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1.
Int J Mol Sci ; 22(9)2021 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-34066955

RESUMO

Peptidoglycan recognition proteins (PGRPs) are ubiquitous among animals and play pivotal functions in insect immunity. Non-catalytic PGRPs are involved in the activation of immune pathways by binding to the peptidoglycan (PGN), whereas amidase PGRPs are capable of cleaving the PGN into non-immunogenic compounds. Drosophila PGRP-LB belongs to the amidase PGRPs and downregulates the immune deficiency (IMD) pathway by cleaving meso-2,6-diaminopimelic (meso-DAP or DAP)-type PGN. While the recognition process is well analyzed for the non-catalytic PGRPs, little is known about the enzymatic mechanism for the amidase PGRPs, despite their essential function in immune homeostasis. Here, we analyzed the specific activity of different isoforms of Drosophila PGRP-LB towards various PGN substrates to understand their specificity and role in Drosophila immunity. We show that these isoforms have similar activity towards the different compounds. To analyze the mechanism of the amidase activity, we performed site directed mutagenesis and solved the X-ray structures of wild-type Drosophila PGRP-LB and its mutants, with one of these structures presenting a protein complexed with the tracheal cytotoxin (TCT), a muropeptide derived from the PGN. Only the Y78F mutation abolished the PGN cleavage while other mutations reduced the activity solely. Together, our findings suggest the dynamic role of the residue Y78 in the amidase mechanism by nucleophilic attack through a water molecule to the carbonyl group of the amide function destabilized by Zn2+.


Assuntos
Amidoidrolases/metabolismo , Proteínas de Transporte/metabolismo , Drosophila melanogaster/metabolismo , Amidoidrolases/química , Sequência de Aminoácidos , Animais , Proteínas de Transporte/química , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Peptidoglicano , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Estrutura Secundária de Proteína , Açúcares/metabolismo , Fatores de Virulência de Bordetella , Zinco/metabolismo
2.
J Med Chem ; 64(11): 7555-7564, 2021 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-34008968

RESUMO

RFamide-related peptide-3 (RFRP-3) and neuropeptide FF (NPFF) target two different receptor subtypes called neuropeptide FF1 (NPFF1R) and neuropeptide FF2 (NPFF2R) that modulate several functions. However, the study of their respective role is severely limited by the absence of selective blockers. We describe here the design of a highly selective NPFF1R antagonist called RF3286, which potently blocks RFRP-3-induced hyperalgesia in mice and luteinizing hormone release in hamsters. We then showed that the pharmacological blockade of NPFF1R in mice prevents the development of fentanyl-induced hyperalgesia while preserving its analgesic effect. Altogether, our data indicate that RF3286 represents a useful pharmacological tool to study the involvement of the NPFF1R/RFRP-3 system in different functions and different species. Thanks to this compound, we showed that this system is critically involved in the development of opioid-induced hyperalgesia, suggesting that NPFF1R antagonists might represent promising therapeutic tools to improve the use of opioids in the treatment of chronic pain.


Assuntos
Analgésicos Opioides/efeitos adversos , Dipeptídeos/química , Receptores de Neuropeptídeos/antagonistas & inibidores , Animais , Cricetinae , Dipeptídeos/metabolismo , Dipeptídeos/farmacologia , Dipeptídeos/uso terapêutico , Feminino , Fentanila/efeitos adversos , Meia-Vida , Humanos , Hiperalgesia/tratamento farmacológico , Hiperalgesia/etiologia , Hormônio Luteinizante/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neuropeptídeos/química , Neuropeptídeos/metabolismo , Neuropeptídeos/farmacologia , Neuropeptídeos/uso terapêutico , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Receptores de Neuropeptídeos/metabolismo , Receptores Opioides/química , Receptores Opioides/metabolismo , Relação Estrutura-Atividade
3.
J Med Chem ; 64(11): 7453-7467, 2021 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-34032427

RESUMO

There has been significant attention concerning the biased agonism of G protein-coupled receptors (GPCRs), and it has resulted in various pharmacological benefits. 5-HT7R belongs to a GPCR, and it is a promising pharmaceutical target for the treatment of neurodevelopmental and neuropsychiatric disorders. Based on our previous research, we synthesized a series of 6-chloro-2'-methoxy biphenyl derivatives 1, 2, and 3 with a variety of amine scaffolds. These compounds were evaluated for their binding affinities to 5-HTR subtypes and their functional selectivity toward the Gs protein and the ß-arrestin signaling pathways of 5-HT7R. Among them, 2-(6-chloro-2'-methoxy-[1,1'-biphenyl]-3-yl)-N-ethylethan-1-amine, 2b, was found to be a G-protein-biased ligand of 5-HT7R. In an in vivo study with Shank3 transgenic mice, the self-grooming behavior test was performed with 2b, which increased the duration of self-grooming. The experiments further suggested that 5-HT7R is associated with autism spectrum disorders (ASDs) and could be a therapeutic target for the treatment of stereotypy in ASDs.


Assuntos
Compostos de Bifenilo/química , Ligantes , Receptores de Serotonina/metabolismo , Animais , Comportamento Animal/efeitos dos fármacos , Compostos de Bifenilo/metabolismo , Compostos de Bifenilo/farmacologia , Avaliação Pré-Clínica de Medicamentos , Estabilidade de Medicamentos , Meia-Vida , Humanos , Masculino , Camundongos , Camundongos Endogâmicos ICR , Camundongos Transgênicos , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Microssomos/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Serotonina/química , Relação Estrutura-Atividade
4.
Int J Mol Sci ; 22(6)2021 Mar 13.
Artigo em Inglês | MEDLINE | ID: mdl-33805725

RESUMO

Monocarboxylate transporters (MCTs) are of great research interest for their role in cancer cell metabolism and their potential ability to transport pharmacologically relevant compounds across the membrane. Each member of the MCT family could potentially provide novel therapeutic approaches to various diseases. The major differences among MCTs are related to each of their specific metabolic roles, their relative substrate and inhibitor affinities, the regulation of their expression, their intracellular localization, and their tissue distribution. MCT4 is the main mediator for the efflux of L-lactate produced in the cell. Thus, MCT4 maintains the glycolytic phenotype of the cancer cell by supplying the molecular resources for tumor cell proliferation and promotes the acidification of the extracellular microenvironment from the co-transport of protons. A promising therapeutic strategy in anti-cancer drug design is the selective inhibition of MCT4 for the glycolytic suppression of solid tumors. A small number of studies indicate molecules for dual inhibition of MCT1 and MCT4; however, no selective inhibitor with high-affinity for MCT4 has been identified. In this study, we attempt to approach the structural characteristics of MCT4 through an in silico pipeline for molecular modelling and pharmacophore elucidation towards the identification of specific inhibitors as a novel anti-cancer strategy.


Assuntos
Antineoplásicos/química , Transportadores de Ácidos Monocarboxílicos/química , Proteínas Musculares/química , Floretina/química , Pirimidinonas/química , Quercetina/química , Reserpina/análogos & derivados , Tiofenos/química , Uracila/análogos & derivados , Animais , Antineoplásicos/metabolismo , Sítios de Ligação , Transporte Biológico , Desenho de Fármacos , Glicólise/fisiologia , Humanos , Ácido Láctico/química , Ácido Láctico/metabolismo , Simulação de Acoplamento Molecular , Transportadores de Ácidos Monocarboxílicos/antagonistas & inibidores , Transportadores de Ácidos Monocarboxílicos/genética , Transportadores de Ácidos Monocarboxílicos/metabolismo , Proteínas Musculares/antagonistas & inibidores , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Floretina/metabolismo , Filogenia , Ligação Proteica , Conformação Proteica em alfa-Hélice , Domínios e Motivos de Interação entre Proteínas , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Pirimidinonas/metabolismo , Quercetina/metabolismo , Reserpina/química , Reserpina/metabolismo , Homologia Estrutural de Proteína , Especificidade por Substrato , Tiofenos/metabolismo , Uracila/química , Uracila/metabolismo
5.
Nat Plants ; 7(4): 437-444, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33875834

RESUMO

Chlorophyll biosynthesis, crucial to life on Earth, is tightly regulated because its precursors are phototoxic1. In flowering plants, the enzyme light-dependent protochlorophyllide oxidoreductase (LPOR) captures photons to catalyse the penultimate reaction: the reduction of a double bond within protochlorophyllide (Pchlide) to generate chlorophyllide (Chlide)2,3. In darkness, LPOR oligomerizes to facilitate photon energy transfer and catalysis4,5. However, the complete three-dimensional structure of LPOR, the higher-order architecture of LPOR oligomers and the implications of these self-assembled states for catalysis, including how LPOR positions Pchlide and the co-factor NADPH, remain unknown. Here, we report the atomic structure of LPOR assemblies by electron cryo-microscopy. LPOR polymerizes with its substrates into helical filaments around constricted lipid bilayer tubes. Portions of LPOR and Pchlide insert into the outer membrane leaflet, targeting the product, Chlide, to the membrane for the final reaction site of chlorophyll biosynthesis. In addition to its crucial photocatalytic role, we show that in darkness LPOR filaments directly shape membranes into high-curvature tubules with the spectral properties of the prolamellar body, whose light-triggered disassembly provides lipids for thylakoid assembly. Moreover, our structure of the catalytic site challenges previously proposed reaction mechanisms6. Together, our results reveal a new and unexpected synergy between photosynthetic membrane biogenesis and chlorophyll synthesis in plants, orchestrated by LPOR.


Assuntos
Arabidopsis/genética , Clorofila/biossíntese , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/química , Microscopia Crioeletrônica , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/genética , Isoformas de Proteínas/química , Isoformas de Proteínas/genética
6.
J Med Chem ; 64(9): 5816-5837, 2021 05 13.
Artigo em Inglês | MEDLINE | ID: mdl-33929863

RESUMO

We previously reported that P-retigabine (P-RTG), a retigabine (RTG) analogue bearing a propargyl group at the nitrogen atom in the linker of RTG, displayed moderate anticonvulsant efficacy. Recently, our further efforts led to the discovery of HN37 (pynegabine), which demonstrated satisfactory chemical stability upon deleting the ortho liable -NH2 group and installing two adjacent methyl groups to the carbamate motif. HN37 exhibited enhanced activation potency toward neuronal Kv7 channels and high in vivo efficacy in a range of pre-clinical seizure models, including the maximal electroshock test and a 6 Hz model of pharmacoresistant limbic seizures. With its improved chemical stability, strong efficacy, and better safety margin, HN37 has progressed to clinical trial in China for epilepsy treatment.


Assuntos
Anticonvulsivantes/química , Carbamatos/química , Desenho de Fármacos , Animais , Anticonvulsivantes/uso terapêutico , Carbamatos/metabolismo , Carbamatos/uso terapêutico , Modelos Animais de Doenças , Cães , Avaliação Pré-Clínica de Medicamentos , Estabilidade de Medicamentos , Eletrochoque , Meia-Vida , Humanos , Canais de Potássio KCNQ/química , Canais de Potássio KCNQ/metabolismo , Camundongos , Fenilenodiaminas/química , Fenilenodiaminas/metabolismo , Fenilenodiaminas/uso terapêutico , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Ratos , Ratos Sprague-Dawley , Convulsões/tratamento farmacológico , Convulsões/etiologia , Relação Estrutura-Atividade
7.
Nat Commun ; 12(1): 1028, 2021 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-33589610

RESUMO

Upon binding to DNA breaks, poly(ADP-ribose) polymerase 1 (PARP1) ADP-ribosylates itself and other factors to initiate DNA repair. Serine is the major residue for ADP-ribosylation upon DNA damage, which strictly depends on HPF1. Here, we report the crystal structures of human HPF1/PARP1-CAT ΔHD complex at 1.98 Å resolution, and mouse and human HPF1 at 1.71 Å and 1.57 Å resolution, respectively. Our structures and mutagenesis data confirm that the structural insights obtained in a recent HPF1/PARP2 study by Suskiewicz et al. apply to PARP1. Moreover, we quantitatively characterize the key residues necessary for HPF1/PARP1 binding. Our data show that through salt-bridging to Glu284/Asp286, Arg239 positions Glu284 to catalyze serine ADP-ribosylation, maintains the local conformation of HPF1 to limit PARP1 automodification, and facilitates HPF1/PARP1 binding by neutralizing the negative charge of Glu284. These findings, along with the high-resolution structural data, may facilitate drug discovery targeting PARP1.


Assuntos
Proteínas de Transporte/química , DNA/química , Histonas/química , Proteínas Nucleares/química , Poli(ADP-Ribose) Polimerase-1/química , Serina/metabolismo , ADP-Ribosilação , Sequência de Aminoácidos , Animais , Sítios de Ligação , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Clonagem Molecular , Cristalografia por Raios X , DNA/genética , DNA/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Glutamina/metabolismo , Histonas/genética , Histonas/metabolismo , Humanos , Camundongos , Modelos Moleculares , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Poli(ADP-Ribose) Polimerase-1/genética , Poli(ADP-Ribose) Polimerase-1/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Eletricidade Estática
8.
Nat Commun ; 12(1): 819, 2021 02 05.
Artigo em Inglês | MEDLINE | ID: mdl-33547302

RESUMO

Regulated cell death is essential in development and cellular homeostasis. Multi-protein platforms, including the Death-Inducing Signaling Complex (DISC), co-ordinate cell fate via a core FADD:Caspase-8 complex and its regulatory partners, such as the cell death inhibitor c-FLIP. Here, using electron microscopy, we visualize full-length procaspase-8 in complex with FADD. Our structural analysis now reveals how the FADD-nucleated tandem death effector domain (tDED) helical filament is required to orientate the procaspase-8 catalytic domains, enabling their activation via anti-parallel dimerization. Strikingly, recruitment of c-FLIPS into this complex inhibits Caspase-8 activity by altering tDED triple helix architecture, resulting in steric hindrance of the canonical tDED Type I binding site. This prevents both Caspase-8 catalytic domain assembly and tDED helical filament elongation. Our findings reveal how the plasticity, composition and architecture of the core FADD:Caspase-8 complex critically defines life/death decisions not only via the DISC, but across multiple key signaling platforms including TNF complex II, the ripoptosome, and RIPK1/RIPK3 necrosome.


Assuntos
Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/química , Caspase 8/química , Proteína de Domínio de Morte Associada a Fas/química , Proteína Serina-Treonina Quinases de Interação com Receptores/química , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/genética , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/metabolismo , Caspase 8/genética , Caspase 8/metabolismo , Domínio Catalítico , Clonagem Molecular , Microscopia Crioeletrônica , Proteínas Adaptadoras de Sinalização de Receptores de Domínio de Morte/química , Proteínas Adaptadoras de Sinalização de Receptores de Domínio de Morte/genética , Proteínas Adaptadoras de Sinalização de Receptores de Domínio de Morte/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteína de Domínio de Morte Associada a Fas/genética , Proteína de Domínio de Morte Associada a Fas/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Células HEK293 , Humanos , Modelos Moleculares , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Multimerização Proteica , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Morte Celular Regulada/genética , Fator de Necrose Tumoral alfa/química , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo
9.
Cell Mol Life Sci ; 78(9): 4335-4364, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33619613

RESUMO

The Synaptotagmin (SYT) family of proteins play key roles in regulating membrane trafficking at neuronal synapses. Using both Ca2+-dependent and Ca2+-independent interactions, several SYT isoforms participate in synchronous and asynchronous fusion of synaptic vesicles (SVs) while preventing spontaneous release that occurs in the absence of stimulation. Changes in the function or abundance of the SYT1 and SYT7 isoforms alter the number and route by which SVs fuse at nerve terminals. Several SYT family members also regulate trafficking of other subcellular organelles at synapses, including dense core vesicles (DCV), exosomes, and postsynaptic vesicles. Although SYTs are linked to trafficking of multiple classes of synaptic membrane compartments, how and when they interact with lipids, the SNARE machinery and other release effectors are still being elucidated. Given mutations in the SYT family cause disorders in both the central and peripheral nervous system in humans, ongoing efforts are defining how these proteins regulate vesicle trafficking within distinct neuronal compartments. Here, we review the Drosophila SYT family and examine their role in synaptic communication. Studies in this invertebrate model have revealed key similarities and several differences with the predicted activity of their mammalian counterparts. In addition, we highlight the remaining areas of uncertainty in the field and describe outstanding questions on how the SYT family regulates membrane trafficking at nerve terminals.


Assuntos
Proteínas de Drosophila/metabolismo , Drosophila/metabolismo , Sinaptotagminas/metabolismo , Animais , Cálcio/metabolismo , Proteínas de Drosophila/química , Proteínas de Drosophila/classificação , Exocitose , Humanos , Neurotransmissores/metabolismo , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Vesículas Sinápticas/metabolismo , Sinaptotagminas/química , Sinaptotagminas/classificação
10.
J Mol Biol ; 433(8): 166875, 2021 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-33556408

RESUMO

The coronavirus nucleocapsid protein (N) controls viral genome packaging and contains numerous phosphorylation sites located within unstructured regions. Binding of phosphorylated SARS-CoV N to the host 14-3-3 protein in the cytoplasm was reported to regulate nucleocytoplasmic N shuttling. All seven isoforms of the human 14-3-3 are abundantly present in tissues vulnerable to SARS-CoV-2, where N can constitute up to ~1% of expressed proteins during infection. Although the association between 14-3-3 and SARS-CoV-2 N proteins can represent one of the key host-pathogen interactions, its molecular mechanism and the specific critical phosphosites are unknown. Here, we show that phosphorylated SARS-CoV-2 N protein (pN) dimers, reconstituted via bacterial co-expression with protein kinase A, directly associate, in a phosphorylation-dependent manner, with the dimeric 14-3-3 protein, but not with its monomeric mutant. We demonstrate that pN is recognized by all seven human 14-3-3 isoforms with various efficiencies and deduce the apparent KD to selected isoforms, showing that these are in a low micromolar range. Serial truncations pinpointed a critical phosphorylation site to Ser197, which is conserved among related zoonotic coronaviruses and located within the functionally important, SR-rich region of N. The relatively tight 14-3-3/pN association could regulate nucleocytoplasmic shuttling and other functions of N via occlusion of the SR-rich region, and could also hijack cellular pathways by 14-3-3 sequestration. As such, the assembly may represent a valuable target for therapeutic intervention.


Assuntos
Proteínas 14-3-3/química , Proteínas 14-3-3/metabolismo , Proteínas do Nucleocapsídeo de Coronavírus/química , Proteínas do Nucleocapsídeo de Coronavírus/metabolismo , Sequência de Aminoácidos , Sítios de Ligação/genética , Proteínas do Nucleocapsídeo de Coronavírus/genética , Proteínas Quinases Dependentes de AMP Cíclico/genética , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Escherichia coli , Humanos , Mutação , Fosfopeptídeos/química , Fosfopeptídeos/metabolismo , Fosfoproteínas/química , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fosforilação , Fosfosserina/metabolismo , Ligação Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , RNA Viral/metabolismo , Especificidade por Substrato
11.
J Mol Biol ; 433(5): 166793, 2021 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-33388290

RESUMO

Many proteins are composed of independently-folded domains connected by flexible linkers. The primary sequence and length of such linkers can set the effective concentration for the tethered domains, which impacts rates of association and enzyme activity. The length of such linkers can be sensitive to environmental conditions, which raises questions as to how studies in dilute buffer relate to the highly-crowded cellular environment. To examine the role of linkers in domain separation, we measured Fluorescent Protein-Fluorescence Resonance Energy Transfer (FP-FRET) for a series of tandem FPs that varied in the length of their interdomain linkers. We used discrete molecular dynamics to map the underlying conformational distribution, which revealed intramolecular contact states that we confirmed with single molecule FRET. Simulations found that attached FPs increased linker length and slowed conformational dynamics relative to the bare linkers. This makes the CLYs poor sensors of inherent linker properties. However, we also showed that FP-FRET in CLYs was sensitive to solvent quality and macromolecular crowding making them potent environmental sensors. Finally, we targeted the same proteins to the plasma membrane of living mammalian cells to measure FP-FRET in cellulo. The measured FP-FRET when tethered to the plasma membrane was the same as that in dilute buffer. While caveats remain regarding photophysics, this suggests that the supertertiary conformational ensemble of these CLY proteins may not be affected by this specific cellular environment.


Assuntos
Proteínas de Bactérias/química , Proteínas de Fluorescência Verde/química , Proteínas Luminescentes/química , Simulação de Dinâmica Molecular , Proteínas Recombinantes de Fusão/química , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Células CHO , Cricetulus , Transferência Ressonante de Energia de Fluorescência , Expressão Gênica , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Modelos Moleculares , Polietilenoglicóis/química , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Imagem Individual de Molécula , Cloreto de Sódio/química , Ureia/química
12.
J Mol Biol ; 433(6): 166789, 2021 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-33387534

RESUMO

Centromeric loci of chromosomes are defined by nucleosomes containing the histone H3 variant CENP-A, which bind their DNA termini more permissively than their canonical counterpart, a feature that is critical for the mitotic fidelity. A recent cryo-EM study demonstrated that the DNA termini of CENP-A nucleosomes, reconstituted with the Widom 601 DNA sequence, are asymmetrically flexible, meaning one terminus is more clearly resolved than the other. However, an earlier work claimed that both ends could be resolved in the presence of two stabilizing single chain variable fragment (scFv) antibodies per nucleosome, and thus are likely permanently bound to the histone octamer. This suggests that the binding of scFv antibodies to the histone octamer surface would be associated with CENP-A nucleosome conformational changes, including stable binding of the DNA termini. Here, we present computational evidence that allows to explain at atomistic level the structural rearrangements of CENP-A nucleosomes resulting from the antibody binding. The antibodies, while they only bind the octamer façades, are capable of altering the dynamics of the nucleosomal core, and indirectly also the surrounding DNA. This effect has more drastic implications for the structure and the dynamics of the CENP-A nucleosome in comparison to its canonical counterpart. Furthermore, we find evidence that the antibodies bind the left and the right octamer façades at different affinities, another manifestation of the DNA sequence. We speculate that the cells could use induction of similar allosteric effects to control centromere function.


Assuntos
Proteína Centromérica A/química , DNA/ultraestrutura , Heterocromatina/ultraestrutura , Histonas/química , Nucleossomos/ultraestrutura , Sequência de Aminoácidos , Pareamento de Bases , Sítios de Ligação , Proteína Centromérica A/genética , Proteína Centromérica A/metabolismo , Regiões Determinantes de Complementaridade/química , Regiões Determinantes de Complementaridade/metabolismo , DNA/genética , DNA/metabolismo , Heterocromatina/genética , Heterocromatina/metabolismo , Histonas/genética , Histonas/metabolismo , Humanos , Modelos Moleculares , Simulação de Dinâmica Molecular , Conformação de Ácido Nucleico , Nucleossomos/genética , Nucleossomos/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Anticorpos de Cadeia Única/química , Anticorpos de Cadeia Única/metabolismo
13.
Neurosci Lett ; 746: 135666, 2021 02 16.
Artigo em Inglês | MEDLINE | ID: mdl-33493646

RESUMO

Glutamate (Glu) and Acetylcholine (ACh), are excitatory neurotransmitters, acting through ionotropic (iR) and metabotropic receptors (mR). Importantly, both neurotransmitters and their signalling are impaired in the prevalent neurodegenerative disease-Alzheimer disease (AD). Glu and its signalling cascade's influence on ACh-neurotransmission (NT) are sparsely understood. The mGluRs coupled to G-protein signalling acting through PI3K cascade (GrpI) or inhibition of adenylate cyclase-cAMP cascade (GrpII and GrpIII) brings about long-lasting structural/functional changes. These complexities are challenging to decipher. Here, we report that human/mouse mGluRs when compared with their Caenorhabditis elegans homologs, MGL-1-3 showed overall of homology of ∼31-39 %. Phylogeneitc analysis revealed homology of MGL-2 to GrpI, MGL-3 with Grp1 &II and GRM6 of GrpIII and MGL-1, a low homology that falls between GrpI & GrpII. Then, alteration of ACh-NT in C. elegans loss-of-function mutants of mgl-1, mgl-2, mgl-3, PI3K (age-1) and iGluR (NMDA)(nmr-1) was estimated by well-established acute aldicarb (Ald), that increases ACh at synapse, and levamisole (Lev) (postsynaptic activation of levamisole sensitive iAChR) induced time-dependent paralysis assays. Surprisingly, all of them were hypersensitive to Ald and Lev compared to wildtype (in percentage), namely, mgl-1 -17, 54; mgl-2 - 7.2, 24; mgl-3 -52, 64; age-1 - 27, 32; nmr-1- 24, 48; respectively. Of the three, mgl-3 contributes to maximal overall acceleration of ACh-NT. Adenylate cyclase, acy-1 gain-of-function mutant showed less hypersensitivity, Ald - 7% and Lev- 25 %. Together, Glu receptors and signalling cascades are altering ACh-NT permanently, thus establishing the interplay between them thereby provide potential drug targets to be considered for AD.


Assuntos
Acetilcolina/metabolismo , Receptores de Glutamato Metabotrópico/química , Receptores de Glutamato Metabotrópico/metabolismo , Transmissão Sináptica/fisiologia , Animais , Animais Geneticamente Modificados , Caenorhabditis elegans , Humanos , Camundongos , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo
14.
Biochemistry ; 60(5): 381-397, 2021 02 09.
Artigo em Inglês | MEDLINE | ID: mdl-33491458

RESUMO

Polyacylated trehaloses in Mycobacterium tuberculosis play important roles in pathogenesis and structural roles in the cell envelope, promoting the intracellular survival of the bacterium, and are potential targets for drug development. Herein, we describe a linear ion-trap multiple-stage mass spectrometric approach (LIT MSn) with high-resolution mass spectrometry to the structural characterization of a glycolipid family that includes a 2,3-diacyltrehalose, 2,3,6-triacyltrehalose, 2,3,6,2',4'-petaacyltrehalose, and a novel 2,3,6,2'-tetraacyltrehalose (TetraAT) subfamily isolated from biofilm cultures of M. tuberculosis H37Rv. The LIT MSn spectra (n = 2, 3, or 4) provide structural information to unveil the location of the palmitoyl/stearoyl and one to four multiple methyl-branched fatty acyl substituents attached to the trehalose backbone, leading to the identification of hundreds of glycolipid species with many isomeric structures. We identified a new TetraAT subfamily whose structure has not been previously defined. We also developed a strategy for defining the structures of the multiple methyl-branched fatty acid substituents, leading to the identification of mycosanoic acid, mycolipenic acid, mycolipodienoic acid, mycolipanolic acid, and a new cyclopropyl-containing acid. The observation of the new TetraAT family, and the realization of the structural similarity between the various subfamilies, may have significant implications in the biosynthetic pathways of this glycolipid family.


Assuntos
Mycobacterium tuberculosis/química , Mycobacterium tuberculosis/enzimologia , Trealose/química , Biofilmes , Parede Celular/química , Ácidos Graxos/química , Cromatografia Gasosa-Espectrometria de Massas/métodos , Glicolipídeos/química , Mycobacterium tuberculosis/metabolismo , Isoformas de Proteínas/química , Espectrometria de Massas por Ionização por Electrospray/métodos , Trealose/metabolismo
15.
Nucleic Acids Res ; 49(2): 745-759, 2021 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-33367825

RESUMO

Gcr1, an important transcription factor for glycolytic genes in Saccharomyces cerevisiae, was recently revealed to have two isoforms, Gcr1U and Gcr1S, produced from un-spliced and spliced transcripts, respectively. In this study, by generating strains expressing only Gcr1U or Gcr1S using the CRISPR/Cas9 system, we elucidate differential activation mechanisms of these two isoforms. The Gcr1U monomer forms an active complex with its coactivator Gcr2 homodimer, whereas Gcr1S acts as a homodimer without Gcr2. The USS domain, 55 residues at the N-terminus existing only in Gcr1U, inhibits dimerization of Gcr1U and even acts in trans to inhibit Gcr1S dimerization. The Gcr1S monomer inhibits the metabolic switch from fermentation to respiration by directly binding to the ALD4 promoter, which can be restored by overexpression of the ALD4 gene, encoding a mitochondrial aldehyde dehydrogenase required for ethanol utilization. Gcr1U and Gcr1S regulate almost the same target genes, but show unique activities depending on growth phase, suggesting that these isoforms play differential roles through separate activation mechanisms depending on environmental conditions.


Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/enzimologia , Fatores de Transcrição/genética , Sistemas CRISPR-Cas , Respiração Celular , Imunoprecipitação da Cromatina , Proteínas de Ligação a DNA/química , Ativação Enzimática , Etanol/metabolismo , Glicerol/metabolismo , Glicólise , Ligação Proteica , Domínios Proteicos , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , RNA-Seq , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/química , Fatores de Transcrição/química , Fatores de Transcrição/deficiência
16.
Cell Prolif ; 54(2): e12974, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33382511

RESUMO

Signal transducer and activator of transcription 3 (STAT3) is one of seven STAT family members involved with the regulation of cellular growth, differentiation and survival. STAT proteins are conserved among eukaryotes and are important for biological functions of embryogenesis, immunity, haematopoiesis and cell migration. STAT3 is widely expressed and located in the cytoplasm in an inactive form. STAT3 is rapidly and transiently activated by tyrosine phosphorylation by a range of signalling pathways, including cytokines from the IL-6 family and growth factors, such as EGF and PDGF. STAT3 activation and subsequent dimer formation initiates nuclear translocation of STAT3 for the regulation of target gene transcription. Four STAT3 isoforms have been identified, which have distinct biological functions. STAT3 is considered a proto-oncogene and constitutive activation of STAT3 is implicated in the development of various cancers, including multiple myeloma, leukaemia and lymphomas. In this review, we focus on recent progress on STAT3 and osteosarcoma (OS). Notably, STAT3 is overexpressed and associated with the poor prognosis of OS. Constitutive activation of STAT3 in OS appears to upregulate the expression of target oncogenes, leading to OS cell transformation, proliferation, tumour formation, invasion, metastasis, immune evasion and drug resistance. Taken together, STAT3 is a target for cancer therapy, and STAT3 inhibitors represent potential therapeutic candidates for the treatment of OS.


Assuntos
Neoplasias Ósseas/patologia , Osteossarcoma/patologia , Fator de Transcrição STAT3/metabolismo , Neoplasias Ósseas/tratamento farmacológico , Neoplasias Ósseas/metabolismo , Citocinas/genética , Citocinas/metabolismo , Resistencia a Medicamentos Antineoplásicos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/uso terapêutico , Humanos , Metástase Neoplásica , Osteossarcoma/tratamento farmacológico , Osteossarcoma/metabolismo , Prognóstico , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Fator de Transcrição STAT3/antagonistas & inibidores , Fator de Transcrição STAT3/química , Fator de Transcrição STAT3/genética
17.
Proc Natl Acad Sci U S A ; 117(38): 23527-23538, 2020 09 22.
Artigo em Inglês | MEDLINE | ID: mdl-32907943

RESUMO

Clathrin light chain (CLC) subunits in vertebrates are encoded by paralogous genes CLTA and CLTB, and both gene products are alternatively spliced in neurons. To understand how this CLC diversity influences neuronal clathrin function, we characterized the biophysical properties of clathrin comprising individual CLC variants for correlation with neuronal phenotypes of mice lacking either CLC-encoding gene. CLC splice variants differentially influenced clathrin knee conformation within assemblies, and clathrin with neuronal CLC mixtures was more effective in membrane deformation than clathrin with single neuronal isoforms nCLCa or nCLCb. Correspondingly, electrophysiological recordings revealed that neurons from mice lacking nCLCa or nCLCb were both defective in synaptic vesicle replenishment. Mice with only nCLCb had a reduced synaptic vesicle pool and impaired neurotransmission compared to WT mice, while nCLCa-only mice had increased synaptic vesicle numbers, restoring normal neurotransmission. These findings highlight differences between the CLC isoforms and show that isoform mixing influences tissue-specific clathrin activity in neurons, which requires their functional balance.


Assuntos
Cadeias Leves de Clatrina , Vesículas Sinápticas/química , Vesículas Sinápticas/metabolismo , Animais , Região CA1 Hipocampal/citologia , Região CA1 Hipocampal/metabolismo , Células Cultivadas , Cadeias Leves de Clatrina/química , Cadeias Leves de Clatrina/genética , Cadeias Leves de Clatrina/metabolismo , Camundongos , Camundongos Knockout , Neurônios/citologia , Neurônios/metabolismo , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo
18.
Mol Pharmacol ; 98(4): 518-527, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32723770

RESUMO

The µ-opioid receptor gene undergoes extensive alternative splicing to generate an array of splice variants. One group of splice variants excludes the first transmembrane (TM) domain and contains six TM domains. These 6TM variants are essential for the action of a novel class of analgesic drugs, including 3-iodobenzoyl-6ß-naltrexamide, which is potent against a spectrum of pain models without exhibiting the adverse side effects of traditional opiates. The 6TM variants are also involved in analgesic action through other drug classes, including δ-opioid and κ-opioids and α 2-adrenergic drugs. Of the five 6TM variants in mouse, mouse µ-opioid receptor (mMOR)-1G is abundant and conserved from rodent to human. In the present study, we demonstrate a new function of mMOR-1G in enhancing expression of the full-length 7TM µ-opioid receptor, mMOR-1. When coexpressed with mMOR-1 in a Tet-Off inducible CHO cell line, mMOR-1G has no effect on mMOR-1 mRNA expression but greatly increases mMOR-1 protein expression in a dose-dependent manner determined by opioid receptor binding and [35S] guanosine 5'-3-O-(thio)triphosphate binding. Subcellular fractionation analysis using OptiPrep density gradient centrifugation shows an increase of functional mMOR-1 receptor in plasma membrane-enriched fractions. Using a coimmunoprecipitation approach, we further demonstrate that mMOR-1G physically associates with mMOR-1 starting at the endoplasmic reticulum, suggesting a chaperone-like function. These data provide a molecular mechanism for how mMOR-1G regulates expression and function of the full-length 7TM µ-opioid receptor. SIGNIFICANCE STATEMENT: The current study establishes a novel function of mouse µ-opioid receptor (mMOR)-1G, a truncated splice variant with six transmembrane (TM) domains of the mouse µ-opioid receptor gene, in enhancing expression of the full-length 7TM mMOR-1 through a chaperone-like function.


Assuntos
Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Receptores Opioides mu/genética , Receptores Opioides mu/metabolismo , Processamento Alternativo , Animais , Células CHO , Linhagem Celular , Cricetulus , Retículo Endoplasmático/metabolismo , Variação Genética , Humanos , Ligação Proteica , Domínios Proteicos , Isoformas de Proteínas/química , Multimerização Proteica , Receptores Opioides mu/química
19.
Nat Struct Mol Biol ; 27(8): 763-767, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32647346

RESUMO

SARS-CoV-2 is thought to have emerged from bats, possibly via a secondary host. Here, we investigate the relationship of spike (S) glycoprotein from SARS-CoV-2 with the S protein of a closely related bat virus, RaTG13. We determined cryo-EM structures for RaTG13 S and for both furin-cleaved and uncleaved SARS-CoV-2 S; we compared these with recently reported structures for uncleaved SARS-CoV-2 S. We also biochemically characterized their relative stabilities and affinities for the SARS-CoV-2 receptor ACE2. Although the overall structures of human and bat virus S proteins are similar, there are key differences in their properties, including a more stable precleavage form of human S and about 1,000-fold tighter binding of SARS-CoV-2 to human receptor. These observations suggest that cleavage at the furin-cleavage site decreases the overall stability of SARS-CoV-2 S and facilitates the adoption of the open conformation that is required for S to bind to the ACE2 receptor.


Assuntos
Betacoronavirus/genética , Interações Hospedeiro-Patógeno/genética , Peptidil Dipeptidase A/química , Receptores Virais/química , Glicoproteína da Espícula de Coronavírus/química , Animais , Betacoronavirus/metabolismo , Betacoronavirus/ultraestrutura , Sítios de Ligação , Quirópteros/virologia , Infecções por Coronavirus/virologia , Microscopia Crioeletrônica , Evolução Molecular , Furina/química , Expressão Gênica , Células HEK293 , Humanos , Modelos Moleculares , Pandemias , Peptidil Dipeptidase A/genética , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/virologia , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estabilidade Proteica , Proteólise , Receptores Virais/genética , Receptores Virais/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismo , Homologia Estrutural de Proteína
20.
Proc Natl Acad Sci U S A ; 117(27): 15554-15564, 2020 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-32561649

RESUMO

The serum haptoglobin protein (Hp) scavenges toxic hemoglobin (Hb) leaked into the bloodstream from erythrocytes. In humans, there are two frequently occurring allelic forms of Hp, resulting in three genotypes: Homozygous Hp 1-1 and Hp 2-2, and heterozygous Hp 2-1. The Hp genetic polymorphism has an intriguing effect on the quaternary structure of Hp. The simplest form, Hp 1-1, forms dimers consisting of two α1ß units, connected by disulfide bridges. Hp 2-1 forms mixtures of linear (α1)2(α2)n-2(ß)n oligomers (n > 1) while Hp 2-2 occurs in cyclic (α2)n(ß)n oligomers (n > 2). Different Hp genotypes bind Hb with different affinities, with Hp 2-2 being the weakest binder. This behavior has a significant influence on Hp's antioxidant capacity, with potentially distinctive personalized clinical consequences. Although Hp has been studied extensively in the past, the finest molecular details of the observed differences in interactions between Hp and Hb are not yet fully understood. Here, we determined the full proteoform profiles and proteoform assemblies of all three most common genetic Hp variants. We combined several state-of-the-art analytical methods, including various forms of chromatography, mass photometry, and different tiers of mass spectrometry, to reveal how the tens to hundreds distinct proteoforms and their assemblies influence Hp's capacity for Hb binding. We extend the current knowledge by showing that Hb binding does not just depend on the donor's genotype, but is also affected by variations in Hp oligomerization, glycosylation, and proteolytic processing of the Hp α-chain.


Assuntos
Haptoglobinas/genética , Hemoglobinas/metabolismo , Alelos , Antioxidantes/química , Antioxidantes/isolamento & purificação , Antioxidantes/metabolismo , Glicosilação , Haptoglobinas/química , Haptoglobinas/isolamento & purificação , Haptoglobinas/metabolismo , Hemoglobinas/toxicidade , Humanos , Espectrometria de Massas , Modelos Moleculares , Estrutura Molecular , Polimorfismo Genético , Ligação Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/isolamento & purificação , Isoformas de Proteínas/metabolismo , Relação Estrutura-Atividade
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