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1.
Nat Commun ; 12(1): 5063, 2021 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-34417466

RESUMO

Depression is a common mental disorder. The standard medical treatment is the selective serotonin reuptake inhibitors (SSRIs). All characterized SSRIs are competitive inhibitors of the serotonin transporter (SERT). A non-competitive inhibitor may produce a more favorable therapeutic profile. Vilazodone is an antidepressant with limited information on its molecular interactions with SERT. Here we use molecular pharmacology and cryo-EM structural elucidation to characterize vilazodone binding to SERT. We find that it exhibits non-competitive inhibition of serotonin uptake and impedes dissociation of [3H]imipramine at low nanomolar concentrations. Our SERT structure with bound imipramine and vilazodone reveals a unique binding pocket for vilazodone, expanding the boundaries of the extracellular vestibule. Characterization of the binding site is substantiated with molecular dynamics simulations and systematic mutagenesis of interacting residues resulting in decreased vilazodone binding to the allosteric site. Our findings underline the versatility of SERT allosteric ligands and describe the unique binding characteristics of vilazodone.


Assuntos
Antidepressivos/farmacologia , Proteínas da Membrana Plasmática de Transporte de Serotonina/metabolismo , Inibidores de Captação de Serotonina/farmacologia , Cloridrato de Vilazodona/farmacologia , Regulação Alostérica/efeitos dos fármacos , Animais , Sítios de Ligação , Células COS , Chlorocebus aethiops , Humanos , Cinética , Simulação de Dinâmica Molecular , Proteínas Mutantes/metabolismo , Mutação/genética , Serotonina/metabolismo , Proteínas da Membrana Plasmática de Transporte de Serotonina/ultraestrutura
2.
Int J Mol Sci ; 22(13)2021 Jun 23.
Artigo em Inglês | MEDLINE | ID: mdl-34201785

RESUMO

Alpha-synuclein (α-syn) and leucine-rich repeat kinase 2 (LRRK2) play crucial roles in Parkinson's disease (PD). They may functionally interact to induce the degeneration of dopaminergic (DA) neurons via mechanisms that are not yet fully understood. We previously showed that the C-terminal portion of LRRK2 (ΔLRRK2) with the G2019S mutation (ΔLRRK2G2019S) was sufficient to induce neurodegeneration of DA neurons in vivo, suggesting that mutated LRRK2 induces neurotoxicity through mechanisms that are (i) independent of the N-terminal domains and (ii) "cell-autonomous". Here, we explored whether ΔLRRK2G2019S could modify α-syn toxicity through these two mechanisms. We used a co-transduction approach in rats with AAV vectors encoding ΔLRRK2G2019S or its "dead" kinase form, ΔLRRK2DK, and human α-syn with the A53T mutation (AAV-α-synA53T). Behavioral and histological evaluations were performed at 6- and 15-weeks post-injection. Results showed that neither form of ΔLRRK2 alone induced the degeneration of neurons at these post-injection time points. By contrast, injection of AAV-α-synA53T alone resulted in motor signs and degeneration of DA neurons. Co-injection of AAV-α-synA53T with AAV-ΔLRRK2G2019S induced DA neuron degeneration that was significantly higher than that induced by AAV-α-synA53T alone or with AAV-ΔLRRK2DK. Thus, mutated α-syn neurotoxicity can be enhanced by the C-terminal domain of LRRK2G2019 alone, through cell-autonomous mechanisms.


Assuntos
Modelos Animais de Doenças , Neurônios Dopaminérgicos/patologia , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/metabolismo , Proteínas Mutantes/metabolismo , Mutação , alfa-Sinucleína/metabolismo , Animais , Neurônios Dopaminérgicos/metabolismo , Humanos , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Proteínas Mutantes/genética , Domínios Proteicos , Ratos , alfa-Sinucleína/genética
3.
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
4.
Biochem Biophys Res Commun ; 562: 15-20, 2021 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-34030040

RESUMO

Bile acids play essential roles in facilitating the intestinal absorption of lipophilic nutrients as well as regulation of glucose, lipid, and energy homeostasis via activation of some receptors. Bile acids are cytotoxic, and consequently their concentrations are tightly controlled. A critical pathway for bile acid elimination and detoxification is sulfation. The pattern of bile acid sulfation differs by species. Sulfation preferentially occurs at the 3α-OH of bile acids in humans, but at the 7α-OH in mice. A recent study identified mouse cytosolic sulfotransferase 2A8 (mSULT2A8) as the major hepatic 7α-hydroxyl bile acid-sulfating enzyme. To elucidate the 7α-OH specific sulfation mechanism of mSULT2A8, instead of 3α-OH specific sulfation in humans, we determined a crystal structure of mSULT2A8 in complex with cholic acid, a major bile acid, and 3'-phosphoadenosine-5'-phosphate, the sulfate donor product. Our study shows that bile acid-binding mode of mSULT2A8 and how the enzyme holds the 7α-OH group of bile acids at the catalytic center, revealing that the mechanism underlying 7α-OH specific sulfation. The structure shows the substrate binds to mSULT2A8 in an orientation perpendicular to that of human 3α-hydroxyl bile acid-sulfotransferase (hSULT2A1). The structure of the complex provides new insight into species different bile acid metabolism.


Assuntos
Ácidos e Sais Biliares/metabolismo , Sulfatos/metabolismo , Sulfotransferases/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Biocatálise , Cristalografia por Raios X , Humanos , Cinética , Camundongos , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Especificidade por Substrato , Sulfotransferases/metabolismo
5.
Biochem Biophys Res Commun ; 562: 127-132, 2021 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-34051576

RESUMO

A novel nitrogen mustard CBISC has been synthesized and evaluated as an anticancer agent. CBISC has been shown to exhibit enhanced cell proliferation inhibition properties against mutant p53 cell lines colorectal cancer WiDr, pancreatic cancer (MIAPaCa-2 and PANC-1), and triple negative breast cancer (MDA-MB-231 and MDA-MB-468). In vitro mechanism of action studies revealed perturbations in the p53 pathway and increased cell death as evidenced by western blotting, immunofluorescent microscopy and MTT assay. Further, in vivo studies revealed that CBISC is well tolerated in healthy mice and exhibited significant in vivo tumor growth inhibition properties in WiDr and MIAPaCa-2 xenograft models. These studies illustrate the potential utility of CBISC as an anticancer agent.


Assuntos
Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Dano ao DNA , Proteínas Mutantes/metabolismo , Proteína Supressora de Tumor p53/genética , Animais , Apoptose/efeitos dos fármacos , Sistemas CRISPR-Cas/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Clorambucila/química , Clorambucila/farmacologia , Cloranfenicol/química , Cloranfenicol/farmacologia , Feminino , Camundongos Nus , Poli(ADP-Ribose) Polimerases/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
6.
J Biol Chem ; 297(1): 100836, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34051236

RESUMO

Hypertrophic cardiomyopathy (HCM) is an inherited cardiovascular disorder primarily caused by mutations in the ß-myosin heavy-chain gene. The proximal subfragment 2 region (S2), 126 amino acids of myosin, binds with the C0-C2 region of cardiac myosin-binding protein-C to regulate cardiac muscle contractility in a manner dependent on PKA-mediated phosphorylation. However, it is unknown if HCM-associated mutations within S2 dysregulate actomyosin dynamics by disrupting its interaction with C0-C2, ultimately leading to HCM. Herein, we study three S2 mutations known to cause HCM: R870H, E924K, and E930Δ. First, experiments using recombinant proteins, solid-phase binding, and isothermal titrating calorimetry assays independently revealed that mutant S2 proteins displayed significantly reduced binding with C0-C2. In addition, CD revealed greater instability of the coiled-coil structure in mutant S2 proteins compared with S2Wt proteins. Second, mutant S2 exhibited 5-fold greater affinity for PKA-treated C0-C2 proteins. Third, skinned papillary muscle fibers treated with mutant S2 proteins showed no change in the rate of force redevelopment as a measure of actin-myosin cross-bridge kinetics, whereas S2Wt showed increased the rate of force redevelopment. In summary, S2 and C0-C2 interaction mediated by phosphorylation is altered by mutations in S2, which augment the speed and force of contraction observed in HCM. Modulating this interaction could be a potential strategy to treat HCM in the future.


Assuntos
Cardiomiopatia Hipertrófica/metabolismo , Proteínas de Transporte/metabolismo , Mutação/genética , Miosinas/genética , Animais , Bovinos , Humanos , Cinética , Camundongos Transgênicos , Proteínas Mutantes/metabolismo , Miosinas/metabolismo , Peptídeos/metabolismo , Fosforilação , Ligação Proteica , Reprodutibilidade dos Testes
8.
Nat Commun ; 12(1): 2939, 2021 05 19.
Artigo em Inglês | MEDLINE | ID: mdl-34011960

RESUMO

Elucidation of non-canonical protein functions can identify novel tissue homeostasis pathways. Herein, we describe a role for the Bcl-2 family member BAD in postnatal mammary gland morphogenesis. In Bad3SA knock-in mice, where BAD cannot undergo phosphorylation at 3 key serine residues, pubertal gland development is delayed due to aberrant tubulogenesis of the ductal epithelium. Proteomic and RPPA analyses identify that BAD regulates focal adhesions and the mRNA translation repressor, 4E-BP1. These results suggest that BAD modulates localized translation that drives focal adhesion maturation and cell motility. Consistent with this, cells within Bad3SA organoids contain unstable protrusions with decreased compartmentalized mRNA translation and focal adhesions, and exhibit reduced cell migration and tubulogenesis. Critically, protrusion stability is rescued by 4E-BP1 depletion. Together our results confirm an unexpected role of BAD in controlling localized translation and cell migration during mammary gland development.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Ciclo Celular/metabolismo , Glândulas Mamárias Animais/crescimento & desenvolvimento , Glândulas Mamárias Animais/metabolismo , Glândulas Mamárias Humanas/crescimento & desenvolvimento , Glândulas Mamárias Humanas/metabolismo , Proteína de Morte Celular Associada a bcl/metabolismo , Substituição de Aminoácidos , Animais , Linhagem Celular , Movimento Celular/genética , Feminino , Técnicas de Introdução de Genes , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Animais , Morfogênese , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Organoides/crescimento & desenvolvimento , Organoides/metabolismo , Fosforilação , Biossíntese de Proteínas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Serina/química , Proteína de Morte Celular Associada a bcl/deficiência , Proteína de Morte Celular Associada a bcl/genética
9.
Nat Commun ; 12(1): 2954, 2021 05 19.
Artigo em Inglês | MEDLINE | ID: mdl-34012010

RESUMO

How cancer cells cope with high levels of replication stress during rapid proliferation is currently unclear. Here, we show that macrophage migration inhibitory factor (MIF) is a 3' flap nuclease that translocates to the nucleus in S phase. Poly(ADP-ribose) polymerase 1 co-localizes with MIF to the DNA replication fork, where MIF nuclease activity is required to resolve replication stress and facilitates tumor growth. MIF loss in cancer cells leads to mutation frequency increases, cell cycle delays and DNA synthesis and cell growth inhibition, which can be rescued by restoring MIF, but not nuclease-deficient MIF mutant. MIF is significantly upregulated in breast tumors and correlates with poor overall survival in patients. We propose that MIF is a unique 3' nuclease, excises flaps at the immediate 3' end during DNA synthesis and favors cancer cells evading replication stress-induced threat for their growth.


Assuntos
Neoplasias da Mama/metabolismo , Replicação do DNA/fisiologia , Endonucleases Flap/metabolismo , Oxirredutases Intramoleculares/metabolismo , Fatores Inibidores da Migração de Macrófagos/metabolismo , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células , DNA/química , DNA/metabolismo , Dano ao DNA , DNA Polimerase III/genética , DNA Polimerase III/metabolismo , Replicação do DNA/genética , Feminino , Endonucleases Flap/deficiência , Endonucleases Flap/genética , Técnicas de Inativação de Genes , Instabilidade Genômica , Células HCT116 , Humanos , Oxirredutases Intramoleculares/deficiência , Oxirredutases Intramoleculares/genética , Fatores Inibidores da Migração de Macrófagos/deficiência , Fatores Inibidores da Migração de Macrófagos/genética , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Conformação de Ácido Nucleico , Poli(ADP-Ribose) Polimerase-1/metabolismo , Fase S , Especificidade por Substrato
10.
Int J Mol Sci ; 22(9)2021 Apr 29.
Artigo em Inglês | MEDLINE | ID: mdl-33946750

RESUMO

Genetic testing in Brugada syndrome (BrS) is still not considered to be useful for clinical management of patients in the majority of cases, due to the current lack of understanding about the effect of specific variants. Additionally, family history of sudden death is generally not considered useful for arrhythmic risk stratification. We sought to demonstrate the usefulness of genetic testing and family history in diagnosis and risk stratification. The family history was collected for a proband who presented with a personal history of aborted cardiac arrest and in whom a novel variant in the SCN5A gene was found. Living family members underwent ajmaline testing, electrophysiological study, and genetic testing to determine genotype-phenotype segregation, if any. Patch-clamp experiments on transfected human embryonic kidney 293 cells enabled the functional characterization of the SCN5A novel variant in vitro. In this study, we provide crucial human data on the novel heterozygous variant NM_198056.2:c.5000T>A (p.Val1667Asp) in the SCN5A gene, and demonstrate its segregation with a severe form of BrS and multiple sudden deaths. Functional data revealed a loss of function of the protein affected by the variant. These results provide the first disease association with this variant and demonstrate the usefulness of genetic testing for diagnosis and risk stratification in certain patients. This study also demonstrates the usefulness of collecting the family history, which can assist in understanding the severity of the disease in certain situations and confirm the importance of the functional studies to distinguish between pathogenic mutations and harmless genetic variants.


Assuntos
Síndrome de Brugada/genética , Mutação de Sentido Incorreto , Canal de Sódio Disparado por Voltagem NAV1.5/genética , Adolescente , Adulto , Idoso , Ajmalina/farmacologia , Substituição de Aminoácidos , Síndrome de Brugada/complicações , Síndrome de Brugada/metabolismo , Morte Súbita Cardíaca/etiologia , Eletrocardiografia , Feminino , Testes Genéticos , Células HEK293 , Heterozigoto , Humanos , Mutação com Perda de Função , Masculino , Pessoa de Meia-Idade , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.5/metabolismo , Técnicas de Patch-Clamp , Linhagem , Polimorfismo de Nucleotídeo Único , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
11.
Nat Commun ; 12(1): 2656, 2021 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-33976200

RESUMO

Activating mutants of RAS are commonly found in human cancers, but to date selective targeting of RAS in the clinic has been limited to KRAS(G12C) through covalent inhibitors. Here, we report a monobody, termed 12VC1, that recognizes the active state of both KRAS(G12V) and KRAS(G12C) up to 400-times more tightly than wild-type KRAS. The crystal structures reveal that 12VC1 recognizes the mutations through a shallow pocket, and 12VC1 competes against RAS-effector interaction. When expressed intracellularly, 12VC1 potently inhibits ERK activation and the proliferation of RAS-driven cancer cell lines in vitro and in mouse xenograft models. 12VC1 fused to VHL selectively degrades the KRAS mutants and provides more extended suppression of mutant RAS activity than inhibition by 12VC1 alone. These results demonstrate the feasibility of selective targeting and degradation of KRAS mutants in the active state with noncovalent reagents and provide a starting point for designing noncovalent therapeutics against oncogenic RAS mutants.


Assuntos
Anticorpos Monoclonais/farmacologia , Proteínas Mutantes/antagonistas & inibidores , Mutação , Neoplasias/tratamento farmacológico , Proteínas Proto-Oncogênicas p21(ras)/genética , Ensaios Antitumorais Modelo de Xenoenxerto/métodos , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Feminino , Células HEK293 , Humanos , Camundongos Nus , Proteínas Mutantes/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Ligação Proteica , Proteólise/efeitos dos fármacos , Proteínas Proto-Oncogênicas p21(ras)/imunologia , Proteínas Proto-Oncogênicas p21(ras)/metabolismo
12.
J Mol Biol ; 433(13): 166993, 2021 06 25.
Artigo em Inglês | MEDLINE | ID: mdl-33865867

RESUMO

It is known that insulin-degrading-enzyme (IDE) plays a crucial role in the clearance of Alzheimer's amyloid-ß (Aß). The cysteine-free IDE mutant (cf-E111Q-IDE) is catalytically inactive against insulin, but its effect on Aß degradation is unknown that would help in the allosteric modulation of the enzyme activity. Herein, the degradation of Aß(1-40) by cf-E111Q-IDE via a non-chaperone mechanism is demonstrated by NMR and LC-MS, and the aggregation of fragmented peptides is characterized using fluorescence and electron microscopy. cf-E111Q-IDE presented a reduced effect on the aggregation kinetics of Aß(1-40) when compared with the wild-type IDE. Whereas LC-MS and diffusion ordered NMR spectroscopy revealed the generation of Aß fragments by both wild-type and cf-E111Q-IDE. The aggregation propensities and the difference in the morphological phenotype of the full-length Aß(1-40) and its fragments are explained using multi-microseconds molecular dynamics simulations. Notably, our results reveal that zinc binding to Aß(1-40) inactivates cf-E111Q-IDE's catalytic function, whereas zinc removal restores its function as evidenced from high-speed AFM, electron microscopy, chromatography, and NMR results. These findings emphasize the catalytic role of cf-E111Q-IDE on Aß degradation and urge the development of zinc chelators as an alternative therapeutic strategy that switches on/off IDE's function.


Assuntos
Doença de Alzheimer/metabolismo , Insulisina/metabolismo , Proteínas Mutantes/metabolismo , Sequência de Aminoácidos , Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/metabolismo , Peptídeos beta-Amiloides/ultraestrutura , Biocatálise , Cromatografia Líquida de Alta Pressão , Humanos , Insulisina/química , Insulisina/genética , Espectrometria de Massas , Microscopia Eletrônica de Transmissão , Simulação de Dinâmica Molecular , Proteínas Mutantes/química , Proteínas Mutantes/genética , Mutação de Sentido Incorreto , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/ultraestrutura , Ligação Proteica , Proteólise , Especificidade por Substrato , Zinco/química , Zinco/metabolismo
13.
PLoS Biol ; 19(4): e3001207, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33909605

RESUMO

Missense variants are present amongst the healthy population, but some of them are causative of human diseases. A classification of variants associated with "healthy" or "diseased" states is therefore not always straightforward. A deeper understanding of the nature of missense variants in health and disease, the cellular processes they may affect, and the general molecular principles which underlie these differences is essential to offer mechanistic explanations of the true impact of pathogenic variants. Here, we have formalised a statistical framework which enables robust probabilistic quantification of variant enrichment across full-length proteins, their domains, and 3D structure-defined regions. Using this framework, we validate and extend previously reported trends of variant enrichment in different protein structural regions (surface/core/interface). By examining the association of variant enrichment with available functional pathways and transcriptomic and proteomic (protein half-life, thermal stability, abundance) data, we have mined a rich set of molecular features which distinguish between pathogenic and population variants: Pathogenic variants mainly affect proteins involved in cell proliferation and nucleotide processing and are enriched in more abundant proteins. Additionally, rare population variants display features closer to common than pathogenic variants. We validate the association between these molecular features and variant pathogenicity by comparing against existing in silico variant impact annotations. This study provides molecular details into how different proteins exhibit resilience and/or sensitivity towards missense variants and provides the rationale to prioritise variant-enriched proteins and protein domains for therapeutic targeting and development. The ZoomVar database, which we created for this study, is available at fraternalilab.kcl.ac.uk/ZoomVar. It allows users to programmatically annotate missense variants with protein structural information and to calculate variant enrichment in different protein structural regions.


Assuntos
Doenças Genéticas Inatas/genética , Mutação de Sentido Incorreto/fisiologia , Proteoma , Sequência de Aminoácidos/genética , Biologia Computacional/métodos , Bases de Dados de Proteínas , Doenças Genéticas Inatas/metabolismo , Predisposição Genética para Doença , Mutação em Linhagem Germinativa , Saúde , Humanos , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Conformação Proteica , Dobramento de Proteína , Domínios e Motivos de Interação entre Proteínas/genética , Processamento de Proteína Pós-Traducional/genética , Proteínas/genética , Proteínas/metabolismo , Proteoma/química , Proteoma/genética , Proteoma/metabolismo , Proteômica , Transdução de Sinais/genética , Software
14.
J Biol Chem ; 296: 100662, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33862085

RESUMO

Photoactive biological systems modify the optical properties of their chromophores, known as spectral tuning. Determining the molecular origin of spectral tuning is instrumental for understanding the function and developing applications of these biomolecules. Spectral tuning in flavin-binding fluorescent proteins (FbFPs), an emerging class of fluorescent reporters, is limited by their dependency on protein-bound flavins, whose structure and hence electronic properties cannot be altered by mutation. A blue-shifted variant of the plant-derived improved light, oxygen, voltage FbFP has been created by introducing a lysine within the flavin-binding pocket, but the molecular basis of this shift remains unconfirmed. We here structurally characterize the blue-shifted improved light, oxygen, voltage variant and construct a new blue-shifted CagFbFP protein by introducing an analogous mutation. X-ray structures of both proteins reveal displacement of the lysine away from the chromophore and opening up of the structure as instrumental for the blue shift. Site saturation mutagenesis and high-throughput screening yielded a red-shifted variant, and structural analysis revealed that the lysine side chain of the blue-shifted variant is stabilized close to the flavin by a secondary mutation, accounting for the red shift. Thus, a single additional mutation in a blue-shifted variant is sufficient to generate a red-shifted FbFP. Using spectroscopy, X-ray crystallography, and quantum mechanics molecular mechanics calculations, we provide a firm structural and functional understanding of spectral tuning in FbFPs. We also show that the identified blue- and red-shifted variants allow for two-color microscopy based on spectral separation. In summary, the generated blue- and red-shifted variants represent promising new tools for application in life sciences.


Assuntos
Proteínas de Bactérias/química , Chloroflexus/metabolismo , Flavinas/metabolismo , Proteínas Luminescentes/química , Proteínas Mutantes/química , Mutação , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Chloroflexus/crescimento & desenvolvimento , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Simulação de Dinâmica Molecular , Mutagênese , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Fotoquímica , Conformação Proteica , Teoria Quântica
15.
Curr Opin Chem Biol ; 62: 101-108, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33838397

RESUMO

The protein KRAS has for decades been considered a holy grail of cancer drug discovery. For most of that time, it has also been considered undruggable. Since 2018, five compounds have entered the clinic targeting a single mutant form of KRAS, G12C. Here, we review each of these compounds along with additional approaches to targeting this and other mutants. Remaining challenges include expanding the identification of inhibitors to a broader range of known mutants and to conformations of the protein more likely to avoid development of resistance.


Assuntos
Antineoplásicos/química , Inibidores Enzimáticos/química , Proteínas Mutantes/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Acetonitrilas/química , Acetonitrilas/farmacologia , Animais , Antineoplásicos/farmacologia , Desenho de Fármacos , Resistencia a Medicamentos Antineoplásicos , Inibidores Enzimáticos/metabolismo , Humanos , Proteínas Mutantes/metabolismo , Mutação/genética , Piperazinas/química , Piperazinas/farmacologia , Medicina de Precisão , Ligação Proteica , Conformação Proteica , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Piridinas/química , Piridinas/farmacologia , Pirimidinas/química , Pirimidinas/farmacologia , Quinazolinas/química , Quinazolinas/farmacologia , Relação Estrutura-Atividade
16.
J Biol Chem ; 296: 100631, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33823153

RESUMO

TREM2 is a pattern recognition receptor, expressed on microglia and myeloid cells, detecting lipids and Aß and inducing an innate immune response. Missense mutations (e.g., R47H) of TREM2 increase risk of Alzheimer's disease (AD). The soluble ectodomain of wild-type TREM2 (sTREM2) has been shown to protect against AD in vivo, but the underlying mechanisms are unclear. We show that Aß oligomers bind to cellular TREM2, inducing shedding of the sTREM2 domain. Wild-type sTREM2 bound to Aß oligomers (measured by single-molecule imaging, dot blots, and Bio-Layer Interferometry) inhibited Aß oligomerization and disaggregated preformed Aß oligomers and protofibrils (measured by transmission electron microscopy, dot blots, and size-exclusion chromatography). Wild-type sTREM2 also inhibited Aß fibrillization (measured by imaging and thioflavin T fluorescence) and blocked Aß-induced neurotoxicity (measured by permeabilization of artificial membranes and by loss of neurons in primary neuronal-glial cocultures). In contrast, the R47H AD-risk variant of sTREM2 is less able to bind and disaggregate oligomeric Aß but rather promotes Aß protofibril formation and neurotoxicity. Thus, in addition to inducing an immune response, wild-type TREM2 may protect against amyloid pathology by the Aß-induced release of sTREM2, which blocks Aß aggregation and neurotoxicity. In contrast, R47H sTREM2 promotes Aß aggregation into protofibril that may be toxic to neurons. These findings may explain how wild-type sTREM2 apparently protects against AD in vivo and why a single copy of the R47H variant gene is associated with increased AD risk.


Assuntos
Peptídeos beta-Amiloides/química , Amiloide/química , Glicoproteínas de Membrana/fisiologia , Proteínas Mutantes/metabolismo , Mutação , Neurônios/patologia , Síndromes Neurotóxicas/patologia , Receptores Imunológicos/fisiologia , Doença de Alzheimer , Amiloide/metabolismo , Animais , Camundongos , Camundongos Knockout , Proteínas Mutantes/genética , Neurônios/metabolismo , Síndromes Neurotóxicas/etiologia
17.
Nat Commun ; 12(1): 2073, 2021 04 06.
Artigo em Inglês | MEDLINE | ID: mdl-33824313

RESUMO

Phenylketonuria (PKU) is caused by autosomal recessive variants in phenylalanine hydroxylase (PAH), leading to systemic accumulation of L-phenylalanine (L-Phe) that may reach neurotoxic levels. A homozygous Pah-R261Q mouse, with a highly prevalent misfolding variant in humans, reveals the expected hepatic PAH activity decrease, systemic L-Phe increase, L-tyrosine and L-tryptophan decrease, and tetrahydrobiopterin-responsive hyperphenylalaninemia. Pah-R261Q mice also present unexpected traits, including altered lipid metabolism, reduction of liver tetrahydrobiopterin content, and a metabolic profile indicative of oxidative stress. Pah-R261Q hepatic tissue exhibits large ubiquitin-positive, amyloid-like oligomeric aggregates of mutant PAH that colocalize with selective autophagy markers. Together, these findings reveal that PKU, customarily considered a loss-of-function disorder, can also have toxic gain-of-function contribution from protein misfolding and aggregation. The proteostasis defect and concomitant oxidative stress may explain the prevalence of comorbid conditions in adult PKU patients, placing this mouse model in an advantageous position for the discovery of mutation-specific biomarkers and therapies.


Assuntos
Amiloide/metabolismo , Fígado/enzimologia , Mutação/genética , Estresse Oxidativo , Fenilalanina Hidroxilase/genética , Agregados Proteicos , Animais , Autofagia , Biomarcadores/metabolismo , Peso Corporal , Cruzamento , Feminino , Regulação da Expressão Gênica , Genótipo , Metabolismo dos Lipídeos , Fígado/patologia , Masculino , Metaboloma , Camundongos , Proteínas Mutantes/metabolismo , Neurotransmissores/metabolismo , Estresse Oxidativo/genética , Fenilalanina/metabolismo , Fenilalanina Hidroxilase/metabolismo , Fenilcetonúrias/enzimologia , Pterinas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Respiração , Ubiquitina/metabolismo , Ubiquitinação
18.
Nat Commun ; 12(1): 2061, 2021 04 06.
Artigo em Inglês | MEDLINE | ID: mdl-33824318

RESUMO

Stress can induce cell surface expression of MHC-like ligands, including MICA, that activate NK cells. Human cytomegalovirus (HCMV) glycoprotein US9 downregulates the activating immune ligand MICA*008 to avoid NK cell activation, but the underlying mechanism remains unclear. Here, we show that the N-terminal signal peptide is the major US9 functional domain targeting MICA*008 to proteasomal degradation. The US9 signal peptide is cleaved with unusually slow kinetics and this transiently retained signal peptide arrests MICA*008 maturation in the endoplasmic reticulum (ER), and indirectly induces its degradation via the ER quality control system and the SEL1L-HRD1 complex. We further identify an accessory, signal peptide-independent US9 mechanism that directly binds MICA*008 and SEL1L. Collectively, we describe a dual-targeting immunoevasin, demonstrating that signal peptides can function as protein-integral effector domains.


Assuntos
Evasão da Resposta Imune , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/metabolismo , Sinais Direcionadores de Proteínas , Proteínas Virais/química , Proteínas Virais/metabolismo , Linhagem Celular , Citomegalovirus/imunologia , Citomegalovirus/fisiologia , Infecções por Citomegalovirus/imunologia , Retículo Endoplasmático/metabolismo , Degradação Associada com o Retículo Endoplasmático , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Células Matadoras Naturais/imunologia , Cinética , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Ligação Proteica , Domínios Proteicos , Proteínas/metabolismo , Proteólise , Solubilidade
19.
Genes Cells ; 26(6): 426-446, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33813791

RESUMO

14-3-3 proteins bind to ligands via phospho-serine containing consensus motifs. However, the molecular mechanisms underlying complex formation and dissociation between 14-3-3 proteins and their ligands remain unclear. We identified two conserved acidic residues in the 14-3-3 peptide-binding pocket (D129 and E136) that potentially regulate complex formation and dissociation. Altering these residues to alanine led to opposing effects on centrosome duplication. D129A inhibited centrosome duplication, whereas E136A stimulated centrosome amplification. These results were due to the differing abilities of these mutant proteins to form a complex with NPM1. Inhibiting complex formation between NPM1 and 14-3-3γ led to an increase in centrosome duplication and over-rode the ability of D129A to inhibit centrosome duplication. We identify a novel role of 14-3-3γ in regulating centrosome licensing and a novel mechanism underlying the formation and dissociation of 14-3-3 ligand complexes dictated by conserved residues in the 14-3-3 family.


Assuntos
Proteínas 14-3-3/metabolismo , Centrossomo/metabolismo , Proteínas Nucleares/metabolismo , Fosfopeptídeos/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Centríolos/metabolismo , Células HCT116 , Células HEK293 , Humanos , Modelos Biológicos , Proteínas Mutantes/metabolismo , Fenótipo , Fosfopeptídeos/química , Fosforilação , Multimerização Proteica , Quinases Associadas a rho/metabolismo
20.
Nat Commun ; 12(1): 2173, 2021 04 12.
Artigo em Inglês | MEDLINE | ID: mdl-33846289

RESUMO

The closely related inhibitory killer-cell immunoglobulin-like receptors (KIR), KIR2DL2 and KIR2DL3, regulate the activation of natural killer cells (NK) by interacting with the human leukocyte antigen-C1 (HLA-C1) group of molecules. KIR2DL2, KIR2DL3 and HLA-C1 are highly polymorphic, with this variation being associated with differences in the onset and progression of some human diseases. However, the molecular bases underlying these associations remain unresolved. Here, we determined the crystal structures of KIR2DL2 and KIR2DL3 in complex with HLA-C*07:02 presenting a self-epitope. KIR2DL2 differed from KIR2DL3 in docking modality over HLA-C*07:02 that correlates with variabilty of recognition of HLA-C1 allotypes. Mutagenesis assays indicated differences in the mechanism of HLA-C1 allotype recognition by KIR2DL2 and KIR2DL3. Similarly, HLA-C1 allotypes differed markedly in their capacity to inhibit activation of primary NK cells. These functional differences derive, in part, from KIR2DS2 suggesting KIR2DL2 and KIR2DL3 binding geometries combine with other factors to distinguish HLA-C1 functional recognition.


Assuntos
Antígenos HLA-C/metabolismo , Simulação de Acoplamento Molecular , Receptores KIR2DL2/química , Receptores KIR2DL2/metabolismo , Receptores KIR2DL3/química , Receptores KIR2DL3/metabolismo , Células HEK293 , Humanos , Células Matadoras Naturais/imunologia , Ligantes , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Peptídeos/química , Ligação Proteica , Mapeamento de Interação de Proteínas
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