RESUMO
The stability of eukaryotic mRNAs is dependent on a ribonucleoprotein (RNP) complex of poly(A)-binding proteins (PABPC1/Pab1) organized on the poly(A) tail. This poly(A) RNP not only protects mRNAs from premature degradation but also stimulates the Pan2-Pan3 deadenylase complex to catalyze the first step of poly(A) tail shortening. We reconstituted this process in vitro using recombinant proteins and show that Pan2-Pan3 associates with and degrades poly(A) RNPs containing two or more Pab1 molecules. The cryo-EM structure of Pan2-Pan3 in complex with a poly(A) RNP composed of 90 adenosines and three Pab1 protomers shows how the oligomerization interfaces of Pab1 are recognized by conserved features of the deadenylase and thread the poly(A) RNA substrate into the nuclease active site. The structure reveals the basis for the periodic repeating architecture at the 3' end of cytoplasmic mRNAs. This illustrates mechanistically how RNA-bound Pab1 oligomers act as rulers for poly(A) tail length over the mRNAs' lifetime.
Assuntos
Exorribonucleases/metabolismo , Proteína I de Ligação a Poli(A)/metabolismo , Ribonucleoproteínas/metabolismo , Microscopia Crioeletrônica/métodos , Exorribonucleases/fisiologia , Poli A/metabolismo , Proteína I de Ligação a Poli(A)/fisiologia , Proteínas de Ligação a Poli(A)/metabolismo , RNA/metabolismo , Estabilidade de RNA/fisiologia , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Top-down analysis of intact proteins by mass spectrometry provides an ideal platform for comprehensive proteoform characterization, in particular, for the identification and localization of post-translational modifications (PTM) co-occurring on a protein. One of the main bottlenecks in top-down proteomics is insufficient protein sequence coverage caused by incomplete protein fragmentation. Based on previous work on peptides, increasing sequence coverage and PTM localization by combining sequential ETD and HCD fragmentation in a single fragmentation event, we hypothesized that protein sequence coverage and phospho-proteoform characterization could be equally improved by this new dual fragmentation method termed EThcD, recently been made available on the Orbitrap Fusion. Here, we systematically benchmark the performance of several (hybrid) fragmentation methods for intact protein analysis on an Orbitrap Fusion, using as a model system a 17.5 kDa N-terminal fragment of the mitotic regulator Bora. During cell division Bora becomes multiply phosphorylated by a variety of cell cycle kinases, including Aurora A and Plk1, albeit at distinctive sites. Here, we monitor the phosphorylation of Bora by Aurora A and Plk1, analyzing the generated distinctive phospho-proteoforms by top-down fragmentation. We show that EThcD and ETciD on a Fusion are feasible and capable of providing richer fragmentation spectra compared to HCD or ETD alone, increasing protein sequence coverage, and thereby facilitating phosphosite localization and the determination of kinase specific phosphorylation sites in these phospho-proteoforms. Data are available via ProteomeXchange with identifier PXD001845.
Assuntos
Aurora Quinase B/análise , Proteômica , Aurora Quinase B/metabolismo , Espectrometria de Massas , FosforilaçãoRESUMO
Deubiquitinating enzymes (DUBs) remove ubiquitin from conjugated substrates to regulate various cellular processes. The Zn(2+)-dependent DUBs AMSH and AMSH-LP regulate receptor trafficking by specifically cleaving Lys 63-linked polyubiquitin chains from internalized receptors. Here we report the crystal structures of the human AMSH-LP DUB domain alone and in complex with a Lys 63-linked di-ubiquitin at 1.2 A and 1.6 A resolutions, respectively. The AMSH-LP DUB domain consists of a Zn(2+)-coordinating catalytic core and two characteristic insertions, Ins-1 and Ins-2. The distal ubiquitin interacts with Ins-1 and the core, whereas the proximal ubiquitin interacts with Ins-2 and the core. The core and Ins-1 form a catalytic groove that accommodates the Lys 63 side chain of the proximal ubiquitin and the isopeptide-linked carboxy-terminal tail of the distal ubiquitin. This is the first reported structure of a DUB in complex with an isopeptide-linked ubiquitin chain, which reveals the mechanism for Lys 63-linkage-specific deubiquitination by AMSH family members.
Assuntos
Lisina/metabolismo , Poliubiquitina/química , Poliubiquitina/metabolismo , Ubiquitina Tiolesterase/química , Ubiquitina Tiolesterase/metabolismo , Animais , Catálise , Sequência Conservada , Cristalografia por Raios X , Endopeptidases/química , Endopeptidases/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte , Humanos , Cinética , Camundongos , Modelos Moleculares , Peptídeo Hidrolases , Poliubiquitina/genética , Estrutura Terciária de Proteína , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Relação Estrutura-Atividade , Especificidade por Substrato , Ubiquitina Tiolesterase/genéticaRESUMO
The linear ubiquitin chain assembly complex (LUBAC) is a key nuclear factor-κB (NF-κB) pathway component that produces linear polyubiquitin chains. The HOIL-1L subunit of LUBAC has been shown to bind linear chains; however, detailed structural and functional analyses on the binding between LUBAC and linear chains have not been performed. In this study, we found that the Npl4 zinc finger (NZF) domain of HOIL-1L specifically binds linear polyubiquitin chains and determined the crystal structure of the HOIL-1L NZF domain in complex with linear diubiquitin at 1.7-Å resolution. The HOIL-1L NZF domain consists of a zinc-coordinating "NZF core" region and an additional α-helical "NZF tail" region. The HOIL-1L NZF core binds both the canonical Ile44-centered hydrophobic surface on the distal ubiquitin and a Phe4-centered hydrophobic patch on the proximal ubiquitin, representing a mechanism for the specific recognition of linear chains. The NZF tail binds the proximal ubiquitin to enhance the binding affinity. These recognition mechanisms were supported by the accompanying in vitro and in vivo structure-based mutagenesis experiments.
Assuntos
Proteínas de Transporte/química , Proteínas Nucleares/química , Ubiquitina-Proteína Ligases/química , Sequência de Aminoácidos , Animais , Cristalografia por Raios X/métodos , Humanos , Camundongos , Conformação Molecular , Dados de Sequência Molecular , NF-kappa B/metabolismo , Conformação Proteica , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Ressonância de Plasmônio de Superfície/métodos , Fatores de TranscriçãoRESUMO
Protein phosphorylation is a widespread process forming the mechanistic basis of cellular signaling. Up to now, different aspects, for example, site-specificity, kinetics, role of co-factors, and structure-function relationships have been typically investigated by multiple techniques that are incompatible with one another. The approach introduced here maximizes the amount of information gained on protein (complex) phosphorylation while minimizing sample handling. Using high-resolution native mass spectrometry on intact protein (assemblies) up to 150â kDa we track the sequential incorporation of phosphate groups and map their localization by peptide LC-MS/MS. On two model systems, the protein kinaseâ G and the interplay between Aurora kinaseâ A and Bora, we demonstrate the simultaneous monitoring of various aspects of the phosphorylation process, namely the effect of different cofactors on PKG autophosphorylation and the interaction of AurA and Bora as both an enzyme-substrate pair and physical binding partners.
Assuntos
Fosforilação/fisiologia , Proteínas/metabolismo , Animais , Aurora Quinase A/metabolismo , Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , Humanos , Cinética , Espectrometria de Massas , Especificidade por SubstratoRESUMO
BACKGROUND: Satralizumab, a humanized anti-interleukin-6 receptor monoclonal antibody, has been approved globally for the treatment of neuromyelitis optica spectrum disorder (NMOSD), based on positive results from two randomized, double-blind, phase 3 studies: SAkuraSky (NCT02028884) and SAkuraStar (NCT02073279). There remains an unmet need to understand the real-world management of NMOSD, especially in patients undergoing tapering of concomitant therapy. We examined real-world treatment patterns, including concomitant glucocorticoids and immunosuppressants, and relapse in satralizumab-treated patients with NMOSD, using a Japanese administrative hospital claims database. METHODS: We used retrospective data from the Medical Data Vision hospital-based administrative claims database. The index date was the date of first satralizumab prescription and the study period was set between August 2018 and March 2022. Patients were included in the overall population if they had a first prescription for satralizumab between August 2020 and March 2022, an International Classification of Disease, Version10 code of G36.0 prior to March 2022, and were observable for ≥90 days prior to the index date. The primary endpoint was the percentage of patients with relapse-free reduction of oral glucocorticoids to 0 mg/day at 360 days of continued satralizumab treatment. Secondary endpoints included time to relapse, number of relapses after the index date while being on continuous satralizumab treatment, annualized relapse rate before and after the index date, and concomitant medication use. Relapse and dose reduction were identified using definition specifically developed for this study. RESULTS: Of the 131 patients included in the overall population, most were female (90.8 %), aged 18-65 years (75.6 %), and were prescribed oral glucocorticoids (93.1 %). Azathioprine (19.1 %) and tacrolimus, a calcineurin inhibitor (18.3 %), were the most common immunosuppressants at index date. Six (4.6 %) patients had a history of biologic use (tocilizumab, 1 [0.8 %]; eculizumab, 5 [3.8 %]). Among 111 patients observable for 360 days pre-index, there were 0.6 ± 0.8 (mean ± SD) relapses during 360 days before the index date. The median (interquartile range) duration of satralizumab exposure was 197.0 (57.0-351.0) days. Most (125/131; 95.4 %) patients were relapse-free post-index; 6 (4.6 %) patients relapsed within 90 days after the index date, of which 2 had the first relapse within 7 days after the index date. Among 21 patients with 360-day follow-up, 6 (28.6 %) patients were on 0 mg/day dose of glucocorticoid prescription without relapse 360 days post-index. Of these 6 patients, 2 had no prescription of oral glucocorticoids at the index date and remained glucocorticoid- and relapse-free 360 days after the index date. CONCLUSION: These real-world data support the phase 3 clinical trials. Our results, over a median duration of satralizumab exposure of 197.0 days, showed that a majority (125/131, 95.4 %) of patients were relapse-free after initiating satralizumab treatment. The number of glucocorticoid-free patients without relapse increased over time under continuous satralizumab prescription. Further studies are needed to confirm if satralizumab can be used as a potential immunosuppressant- and glucocorticoid-sparing agent.
Assuntos
Anticorpos Monoclonais Humanizados , Neuromielite Óptica , Feminino , Humanos , Masculino , Anticorpos Monoclonais/uso terapêutico , Aquaporina 4 , Doença Crônica , Glucocorticoides/uso terapêutico , Imunossupressores/uso terapêutico , Japão , Recidiva , Estudos RetrospectivosRESUMO
RAP80 has a key role in the recruitment of the Abraxas-BRCC36-BRCA1-BARD1 complex to DNA-damage foci for DNA repair through specific recognition of Lys 63-linked polyubiquitinated proteins by its tandem ubiquitin-interacting motifs (UIMs). Here, we report the crystal structure of the RAP80 tandem UIMs (RAP80-UIM1-UIM2) in complex with Lys 63-linked di-ubiquitin at 2.2 A resolution. The two UIMs, UIM1 and UIM2, and the alpha-helical inter-UIM region together form a continuous 60 A-long alpha-helix. UIM1 and UIM2 bind to the proximal and distal ubiquitin moieties, respectively. Both UIM1 and UIM2 of RAP80 recognize an Ile 44-centered hydrophobic patch on ubiquitin but neither UIM interacts with the Lys 63-linked isopeptide bond. Our structure suggests that the inter-UIM region forms a 12 A-long alpha-helix that ensures that the UIMs are arranged to enable specific binding of Lys 63-linked di-ubiquitin. This was confirmed by pull-down analyses using RAP80-UIM1-UIM2 mutants of various length inter-UIM regions. Further, we show that the Epsin1 tandem UIM, which has an inter-UIM region similar to that of RAP80-UIM1-UIM2, also selectively binds Lys 63-linked di-ubiquitin.
Assuntos
Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Lisina/metabolismo , Poliubiquitina/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Motivos de Aminoácidos , Animais , Proteínas de Transporte/química , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/genética , Cristalografia por Raios X , Proteínas de Ligação a DNA , Chaperonas de Histonas , Lisina/química , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Poliubiquitina/química , Ligação Proteica , Conformação Proteica , Alinhamento de Sequência , Fatores de Transcrição/genéticaRESUMO
TAB2 and TAB3 activate the Jun N-terminal kinase and nuclear factor-kappaB pathways through the specific recognition of Lys 63-linked polyubiquitin chains by its Npl4 zinc-finger (NZF) domain. Here we report crystal structures of the TAB2 and TAB3 NZF domains in complex with Lys 63-linked diubiquitin at 1.18 and 1.40 A resolutions, respectively. Both NZF domains bind to the distal ubiquitin through a conserved Thr-Phe dipeptide that has been shown to be important for the interaction of the NZF domain of Npl4 with monoubiquitin. In contrast, a surface specific to TAB2 and TAB3 binds the proximal ubiquitin. Both the distal and proximal binding sites of the TAB2 and TAB3 NZF domains recognize the Ile 44-centred hydrophobic patch on ubiquitin but do not interact with the Lys 63-linked isopeptide bond. Mutagenesis experiments show that both binding sites are required to enable binding of Lys 63-linked diubiquitin. We therefore propose a mechanism for the recognition of Lys 63-linked polyubiquitin chains by TAB2 and TAB3 NZF domains in which diubiquitin units are specifically recognized by a single NZF domain.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/química , Poliubiquitina/química , Animais , Sítios de Ligação , Cristalografia por Raios X/métodos , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Lisina/química , Camundongos , Modelos Moleculares , NF-kappa B/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Ubiquitina/química , Dedos de ZincoRESUMO
BACKGROUND: Neuromyelitis optica spectrum disorder (NMOSD) is a rare, chronic, autoimmune disease, characterized by astrocytopathic lesions in the central nervous system (Beekman et al., 2019; Fujihara et al., 2020). The main aim of NMOSD maintenance therapy is to reduce the frequency and severity of relapses and minimize future disability (Fujihara et al., 2020). Oral corticosteroids are used long-term to prevent relapses, but are associated with serious complications (Kessler et al., 2016; Kimbrough et al., 2012). In the SAkuraSky study, satralizumab reduced the risk of relapse in patients with NMOSD compared with placebo, with comparable rates of serious adverse events and infections between treatment arms (Yamamura et al., 2019). Here, we report on 16 patients who tapered their steroid dose during the openlabel extension (OLE) period of SAkuraSky. METHODS: SAkuraSky was a phase 3, multicenter, randomized, double-blind (DB), placebo-controlled study of satralizumab in combination with immunosuppressive therapies (ISTs) in patients with NMOSD. Patients were randomized 1:1 to receive 120 mg subcutaneous satralizumab or placebo in addition to a stable dose of their baseline IST. After completing the DB period or experiencing relapse, patients could enter the OLE period. In the OLE, all patients received satralizumab, and IST doses could be tapered at the investigator's discretion. We assessed the different steroid tapering patterns and their impact on relapse and safety. Patients were considered to have tapered their steroids if their steroid dose at the clinical cut-off date (CCOD: February 18, 2020) was lower than on the first day of the OLE. Annualized relapse rate (ARR) was calculated as the number of relapses divided by the total number of patientyears at risk. RESULTS: Overall, 36 patients receiving oral corticosteroids entered the OLE, of whom 16 tapered their steroid dose. The mean age (range) at baseline was 44.9 (16-73) years, all 16 were female, 14 (88%) were Japanese, and 15 (94%) were AQP4-IgG seropositive. None were receiving any additional ISTs. Patients tapered their steroids from a median of 10 (range: 5-25) mg/day at OLE baseline to 2.75 (0-15) mg/day at the CCOD. Three patients discontinued steroids entirely, and all three remained relapse free. One patient who remained relapse free had temporary increases in steroid dose. Three relapses were observed in two patients who tapered steroids during the OLE; all three relapses required treatment. One of the relapses occurred shortly after a drop in steroid dose from 25 to 10 mg/day. The ARR for steroid-tapered patients was numerically lower in the OLE period than the satralizumab group in the DB period. The safety profile of satralizumab was in line with the overall SAkuraSky population. Two serious infections were observed in steroid-tapered patients in the OLE, both in the same patient: one event (hepatitis E) occurred before the patient began tapering their steroid dose; and one event (influenza) occurred while the patient was tapering. CONCLUSION: During the OLE of SAkuraSky, 16 patients tapered steroids and the ARR did not increase from the DB period. Patient numbers limit interpretation.
Assuntos
Neuromielite Óptica , Adulto , Anticorpos Monoclonais Humanizados/uso terapêutico , Aquaporina 4 , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Recidiva , Esteroides/uso terapêuticoRESUMO
Tail anchored (TA) proteins, which are important for numerous cellular processes, are defined by a single transmembrane domain (TMD) near the C-terminus. The membrane insertion of TA proteins is mediated by the highly conserved ATPase Get3. Here we report the crystal structures of Get3 in ADP-bound and nucleotide-free forms at 3.0 A and 2.8 A resolutions, respectively. Get3 consists of a nucleotide binding domain and a helical domain. Both structures exhibit a Zn(2+)-mediated homodimer in a head-to-head orientation, representing an open dimer conformation. Our cross-link experiments indicated the closed dimer-stimulating ATP hydrolysis, which might be coupled with TA-protein release. Further, our coexpression-based binding assays using a model TA protein Sec22p revealed the direct interaction between the helical domain of Get3 and the Sec22p TMD. This interaction is independent of ATP and dimer formation. Finally, we propose a structural mechanism that links ATP hydrolysis with the TA-protein insertion mediated by the conserved DTAPTGH motif.
Assuntos
Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , Membrana Celular/metabolismo , Fatores de Troca do Nucleotídeo Guanina/química , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismo , Trifosfato de Adenosina/metabolismo , Sítios de Ligação , Membrana Celular/efeitos dos fármacos , Reagentes de Ligações Cruzadas/farmacologia , Cristalografia por Raios X , Hidrólise/efeitos dos fármacos , Modelos Biológicos , Mutação/genética , Ligação Proteica/efeitos dos fármacos , Multimerização Proteica/efeitos dos fármacos , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Saccharomyces cerevisiae/efeitos dos fármacos , Zinco/metabolismoRESUMO
The tunneling nanotube (TNT) is a structure used for intercellular communication, and is a thin membrane protrusion mediating transport of various signaling molecules and cellular components. M-Sec has potent membrane deformation ability and induces TNT formation in cooperation with the Ral/exocyst complex. Here, we show that the N-terminal polybasic region of M-Sec directly binds phosphatidylinositol (4,5)-bisphosphate for its localization to the plasma membrane during the initial stage of TNT formation. We further report a crystal structure of M-Sec, which consists of helix bundles arranged in a straight rod-like shape, similar to the membrane tethering complex subunits. A positively charged surface in the C-terminal domains is required for M-Sec interaction with active RalA to extend the plasma membrane protrusions. Our results suggest that the membrane-associated M-Sec recruits active RalA, which directs the exocyst complex to form TNTs.
RESUMO
Multisite phosphorylation is a common pathway to regulate protein function, activity, and interaction pattern in vivo, but routine biochemical analysis is often insufficient to identify the number and order of individual phosphorylation reactions and their mechanistic impact on the protein behavior. Here, we integrate complementary mass spectrometry (MS)-based approaches to characterize a multisite phosphorylation-regulated protein system comprising Polo-like kinase 1 (Plk1) and its coactivators Aurora kinase A (Aur-A) and Bora, the interplay of which is essential for mitotic entry after DNA damage-induced cell cycle arrest. Native MS and cross-linking-MS revealed that Aur-A/Bora-mediated Plk1 activation is accompanied by the formation of Aur-A/Bora and Plk1/Bora heterodimers. We found that the Aur-A/Bora interaction is independent of the Bora phosphorylation state, whereas the Plk1/Bora interaction is dependent on extensive Bora multisite phosphorylation. Bottom-up and top-down proteomics analyses showed that Bora multisite phosphorylation proceeds via a well-ordered sequence of site-specific phosphorylation reactions, whereby we could reveal the involvement of up to 16 phosphorylated Bora residues. Ion mobility spectrometry-MS demonstrated that this multisite phosphorylation primes a substantial structural rearrangement of Bora, explaining the interdependence between extensive Bora multisite phosphorylation and Plk1/Bora complex formation. These results represent a first benchmark of our multipronged MS strategy, highlighting its potential to elucidate the mechanistic and structural implications of multisite protein phosphorylation.
RESUMO
The steroidal glucocorticoid antagonist mifepristone has been reported to improve the symptoms of depression. We report the discovery of 6-(3,5-dimethylisoxazol-4-yl)-2,2,4,4-tetramethyl-2,3,4,7,8,9-hexahydro-1H-cyclopenta[h]quinolin-3-one 3d (QCA-1093) as a novel nonsteroidal glucocorticoid receptor antagonist. The compound displayed potent in vitro activity, high selectivity over other steroid hormone receptors, and significant antidepressant-like activity in vivo.
Assuntos
Antidepressivos/química , Antidepressivos/uso terapêutico , Depressão/tratamento farmacológico , Quinolinas/química , Quinolinas/uso terapêutico , Receptores de Glucocorticoides/antagonistas & inibidores , Animais , Antidepressivos/síntese química , Antidepressivos/farmacologia , Linhagem Celular , Humanos , Masculino , Simulação de Acoplamento Molecular , Quinolinas/síntese química , Quinolinas/farmacologia , Ratos , Ratos Wistar , Receptores de Glucocorticoides/metabolismo , Relação Estrutura-AtividadeRESUMO
The exocyst complex is a hetero-octameric protein complex that functions during cell polarization by tethering the secretory vesicle to the target membrane. The yeast exocyst subunit Sec3 binds to phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)) and the small GTPases Rho1 and Cdc42 via its N-terminal domain (Sec3-N), and these interactions target Sec3 to the plasma membrane. Here we report the crystal structure of the Sec3-N in complex with Rho1 at 2.6-A resolution. Sec3-N adopts a pleckstrin homology (PH) fold, despite having no detectable sequence homology with other PH domains of known structure. Clusters of conserved basic residues constitute a positively charged cleft, which was identified as a binding site for PtdIns(4,5)P(2). Residues Phe77, Ile115 and Leu131 of Sec3 bind to an extended hydrophobic surface formed around switch regions I and II of Rho1. To our knowledge, these are the first structural insights into how an exocyst subunit might interact with both protein and phospholipid factors on the target membrane.
Assuntos
Fosfatidilinositóis/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Leveduras/química , Leveduras/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Cristalografia por Raios X , Modelos Moleculares , Estrutura Quaternária de Proteína , Subunidades Proteicas/química , Subunidades Proteicas/metabolismoRESUMO
NEMO is essential for activation of the NF-kappaB signaling pathway, which is regulated by ubiquitination of proteins. The C-terminal leucine zipper of NEMO and its adjacent coiled-coil region (CC2-LZ) reportedly bind to linear ubiquitin chains with 1 microM affinity and to Lys 63-linked chains with 100 microM affinity. Here we report the crystal structure of the CC2-LZ region of mouse NEMO in complex with Lys 63-linked di-ubiquitin (K63-Ub(2)) at 2.7A resolution. The ubiquitin-binding region consists of a 130A-long helix and forms a parallel coiled-coil dimer. The Ile 44-centered hydrophobic patch of ubiquitin is recognized in the middle of the NEMO ubiquitin-binding region. NEMO interacts with each K63-Ub(2)via a single ubiquitin-binding site, consistent with low affinity binding with K63-Ub(2).
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/química , Lisina/química , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Ubiquitina/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Cristalografia por Raios X , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Lisina/metabolismo , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Alinhamento de Sequência , Ubiquitina/genética , Ubiquitina/metabolismoRESUMO
We attempted to determine natural agonists of GPR39 in fetal bovine serum (FBS). FBS was conditioned to extract peptides and fractionated by two types of HPLC. The activity of each fraction was monitored by intracellular calcium mobilization. Then the purified active ingredient was analyzed by inductively coupled plasma mass spectrometry. In this fashion, Zn2+ ion was identified as an agonist of GPR39, though no peptidergic molecules were found. The calcium-mobilizing activity of Zn2+ was not abolished by pertussis toxin but was by a phospholipase C (PLC) inhibitor, U73122, indicating that the activity of GPR39 is mediated through the Gqalpha -PLC pathway. In addition, Zn2+ also activated mouse and rat GPR39, showing that the function of GPR39 as a Zn2+ receptor is conserved across species. This study is the first exploration of GPR39 agonists in FBS and indicates that GPR39 functions as a Gq-coupled Zn2+-sensing receptor.
Assuntos
Receptores Acoplados a Proteínas G/química , Zinco/química , Animais , Células CHO , Bovinos , Cricetinae , Cricetulus , Inibidores Enzimáticos/farmacologia , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Camundongos , Hormônios Peptídicos/química , Ratos , Receptores Acoplados a Proteínas G/sangue , Receptores Acoplados a Proteínas G/metabolismoRESUMO
Reorganization of the actin filament is an essential process for cell motility, cell-cell attachment and intracellular transport. Formin proteins promote nucleation and elongation of the actin filament, and thus are key regulators for this process. The formin homology 2 (FH2) domain forms a head-to-tail ring-shaped dimer, and processively moves towards the barbed end. Dishevelled-associated activator of morphogenesis (DAAM) is a Rho-regulated formin implicated in neuronal development. Here, we present the crystal structure of human DAAM1 FH2 dimer at 2.8 A resolution. This is the first dimeric structure of the mammalian formin. The core structure of human DAAM1 is similar to those of mouse mDia1 and yeast Bni1p, whereas the orientations of the FH2 dimeric rings are different between human DAAM1 and yeast Bni1p, despite their similar dimer interactions. This difference supports the previous prediction that the dimer architecture of the formin is highly flexible in the actin-free state. The results of the actin assembly assays using the DAAM1 mutants demonstrated that the length of the linker connecting the N-terminal domain and the core region is crucial for the activity.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/química , Modelos Moleculares , Sequência de Aminoácidos , Animais , Proteínas de Transporte/química , Cristalografia por Raios X , Dimerização , Forminas , Humanos , Camundongos , Proteínas dos Microfilamentos/química , Dados de Sequência Molecular , Conformação Proteica , Proteínas de Saccharomyces cerevisiae/química , Alinhamento de Sequência , Homologia Estrutural de Proteína , Proteínas rho de Ligação ao GTPRESUMO
OBJECTIVE: To investigate whether tin chloride pretreatment ameliorates renal injury in rats with ischemic acute renal failure (IARF) by virtue of its kidney-specific heme oxygenase-1 induction. DESIGN: Randomized, masked, controlled animal study. SETTING: University-based animal research facility. SUBJECTS: Sprague-Dawley male rats, weighing 200-230 g (n = 359). INTERVENTIONS: Rats were injected with tin chloride subcutaneously, because subcutaneous administration of tin chloride is known to specifically and potently induce renal heme oxygenase activity in the rat. Anesthetized rats were subjected to bilateral flank incisions, and the right kidney was removed. Renal ischemia for 40 mins was performed by left renal microvascular clamping, followed by reflow of the blood. MEASUREMENTS AND MAIN RESULTS: Tin chloride treatment specifically induced heme oxygenase-1 mRNA and protein in the proximal tubular epithelial cells of the kidney without apparent cell injury in the rat. Tin chloride treatment before renal ischemia augmented the induction of heme oxygenase-1 in IARF rats at both transcriptional and protein concentrations in the renal epithelial cells compared with IARF animals. Tin chloride pretreatment, which decreased microsomal heme concentration, ameliorated the ischemic renal injury as judged by the significant decrease in serum creatinine and blood urea nitrogen concentrations and the lesser tubular epithelial cell injuries. In contrast, inhibition of heme oxygenase activity by treatment with tin mesoporphyrin, which increased microsomal heme concentration, abolished the beneficial effect of tin chloride pretreatment. CONCLUSION: These findings indicate that tin chloride pretreatment significantly ameliorates renal injury in rats with IARF by virtue of its specific heme oxygenase-1 induction in renal epithelial cells. These findings also suggest that heme oxygenase-1 induction plays an important role in protecting renal cells from oxidative damage caused by heme.