL-ATPS at different pH values, the ionic liquid Ammoeng 110™ and K2HPO4 have been chosen as a test system. To quantify the concentration of amino acids and peptides in the different phases, liquid chromatography and mass spectrometry (LC-MS) technologies were used. Therefore the peptides and amino acids have been processed with EZ:faast™-Kit from Phenomenex for an easy and reliable quantification method even in complex sample matrices. Partitioning is a surface-dependent phenomenon, investigations were focused on surface-related amino acid respectively peptide properties such as charge and hydrophobicity. Only a very low dependence between the amino acids or peptides hydrophobicity and the partition coefficient was found. Nevertheless, the presented results show that electrostatic respectively ionic interactions between the ionic liquid and the amino acids or peptides have a strong impact on their partitioning behavior.
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Checkpoint kinases sense replicative stress to prevent DNA damage. Here we show that the histone deacetylases HDAC1/HDAC2 sustain the phosphorylation of the checkpoint kinases ATM, CHK1 and CHK2, activity of the cell cycle gatekeeper kinases WEE1 and CDK1, and induction of the tumour suppressor p53 in response to stalled DNA replication. Consequently, HDAC inhibition upon replicative stress promotes mitotic catastrophe. Mechanistically, HDAC1 and HDAC2 suppress the expression of PPP2R3A/PR130, a regulatory subunit of the trimeric serine/threonine phosphatase 2 (PP2A). Genetic elimination of PR130 reveals that PR130 promotes dephosphorylation of ATM by PP2A. Moreover, the ablation of PR130 slows G1/S phase transition and increases the levels of phosphorylated CHK1, replication protein A foci and DNA damage upon replicative stress. Accordingly, stressed PR130 null cells are very susceptible to HDAC inhibition, which abrogates the S phase checkpoint, induces apoptosis and reduces the homologous recombination protein RAD51. Thus, PR130 controls cell fate decisions upon replicative stress.
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Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/metabolismo , Quinasa de Punto de Control 2/metabolismo , Histona Desacetilasa 1/metabolismo , Histona Desacetilasa 2/metabolismo , Proteína Fosfatasa 2/metabolismo , Proteínas de la Ataxia Telangiectasia Mutada/genética , Proteína Quinasa CDC2/genética , Proteína Quinasa CDC2/metabolismo , Ciclo Celular , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/genética , Quinasa de Punto de Control 2/genética , Regulación de la Expresión Génica , Histona Desacetilasa 1/genética , Histona Desacetilasa 2/genética , Humanos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosforilación , Proteína Fosfatasa 2/genética , Proteínas Tirosina Quinasas/genética , Proteínas Tirosina Quinasas/metabolismo , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
G protein-independent, arrestin-dependent signaling is a paradigm that broadens the signaling scope of G protein-coupled receptors (GPCRs) beyond G proteins for numerous biological processes. However, arrestin signaling in the collective absence of functional G proteins has never been demonstrated. Here we achieve a state of "zero functional G" at the cellular level using HEK293 cells depleted by CRISPR/Cas9 technology of the Gs/q/12 families of Gα proteins, along with pertussis toxin-mediated inactivation of Gi/o. Together with HEK293 cells lacking ß-arrestins ("zero arrestin"), we systematically dissect G protein- from arrestin-driven signaling outcomes for a broad set of GPCRs. We use biochemical, biophysical, label-free whole-cell biosensing and ERK phosphorylation to identify four salient features for all receptors at "zero functional G": arrestin recruitment and internalization, but-unexpectedly-complete failure to activate ERK and whole-cell responses. These findings change our understanding of how GPCRs function and in particular of how they activate ERK1/2.
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Proteínas de Unión al GTP/genética , Sistema de Señalización de MAP Quinasas , Receptores Acoplados a Proteínas G/metabolismo , beta-Arrestina 1/metabolismo , Arrestina beta 2/metabolismo , Sistemas CRISPR-Cas , Subunidades alfa de la Proteína de Unión al GTP/genética , Subunidades alfa de la Proteína de Unión al GTP G12-G13/genética , Subunidades alfa de la Proteína de Unión al GTP Gs/genética , Proteínas de Unión al GTP/metabolismo , Técnicas de Inactivación de Genes , Células HEK293 , Humanos , Fosforilación , Transducción de Señal , beta-Arrestinas/metabolismoRESUMEN
Labile heme has been suggested to have an impact in several severe diseases. In the context of Alzheimer's disease (AD), however, decreased levels of free heme have been reported. Therefore, we were looking for an assay system that can be used for heme concentration determination. From a biochemical point of view the peroxidase activity of the Aß-heme complex seemed quite attractive to pursue this goal. As a consequence, a peptide that is able to increase the readout even in the case of a low heme concentration is favorable. The examination of Aß- and non-Aß-derived peptides in complex with heme revealed that the peroxidase-like activity significantly depends on the peptide sequence and length. A 23mer His-based peptide derived from human fatty acyl-CoA reductase 1 in complex with heme exhibited a significantly higher peroxidase activity than Aß(40)-heme. Structural modeling of both complexes demonstrated that heme binding via a histidine can be supported by hydrogen bond interactions of a basic residue near the propionate carboxyl function of protoporphyrin IX. Furthermore, the interplay of Aß-heme and the lipoprotein LDL as a potential physiological effector of Aß was examined.