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1.
Phys Chem Chem Phys ; 22(44): 25455-25466, 2020 Nov 18.
Artigo em Inglês | MEDLINE | ID: mdl-33103678

RESUMO

Dynamic nuclear polarization (DNP) of a biomolecule tagged with a polarizing agent has the potential to not only increase NMR sensitivity but also to provide specificity towards the tagging site. Although the general concept has been often discussed, the observation of true site-specific DNP and its dependence on the electron-nuclear distance has been elusive. Here, we demonstrate site-specific DNP in a uniformly isotope-labeled ubiquitin. By recombinant expression of three different ubiquitin point mutants (F4C, A28C, and G75C) post-translationally modified with a Gd3+-chelator tag, localized metal-ion DNP of 13C and 15N is investigated. Effects counteracting the site-specificity of DNP such as nuclear spin-lattice relaxation and proton-driven spin diffusion have been attenuated by perdeuteration of the protein. Particularly for 15N, large DNP enhancement factors on the order of 100 and above as well as localized effects within side-chain resonances differently distributed over the protein are observed. By analyzing the experimental DNP built-up dynamics combined with structural modeling of Gd3+-tags in ubiquitin supported by paramagnetic relaxation enhancement (PRE) in solution, we provide, for the first time, quantitative information on the distance dependence of the initial DNP transfer. We show that the direct 15N DNP transfer rate indeed linearly depends on the square of the hyperfine interaction between the electron and the nucleus following Fermi's golden rule, however, below a certain distance cutoff paramagnetic signal bleaching may dramatically skew the correlation.


Assuntos
Gadolínio/química , Ressonância Magnética Nuclear Biomolecular , Marcação por Isótopo , Mutação , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Ubiquitina/química , Ubiquitina/genética
2.
Sheng Wu Gong Cheng Xue Bao ; 36(8): 1484-1492, 2020 Aug 25.
Artigo em Chinês | MEDLINE | ID: mdl-32924347

RESUMO

Ubiquitination, one type of the most common post-translational modification, mediates the regulation of protein homeostasis in vivo. Since ubiquitin itself contains multiple lysine residues and one N-terminal free amino group, eight types of ubiquitin chains can be formed. The K27 ubiquitin chain is formed through the ubiquitination of the ubiquitin Lys27 (K27), which adopts a compact conformation. In recent years, biological function of the K27 ubiquitin chain in innate immunity, protein homeostasis and DNA damage has been discovered, but the molecular mechanisms of K27 ubiquitin chain assembly, recognition and hydrolysis are still poorly understood. Here we review the structural features and biological functions of K27 ubiquitin chain, to provide a reference for future studies.


Assuntos
Ubiquitina , Imunidade Inata , Lisina , Processamento de Proteína Pós-Traducional , Ubiquitina/química , Ubiquitina/metabolismo , Ubiquitinação
3.
Nat Chem Biol ; 16(11): 1170-1178, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32778845

RESUMO

The RAF family kinases function in the RAS-ERK pathway to transmit signals from activated RAS to the downstream kinases MEK and ERK. This pathway regulates cell proliferation, differentiation and survival, enabling mutations in RAS and RAF to act as potent drivers of human cancers. Drugs targeting the prevalent oncogenic mutant BRAF(V600E) have shown great efficacy in the clinic, but long-term effectiveness is limited by resistance mechanisms that often exploit the dimerization-dependent process by which RAF kinases are activated. Here, we investigated a proteolysis-targeting chimera (PROTAC) approach to BRAF inhibition. The most effective PROTAC, termed P4B, displayed superior specificity and inhibitory properties relative to non-PROTAC controls in BRAF(V600E) cell lines. In addition, P4B displayed utility in cell lines harboring alternative BRAF mutations that impart resistance to conventional BRAF inhibitors. This work provides a proof of concept for a substitute to conventional chemical inhibition to therapeutically constrain oncogenic BRAF.


Assuntos
Antineoplásicos/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Proteínas Proto-Oncogênicas B-raf/genética , Talidomida/análogos & derivados , Ubiquitina/química , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Quimera/metabolismo , Desenho de Fármacos , Resistencia a Medicamentos Antineoplásicos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Regulação da Expressão Gênica , Humanos , Sistema de Sinalização das MAP Quinases , Camundongos , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Modelos Moleculares , Estrutura Molecular , Terapia de Alvo Molecular , Mutação , Fosforilação/efeitos dos fármacos , Ligação Proteica , Proteólise , Transdução de Sinais , Relação Estrutura-Atividade , Talidomida/química
4.
Nat Chem Biol ; 16(11): 1218-1226, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32807965

RESUMO

The ubiquitin-proteasome system (UPS) is a highly regulated protein disposal process critical to cell survival. Inhibiting the pathway induces proteotoxic stress and can be an effective cancer treatment. The therapeutic window observed upon proteasomal blockade has motivated multiple UPS-targeting strategies, including preventing ubiquitination altogether. E1 initiates the cascade by transferring ubiquitin to E2 enzymes. A small molecule that engages the E1 ATP-binding site and derivatizes ubiquitin disrupts enzymatic activity and kills cancer cells. However, binding-site mutations cause resistance, motivating alternative approaches to block this promising target. We identified an interaction between the E2 N-terminal alpha-1 helix and a pocket within the E1 ubiquitin-fold domain as a potentially druggable site. Stapled peptides modeled after the E2 alpha-1 helix bound to the E1 groove, induced a consequential conformational change and inhibited E1 ubiquitin thiotransfer, disrupting E2 ubiquitin charging and ubiquitination of cellular proteins. Thus, we provide a blueprint for a distinct E1-targeting strategy to treat cancer.


Assuntos
Complexo de Endopeptidases do Proteassoma/metabolismo , Enzimas Ativadoras de Ubiquitina/metabolismo , Ubiquitina/metabolismo , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Linhagem Celular Tumoral , Desenho de Fármacos , Resistencia a Medicamentos Antineoplásicos , Humanos , Conformação Molecular , Simulação de Acoplamento Molecular , Peptídeos/química , Ligação Proteica , Relação Estrutura-Atividade , Ubiquitina/química , Ubiquitina/genética , Ubiquitinação
5.
J Vis Exp ; (160)2020 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-32628175

RESUMO

Assessment of the global profile of ubiquitin chain topologies within a proteome is of interest to answer a wide range of biological questions. The protocol outlined here takes advantage of the di-glycine (-GG) modification left after the tryptic digestion of ubiquitin incorporated in a chain. By quantifying these topology-characteristic peptides the relative abundance of each ubiquitin chain topology can be determined. The steps required to quantify these peptides by a parallel reaction monitoring experiment are reported taking into consideration the stabilization of ubiquitin chains. Preparation of heavy controls, cell lysis, and digestion are described along with the appropriate mass spectrometer setup and data analysis workflow. An example data set with perturbations in ubiquitin topology is presented, accompanied by examples of how optimization of the protocol can affect results. By following the steps outlined, a user will be able to perform a global assessment of the ubiquitin topology landscape within their biological context.


Assuntos
Espectrometria de Massas/métodos , Ubiquitina/análise , Alquilação , Sequência de Aminoácidos , Animais , Linhagem Celular Tumoral , Cromatografia Líquida , Humanos , Leupeptinas/farmacologia , Lisina/metabolismo , Camundongos , Peptídeos/análise , Proteoma , Padrões de Referência , Software , Ubiquitina/química
6.
Nat Chem Biol ; 16(8): 866-875, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32483380

RESUMO

Changes in the cellular environment modulate protein energy landscapes to drive important biology, with consequences for signaling, allostery and other vital processes. The effects of ubiquitination are particularly important because of their potential influence on degradation by the 26S proteasome. Moreover, proteasomal engagement requires unstructured initiation regions that many known proteasome substrates lack. To assess the energetic effects of ubiquitination and how these manifest at the proteasome, we developed a generalizable strategy to produce isopeptide-linked ubiquitin within structured regions of a protein. The effects on the energy landscape vary from negligible to dramatic, depending on the protein and site of ubiquitination. Ubiquitination at sensitive sites destabilizes the native structure and increases the rate of proteasomal degradation. In well-folded proteins, ubiquitination can even induce the requisite unstructured regions needed for proteasomal engagement. Our results indicate a biophysical role of site-specific ubiquitination as a potential regulatory mechanism for energy-dependent substrate degradation.


Assuntos
Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitina/química , Ubiquitinação/genética , Animais , Proteínas de Bactérias/metabolismo , Humanos , Camundongos , Poliubiquitina/metabolismo , Complexo de Endopeptidases do Proteassoma/química , Complexo de Endopeptidases do Proteassoma/genética , Ligação Proteica/fisiologia , Proteólise , Ribonucleases/metabolismo , Ubiquitina/metabolismo , Ubiquitinação/fisiologia
7.
Inorg Chem ; 59(14): 9739-9755, 2020 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-32585093

RESUMO

In this study, the binding to lysozyme (Lyz) of four important VIV compounds with antidiabetic and/or anticancer activity, [VIVO(pic)2(H2O)], [VIVO(ma)2], [VIVO(dhp)2], and [VIVO(acac)2], where pic-, ma-, dhp-, and acac- are picolinate, maltolate, 1,2-dimethyl-3-hydroxy-4(1H)-pyridinonate, and acetylacetonate anions, and of the vanadium-containing natural product amavadin ([VIV(hidpa)2]2-, with hidpa3- N-hydroxyimino-2,2'-diisopropionate) was investigated by ElectroSpray Ionization-Mass Spectrometry (ESI-MS). Moreover, the interaction of [VIVO(pic)2(H2O)], chosen as a representative VIVO2+ complex, was examined with two additional proteins, myoglobin (Mb) and ubiquitin (Ub), to compare the data. The examined vanadium concentration was in the range 15-150 µM, i.e., very close to that found under physiological conditions. With pic-, dhp-, and hidpa3-, the formation of adducts n[VIVOL2]-Lyz or n[VIVL2]-Lyz is favored, while with ma- and acac- the species n[VIVOL]-Lyz are detected, with n dependent on the experimental VIV/protein ratio. The behavior of the systems with [VIVO(pic)2(H2O)] and Mb or Ub is very similar to that of Lyz. The results suggested that under physiological conditions, the moiety cis-VIVOL2 (L = pic-, dhp-) is bound by only one accessible side-chain protein residue that can be Asp, Glu, or His, while VIVOL+ (L = ma-, acac-) can interact with the two equatorial and axial sites. If the VIV complex is thermodynamically stable and does not have available coordination positions, such as amavadin, the protein cannot interact with it through the formation of coordination bonds and, in such cases, noncovalent interactions are predicted. The formation of the adducts is dependent on the thermodynamic stability and geometry in aqueous solution of the VIVO2+ complex and affects the transport, uptake, and mechanism of action of potential V drugs.


Assuntos
Alanina/análogos & derivados , Antineoplásicos/química , Complexos de Coordenação/química , Ácidos Hidroxâmicos/química , Hipoglicemiantes/química , Proteínas/química , Alanina/química , Animais , Bovinos , Galinhas , Cavalos , Muramidase/química , Mioglobina/química , Espectrometria de Massas por Ionização por Electrospray , Ubiquitina/química , Vanádio/química
8.
Nucleic Acids Res ; 48(11): 6310-6325, 2020 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-32356875

RESUMO

Tyrosyl-DNA phosphodiesterase 2 (TDP2) reverses Topoisomerase 2 DNA-protein crosslinks (TOP2-DPCs) in a direct-reversal pathway licensed by ZATTZNF451 SUMO2 E3 ligase and SUMOylation of TOP2. TDP2 also binds ubiquitin (Ub), but how Ub regulates TDP2 functions is unknown. Here, we show that TDP2 co-purifies with K63 and K27 poly-Ubiquitinated cellular proteins independently of, and separately from SUMOylated TOP2 complexes. Poly-ubiquitin chains of ≥ Ub3 stimulate TDP2 catalytic activity in nuclear extracts and enhance TDP2 binding of DNA-protein crosslinks in vitro. X-ray crystal structures and small-angle X-ray scattering analysis of TDP2-Ub complexes reveal that the TDP2 UBA domain binds K63-Ub3 in a 1:1 stoichiometric complex that relieves a UBA-regulated autoinhibitory state of TDP2. Our data indicates that that poly-Ub regulates TDP2-catalyzed TOP2-DPC removal, and TDP2 single nucleotide polymorphisms can disrupt the TDP2-Ubiquitin interface.


Assuntos
DNA Topoisomerases Tipo II/metabolismo , Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Diester Fosfórico Hidrolases/metabolismo , Ubiquitina/metabolismo , Sítios de Ligação/genética , Domínio Catalítico , Cristalografia por Raios X , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Humanos , Modelos Moleculares , Mutação , Diester Fosfórico Hidrolases/química , Diester Fosfórico Hidrolases/genética , Poliubiquitina/química , Poliubiquitina/genética , Poliubiquitina/metabolismo , Ligação Proteica , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Especificidade por Substrato , Sumoilação , Ubiquitina/química , Ubiquitina/genética
9.
BMC Mol Cell Biol ; 21(1): 21, 2020 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-32228444

RESUMO

BACKGROUND: Ubiquitination and ubiquitin-like protein post-translational modifications play an enormous number of roles in cellular processes. These modifications are constituted of multistep reaction cascades. Readily implementable and robust methods to evaluate each step of the overall process, while presently limited, are critical to the understanding and modulation of the reaction sequence at any desired level, both in terms of basic research and potential therapeutic drug discovery and development. RESULTS: We developed multiple robust and reliable high-throughput assays to interrogate each of the sequential discrete steps in the reaction cascade leading to protein ubiquitination. As models for the E1 ubiquitin-activating enzyme, the E2 ubiquitin-conjugating enzyme, the E3 ubiquitin ligase, and their ultimate substrate of ubiquitination in a cascade, we examined Uba1, Rad6, Rad18, and proliferating cell nuclear antigen (PCNA), respectively, in reconstituted systems. Identification of inhibitors of this pathway holds promise in cancer therapy since PCNA ubiquitination plays a central role in DNA damage tolerance and resulting mutagenesis. The luminescence-based assays we developed allow for the quantitative determination of the degree of formation of ubiquitin thioester conjugate intermediates with both E1 and E2 proteins, autoubiquitination of the E3 protein involved, and ubiquitination of the final substrate. Thus, all covalent adducts along the cascade can be individually probed. We tested previously identified inhibitors of this ubiquitination cascade, finding generally good correspondence between compound potency trends determined by more traditional low-throughput methods and the present high-throughput ones. CONCLUSIONS: These approaches are readily adaptable to other E1, E2, and E3 systems, and their substrates in both ubiquitination and ubiquitin-like post-translational modification cascades.


Assuntos
Antígeno Nuclear de Célula em Proliferação , Processamento de Proteína Pós-Traducional , Ubiquitinação , Dano ao DNA , Antígeno Nuclear de Célula em Proliferação/análise , Antígeno Nuclear de Célula em Proliferação/metabolismo , Ubiquitina/química , Ubiquitina/metabolismo , Enzimas de Conjugação de Ubiquitina/química , Ubiquitina-Proteína Ligases/química , Ubiquitinas/química , Ubiquitinas/metabolismo
10.
Nature ; 580(7802): 278-282, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32269332

RESUMO

The ID complex, involving the proteins FANCI and FANCD2, is required for the repair of DNA interstrand crosslinks (ICL) and related lesions1. These proteins are mutated in Fanconi anaemia, a disease in which patients are predisposed to cancer. The Fanconi anaemia pathway of ICL repair is activated when a replication fork stalls at an ICL2; this triggers monoubiquitination of the ID complex, in which one ubiquitin molecule is conjugated to each of FANCI and FANCD2. Monoubiquitination of ID is essential for ICL repair by excision, translesion synthesis and homologous recombination; however, its function remains unknown1,3. Here we report a cryo-electron microscopy structure of the monoubiquitinated human ID complex bound to DNA, and reveal that it forms a closed ring that encircles the DNA. By comparison with the structure of the non-ubiquitinated ID complex bound to ICL DNA-which we also report here-we show that monoubiquitination triggers a complete rearrangement of the open, trough-like ID structure through the ubiquitin of one protomer binding to the other protomer in a reciprocal fashion. These structures-together with biochemical data-indicate that the monoubiquitinated ID complex loses its preference for ICL and related branched DNA structures, and becomes a sliding DNA clamp that can coordinate the subsequent repair reactions. Our findings also reveal how monoubiquitination in general can induce an alternative protein structure with a new function.


Assuntos
Microscopia Crioeletrônica , DNA/metabolismo , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/química , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/metabolismo , Proteínas de Grupos de Complementação da Anemia de Fanconi/química , Proteínas de Grupos de Complementação da Anemia de Fanconi/metabolismo , Ubiquitina/metabolismo , Ubiquitinação , DNA/química , Anemia de Fanconi/genética , Humanos , Modelos Moleculares , Conformação Proteica , Ubiquitina/química
11.
Chemphyschem ; 21(11): 1087-1091, 2020 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-32246547

RESUMO

Dynamics of protein side chains is one of the principal determinants of conformational entropy in protein structures and molecular recognition events. We describe NMR experiments that rely on the use of magic-angle pulses for efficient isolation of degenerate 1 H transitions of the I=3/2 manifold of 13 CH3 methyl groups, and serve as 'building blocks' for the measurement of transverse spin relaxation rates of the fast- and slow-relaxing 1 H transitions - the primary quantitative reporters of methyl axis dynamics in selectively {13 CH3 }-methyl-labelled, highly deuterated proteins. The magic-angle-pulse driven experiments are technically simpler and, in the absence of relaxation, predicted to be 2.3-fold more sensitive than previously developed analogous schemes. Validation of the methodology on a sample of {13 CH3 }-labeled ubiquitin demonstrates quantitative agreement between order parameters of methyl three-fold symmetry axis obtained with magic-angle-pulse driven experiments and other established NMR techniques, paving the way for studies of methyl axis dynamics in human DNAJB6b chaperone, a protein that undergoes exchange with high-molecular-weight oligomeric species.


Assuntos
Deutério/química , Proteínas/química , Humanos , Ressonância Magnética Nuclear Biomolecular/métodos , Ubiquitina/química
12.
Nat Struct Mol Biol ; 27(4): 323-332, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32203490

RESUMO

Ribosome-associated quality control (RQC) represents a rescue pathway in eukaryotic cells that is triggered upon translational stalling. Collided ribosomes are recognized for subsequent dissociation followed by degradation of nascent peptides. However, endogenous RQC-inducing sequences and the mechanism underlying the ubiquitin-dependent ribosome dissociation remain poorly understood. Here, we identified SDD1 messenger RNA from Saccharomyces cerevisiae as an endogenous RQC substrate and reveal the mechanism of its mRNA-dependent and nascent peptide-dependent translational stalling. In vitro translation of SDD1 mRNA enabled the reconstitution of Hel2-dependent polyubiquitination of collided disomes and, preferentially, trisomes. The distinct trisome architecture, visualized using cryo-EM, provides the structural basis for the more-efficient recognition by Hel2 compared with that of disomes. Subsequently, the Slh1 helicase subunit of the RQC trigger (RQT) complex preferentially dissociates the first stalled polyubiquitinated ribosome in an ATP-dependent manner. Together, these findings provide fundamental mechanistic insights into RQC and its physiological role in maintaining cellular protein homeostasis.


Assuntos
Proteínas de Ciclo Celular/ultraestrutura , Biossíntese de Proteínas , Ribossomos/genética , Proteínas de Saccharomyces cerevisiae/ultraestrutura , Serina Endopeptidases/ultraestrutura , Ubiquitina-Proteína Ligases/ultraestrutura , Trifosfato de Adenosina/química , Trifosfato de Adenosina/genética , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Peptídeos/química , Peptídeos/genética , RNA Mensageiro/genética , Ribossomos/química , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Serina Endopeptidases/química , Serina Endopeptidases/genética , Ubiquitina/química , Ubiquitina/genética , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/genética , Ubiquitinação/genética
13.
Molecules ; 25(6)2020 Mar 12.
Artigo em Inglês | MEDLINE | ID: mdl-32178473

RESUMO

Protein degradation is a fundamental process in all living organisms. An important part of this system is a multisubunit, barrel-shaped protease complex called the proteasome. This enzyme is directly responsible for the proteolysis of ubiquitin- or pup-tagged proteins to smaller peptides. In this study, we present a series of 92 psoralen derivatives, of which 15 displayed inhibitory potency against the Mycobacterium tuberculosis proteasome in low micromolar concentrations. The best inhibitors, i.e., 8, 11, 13 and 15, exhibited a mixed type of inhibition and overall good inhibitory potency in biochemical assays. N-(cyanomethyl)acetamide 8 (Ki = 5.6 µM) and carboxaldehyde-based derivative 15 (Ki = 14.9 µM) were shown to be reversible inhibitors of the enzyme. On the other hand, pyrrolidine-2,5-dione esters 11 and 13 irreversibly inhibited the enzyme with Ki values of 4.2 µM and 1.1 µM, respectively. In addition, we showed that an established immunoproteasome inhibitor, PR-957, is a noncompetitive irreversible inhibitor of the mycobacterial proteasome (Ki = 5.2 ± 1.9 µM, kinact/Ki = 96 ± 41 M-1·s-1). These compounds represent interesting hit compounds for further optimization in the development of new drugs for the treatment of tuberculosis.


Assuntos
Ficusina/farmacologia , Inibidores de Proteassoma/farmacologia , Tuberculose/tratamento farmacológico , Ficusina/química , Humanos , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/patogenicidade , Inibidores de Proteassoma/química , Proteólise/efeitos dos fármacos , Tuberculose/microbiologia , Ubiquitina/química , Ubiquitinas/química
14.
J Chem Theory Comput ; 16(5): 3205-3220, 2020 May 12.
Artigo em Inglês | MEDLINE | ID: mdl-32196332

RESUMO

Understanding the conformational characteristics of protein complexes in solution is crucial for a deeper insight in their biological function. Molecular dynamics simulations performed on high performance computing plants and with modern simulation techniques can be used to obtain large data sets that contain conformational and thermodynamic information about biomolecular systems. While this can in principle give a detailed picture of protein-protein interactions in solution and therefore complement experimental data, it also raises the challenge of processing exceedingly large high-dimensional data sets with several million samples. Here we present a novel method for the characterization of protein-protein interactions, which combines a neural network based dimensionality reduction technique to obtain a two-dimensional representation of the conformational space with a density based clustering algorithm for state detection and a metric which assesses the (dis)similarity between different conformational spaces. This method is highly scalable and therefore makes the analysis of massive data sets computationally tractable. We demonstrate the power of this approach to large scale data analysis by characterizing highly dynamic conformational phase spaces of differently linked ubiquitin (Ub) oligomers from coarse-grained simulations. We are able to extract a protein-protein interaction model for two unlinked Ub proteins which is then used to determine how the Ub-Ub interaction pattern is altered in Ub oligomers by the introduction of a covalent linkage. We find that the Ub chain conformational ensemble depends highly on the linkage type and for some cases also on the Ub chain length. By this, we obtain insight into the conformational characteristics of different Ub chains and how this may contribute to linkage type and chain length specific recognition.


Assuntos
Aprendizado de Máquina , Simulação de Dinâmica Molecular , Ubiquitina/química , Conformação Proteica
15.
J Biomol NMR ; 74(2-3): 161-171, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32040802

RESUMO

Signal enhancements of up to two orders of magnitude in protein NMR can be achieved by employing HDO as a vector to introduce hyperpolarization into folded or intrinsically disordered proteins. In this approach, hyperpolarized HDO produced by dissolution-dynamic nuclear polarization (D-DNP) is mixed with a protein solution waiting in a high-field NMR spectrometer, whereupon amide proton exchange and nuclear Overhauser effects (NOE) transfer hyperpolarization to the protein and enable acquisition of a signal-enhanced high-resolution spectrum. To date, the use of this strategy has been limited to 1D and 1H-15N 2D correlation experiments. Here we introduce 2D 13C-detected D-DNP, to reduce exchange-induced broadening and other relaxation penalties that can adversely affect proton-detected D-DNP experiments. We also introduce hyperpolarized 3D spectroscopy, opening the possibility of D-DNP studies of larger proteins and IDPs, where assignment and residue-specific investigation may be impeded by spectral crowding. The signal enhancements obtained depend in particular on the rates of chemical and magnetic exchange of the observed residues, thus resulting in non-uniform 'hyperpolarization-selective' signal enhancements. The resulting spectral sparsity, however, makes it possible to resolve and monitor individual amino acids in IDPs of over 200 residues at acquisition times of just over a minute. We apply the proposed experiments to two model systems: the compactly folded protein ubiquitin, and the intrinsically disordered protein (IDP) osteopontin (OPN).


Assuntos
Proteínas Intrinsicamente Desordenadas/química , Ressonância Magnética Nuclear Biomolecular , Osteopontina/química , Ubiquitina/química , Água/química , Humanos
16.
Proc Natl Acad Sci U S A ; 117(9): 4664-4674, 2020 03 03.
Artigo em Inglês | MEDLINE | ID: mdl-32071216

RESUMO

During protein degradation by the ubiquitin-proteasome pathway, latent 26S proteasomes in the cytosol must assume an active form. Proteasomes are activated when ubiquitylated substrates bind to them and interact with the proteasome-bound deubiquitylase Usp14/Ubp6. The resulting increase in the proteasome's degradative activity was recently shown to be mediated by Usp14's ubiquitin-like (Ubl) domain, which, by itself, can trigger proteasome activation. Many other proteins with diverse cellular functions also contain Ubl domains and can associate with 26S proteasomes. We therefore tested if various Ubl-containing proteins that have important roles in protein homeostasis or disease also activate 26S proteasomes. All seven Ubl-containing proteins tested-the shuttling factors Rad23A, Rad23B, and Ddi2; the deubiquitylase Usp7, the ubiquitin ligase Parkin, the cochaperone Bag6, and the protein phosphatase UBLCP1-stimulated peptide hydrolysis two- to fivefold. Rather than enhancing already active proteasomes, Rad23B and its Ubl domain activated previously latent 26S particles. Also, Ubl-containing proteins (if present with an unfolded protein) increased proteasomal adenosine 5'-triphosphate (ATP) hydrolysis, the step which commits substrates to degradation. Surprisingly, some of these proteins also could stimulate peptide hydrolysis even when their Ubl domains were deleted. However, their Ubl domains were required for the increased ATPase activity. Thus, upon binding to proteasomes, Ubl-containing proteins not only deliver substrates (e.g., the shuttling factors) or provide additional enzymatic activities (e.g., Parkin) to proteasomes, but also increase their capacity for proteolysis.


Assuntos
Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Ubiquitina/metabolismo , Sítios de Ligação , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Endopeptidases/química , Endopeptidases/metabolismo , Complexo de Endopeptidases do Proteassoma/química , Ligação Proteica , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Ubiquitina/química , Ubiquitina Tiolesterase/química , Ubiquitina Tiolesterase/metabolismo , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/metabolismo , Peptidase 7 Específica de Ubiquitina/química , Peptidase 7 Específica de Ubiquitina/metabolismo
17.
Nat Commun ; 11(1): 982, 2020 02 20.
Artigo em Inglês | MEDLINE | ID: mdl-32080186

RESUMO

Although peptide chemistry has made great progress, the frequent occurrence of aspartimide formation during peptide synthesis remains a formidable challenge. Aspartimide formation leads to low yields in addition to costly purification or even inaccessible peptide sequences. Here, we report an alternative approach to address this longstanding challenge of peptide synthesis by utilizing cyanosulfurylides to mask carboxylic acids by a stable C-C bond. These functional groups-formally zwitterionic species-are exceptionally stable to all common manipulations and impart improved solubility during synthesis. Deprotection is readily and rapidly achieved under aqueous conditions with electrophilic halogenating agents via a highly selective C-C bond cleavage reaction. This protecting group is employed for the synthesis of a range of peptides and proteins including teduglutide, ubiquitin, and the low-density lipoprotein class A. This protecting group strategy has the potential to overcome one of the most difficult aspects of modern peptide chemistry.


Assuntos
Peptídeos/química , Peptídeos/síntese química , Técnicas de Síntese em Fase Sólida/métodos , Ácido Aspártico/análogos & derivados , Ácido Aspártico/síntese química , Ácido Aspártico/química , Ácidos Carboxílicos/química , Cianetos/química , Lipoproteínas LDL/síntese química , Lipoproteínas LDL/química , Dobramento de Proteína , Ubiquitina/química
18.
PLoS One ; 15(2): e0229000, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32092106

RESUMO

Site-specific conjugation of ubiquitin onto a range of DNA repair proteins regulates their critical functions in the DNA damage response. Biochemical and structural characterization of these functions are limited by an absence of tools for the purification of DNA repair proteins in purely the ubiquitinated form. To overcome this barrier, we designed a ubiquitin fusion protein that is N-terminally biotinylated and can be conjugated by E3 RING ligases onto various substrates. Biotin affinity purification of modified proteins, followed by cleavage of the affinity tag leads to release of natively-mono-ubiquitinated substrates. As proof-of-principle, we applied this method to several substrates of mono-ubiquitination in the Fanconi anemia (FA)-BRCA pathway of DNA interstrand crosslink repair. These include the FANCI:FANCD2 complex, the PCNA trimer and BRCA1 modified nucleosomes. This method provides a simple approach to study the role of mono-ubiquitination in DNA repair or any other mono-ubiquitination signaling pathways.


Assuntos
Avidina/química , Proteína do Grupo de Complementação D2 da Anemia de Fanconi , Proteínas de Grupos de Complementação da Anemia de Fanconi , Antígeno Nuclear de Célula em Proliferação , Ubiquitina-Proteína Ligases , Ubiquitina , Animais , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/química , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/isolamento & purificação , Proteínas de Grupos de Complementação da Anemia de Fanconi/química , Proteínas de Grupos de Complementação da Anemia de Fanconi/isolamento & purificação , Humanos , Antígeno Nuclear de Célula em Proliferação/química , Antígeno Nuclear de Célula em Proliferação/isolamento & purificação , Células Sf9 , Spodoptera , Ubiquitina/química , Ubiquitina/isolamento & purificação , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/isolamento & purificação , Proteínas Ubiquitinadas/química , Proteínas Ubiquitinadas/isolamento & purificação
19.
Sci Rep ; 10(1): 3163, 2020 02 21.
Artigo em Inglês | MEDLINE | ID: mdl-32081948

RESUMO

Magnetic resonance imaging can be used to track cellular activities in the body using iron-based contrast agents. However, multiple intrinsic cellular iron handling mechanisms may also influence the detection of magnetic resonance (MR) contrast: a need to differentiate among those mechanisms exists. In hepcidin-mediated inflammation, for example, downregulation of iron export in monocytes and macrophages involves post-translational degradation of ferroportin. We examined the influence of hepcidin endocrine activity on iron regulation and MR transverse relaxation rates in multi-potent P19 cells, which display high iron import and export activities, similar to alternatively-activated macrophages. Iron import and export were examined in cultured P19 cells in the presence and absence of iron-supplemented medium, respectively. Western blots indicated the levels of transferrin receptor, ferroportin and ubiquitin in the presence and absence of extracellular hepcidin. Total cellular iron was measured by inductively-coupled plasma mass spectrometry and correlated to transverse relaxation rates at 3 Tesla using a gelatin phantom. Under varying conditions of iron supplementation, the level of ferroportin in P19 cells responds to hepcidin regulation, consistent with degradation through a ubiquitin-mediated pathway. This response of P19 cells to hepcidin is similar to that of classically-activated macrophages. The correlation between total cellular iron content and MR transverse relaxation rates was different in hepcidin-treated and untreated P19 cells: slope, Pearson correlation coefficient and relaxation rate were all affected. These findings may provide a tool to non-invasively distinguish changes in endogenous iron contrast arising from hepcidin-ferroportin interactions, with potential utility in monitoring of different macrophage phenotypes involved in pro- and anti-inflammatory signaling. In addition, this work demonstrates that transverse relaxivity is not only influenced by the amount of cellular iron but also by its metabolism.


Assuntos
Hepcidinas/metabolismo , Ferro/metabolismo , Macrófagos/metabolismo , Animais , Proteínas de Transporte de Cátions/metabolismo , Linhagem Celular Tumoral , Suplementos Nutricionais , Inflamação , Imagem por Ressonância Magnética , Camundongos , Imagens de Fantasmas , Fenótipo , Ondas de Rádio , Software , Oligoelementos , Ubiquitina/química
20.
Nat Struct Mol Biol ; 27(3): 240-248, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32066963

RESUMO

Vertebrate DNA crosslink repair excises toxic replication-blocking DNA crosslinks. Numerous factors involved in crosslink repair have been identified, and mutations in their corresponding genes cause Fanconi anemia (FA). A key step in crosslink repair is monoubiquitination of the FANCD2-FANCI heterodimer, which then recruits nucleases to remove the DNA lesion. Here, we use cryo-EM to determine the structures of recombinant chicken FANCD2 and FANCI complexes. FANCD2-FANCI adopts a closed conformation when the FANCD2 subunit is monoubiquitinated, creating a channel that encloses double-stranded DNA (dsDNA). Ubiquitin is positioned at the interface of FANCD2 and FANCI, where it acts as a covalent molecular pin to trap the complex on DNA. In contrast, isolated FANCD2 is a homodimer that is unable to bind DNA, suggestive of an autoinhibitory mechanism that prevents premature activation. Together, our work suggests that FANCD2-FANCI is a clamp that is locked onto DNA by ubiquitin, with distinct interfaces that may recruit other DNA repair factors.


Assuntos
Reparo do DNA , DNA/química , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/química , Proteínas de Grupos de Complementação da Anemia de Fanconi/química , Ubiquitina/química , Animais , Sítios de Ligação , Galinhas , Microscopia Crioeletrônica , Cristalografia por Raios X , DNA/genética , DNA/metabolismo , Dano ao DNA , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/genética , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/metabolismo , Proteínas de Grupos de Complementação da Anemia de Fanconi/genética , Proteínas de Grupos de Complementação da Anemia de Fanconi/metabolismo , Expressão Gênica , Modelos Moleculares , Ligação Proteica , Conformação Proteica em alfa-Hélice , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Células Sf9 , Spodoptera , Ubiquitina/genética , Ubiquitina/metabolismo , Ubiquitinação
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