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Biochemical crosstalk between two or more histone modifications is often observed in epigenetic enzyme regulation, but its functional significance in cells has been difficult to discern. Previous enzymatic studies revealed that Lys14 acetylation of histone H3 can inhibit Lys4 demethylation by lysine-specific demethylase 1 (LSD1). In the present study, we engineered a mutant form of LSD1, Y391K, which renders the nucleosome demethylase activity of LSD1 insensitive to Lys14 acetylation. K562 cells with the Y391K LSD1 CRISPR knockin show decreased expression of a set of genes associated with cellular adhesion and myeloid leukocyte activation. Chromatin profiling revealed that the cis-regulatory regions of these silenced genes display a higher level of H3 Lys14 acetylation, and edited K562 cells show diminished H3 mono-methyl Lys4 near these silenced genes, consistent with a role for enhanced LSD1 demethylase activity. These findings illuminate the functional consequences of disconnecting histone modification crosstalk for a key epigenetic enzyme.
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NEDD4L is a HECT-type E3 ligase that catalyzes the addition of ubiquitin to intracellular substrates such as the cardiac voltage-gated sodium channel, NaV1.5. The intramolecular interactions of NEDD4L regulate its enzymatic activity which is essential for proteostasis. For NaV1.5, this process is critical as alterations in Na+ current is involved in cardiac diseases including arrhythmias and heart failure. In this study, we perform extensive biochemical and functional analyses that implicate the C2 domain and the first WW-linker (1,2-linker) in the autoregulatory mechanism of NEDD4L. Through in vitro and electrophysiological experiments, the NEDD4L 1,2-linker was determined to be important in substrate ubiquitination of NaV1.5. We establish the preferred sites of ubiquitination of NEDD4L to be in the second WW-linker (2,3-linker). Interestingly, NEDD4L ubiquitinates the cytoplasmic linker between the first and second transmembrane domains of the channel (DI-DII) of NaV1.5. Moreover, we design a genetically encoded modulator of Nav1.5 that achieves Na+ current reduction using the NEDD4L HECT domain as cargo of a NaV1.5-binding nanobody. These investigations elucidate the mechanisms regulating the NEDD4 family and furnish a new molecular framework for understanding NaV1.5 ubiquitination.
Assuntos
Complexos Endossomais de Distribuição Requeridos para Transporte , Canal de Sódio Disparado por Voltagem NAV1.5 , Ubiquitina-Proteína Ligases Nedd4 , Ubiquitinação , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Ubiquitina-Proteína Ligases Nedd4/genética , Ubiquitina-Proteína Ligases Nedd4/metabolismo , Ubiquitina/metabolismo , Humanos , Canal de Sódio Disparado por Voltagem NAV1.5/metabolismo , Células HEK293RESUMO
The HECT E3 ligases ubiquitinate numerous transcription factors and signaling molecules, and their activity must be tightly controlled to prevent cancer, immune disorders, and other diseases. In this study, we have found unexpectedly that peptide linkers tethering WW domains in several HECT family members are key regulatory elements of their catalytic activities. Biochemical, structural, and cellular analyses have revealed that the linkers can lock the HECT domain in an inactive conformation and block the proposed allosteric ubiquitin binding site. Such linker-mediated autoinhibition of the HECT domain can be relieved by linker post-translational modifications, but complete removal of the brake can induce hyperactive autoubiquitination and E3 self destruction. These results clarify the mechanisms of several HECT protein cancer associated mutations and provide a new framework for understanding how HECT ubiquitin ligases must be finely tuned to ensure normal cellular behavior.
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Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Proteínas Repressoras/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Regulação Alostérica , Complexos Endossomais de Distribuição Requeridos para Transporte/química , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Ativação Enzimática , Estabilidade Enzimática , Células HeLa , Humanos , Modelos Moleculares , Mutação , Ubiquitina-Proteína Ligases Nedd4 , Fosforilação , Domínios Proteicos , Processamento de Proteína Pós-Traducional , Proteólise , Proteínas Repressoras/química , Proteínas Repressoras/genética , Relação Estrutura-Atividade , Transfecção , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/genéticaRESUMO
WWP2 is a HECT E3 ligase that targets protein Lys residues for ubiquitination and is comprised of an N-terminal C2 domain, four central WW domains, and a C-terminal catalytic HECT domain. The peptide segment between the middle WW domains, the 2,3-linker, is known to autoinhibit the catalytic domain, and this autoinhibition can be relieved by phosphorylation at Tyr369. Several protein substrates of WWP2 have been identified, including the tumor suppressor lipid phosphatase PTEN, but the full substrate landscape and biological functions of WWP2 remain to be elucidated. Here, we used protein microarray technology and the activated enzyme phosphomimetic mutant WWP2Y369E to identify potential WWP2 substrates. We identified 31 substrate hits for WWP2Y369E using protein microarrays, of which three were known autophagy receptors (NDP52, OPTN, and SQSTM1). These three hits were validated with in vitro and cell-based transfection assays and the Lys ubiquitination sites on these proteins were mapped by mass spectrometry. Among the mapped ubiquitin sites on these autophagy receptors, many had been previously identified in the endogenous proteins. Finally, we observed that WWP2 KO SH-SH5Y neuroblastoma cells using CRISPR-Cas9 showed a defect in mitophagy, which could be rescued by WWP2Y369E transfection. These studies suggest that WWP2-mediated ubiquitination of the autophagy receptors NDP52, OPTN, and SQSTM1 may positively contribute to the regulation of autophagy.
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Autofagia , Análise Serial de Proteínas , Ubiquitina-Proteína Ligases , Proteínas de Ciclo Celular/metabolismo , Humanos , Proteínas de Membrana Transportadoras/metabolismo , Proteínas Nucleares/metabolismo , Proteínas/metabolismo , Proteína Sequestossoma-1/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , UbiquitinaçãoRESUMO
Phosphatase and tensin homologue (PTEN) tumor suppressor protein is a PIP3 lipid phosphatase that is subject to multifaceted post-translational modifications. One such modification is the monoubiquitination of Lys13 that may alter its cellular localization but is also positioned in a manner that could influence several of its cellular functions. To explore the regulatory influence of ubiquitin on PTEN's biochemical properties and its interaction with ubiquitin ligases and a deubiquitinase, the generation of a site-specifically and stoichiometrically ubiquitinated protein could be beneficial. Here, we describe a semisynthetic method that relies upon sequential expressed protein ligation steps to install ubiquitin at a Lys13 mimic in near full-length PTEN. This approach permits the concurrent installation of C-terminal modifications in PTEN, thereby facilitating an analysis of the interplay between N-terminal ubiquitination and C-terminal phosphorylation. We find that the N-terminal ubiquitination of PTEN inhibits its enzymatic function, reduces its binding to lipid vesicles, modulates its processing by NEDD4-1 E3 ligase, and is efficiently cleaved by the deubiquitinase, USP7. Our ligation approach should motivate related efforts for uncovering the effects of ubiquitination of complex proteins.
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Complexos Endossomais de Distribuição Requeridos para Transporte , Ubiquitina-Proteína Ligases , Ubiquitina-Proteína Ligases/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Ubiquitinação , Ubiquitina-Proteína Ligases Nedd4/genética , Ubiquitina-Proteína Ligases Nedd4/metabolismo , Ubiquitina/química , PTEN Fosfo-Hidrolase/química , Enzimas Desubiquitinantes/metabolismo , LipídeosRESUMO
The reversible acetylation of histone lysine residues is controlled by the action of acetyltransferases and deacetylases (HDACs), which regulate chromatin structure and gene expression. The sirtuins are a family of NAD-dependent HDAC enzymes, and one member, sirtuin 6 (Sirt6), influences DNA repair, transcription, and aging. Here, we demonstrate that Sirt6 is efficient at deacetylating several histone H3 acetylation sites, including its canonical site Lys9, in the context of nucleosomes but not free acetylated histone H3 protein substrates. By installing a chemical warhead at the Lys9 position of histone H3, we trap a catalytically poised Sirt6 in complex with a nucleosome and employ this in cryo-EM structural analysis. The structure of Sirt6 bound to a nucleosome reveals extensive interactions between distinct segments of Sirt6 and the H2A/H2B acidic patch and nucleosomal DNA, which accounts for the rapid deacetylation of nucleosomal H3 sites and the disfavoring of histone H2B acetylation sites. These findings provide a new framework for understanding how HDACs target and regulate chromatin.
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Nucleossomos , Sirtuínas , Histonas/química , Cromatina , Sirtuínas/metabolismo , Acetilação , Glicosiltransferases/metabolismo , CatáliseRESUMO
The restricted active space spin-flip (RAS-SF) formalism is a particular form of single-reference configuration interaction that can describe some forms of strong correlation at a relatively low cost and which has recently been formulated for the description of charge-transfer excited states. Here, we introduce both equilibrium and nonequilibrium versions of a state-specific solvation correction for vertical transition energies computed using RAS-SF wave functions, based on the framework of a polarizable continuum model (PCM). Ground-state polarization is described using the solvent's static dielectric constant and in the nonequilibrium solvation approach that polarization is modified upon vertical excitation using the solvent's optical dielectric constant. Benchmark calculations are reported for well-studied models of photo-induced charge transfer, including naphthalene dimer, C2H4â¯C2F4, pentacene dimer, and perylene diimide (PDI) dimer, several of which are important in organic photovoltaic applications. For the PDI dimer, we demonstrate that the charge-transfer character of the excited states is enhanced in the presence of a low-dielectric medium (static dielectric constant É0 = 3) as compared to a gas-phase calculation (É0 = 1). This stabilizes mechanistic traps for singlet fission and helps to explain experimental singlet fission rates. We also examine the effects of nonequilibrium solvation on charge-separated states in an intramolecular singlet fission chromophore, where we demonstrate that the energetic ordering of the states changes as a function of solvent polarity. The RAS-SF + PCM methodology that is reported here provides a framework to study charge-separated states in solution and in photovoltaic materials.
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WWP1 is an E3 ubiquitin ligase that has been reported to target the tumor suppressor lipid phosphatase PTEN. K740N and N745S are recently identified germline variants of WWP1 that have been linked to PTEN-associated cancers [Lee, Y. R., et al. (2020) N. Engl. J. Med.]. These WWP1 variants have been suggested to release WWP1 from its native autoinhibited state, thereby promoting enhanced PTEN ubiquitination as a mechanism for driving cancer. Using purified proteins and in vitro enzymatic assays, we investigate the possibility that K740N and N745S WWP1 possess enhanced ubiquitin ligase activity and demonstrate that these variants are similar to the wild type (WT) in both autoubiquitination and PTEN ubiquitination. Furthermore, K740N and N745S WWP1 show dependencies similar to those of WT in terms of allosteric activation by an engineered ubiquitin variant, upstream E2 concentration, and substrate ubiquitin concentration. Transfected WWP1 WT and mutants demonstrate comparable effects on cellular PTEN levels. These findings challenge the idea that K740N and N745S WWP1 variants promote cancer by enhanced PTEN ubiquitination.
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PTEN Fosfo-Hidrolase/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Linhagem Celular Tumoral , Células Germinativas/metabolismo , Humanos , Neoplasias/metabolismo , PTEN Fosfo-Hidrolase/química , Ubiquitina-Proteína Ligases/química , Ubiquitinação , Ubiquitinas/metabolismoRESUMO
NEDD4-1 E3 ubiquitin protein ligase (NEDD4-1) and WW domain-containing E3 ubiquitin ligase (WWP2) are HECT family ubiquitin E3 ligases. They catalyze Lys ubiquitination of themselves and other proteins and are important in cell growth and differentiation. Regulation of NEDD4-1 and WWP2 catalytic activities is important for controlling cellular protein homeostasis, and their dysregulation may lead to cancer and other diseases. Previous work has implicated noncatalytic regions, including the C2 domain and/or WW domain linkers in NEDD4-1 and WWP2, in contributing to autoinhibition of the catalytic HECT domains by intramolecular interactions. Here, we explored the molecular mechanisms of these NEDD4-1 and WWP2 regulatory regions and their interplay with allosteric binding proteins such as Nedd4 family-interacting protein (NDFIP1), engineered ubiquitin variants, and linker phosphomimics. We found that in addition to influencing catalytic activities, the WW domain linker regions in NEDD4-1 and WWP2 can impact product distribution, including the degree of polyubiquitination and Lys-48 versus Lys-63 linkages. We show that allosteric activation by NDFIP1 or engineered ubiquitin variants is largely mediated by relief of WW domain linker autoinhibition. WWP2-mediated ubiquitination of WW domain-binding protein 2 (WBP2), phosphatase and tensin homolog (PTEN), and p62 proteins by WWP2 suggests that substrate ubiquitination can also be influenced by WW linker autoinhibition, although to differing extents. Overall, our results provide a deeper understanding of the intricate and multifaceted set of regulatory mechanisms in the control of NEDD4-1-related ubiquitin ligases.
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Proteínas de Transporte/genética , Proteínas de Membrana/genética , Ubiquitina-Proteína Ligases Nedd4/genética , Ubiquitina-Proteína Ligases/genética , Proteínas de Transporte/química , Complexos Endossomais de Distribuição Requeridos para Transporte , Humanos , Lisina/química , Proteínas de Membrana/química , Ubiquitina-Proteína Ligases Nedd4/química , PTEN Fosfo-Hidrolase/química , PTEN Fosfo-Hidrolase/genética , Ligação Proteica/genética , Estrutura Terciária de Proteína , Proteínas de Ligação a RNA/química , Ubiquitina/química , Ubiquitina/genética , Ubiquitina-Proteína Ligases/química , Ubiquitinação/genéticaRESUMO
Charge transfer and multi-exciton states are among the most difficult to characterize using electronic structure theories. Previously, restricted active space-spin flip (RAS-SF) methods have been applied to describe multi-exciton states, but these have not yet been shown to be useful for charge-transfer states. Herein, a variant of RAS-SF is introduced to treat charge-transfer states and electronic couplings. This approach relies on partitioning of the full RAS-SF Hamiltonian into charge-transfer and non-charge-transfer blocks, allowing the different types of diabatic states to be resolved in a straightforward fashion. To demonstrate this approach in practice, model dimer systems, intramolecular charge-transfer dyads, and an intramolecular singlet fission system were examined. Being low-cost and relatively accurate, RAS-SF provides important insight into electron transfer pathways in conventional donor-acceptor systems, as well as characterizations of charge transfer mechanisms involving strongly correlated multi-exciton states. Studies of electron transfer from an intramolecular singlet fission chromophore to an anthraquinone acceptor demonstrate the unique capabilities of the proposed RAS-SF method.
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This work presents the formalism and implementation for calculations of spin-orbit couplings (SOCs) using the Breit-Pauli Hamiltonian and non-relativistic wave functions described by the restricted active space configuration interaction (RASCI) method with general excitation operators of spin-conserving spin-flipping, ionizing, and electron-attaching types. The implementation is based on the application of the Wigner-Eckart theorem within the spin space, which enables the calculation of the entire SOC matrix based on the explicit calculation of just one transition between the two spin multiplets. Numeric results for a diverse set of atoms and molecules highlight the importance of a balanced treatment of correlation and adequate basis sets and illustrate the overall robust performance of RASCI SOCs. The new implementation is a useful addition to the methodological toolkit for studying spin-forbidden processes and molecular magnetism.
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N-Hydroxysuccinimide (NHS)-esters are widely used to label proteins nonselectively on free amino groups. Such broad labeling can be disadvantageous because it can interfere with protein structure or function and because stoichiometry is poorly controlled. Here we describe a simple method to transform NHS-esters into site-specific protein labeling on N-terminal Cys residues. MESNA addition converts NHS-esters to chemoselective thioesters for N-Cys modification. This labeling strategy was applied to clarify mechanistic features of the ubiquitin E3 ligase WWP2 including its interaction with one of its substrates, the tumor suppressor PTEN, as well as its autoubiquitination molecularity. We propose that this convenient protein labeling strategy will allow for an expanded application of NHS-esters in biochemical investigation.
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Ésteres/química , PTEN Fosfo-Hidrolase/metabolismo , Succinimidas/química , Ubiquitina-Proteína Ligases/metabolismo , Animais , Biotina/química , Cisteína/química , Escherichia coli/genética , Fluoresceínas/química , Corantes Fluorescentes/química , Glutationa Transferase/química , Humanos , PTEN Fosfo-Hidrolase/química , Ligação Proteica , Rodaminas/química , Saccharomyces cerevisiae/genética , Spodoptera/genética , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/química , Ubiquitinação , Uracila-DNA Glicosidase/químicaRESUMO
Lys ubiquitination is catalysed by E3 ubiquitin ligases and is central to the regulation of protein stability and cell signalling in normal and disease states. There are gaps in our understanding of E3 mechanisms, and here we use protein semisynthesis, chemical rescue, microscale thermophoresis and other biochemical approaches to dissect the role of catalytic base/acid function and conformational interconversion in HECT-domain E3 catalysis. We demonstrate that there is plasticity in the use of the terminal side chain or backbone carboxylate for proton transfer in HECT E3 ubiquitin ligase reactions, with yeast Rsp5 orthologues appearing to be possible evolutionary intermediates. We also show that the HECT-domain ubiquitin covalent intermediate appears to eject the E2 conjugating enzyme, promoting catalytic turnover. These findings provide key mechanistic insights into how protein ubiquitination occurs and provide a framework for understanding E3 functions and regulation.
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Mind bomb 1 (MIB1) is a RING E3 ligase that ubiquitinates Notch ligands, a necessary step for induction of Notch signaling. The structural basis for binding of the JAG1 ligand by the N-terminal region of MIB1 is known, yet how the ankyrin (ANK) and RING domains of MIB1 cooperate to catalyze ubiquitin transfer from E2â¼Ub to Notch ligands remains unclear. Here, we show that the third RING domain and adjacent coiled coil region (ccRING3) drive MIB1 dimerization and that MIB1 ubiquitin transfer activity relies solely on ccRING3. We report X-ray crystal structures of a UbcH5B-ccRING3 complex and the ANK domain. Directly tethering the MIB1 N-terminal region to ccRING3 forms a minimal MIB1 protein sufficient to induce a Notch response in receiver cells and rescue mib knockout phenotypes in flies. Together, these studies define the functional elements of an E3 ligase needed for ligands to induce a Notch signaling response.
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Mind bomb 1 (MIB1) is a RING E3 ligase that ubiquitinates Notch ligands, a necessary step for induction of Notch signaling. The structural basis for binding of the JAG1 ligand by the N-terminal region of MIB1 is known, yet how the ankyrin (ANK) and RING domains of MIB1 cooperate to catalyze ubiquitin transfer from E2~Ub to Notch ligands remains unclear. Here, we show that the third RING domain and adjacent coiled coil region of MIB1 (ccRING3) drives MIB1 dimerization and that ubiquitin transfer activity of MIB1 relies solely on RING3. We report x-ray crystal structures of a UbcH5B-ccRING3 complex as a fusion protein and of the ANK region. Directly tethering the N-terminal region to ccRING3 forms a minimal MIB1 protein, which is sufficient to induce a Notch response in receiver cells. Together, these studies define the functional elements of an E3 ligase needed for ligands to induce a Notch signaling response.
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Cancer mutations can create neomorphic protein-protein interactions to drive aberrant function 1 . As a substrate receptor of the CULLIN3-RBX1 E3 ubiquitin ligase complex, KBTBD4 is recurrently mutated in medulloblastoma (MB) 2 , the most common embryonal brain tumor in children, and pineoblastoma 3 . These mutations impart gain-of-function to KBTBD4 to induce aberrant degradation of the transcriptional corepressor CoREST 4 . However, their mechanism of action remains unresolved. Here, we elucidate the mechanistic basis by which KBTBD4 mutations promote CoREST degradation through engaging HDAC1/2, the direct neomorphic target of the substrate receptor. Using deep mutational scanning, we systematically map the mutational landscape of the KBTBD4 cancer hotspot, revealing distinct preferences by which insertions and substitutions can promote gain-of-function and the critical residues involved in the hotspot interaction. Cryo-electron microscopy (cryo-EM) analysis of two distinct KBTBD4 cancer mutants bound to LSD1-HDAC1-CoREST reveals that a KBTBD4 homodimer asymmetrically engages HDAC1 with two KELCH-repeat propeller domains. The interface between HDAC1 and one of the KBTBD4 propellers is stabilized by the MB mutations, which directly insert a bulky side chain into the active site pocket of HDAC1. Our structural and mutational analyses inform how this hotspot E3-neo-substrate interface can be chemically modulated. First, our results unveil a converging shape complementarity-based mechanism between gain-of-function E3 mutations and a molecular glue degrader, UM171. Second, we demonstrate that HDAC1/2 inhibitors can block the mutant KBTBD4-HDAC1 interface, the aberrant degradation of CoREST, and the growth of KBTBD4-mutant MB models. Altogether, our work reveals the structural and mechanistic basis of cancer mutation-driven neomorphic protein-protein interactions and pharmacological strategies to modulate their action for therapeutic applications.
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UM171 is a potent small molecule agonist of ex vivo human hematopoietic stem cell (HSC) self-renewal, a process that is tightly controlled by epigenetic regulation. By co-opting KBTBD4, a substrate receptor of the CULLIN3-RING E3 ubiquitin ligase complex, UM171 promotes the degradation of members of the CoREST transcriptional corepressor complex, thereby limiting HSC attrition. However, the direct target and mechanism of action of UM171 remain unclear. Here, we reveal that UM171 acts as a molecular glue to induce high-affinity interactions between KBTBD4 and HDAC1 to promote the degradation of select HDAC1/2 corepressor complexes. Through proteomics and chemical inhibitor studies, we discover that the principal target of UM171 is HDAC1/2. Cryo-electron microscopy (cryo-EM) analysis of dimeric KBTBD4 bound to UM171 and the LSD1-HDAC1-CoREST complex unveils an unexpected asymmetric assembly, in which a single UM171 molecule enables a pair of KBTBD4 KELCH-repeat propeller domains to recruit HDAC1 by clamping on its catalytic domain. One of the KBTBD4 propellers partially masks the rim of the HDAC1 active site pocket, which is exploited by UM171 to extend the E3-neo-substrate interface. The other propeller cooperatively strengthens HDAC1 binding via a separate and distinct interface. The overall neomorphic interaction is further buttressed by an endogenous cofactor of HDAC1-CoREST, inositol hexakisphosphate, which makes direct contacts with KBTBD4 and acts as a second molecular glue. The functional relevance of the quaternary complex interaction surfaces defined by cryo-EM is demonstrated by in situ base editor scanning of KBTBD4 and HDAC1. By delineating the direct target of UM171 and its mechanism of action, our results reveal how the cooperativity offered by a large dimeric CRL E3 family can be leveraged by a small molecule degrader and establish for the first time a dual molecular glue paradigm.
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NaV1.7, the neuronal voltage-gated sodium channel isoform, plays an important role in the human body's ability to feel pain. Mutations within NaV1.7 have been linked to pain-related syndromes, such as insensitivity to pain. To date, the regulation and internalization mechanisms of the NaV1.7 channel are not well known at a biochemical level. In this study, we perform biochemical and biophysical analyses that establish that the HECT-type E3 ligase, NEDD4L, ubiquitinates the cytoplasmic C-terminal (CT) region of NaV1.7. Through in vitro ubiquitination and mass spectrometry experiments, we identify, for the first time, the lysine residues of NaV1.7 within the CT region that get ubiquitinated. Furthermore, binding studies with an NEDD4L E3 ligase modulator (ubiquitin variant) highlight the dynamic partnership between NEDD4L and NaV1.7. These investigations provide a framework for understanding how NEDD4L-dependent regulation of the channel can influence the NaV1.7 function.
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Ubiquitin-proteasome system (UPS) dysfunction is associated with the pathology of a wide range of human diseases, including myopathies and muscular atrophy. However, the mechanistic understanding of specific components of the regulation of protein turnover during development and disease progression in skeletal muscle is unclear. Mutations in KLHL40, an E3 ubiquitin ligase cullin3 (CUL3) substrate-specific adapter protein, result in severe congenital nemaline myopathy, but the events that initiate the pathology and the mechanism through which it becomes pervasive remain poorly understood. To characterize the KLHL40-regulated ubiquitin-modified proteome during skeletal muscle development and disease onset, we used global, quantitative mass spectrometry-based ubiquitylome and global proteome analyses of klhl40a mutant zebrafish during disease progression. Global proteomics during skeletal muscle development revealed extensive remodeling of functional modules linked with sarcomere formation, energy, biosynthetic metabolic processes, and vesicle trafficking. Combined analysis of klh40 mutant muscle proteome and ubiquitylome identified thin filament proteins, metabolic enzymes, and ER-Golgi vesicle trafficking pathway proteins regulated by ubiquitylation during muscle development. Our studies identified a role for KLHL40 as a regulator of ER-Golgi anterograde trafficking through ubiquitin-mediated protein degradation of secretion-associated Ras-related GTPase1a (Sar1a). In KLHL40-deficient muscle, defects in ER exit site vesicle formation and downstream transport of extracellular cargo proteins result in structural and functional abnormalities. Our work reveals that the muscle proteome is dynamically fine-tuned by ubiquitylation to regulate skeletal muscle development and uncovers new disease mechanisms for therapeutic development in patients.
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Proteínas Musculares , Peixe-Zebra , Animais , Humanos , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Peixe-Zebra/metabolismo , Proteoma/metabolismo , Músculo Esquelético/metabolismo , Ubiquitinação , Sarcômeros/metabolismo , Ubiquitina/metabolismo , Retículo Endoplasmático/metabolismo , Desenvolvimento Muscular , Progressão da DoençaRESUMO
In the present work, aluminum chloride and various soluble salts of doping ions were dissolved in water. In addition, urea and polyvinyl pyrrolidone (PVP) were also dissolved in the above aqueous solution under supersonic treatments. Then the solutions were heated to induce the hydrolysis of urea so that soluble aluminum and doping ions convert into insoluble hydroxide or carbonate gels. After calcinations, the obtained gels change to mixed metal oxide pigments whose color is related to type and concentrations of the doping ions. XRD characterization demonstrates that the diffraction patterns of the products are the same as that of alpha-alumina. Diffuse reflectance spectra of samples of the samples in UV-Vis regions show that the absorption bands for d-d transitions of the doping ions undergo considerable change as the coordinate environments change. In addition, L*, a* and b* values of the pigments were measured by using UV-Vis densitometer. SEM results indicate that the size of the pigment powders is in the range 200-300 nm. The pigments are quite stable since no evidence of dissolution was observed after the synthesized pigment is soaked for 24 hours. ICP test shows that very little amount of doped metal occurs in the corresponding filtrate. The above results suggest that these new kinds of mixed metal oxide pigments are stable, non-toxic, environmental friendly and they may be applicable in molten spinning process and provide a new chance for non-aqueous printing and dyeing industry.