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1.
Phys Chem Chem Phys ; 22(1): 212-222, 2019 Dec 18.
Artigo em Inglês | MEDLINE | ID: mdl-31799520

RESUMO

In this computational study, the intermolecular solute-solvent Nuclear Overhauser Effect (NOE) of the model protein ubiquitin in different chemical environments (free, bound to a partner protein and encapsulated) is investigated. Short-ranged NOE observables such as the NOE/ROE ratio reveal hydration phenomena on absolute timescales such as fast hydration sites and slow water clefts. We demonstrate the ability of solute-solvent NOE differences measured of the same protein in different chemical environments to reveal hydration changes on the relative timescale. The resulting NOE/ROE-surface maps are shown to be a central key for analyzing biologically relevant chemical influences such as complexation and confinement: the presence of a complexing macromolecule or a confining surface wall modulates the water mobility in the vicinity of the probe protein, hence revealing which residues of said protein are proximate to the foreign interface and which are chemically unaffected. This way, hydration phenomena can serve to indirectly map the precise influence (position) of other molecules or interfaces onto the protein surface. This proposed one-protein many-solvents approach may offer experimental benefits over classical one-protein other-protein pseudo-intermolecular transient NOEs. Furthermore, combined influences such as complexation and confinement may exert non-additive influences on the protein compared to a reference state. We offer a mathematical method to disentangle the influence of these two different chemical environments.


Assuntos
Ligases/química , Modelos Moleculares , Solventes/química , Ubiquitina/química , Água/química , Simulação por Computador , Soluções
2.
Chem Commun (Camb) ; 55(87): 13093-13095, 2019 Oct 29.
Artigo em Inglês | MEDLINE | ID: mdl-31612161

RESUMO

Ubiquitin monomers functionalized with an azide or multiple alkynes were utilized for the assembly of branched ubiquitin oligomers (K6/K11, K11/K48, K11/K63, K6/K11/K48) by click chemistry. The oligomers resist deubiquitylase-catalysed hydrolysis and exhibit stability in eukaryotic cell lysates.


Assuntos
Ubiquitina/biossíntese , Alquinos/química , Azidas/química , Biocatálise , Química Click , Enzimas Desubiquitinantes/metabolismo , Células Eucarióticas/metabolismo , Humanos , Hidrólise , Ubiquitina/química , Ubiquitina/metabolismo , Ubiquitinação
3.
J Phys Chem Lett ; 10(19): 5917-5922, 2019 Oct 03.
Artigo em Inglês | MEDLINE | ID: mdl-31509419

RESUMO

Nuclear magnetic relaxation provides invaluable quantitative site-specific information on the dynamics of complex systems. Determining dynamics on nanosecond time scales requires relaxation measurements at low magnetic fields incompatible with high-resolution NMR. Here, we use a two-field NMR spectrometer to measure carbon-13 transverse and longitudinal relaxation rates at a field as low as 0.33 T (proton Larmor frequency 14 MHz) in specifically labeled side chains of the protein ubiquitin. The use of radiofrequency pulses enhances the accuracy of measurements as compared to high-resolution relaxometry approaches, where the sample is moved in the stray field of the superconducting magnet. Importantly, we demonstrate that accurate measurements at a single low magnetic field provide enough information to characterize complex motions on low nanosecond time scales, which opens a new window for the determination of site-specific nanosecond motions in complex systems such as proteins.


Assuntos
Ressonância Magnética Nuclear Biomolecular/métodos , Proteínas/química , Isótopos de Carbono , Cinética , Campos Magnéticos , Movimento (Física) , Prótons , Ubiquitina/química
4.
Acta Crystallogr F Struct Biol Commun ; 75(Pt 8): 552-560, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31397327

RESUMO

Endoplasmic reticulum (ER)-associated degradation (ERAD) is a protein quality-control pathway in eukaryotes in which misfolded ER proteins are polyubiquitylated, extracted and ultimately degraded by the proteasome. This process involves ER membrane-embedded ubiquitin E2 and E3 enzymes, as well as a soluble E2 enzyme (Ubc7 in Saccharomyces cerevisiae and UBE2G2 in mammals). E2-binding regions (E2BRs) that recruit these soluble ERAD E2s to the ER have been identified in humans and S. cerevisiae, and structures of E2-E2BR complexes from both species have been determined. In addition to sequence and structural differences between the human and S. cerevisiae E2BRs, the binding of E2BRs also elicits different biochemical outcomes with respect to E2 charging by E1 and E2 discharge. Here, the Schizosaccharomyces pombe E2BR was identified and purified with Ubc7 to resolve a 1.7 Šresolution co-crystal structure of the E2BR in complex with Ubc7. The S. pombe E2BR binds to the back side of the E2 as an α-helix and, while differences exist, it exhibits greater similarity to the human E2BR. Structure-based sequence alignments reveal differences and conserved elements among these species. Structural comparisons and biochemistry reveal that the S. pombe E2BR presents a steric impediment to E1 binding and inhibits E1-mediated charging, respectively.


Assuntos
Cristalografia por Raios X/métodos , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Enzimas de Conjugação de Ubiquitina/química , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitina/metabolismo , Retículo Endoplasmático/metabolismo , Modelos Moleculares , Conformação Proteica , Ubiquitina/química
5.
J Biotechnol ; 304: 38-43, 2019 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-31415789

RESUMO

Efficient coproduction of multiple proteins or their subunits is important in many facets of life sciences and biotechnology. Here, we report a novel approach that exploits the synergy between an engineered mini-intein and an ubiquitin variant to achieve coordinated coexpression of multiple proteins in eukaryotic hosts, from a single open reading frame that encodes a polyprotein precursor consisting of proteins of interest (POIs) connected by an intervening intein-ubiquitin fusion domain. The intein variant mediates highly active autocatalytic cleavage at its N-terminus, whereas the endogenous deubiquitinases cleave at ubiquitin's C-terminus, leading to the release of the POIs. Using fluorescent reporter proteins for proof-of-concept, utility of the intein-ubiquitin domain was validated in higher plants and yeast systems. Essentially complete release of the POIs was achieved as demonstrated with western blots. Proteins expressed using the intein-ubiquitin system potentially preserve their intended sequences, which is important for preventing alteration of POI function.


Assuntos
Engenharia de Proteínas/métodos , Proteínas Recombinantes de Fusão/metabolismo , Ubiquitina/química , Corantes Fluorescentes/metabolismo , Inteínas , Fases de Leitura Aberta , Plantas/genética , Plantas/metabolismo , Domínios Proteicos , Proteínas Recombinantes de Fusão/genética , Ubiquitina/genética , Leveduras/genética , Leveduras/metabolismo
6.
Cell Mol Life Sci ; 76(23): 4589-4611, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31254044

RESUMO

Parkinson's disease (PD) is a degenerative movement disorder resulting from the loss of specific neuron types in the midbrain. Early environmental and pathophysiological studies implicated mitochondrial damage and protein aggregation as the main causes of PD. These findings are now vindicated by the characterization of more than 20 genes implicated in rare familial forms of the disease. In particular, two proteins encoded by the Parkin and PINK1 genes, whose mutations cause early-onset autosomal recessive PD, function together in a mitochondrial quality control pathway. In this review, we will describe recent development in our understanding of their mechanisms of action, structure, and function. We explain how PINK1 acts as a mitochondrial damage sensor via the regulated proteolysis of its N-terminus and the phosphorylation of ubiquitin tethered to outer mitochondrial membrane proteins. In turn, phospho-ubiquitin recruits and activates Parkin via conformational changes that increase its ubiquitin ligase activity. We then describe how the formation of polyubiquitin chains on mitochondria triggers the recruitment of the autophagy machinery or the formation of mitochondria-derived vesicles. Finally, we discuss the evidence for the involvement of these mechanisms in physiological processes such as immunity and inflammation, as well as the links to other PD genes.


Assuntos
Mitocôndrias/metabolismo , Proteínas Quinases/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina/metabolismo , Autofagia , Humanos , Mitocôndrias/genética , Membranas Mitocondriais/metabolismo , Doença de Parkinson/metabolismo , Doença de Parkinson/patologia , Proteínas Quinases/química , Proteínas Quinases/genética , Ubiquitina/química , Ubiquitina-Proteína Ligases/química , Ubiquitinação
7.
J Chem Theory Comput ; 15(8): 4318-4331, 2019 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-31241940

RESUMO

The relative prevalence of native protein-protein interactions (PPIs) are the cornerstone for understanding the structure, dynamics and mechanisms of function of protein complexes. In this study, we develop a scheme for scaling the protein-water interaction in the CHARMM36 force field, in order to better fit the solvation free energy of amino acids side-chain analogues. We find that the molecular dynamics simulation with the scaled force field, CHARMM36s, as well as a recently released version, CHARMM36m, effectively improve on the overly sticky association of proteins, such as ubiquitin. We investigate the formation of a heterodimer protein complex between the SAM domains of the EphA2 receptor and the SHIP2 enzyme by performing a combined total of 48 µs simulations with the different potential functions. While the native SAM heterodimer is only predicted at a low rate of 6.7% with the original CHARMM36 force field, the yield is increased to 16.7% with CHARMM36s, and to 18.3% with CHARMM36m. By analyzing the 25 native SAM complexes formed in the simulations, we find that their formation involves a preorientation guided by Coulomb interactions, consistent with an electrostatic steering mechanism. In 12 cases, the complex could directly transform to the native protein interaction surfaces with only small adjustments in domain orientation. In the other 13 cases, orientational and/or translational adjustments are needed to reach the native complex. Although the tendency for non-native complexes to dissociate has nearly doubled with the modified potential functions, a dissociation followed by a reassociation to the correct complex structure is still rare. Instead, the remaining non-native complexes undergo configurational changes/surface searching, which, however, rarely leads to native structures on a time scale of 250 ns. These observations provide a rich picture of the mechanisms of protein-protein complex formation and suggest that computational predictions of native complex PPIs could be improved further.


Assuntos
Mapas de Interação de Proteínas , Proteínas/metabolismo , Humanos , Simulação de Dinâmica Molecular , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases/química , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases/metabolismo , Ligação Proteica , Conformação Proteica , Multimerização Proteica , Proteínas/química , Receptor EphA2/química , Receptor EphA2/metabolismo , Eletricidade Estática , Termodinâmica , Ubiquitina/química , Ubiquitina/metabolismo , Água/metabolismo
8.
Nanoscale ; 11(20): 9920-9930, 2019 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-31069350

RESUMO

Nanopore based sensors constitute a promising approach to single molecule protein characterization being able, in principle, to detect sequences, structural elements and folding states of proteins and polypeptide chains. In narrow nanopores, one of the open issues concerns the coupling between unfolding and translocation. Here, we studied the ubiquitin translocation in an α-hemolysin nanopore, the most widely used pore for nanopore sensing, via all-atom molecular dynamics simulations. We completely characterize the co-translocational unfolding pathway finding that robust translocation intermediates are associated with the rearrangement of secondary structural elements, as also confirmed by coarse grained simulations. An interesting recurrent pattern is the clogging of the α-hemolysin constriction by an N-terminal ß-hairpin. This region of ubiquitin is the target of several post-translational modifications. We propose a strategy to detect post-translational modifications at the N-terminal using the α-hemolysin nanopore based on the comparison of the co-translocational unfolding signals associated with modified and unmodified proteins.


Assuntos
Proteínas Hemolisinas/metabolismo , Nanoporos , Ubiquitina/metabolismo , Sequência de Aminoácidos , Proteínas Hemolisinas/química , Processamento de Proteína Pós-Traducional , Estrutura Secundária de Proteína , Desdobramento de Proteína , Ubiquitina/química
9.
Methods Mol Biol ; 1977: 25-34, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30980320

RESUMO

Protein homeostasis is essential for the survival of cells. It is closely related to the functioning of the ubiquitin-proteasome system, which utilizes the small protein ubiquitin as a posttranslational modifier (PTM). Clinically, the modification is of great importance as its disruption is the cause of many diseases. Unlike other PTMs, ubiquitin can encode several cellular signals by being attached as a single molecule or as a chain of several ubiquitins in various conformations. Thus, ubiquitin signaling is dependent not only on the site of attachment but also on the chain type, the so-called ubiquitin chain topology.The most reliable quantification method for the chain topology uses a bottom-up targeted mass spectrometry-based proteomics technique. While similar to other targeted proteomics techniques, the measurement of ubiquitination chain topology is complicated. First, the ubiquitin chains in the sample have to be biochemically stabilized. Second, the selection of peptides for the analysis is restricted to a given set harboring the PTMs and does not allow for optimization for amenability to mass spectrometry-based quantification. Instead, the topology-characteristic peptides are fixed. We here present such a methodology, including notes for a successful application.


Assuntos
Espectrometria de Massas , Proteômica , Ubiquitina/química , Análise de Dados , Humanos , Espectrometria de Massas/métodos , Espectrometria de Massas/normas , Processamento de Proteína Pós-Traducional , Proteômica/métodos , Coloração e Rotulagem , Ubiquitina/metabolismo , Ubiquitinação
10.
J Chem Phys ; 150(11): 115103, 2019 Mar 21.
Artigo em Inglês | MEDLINE | ID: mdl-30901983

RESUMO

The ability to separate proteins is desirable for many fields of study, and nanoporous membranes may offer a method for rapid protein filtration at high throughput volume, provided there is an understanding of the protein dynamics involved. In this work, we use Brownian dynamics simulations to study the motion of coarse-grained proteins insulin and ubiquitin in an electrically biased membrane. In our model, the protein is subjected to various biases applied to the silicon membrane equipped with a nanopore of different radii. The time each protein takes to find a cylindrical nanopore embedded in a thin silicon membrane, attempt to translocate it (waiting time), and successfully translocate it in a single attempt (translocation time) is calculated. We observe insulin finding the nanopore and translocating it faster than the electrically neutral ubiquitin due to insulin's slightly smaller size and net negative charge. While ubiquitin's dynamics is also affected by the size of the pore, surprisingly, its translocation process is also noticeably changed by the membrane bias. By investigating the protein's multipole moments, we demonstrate that this behavior is largely due to the protein's dipole and quadrupole interactions with the membrane potential.


Assuntos
Insulina/química , Membranas Artificiais , Nanoporos , Ubiquitina/química , Simulação de Dinâmica Molecular , Movimento (Física) , Silício/química , Eletricidade Estática
11.
Front Biosci (Landmark Ed) ; 24: 1097-1157, 2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-30844733

RESUMO

Diet and environment are two critical regulators that influence an individual's epigenetic profile. Besides the anterograde signaling, mitochondria act as a key regulator of epigenetic alterations in cancer either by controlling the concentration of the cofactors, activity of vital enzymes or by affecting the transcription of NF-kappaB and associated signaling molecules. As epigenetic modifications are the major drivers of aberrant gene expression, designing novel nutri-epigenomic strategies to modulate reversible epigenetic modifications will be important for effective cancer protection. In this regard, nutraceuticals such as flavonoids holds significant promise to modulate the epigenome through a network of interconnected anti-redox mechanisms. However, low solubility, rapid metabolism and poor absorption of flavonoids in gastrointestinal tract hinder their use in clinical settings. Therefore, it is imperative to develop nano-engineered systems which could considerably improve the targeted delivery of these bioactive compounds with better efficacy and pharmacokinetic properties. Concerted efforts in nano-engineering of flavonoids using polymer, lipid and complexation based approaches could provide successful bench-to-bedside translation of flavonoids as broad spectrum anti-cancer agents.


Assuntos
Flavonoides/química , Nanomedicina/métodos , Neoplasias/prevenção & controle , Acetilação , Animais , Linhagem Celular Tumoral , Citosina/química , Metilação de DNA , Suplementos Nutricionais , Sistemas de Liberação de Medicamentos , Epigênese Genética , Epigenômica , Histonas/química , Humanos , Lipídeos/química , Lipossomos/química , Micelas , MicroRNAs/metabolismo , Mitocôndrias/metabolismo , Nanopartículas , Fosforilação , Polímeros/química , Ubiquitina/química
12.
Methods Enzymol ; 618: 1-27, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30850047

RESUMO

Posttranslational modifications of histone proteins regulate all biological processes requiring access to DNA. Monoubiquitination of histone H2B is a mark of actively transcribed genes in all eukaryotes that also plays a role in DNA replication and repair. Solution and structural studies of the mechanism by which histone ubiquitination modulates these processes depend on the ability to generate homogeneous preparations of nucleosomes containing ubiquitin conjugated to a specific lysine residue. We describe here methods for generating milligram quantities of histone H2B with ubiquitin (Ub) conjugated to Lys 120 via either a nonhydrolyzable, dichloroacetone linkage or a cleavable isopeptide bond. H2B-Ub with an isopeptide linkage is generated by a combination of intein-fusion protein derivatization and native chemical ligation, yielding a fully native ubiquitinated lysine that can be cleaved by Ub isopeptidases. We also describe how to reconstitute nucleosomes containing ubiquitinated H2B.


Assuntos
Histonas/síntese química , Ubiquitina/síntese química , Proteínas de Xenopus/síntese química , Xenopus laevis , Animais , Histonas/química , Histonas/genética , Hidrólise , Lisina/síntese química , Lisina/química , Lisina/genética , Modelos Moleculares , Ubiquitina/química , Ubiquitina/genética , Ubiquitinação , Proteínas de Xenopus/química , Proteínas de Xenopus/genética , Xenopus laevis/genética
13.
Mol Cell ; 74(2): 330-346.e11, 2019 04 18.
Artigo em Inglês | MEDLINE | ID: mdl-30853400

RESUMO

The autophagy cargo receptor p62 facilitates the condensation of misfolded, ubiquitin-positive proteins and their degradation by autophagy, but the molecular mechanism of p62 signaling to the core autophagy machinery is unclear. Here, we show that disordered residues 326-380 of p62 directly interact with the C-terminal region (CTR) of FIP200. Crystal structure determination shows that the FIP200 CTR contains a dimeric globular domain that we designated the "Claw" for its shape. The interaction of p62 with FIP200 is mediated by a positively charged pocket in the Claw, enhanced by p62 phosphorylation, mutually exclusive with the binding of p62 to LC3B, and it promotes degradation of ubiquitinated cargo by autophagy. Furthermore, the recruitment of the FIP200 CTR slows the phase separation of ubiquitinated proteins by p62 in a reconstituted system. Our data provide the molecular basis for a crosstalk between cargo condensation and autophagosome formation.


Assuntos
Autofagossomos/metabolismo , Conformação Proteica , Proteínas Tirosina Quinases/química , Proteína Sequestossoma-1/química , Autofagossomos/química , Autofagia/genética , Cristalografia por Raios X , Humanos , Proteínas Associadas aos Microtúbulos/química , Proteínas Associadas aos Microtúbulos/genética , Mapas de Interação de Proteínas/genética , Proteínas Tirosina Quinases/genética , Proteólise , Proteína Sequestossoma-1/genética , Transdução de Sinais/genética , Ubiquitina/química , Ubiquitina/genética
14.
Int J Mol Sci ; 20(5)2019 Mar 08.
Artigo em Inglês | MEDLINE | ID: mdl-30857167

RESUMO

The newly identified CUBAN (Cullin binding domain associating with NEDD8) domain recognizes both ubiquitin and the ubiquitin-like NEDD8. Despite the high similarity between the two molecules, CUBAN shows a clear preference for NEDD8, free and conjugated to cullins. We previously characterized the domain structure, both alone and in complex with NEDD8. The results here reported are addressed to investigate the determinants that drive the selective binding of CUBAN towards NEDD8 and ubiquitin. The 15N HSQC NMR perturbation pattern of the labeled CUBAN domain, when combined with either NEDD8 or ubiquitin, shows a clear involvement of hydrophobic residues that characterize the early stages of these interactions. After a slow conformational selection step, hydrophobic and then neutral and polar interactions take place, which drive the correct orientation of the CUBAN domain, leading to differences in the recognition scheme of NEDD8 and ubiquitin. As a result, a cascade of induced fit steps seems to determine the structural preference shown for NEDD8 and therefore the basis of the selectivity of the CUBAN domain. Finally, molecular dynamics analysis was performed to determine by fluctuations the internal flexibility of the CUBAN/NEDD8 complex. We consider that our results, based on a structural investigation mainly focused on the early stages of the recognition, provide a fruitful opportunity to report the different behavior of the same protein with two highly similar binding partners.


Assuntos
Proteína NEDD8/metabolismo , Ubiquitina/metabolismo , Humanos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Proteína NEDD8/química , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Mapas de Interação de Proteínas , Ubiquitina/química , Ubiquitinação
15.
Nucleic Acids Res ; 47(7): 3784-3794, 2019 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-30753618

RESUMO

Cockayne syndrome group B (CSB, also known as ERCC6) protein is involved in many DNA repair processes and essential for transcription-coupled repair (TCR). The central region of CSB has the helicase motif, whereas the C-terminal region contains important regulatory elements for repair of UV- and oxidative stress-induced damages and double-strand breaks (DSBs). A previous study suggested that a small part (∼30 residues) within this region was responsible for binding to ubiquitin (Ub). Here, we show that the Ub-binding of CSB requires a larger part of CSB, which was previously identified as a winged-helix domain (WHD) and is involved in the recruitment of CSB to DSBs. We also present the crystal structure of CSB WHD in complex with Ub. CSB WHD folds as a single globular domain, defining a class of Ub-binding domains (UBDs) different from 23 UBD classes identified so far. The second α-helix and C-terminal extremity of CSB WHD interact with Ub. Together with structure-guided mutational analysis, we identified the residues critical for the binding to Ub. CSB mutants defective in the Ub binding reduced repair of UV-induced damage. This study supports the notion that DSB repair and TCR may be associated with the Ub-binding of CSB.


Assuntos
Quebras de DNA de Cadeia Dupla , DNA Helicases/química , Enzimas Reparadoras do DNA/química , Proteínas de Ligação a Poli-ADP-Ribose/química , Ubiquitina/química , Ubiquitinas/química , Fatores de Transcrição Winged-Helix/química , Sequência de Aminoácidos/genética , Sobrevivência Celular , Síndrome de Cockayne/genética , Síndrome de Cockayne/metabolismo , Dano ao DNA/genética , Dano ao DNA/efeitos da radiação , DNA Helicases/genética , Reparo do DNA/genética , Reparo do DNA/efeitos da radiação , Enzimas Reparadoras do DNA/genética , Humanos , Mutação , Proteínas de Ligação a Poli-ADP-Ribose/genética , Conformação Proteica em alfa-Hélice/genética , Ubiquitina/genética , Ubiquitinas/genética , Raios Ultravioleta , Fatores de Transcrição Winged-Helix/genética
16.
J Biol Chem ; 294(15): 6113-6129, 2019 04 12.
Artigo em Inglês | MEDLINE | ID: mdl-30737286

RESUMO

Deregulation of the HECT-type ubiquitin ligase E6AP (UBE3A) is implicated in human papilloma virus-induced cervical tumorigenesis and several neurodevelopmental disorders. Yet the structural underpinnings of activity and specificity in this crucial ligase are incompletely understood. Here, we unravel the determinants of ubiquitin recognition by the catalytic domain of E6AP and assign them to particular steps in the catalytic cycle. We identify a functionally critical interface that is specifically required during the initial formation of a thioester-linked intermediate between the C terminus of ubiquitin and the ligase-active site. This interface resembles the one utilized by NEDD4-type enzymes, indicating that it is widely conserved across HECT ligases, independent of their linkage specificities. Moreover, we uncover surface regions in ubiquitin and E6AP, both in the N- and C-terminal portions of the catalytic domain, that are important for the subsequent reaction step of isopeptide bond formation between two ubiquitin molecules. We decipher key elements of linkage specificity, including the C-terminal tail of E6AP and a hydrophilic surface region of ubiquitin in proximity to the acceptor site Lys-48. Intriguingly, mutation of Glu-51, a single residue within this region, permits formation of alternative chain types, thus pointing to a key role of ubiquitin in conferring linkage specificity to E6AP. We speculate that substrate-assisted catalysis, as described previously for certain RING-associated ubiquitin-conjugating enzymes, constitutes a common principle during linkage-specific ubiquitin chain assembly by diverse classes of ubiquitination enzymes, including HECT ligases.


Assuntos
Ubiquitina-Proteína Ligases/química , Ubiquitina/química , Substituição de Aminoácidos , Catálise , Domínio Catalítico , Humanos , Mutação de Sentido Incorreto , Especificidade por Substrato , Ubiquitina/genética , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo
17.
J Biomol NMR ; 73(1-2): 59-70, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30771052

RESUMO

A flexible and scalable approach for protein NMR is introduced that builds on rapid data collection via projection spectroscopy and analysis of the spectral input data via joint decomposition. Input data may originate from various types of spectra, depending on the ultimate goal: these may result from experiments based on triple-resonance pulse sequences, or on TOCSY or NOESY sequences, or mixtures thereof. Flexible refers to the free choice of spectra for the joint decompositions depending on the purpose: assignments, structure, dynamics, interactions. Scalable means that the approach is open to the addition of similar or different experiments, e.g. larger proteins may require a wider selection of triple-resonance based experiments. Central to the proposed approach is the mutual support among the different spectra during the spectral analysis: for example, sparser triple-resonance spectra may help decomposing (separating) spin systems in a TOCSY or identifying unique NOEs. In the example presented, backbone plus side chain assignments of ubiquitin were obtained from the combination of either two or three of the following projection experiments: a 4D HCCCONH, a 4D HNCACO and a 3D HNCACB. In all cases, TOCSY data (4D HCCCONH) proved crucial not only for the side chain assignments, but also for the sequential assignment. Even when total recording time was reduced to about 10 h, nearly complete assignments were obtained, with very few missing assignments and even fewer differences to a reference.


Assuntos
Ressonância Magnética Nuclear Biomolecular/métodos , Proteínas/química , Sequência de Aminoácidos , Ubiquitina/química
18.
J Biomol NMR ; 73(1-2): 19-29, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30680507

RESUMO

We report linewidth and proton T1, T1ρ and T2' relaxation data of the model protein ubiquitin acquired at MAS frequencies up to 126 kHz. We find a predominantly linear improvement in linewidths and coherence decay times of protons with increasing spinning frequency in the range from 93 to 126 kHz. We further attempt to gain insight into the different contributions to the linewidth at fast MAS using site-specific analysis of proton relaxation parameters and present bulk relaxation times as a function of the MAS frequency. For microcrystalline fully-protonated ubiquitin, inhomogeneous contributions are only a minor part of the proton linewidth, and at 126 kHz MAS coherent effects are still dominating. We furthermore present site-specific proton relaxation rate constants during a spinlock at 126 kHz MAS, as well as MAS-dependent bulk T1ρ (1HN).


Assuntos
Ressonância Magnética Nuclear Biomolecular/métodos , Ressonância Magnética Nuclear Biomolecular/instrumentação , Proteínas , Prótons , Ubiquitina/química
19.
Biochemistry ; 58(7): 883-886, 2019 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-30668904

RESUMO

Ubiquitin-mediated signaling pathways regulate essentially every aspect of cell biology in eukaryotes. Ubiquitin receptors typically contain ubiquitin-binding domains (UBDs) that have the ability to recognize monomeric ubiquitin (Ub) and polymeric Ub (polyUb) chains. However, how signaling specificity is achieved remains poorly understood, and many of the UBDs that selectively recognize polyUb chains of particular linkages still need to be identified and characterized. Here we report the incorporation of a genetically encoded photo-cross-linker, p-benzoyl-l-phenylalanine (Bpa), into recombinant Ub and enzymatically synthesized polyUb chains. This allows photo-cross-linking (covalent bond formation) of monoUb and K48- and K63-linked diUb chains to UBDs. This approach provides a framework for understanding Ub cellular signaling through the capture and identification of (poly)Ub-binding proteins.


Assuntos
Fenilalanina/análogos & derivados , Poliubiquitina/metabolismo , Ubiquitina/genética , Benzofenonas/química , Sítios de Ligação , Proteínas de Transporte/metabolismo , Reagentes para Ligações Cruzadas/química , Proteínas de Ligação a DNA , Chaperonas de Histonas , Mutação , Proteínas Nucleares/metabolismo , Fenilalanina/química , Fenilalanina/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Domínios Proteicos , RNA de Transferência de Tirosina , Proteínas de Saccharomyces cerevisiae/metabolismo , Ubiquitina/química , Ubiquitina/metabolismo , Enzimas Ativadoras de Ubiquitina/metabolismo
20.
Nat Commun ; 10(1): 231, 2019 01 16.
Artigo em Inglês | MEDLINE | ID: mdl-30651545

RESUMO

USP7 is a highly abundant deubiquitinating enzyme (DUB), involved in cellular processes including DNA damage response and apoptosis. USP7 has an unusual catalytic mechanism, where the low intrinsic activity of the catalytic domain (CD) increases when the C-terminal Ubl domains (Ubl45) fold onto the CD, allowing binding of the activating C-terminal tail near the catalytic site. Here we delineate how the target protein promotes the activation of USP7. Using NMR analysis and biochemistry we describe the order of activation steps, showing that ubiquitin binding is an instrumental step in USP7 activation. Using chemically synthesised p53-peptides we also demonstrate how the correct ubiquitinated substrate increases catalytic activity. We then used transient reaction kinetic modelling to define how the USP7 multistep mechanism is driven by target recognition. Our data show how this pleiotropic DUB can gain specificity for its cellular targets.


Assuntos
Processamento de Proteína Pós-Traducional , Peptidase 7 Específica de Ubiquitina/metabolismo , Ubiquitina/metabolismo , Isótopos de Carbono/química , Domínio Catalítico/genética , Ensaios Enzimáticos/métodos , Cinética , Modelos Químicos , Mutagênese Sítio-Dirigida , Isótopos de Nitrogênio/química , Ressonância Magnética Nuclear Biomolecular/métodos , Peptídeos/química , Peptídeos/metabolismo , Ligação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Ressonância de Plasmônio de Superfície , Proteína Supressora de Tumor p53/química , Ubiquitina/química , Peptidase 7 Específica de Ubiquitina/química , Peptidase 7 Específica de Ubiquitina/genética , Peptidase 7 Específica de Ubiquitina/isolamento & purificação
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