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1.
Cell ; 167(7): 1762-1773.e12, 2016 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-27984726

RESUMO

Overlapping genes pose an evolutionary dilemma as one DNA sequence evolves under the selection pressures of multiple proteins. Here, we perform systematic statistical and mutational analyses of the overlapping HIV-1 genes tat and rev and engineer exhaustive libraries of non-overlapped viruses to perform deep mutational scanning of each gene independently. We find a "segregated" organization in which overlapped sites encode functional residues of one gene or the other, but never both. Furthermore, this organization eliminates unfit genotypes, providing a fitness advantage to the population. Our comprehensive analysis reveals the extraordinary manner in which HIV minimizes the constraint of overlapping genes and repurposes that constraint to its own advantage. Thus, overlaps are not just consequences of evolutionary constraints, but rather can provide population fitness advantages.


Assuntos
Evolução Biológica , HIV-1/genética , Produtos do Gene tat do Vírus da Imunodeficiência Humana/genética , Entropia , Aptidão Genética , Infecções por HIV/virologia , Humanos , Mutação , Fases de Leitura Aberta , Produtos do Gene rev do Vírus da Imunodeficiência Humana/genética
2.
J Biol Chem ; 299(9): 104927, 2023 09.
Artigo em Inglês | MEDLINE | ID: mdl-37330175

RESUMO

Methicillin-resistant Staphylococcus aureus, or MRSA, is one of the major causative agents of hospital-acquired infections worldwide. Novel antimicrobial strategies efficient against antibiotic-resistant strains are necessary and not only against S. aureus. Among those, strategies that aim at blocking or dismantling proteins involved in the acquisition of essential nutrients, helping the bacteria to colonize the host, are intensively studied. A major route for S. aureus to acquire iron from the host organism is the Isd (iron surface determinant) system. In particular, the hemoglobin receptors IsdH and IsdB located on the surface of the bacterium are necessary to acquire the heme moiety containing iron, making them a plausible antibacterial target. Herein, we obtained an antibody of camelid origin that blocked heme acquisition. We determined that the antibody recognized the heme-binding pocket of both IsdH and IsdB with nanomolar order affinity through its second and third complementary-determining regions. The mechanism explaining the inhibition of acquisition of heme in vitro could be described as a competitive process in which the complementary-determining region 3 from the antibody blocked the acquisition of heme by the bacterial receptor. Moreover, this antibody markedly reduced the growth of three different pathogenic strains of MRSA. Collectively, our results highlight a mechanism for inhibiting nutrient uptake as an antibacterial strategy against MRSA.


Assuntos
Anticorpos Antibacterianos , Staphylococcus aureus Resistente à Meticilina , Receptores de Superfície Celular , Anticorpos de Domínio Único , Humanos , Antibacterianos/farmacologia , Heme/metabolismo , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Receptores de Superfície Celular/metabolismo , Receptores de Superfície Celular/uso terapêutico , Anticorpos de Domínio Único/biossíntese , Anticorpos de Domínio Único/química , Anticorpos de Domínio Único/metabolismo , Anticorpos de Domínio Único/farmacologia , Infecções Estafilocócicas/tratamento farmacológico , Antígenos de Bactérias/imunologia , Anticorpos Antibacterianos/genética , Anticorpos Antibacterianos/imunologia , Camelídeos Americanos , Animais , Ligação Proteica/efeitos dos fármacos , Modelos Moleculares , Simulação de Dinâmica Molecular
3.
Biochem Biophys Res Commun ; 691: 149316, 2024 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-38039832

RESUMO

For certain industrial applications, the stability of protein oligomers is important. In this study, we demonstrated an efficient method to improve the thermal stability of oligomers using the trimeric protein chloramphenicol acetyltransferase (CAT) as the model. We substituted all interfacial residues of CAT with alanine to detect residues critical for oligomer stability. Mutation of six of the forty-nine interfacial residues enhanced oligomer thermal stability. Site saturation mutagenesis was performed on these six residues to optimize the side chains. About 15% of mutations enhanced thermal stability by more than 0.5 °C and most did not disrupt activity of CAT. Certain combinations of mutations further improved thermal stability and resistance against heat treatment. The quadruple mutant, H17V/N34S/F134A/D157C, retained the same activity as the wild-type after heat treatment at 9 °C higher temperature than the wild-type CAT. Furthermore, combinations with only alanine substitutions also improved thermal stability, suggesting the method we developed can be used for rapid modification of industrially important proteins.


Assuntos
Alanina , Alanina/genética , Mutagênese , Mutação , Mutagênese Sítio-Dirigida , Cloranfenicol O-Acetiltransferase , Estabilidade Enzimática
4.
J Med Virol ; 96(2): e29443, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38373154

RESUMO

Cross-neutralizing activity of human antibody response against Dengue virus complex (DENV) changes importantly over time. Domain III (DIII) of the envelope protein of DENV elicits a potently neutralizing and mostly type-specific IgG response. We used sera from 24 individuals from early- or late convalescence of DENV1 infection to investigate the evolution of anti-DIII human IgG with the time lapse since the infection. We evaluated the correlation between the serotype-specific reactivity against recombinant DIII proteins and the neutralization capacity against the four serotypes, and examined its behavior with the time of convalescence. Also, we use a library of 71 alanine mutants of surface-exposed amino acid residues to investigate the dominant epitopes. In early convalescence anti-DIII titers and potency of virus neutralization were positively associated with correlation coefficients from 0.82 to 1.0 for the four serotypes. For late convalescence, a positive correlation (r = 0.69) was found only for DENV1. The dominant epitope of the type-specific response is centered in the FG-loop (G383, E384, and K385) and includes most of the lateral ridge. The dominant epitope of the anti-DIII cross-reactive IgG in secondary infections shifts from the A-strand during early convalescence to a site centered in residues E314-H317 of the AB-loop and I352-E368 of the DI/DIII interface, in late convalescence. An immunoassay based on the detection of IgG anti-DIII response can be implemented for detection of infecting serotype in diagnosis of DENV infection, either primary or secondary. Human dominant epitopes of the cross-reactive circulating antibodies change with time of convalescence.


Assuntos
Vírus da Dengue , Dengue , Humanos , Epitopos , Anticorpos Neutralizantes , Anticorpos Antivirais , Formação de Anticorpos , Convalescença , Proteínas do Envelope Viral , Proteínas Recombinantes/metabolismo , Imunoglobulina G/metabolismo , Reações Cruzadas
5.
Bioorg Med Chem Lett ; 104: 129732, 2024 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-38583785

RESUMO

Osteoporosis is a progressive systemic skeletal disease that decreases bone density and bone quality, making them fragile and easy to break. In spite of effective anti-osteoporosis potency, teriparatide, the first anabolic medications approved for the treatment of osteoporosis, was proven to exhibit various side effects. And the relevant structure-activity relationship (SAR) of teriparatide was in need. In this work, we performed a systematical alanine scanning against teriparatide and synthesized 34 teriparatide derivatives. Their biological activities were evaluated and the importance of each residue for anti-osteoporosis activity was also revealed. A remarkable decrease in activity was observed for alanine replacement of the residue Gly12, His14, Ser17, Arg20 and Leu24, showcasing the important role of these residues in teriparatide on anti-osteoporosis activity. On contrary, when Gly13 and Gln30 were mutated to Ala, the peptide derivatives exhibited the significantly increased activities, demonstrating that these two residues could be readily replaced. Our research expanded the peptide library of teriparatide analogues and presented a potential opportunity for designing the more powerful anti-osteoporosis peptide agents.


Assuntos
Conservadores da Densidade Óssea , Osteoporose , Teriparatida , Humanos , Densidade Óssea , Conservadores da Densidade Óssea/efeitos adversos , Conservadores da Densidade Óssea/química , Osteoporose/tratamento farmacológico , Relação Estrutura-Atividade , Teriparatida/efeitos adversos , Teriparatida/análogos & derivados , Análise Mutacional de DNA , Mutagênese Sítio-Dirigida , Alanina/genética
6.
Molecules ; 29(4)2024 Feb 16.
Artigo em Inglês | MEDLINE | ID: mdl-38398632

RESUMO

The major histocompatibility complex (MHC) can recognize and bind to external peptides to generate effective immune responses by presenting the peptides to T cells. Therefore, understanding the binding modes of peptide-MHC complexes (pMHC) and predicting the binding affinity of pMHCs play a crucial role in the rational design of peptide vaccines. In this study, we employed molecular dynamics (MD) simulations and free energy calculations with an Alanine Scanning with Generalized Born and Interaction Entropy (ASGBIE) method to investigate the protein-peptide interaction between HLA-A*02:01 and the G9209 peptide derived from the melanoma antigen gp100. The energy contribution of individual residue was calculated using alanine scanning, and hotspots on both the MHC and the peptides were identified. Our study shows that the pMHC binding is dominated by the van der Waals interactions. Furthermore, we optimized the ASGBIE method, achieving a Pearson correlation coefficient of 0.91 between predicted and experimental binding affinity for mutated antigens. This represents a significant improvement over the conventional MM/GBSA method, which yields a Pearson correlation coefficient of 0.22. The computational protocol developed in this study can be applied to the computational screening of antigens for the MHC1 as well as other protein-peptide binding systems.


Assuntos
Peptídeos , Proteínas , Peptídeos/química , Proteínas/metabolismo , Ligação Proteica , Complexo Principal de Histocompatibilidade , Antígenos de Histocompatibilidade/metabolismo , Alanina/metabolismo
7.
Chembiochem ; 24(12): e202300165, 2023 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-37170827

RESUMO

We developed a synthetic route for producing 3-amino-2-hydroxy acetophenone (3AHAP) from m-nitroacetophenone (3NAP) using an in vitro approach. Various reaction systems were evaluated, and a direct reaction method with crude enzyme and supersaturated substrates for optimal catalytic efficiency was chosen. The reaction system included three enzymes and was enhanced by adjusting enzyme molar ratios and optimizing ribosomal binding sites. We performed substrate docking and alanine scanning to identify key sites in the enzymes nitrobenzene nitroreductase (nbzA) and hydroxylaminobenzene mutase (habA). The optimal mutant was obtained through site-directed mutagenesis, and incorporated into the reaction system, resulting in increased product yield. After optimization, the yield of 3AHAP increased from 75 mg/L to 580 mg/L within 5 hours, the highest reported yield using biosynthesis. This work provides a promising strategy for the efficient and sustainable production of 3AHAP, which has critical applications in the chemical and pharmaceutical industries.


Assuntos
Acetofenonas , Biossíntese de Proteínas , Catálise , Acetofenonas/metabolismo
8.
Brief Bioinform ; 22(5)2021 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-33406224

RESUMO

Protein-nucleic acid interactions play essential roles in many biological processes, such as transcription, replication and translation. In protein-nucleic acid interfaces, hotspot residues contribute the majority of binding affinity toward molecular recognition. Hotspot residues are commonly regarded as potential binding sites for compound molecules in drug design projects. The dynamic property is a considerable factor that affects the binding of ligands. Computational approaches have been developed to expedite the prediction of hotspot residues on protein-nucleic acid interfaces. However, existing approaches overlook hotspot dynamics, despite their essential role in protein function. Here, we report a web server named Hotspots In silico Scanning on Nucleic Acid and Protein Interface (HISNAPI) to analyze hotspot residue dynamics by integrating molecular dynamics simulation and one-step free energy perturbation. HISNAPI is capable of not only predicting the hotspot residues in protein-nucleic acid interfaces but also providing insights into their intensity and correlation of dynamic motion. Protein dynamics have been recognized as a vital factor that has an effect on the interaction specificity and affinity of the binding partners. We applied HISNAPI to the case of SARS-CoV-2 RNA-dependent RNA polymerase, a vital target of the antiviral drug for the treatment of coronavirus disease 2019. We identified the hotspot residues and characterized their dynamic behaviors, which might provide insight into the target site for antiviral drug design. The web server is freely available via a user-friendly web interface at http://chemyang.ccnu.edu.cn/ccb/server/HISNAPI/ and http://agroda.gzu.edu.cn:9999/ccb/server/HISNAPI/.


Assuntos
Biologia Computacional/métodos , Ácidos Nucleicos/metabolismo , Proteínas/metabolismo , Biologia Computacional/instrumentação , Internet , Ligação Proteica , Interface Usuário-Computador
9.
Bioorg Chem ; 130: 106200, 2023 01.
Artigo em Inglês | MEDLINE | ID: mdl-36332316

RESUMO

Targeting vascular endothelial growth factor receptor (VEFGR) and its co-receptor neuropilin-1 (NRP-1) is an interesting vascular strategy. tLyp-1 is a tumor-homing and penetrating peptide of 7 amino acids (CGNKRTR). It is a truncated form of Lyp-1 (CGNKRTRGC), which is known to target NRP-1 receptor, with high affinity and specificity. It is mediated by endocytosis via C-end rule (CendR) internalization pathway. The aim of this study is to evaluate the importance of each amino acid in the tLyp-1 sequence through alanine-scanning (Ala-scan) technique, during which each of the amino acid in the sequence was systematically replaced by alanine to produce 7 different analogues. In silico approach through molecular docking and molecular dynamics are employed to understand the interaction between the peptide and its analogues with the NRP-1 receptor, followed by in vitro ligand binding assay study. The C-terminal Arg is crucial in the interaction of tLyp-1 with NRP-1 receptor. Substituting this residue dramatically reduces the affinity of this peptide which is clearly seen in this study. Lys-4 is also important in the interaction, which is confirmed via the in vitro study and the MM-PBSA analysis. The finding in this study supports the CendR, in which the presence of R/K-XX-R/K motif is essential in the binding of a ligand with NRP-1 receptor. This presented work will serve as a guide in the future work pertaining the development of active targeting agent towards NRP-1 receptor.


Assuntos
Neuropilina-1 , Fator A de Crescimento do Endotélio Vascular , Alanina , Aminoácidos , Ligantes , Simulação de Acoplamento Molecular , Neuropilina-1/química , Neuropilina-1/metabolismo , Peptídeos/química , Fator A de Crescimento do Endotélio Vascular/metabolismo
10.
Mol Divers ; 27(6): 2577-2603, 2023 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-36400898

RESUMO

The COVID-19 crisis, incited by the zoonotic SARS-CoV-2 virus, has quickly escalated into a catastrophic public health issue and a grave threat to humankind owing to the advent of mutant viruses. Multiple pharmaceutical therapies or biologics envision stopping the virus from spreading further; however, WHO has voiced concerns about the variants of concern (VoCs) inability to respond. Nanobodies are a new class of antibody mimics with binding affinity and specificity similar to classical mAbs, as well as the privileges of a small molecular weight, ease of entry into solid tissues, and binding cryptic epitopes of the antigen. Herein, we investigated multiple putative anti-SARS-CoV-2 nanobodies targeting the Receptor binding domain of the WHO-listed SARS-CoV-2 variants of concern using a comprehensive computational immunoinformatics methodology. With affinity maturation via alanine scanning mutagenesis, we remodeled an ultrapotent nanobody with substantial breadth and potency, exhibiting pico-molar binding affinities against all the VoCs. An antiviral peptide with specificity for ACE-2 receptors was affixed to make it multispecific and discourage viral entry. Collectively, we constructed a broad-spectrum therapeutic biparatopic nanobody-peptide conjugate (NPC) extending coverage to SARS-CoV-2 VoCs RBDs. We PEGylated the biparatopic construct with 20kD maleimide-terminated PEG (MAL-(PEG)n-OMe) to improve its clinical efficacy limiting rapid renal clearance, and performed in silico cloning to facilitate future experimental studies. Our findings suggest that combining biparatopic nanobody conjugate with standard treatment may be a promising bivariate tool for combating viral entry during COVID-19 illness.


Assuntos
COVID-19 , Anticorpos de Domínio Único , Humanos , SARS-CoV-2 , Estudos Prospectivos , COVID-19/terapia , Imunização Passiva
11.
Traffic ; 21(2): 250-264, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31675144

RESUMO

Cyclotriazadisulfonamide (CADA) inhibits the co-translational translocation of human CD4 (huCD4) into the endoplasmic reticulum lumen in a signal peptide (SP)-dependent way. We propose that CADA binds the nascent huCD4 SP in a folded conformation within the translocon resembling a normally transitory state during translocation. Here, we used alanine scanning on the huCD4 SP to identify the signature for full susceptibility to CADA. In accordance with our previous work, we demonstrate that residues in the vicinity of the hydrophobic h-region are critical for sensitivity to CADA. In particular, exchanging Gln-15, Val-17 or Pro-20 in the huCD4 SP for Ala resulted in a resistant phenotype. Together with positively charged residues at the N-terminal portion of the mature protein, these residues mediate full susceptibility to the co-translational translocation inhibitory activity of CADA towards huCD4. In addition, sensitivity to CADA is inversely related to hydrophobicity in the huCD4 SP. In vitro translocation experiments confirmed that the general hydrophobicity of the h-domain and positive charges in the mature protein are key elements that affect both the translocation efficiency of huCD4 and the sensitivity towards CADA. Besides these two general SP parameters that determine the functionality of the signal sequence, unique amino acid pairs (L14/Q15 and L19/P20) in the SP hydrophobic core add specificity to the sensitivity signature for a co-translational translocation inhibitor.


Assuntos
Antígenos CD4 , Sinais Direcionadores de Proteínas , Inibidores da Síntese de Proteínas , Antígenos CD4/metabolismo , Retículo Endoplasmático/metabolismo , Humanos , Interações Hidrofóbicas e Hidrofílicas , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Sinais Direcionadores de Proteínas/efeitos dos fármacos , Inibidores da Síntese de Proteínas/farmacologia
12.
J Biol Chem ; 296: 100284, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33450226

RESUMO

ETV6 is an E26 transformation specific family transcriptional repressor that self-associates by its PNT domain to facilitate cooperative DNA binding. Chromosomal translocations frequently generate constitutively active oncoproteins with the ETV6 PNT domain fused to the kinase domain of one of many protein tyrosine kinases. Although an attractive target for therapeutic intervention, the propensity of the ETV6 PNT domain to polymerize via the tight head-to-tail association of two relatively flat interfaces makes it challenging to identify suitable small molecule inhibitors of this protein-protein interaction. Herein, we provide a comprehensive biophysical characterization of the ETV6 PNT domain interaction interfaces to aid future drug discovery efforts and help define the mechanisms by which its self-association mediates transcriptional repression. Using NMR spectroscopy, X-ray crystallography, and molecular dynamics simulations, along with amide hydrogen exchange measurements, we demonstrate that monomeric PNT domain variants adopt very stable helical bundle folds that do not change in conformation upon self-association into heterodimer models of the ETV6 polymer. Surface plasmon resonance-monitored alanine scanning mutagenesis studies identified hot spot regions within the self-association interfaces. These regions include both central hydrophobic residues and flanking salt-bridging residues. Collectively, these studies indicate that small molecules targeted to these hydrophobic or charged regions within the relatively rigid interfaces could potentially serve as orthosteric inhibitors of ETV6 PNT domain polymerization.


Assuntos
Alanina/química , Ácido Aspártico/química , Ácido Glutâmico/química , Proteínas Proto-Oncogênicas c-ets/química , Proteínas Repressoras/química , Transcrição Gênica , Valina/química , Alanina/metabolismo , Substituição de Aminoácidos , Ácido Aspártico/metabolismo , Sítios de Ligação , Clonagem Molecular , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Ácido Glutâmico/metabolismo , Humanos , Modelos Moleculares , Mutagênese Sítio-Dirigida , Mutação , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Proteínas Proto-Oncogênicas c-ets/genética , Proteínas Proto-Oncogênicas c-ets/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Termodinâmica , Valina/metabolismo , Variante 6 da Proteína do Fator de Translocação ETS
13.
Chembiochem ; 23(16): e202200321, 2022 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-35731601

RESUMO

Nanobodies are becoming increasingly popular as tools for manipulating and visualising proteins in vivo. The ability to control nanobody/antigen interactions using light could provide precise spatiotemporal control over protein function. We develop a general approach to engineer photo-activatable nanobodies using photocaged amino acids that are introduced into the target binding interface by genetic code expansion. Guided by computational alanine scanning and molecular dynamics simulations, we tune nanobody/target binding affinity to eliminate binding before uncaging. Upon photo-activation using 365 nm light, binding is restored. We use this approach to generate improved photocaged variants of two anti-GFP nanobodies that function robustly when directly expressed in a complex intracellular environment together with their antigen. We apply them to control subcellular protein localisation in the nematode worm Caenorhabditis elegans. Our approach applies predictions derived from computational modelling directly in a living animal and demonstrates the importance of accounting for in vivo effects on protein-protein interactions.


Assuntos
Anticorpos de Domínio Único , Animais , Antígenos , Código Genético , Engenharia de Proteínas , Proteínas , Anticorpos de Domínio Único/genética
14.
European J Org Chem ; 2022(28)2022 Jul 27.
Artigo em Inglês | MEDLINE | ID: mdl-36120398

RESUMO

Fluorescently labelled alanine scan analogues of odorranalectin (OL), a cyclic peptide that exhibits lectin like properties, were screened for binding BSA-conjugated monosaccharides using an enzyme-linked lectin assay (ELLA). Results revealed that Lys5, Phe7, Tyr9, Gly12, Leu14, and Thr17 were crucial for binding BSA-L-fucose, BSA-D-galactose and BSA-N-acetyl-D-galactosamine. Notably, Ala substitution of Ser3, Pro4, and Val13 resulted in higher binding affinities compared to the native OL. The obtained data also indicated that Arg8 plays an important role in differentiation of binding for BSA-L-fucose/D-galactose from BSA-N-acetyl-D-galactosamine. The thermodynamics of binding of the selected alanine analogues was evaluated by isothermal titration calorimetry. Low to moderate binding affinities were determined for the tetravalent MUC1 glycopeptide and asialofetuin, respectively, and high for the fucose rich polysaccharide, fucoidan. The thermodynamic profile of interactions with asialofetuin exhibits shift to an entropy-driven mechanism compared to the fucoidan, which displayed an enthalpyentropy compensation, typically associated with the carbohydratelectin recognition process.

15.
Molecules ; 27(10)2022 May 23.
Artigo em Inglês | MEDLINE | ID: mdl-35630830

RESUMO

The accumulation of polyethylene terephthalate (PET) seriously harms the environment because of its high resistance to degradation. The recent discovery of the bacteria-secreted biodegradation enzyme, PETase, sheds light on PET recycling; however, the degradation efficiency is far from practical use. Here, in silico alanine scanning mutagenesis (ASM) and site-saturation mutagenesis (SSM) were employed to construct the protein sequence space from binding energy of the PETase-PET interaction to identify the number and position of mutation sites and their appropriate side-chain properties that could improve the PETase-PET interaction. The binding mechanisms of the potential PETase variant were investigated through atomistic molecular dynamics simulations. The results show that up to two mutation sites of PETase are preferable for use in protein engineering to enhance the PETase activity, and the proper side chain property depends on the mutation sites. The predicted variants agree well with prior experimental studies. Particularly, the PETase variants with S238C or Q119F could be a potential candidate for improving PETase. Our combination of in silico ASM and SSM could serve as an alternative protocol for protein engineering because of its simplicity and reliability. In addition, our findings could lead to PETase improvement, offering an important contribution towards a sustainable future.


Assuntos
Hidrolases , Simulação de Dinâmica Molecular , Proteínas de Bactérias/metabolismo , Hidrolases/química , Plásticos , Polietilenotereftalatos/química , Reprodutibilidade dos Testes
16.
J Cell Mol Med ; 25(5): 2530-2548, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33523598

RESUMO

Excitatory amino acid transporter 2 (EAAT2), the gene of which is known as solute carrier family 1 member 2 (SLC1A2), is an important membrane-bound transporter that mediates approximately 90% of the transport and clearance of l-glutamate at synapses in the central nervous system (CNS). Transmembrane domain 2 (TM2) of EAAT2 is close to hairpin loop 2 (HP2) and far away from HP1 in the inward-facing conformation. In the present study, 14 crucial amino acid residues of TM2 were identified via alanine-scanning mutations. Further analysis in EAAT2-transfected HeLa cells in vitro showed that alanine substitutions of these residues resulted in a decrease in the efficiency of trafficking/targeting to the plasma membrane and/or reduced functionality of membrane-bound, which resulted in impaired transporter activity. After additional mutations, the transporter activities of some alanine-substitution mutants recovered. Specifically, the P95A mutant decreased EAAT2-associated anion currents. The Michaelis constant (Km ) values of the mutant proteins L85A, L92A and L101A were increased significantly, whereas R87 and P95A were decreased significantly, indicating that the mutations L85A, L92A and L101A reduced the affinity of the transporter and the substrate, whereas R87A and P95A enhanced this affinity. The maximum velocity (Vmax) values of all 14 alanine mutant proteins were decreased significantly, indicating that all these mutations reduced the substrate transport rate. These results suggest that critical residues in TM2 affect not only the protein expression and membrane-bound localization of EAAT2, but also its interactions with substrates. Additionally, our findings elucidate that the P95A mutant decreased EAAT2-related anion currents. Our results indicate that the TM2 of EAAT2 plays a vital role in the transport process. The key residues in TM2 affect protein expression in the membrane, substrate transport and the anion currents of EAAT2.


Assuntos
Aminoácidos , Ânions/metabolismo , Transportador 2 de Aminoácido Excitatório/química , Transportador 2 de Aminoácido Excitatório/metabolismo , Domínios e Motivos de Interação entre Proteínas , Aminoácidos/metabolismo , Transporte Biológico , Membrana Celular/metabolismo , Transportador 2 de Aminoácido Excitatório/genética , Células HeLa , Humanos , Cinese , Modelos Moleculares , Mutação , Ligação Proteica , Conformação Proteica , Relação Estrutura-Atividade
17.
Microbiology (Reading) ; 167(10)2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34676818

RESUMO

In Actinobacteria, protein O-mannosyl transferase (Pmt)-mediated protein O-glycosylation has an important role in cell envelope physiology. In S. coelicolor, defective Pmt leads to increased susceptibility to cell wall-targeting antibiotics, including vancomycin and ß-lactams, and resistance to phage ϕC31. The aim of this study was to gain a deeper understanding of the structure and function of S. coelicolor Pmt. Sequence alignments and structural bioinformatics were used to identify target sites for an alanine-scanning mutagenesis study. Mutant alleles were introduced into pmt-deficient S. coelicolor strains using an integrative plasmid and scored for their ability to complement phage resistance and antibiotic hypersusceptibility phenotypes. Twenty-three highly conserved Pmt residues were each substituted for alanine. Six mutant alleles failed to complement the pmt▬ strains in either assay. Mapping the six corresponding residues onto a homology model of the three-dimensional structure of Pmt, indicated that five are positioned close to the predicted catalytic DE motif. Further mutagenesis to produce more conservative substitutions at these six residues produced Pmts that invariably failed to complement the DT1025 pmt▬ strain, indicating that strict residue conservation was necessary to preserve function. Cell fractionation and Western blotting of strains with the non-complementing pmt alleles revealed undetectable levels of the enzyme in either the membrane fractions or whole cell lysates. Meanwhile for all of the strains that complemented the antibiotic hypersusceptibility and phage resistance phenotypes, Pmt was readily detected in the membrane fraction. These data indicate a tight correlation between the activity of Pmt and its stability or ability to localize to the membrane.


Assuntos
Manosiltransferases/química , Manosiltransferases/metabolismo , Streptomyces coelicolor/enzimologia , Alanina/genética , Antibacterianos/farmacologia , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Bacteriófagos/fisiologia , Membrana Celular/metabolismo , Sequência Conservada , Manosiltransferases/genética , Testes de Sensibilidade Microbiana , Modelos Moleculares , Mutagênese Sítio-Dirigida , Mutação , Estabilidade Proteica , Streptomyces coelicolor/efeitos dos fármacos , Streptomyces coelicolor/genética , Streptomyces coelicolor/virologia
18.
Chembiochem ; 22(6): 1012-1019, 2021 03 16.
Artigo em Inglês | MEDLINE | ID: mdl-33125165

RESUMO

Macrophage migration inhibitory factor (MIF) is an inflammatory cytokine and atypical chemokine with a key role in inflammatory diseases including atherosclerosis. Key atherogenic functions of MIF are mediated by noncognate interaction with the chemokine receptor CXCR2. The MIF N-like loop comprising the sequence 47-56 is an important structural determinant of the MIF/CXCR2 interface and MIF(47-56) blocks atherogenic MIF activities. However, the mechanism and critical structure-activity information within this sequence have remained elusive. Here, we show that MIF(47-56) directly binds to CXCR2 to compete with MIF receptor activation. By using alanine scanning, essential and dispensable residues were identified. Moreover, MIF(cyclo10), a designed cyclized variant of MIF(47-56), inhibited key inflammatory and atherogenic MIF activities in vitro and in vivo/ex vivo, and exhibited strongly improved resistance to proteolytic degradation in human plasma in vitro, thus suggesting that it could serve as a promising basis for MIF-derived anti-atherosclerotic peptides.


Assuntos
Fatores Inibidores da Migração de Macrófagos/química , Peptídeos Cíclicos/metabolismo , Receptores de Interleucina-8B/metabolismo , Sequência de Aminoácidos , Animais , Adesão Celular , Fluoresceínas/química , Células HEK293 , Humanos , Leucócitos/química , Leucócitos/citologia , Leucócitos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Peptídeos Cíclicos/sangue , Peptídeos Cíclicos/química , Ligação Proteica , Estabilidade Proteica , Receptores de Interleucina-8B/antagonistas & inibidores , Espectrometria de Fluorescência , Ácidos Sulfônicos/química
19.
Chembiochem ; 22(7): 1196-1200, 2021 04 06.
Artigo em Inglês | MEDLINE | ID: mdl-33174669

RESUMO

Infection and replication of SARS CoV-2 (the virus that causes COVID-19) requires entry to the interior of host cells. In humans, a protein-protein interaction (PPI) between the SARS CoV-2 receptor-binding domain (RBD) and the extracellular peptidase domain of ACE2 on the surface of cells in the lower respiratory tract is an initial step in the entry pathway. Inhibition of the SARS CoV-2 RBD/ACE2 PPI is currently being evaluated as a target for therapeutic and/or prophylactic intervention. However, relatively little is known about the molecular underpinnings of this complex. Employing multiple computational platforms, we predicted "hot-spot" residues in a positive-control PPI (PMI/MDM2) and the CoV-2 RBD/ACE2 complex. Computational alanine scanning mutagenesis was performed to predict changes in Gibbs' free energy that are associated with mutating residues at the positive control (PMI/MDM2) or SARS RBD/ACE2 binding interface to alanine. Additionally, we used the Adaptive Poisson-Boltzmann Solver to calculate macromolecular electrostatic surfaces at the interface of the positive-control PPI and SARS CoV-2/ACE2 PPI. Finally, a comparative analysis of hot-spot residues for SARS-CoV and SARS-CoV-2, in complex with ACE2, is provided. Collectively, this study illuminates predicted hot-spot residues, and clusters, at the SARS CoV-2 RBD/ACE2 binding interface, potentially guiding the development of reagents capable of disrupting this complex and halting COVID-19.


Assuntos
Enzima de Conversão de Angiotensina 2/metabolismo , Glicoproteína da Espícula de Coronavírus/metabolismo , Enzima de Conversão de Angiotensina 2/química , Enzima de Conversão de Angiotensina 2/genética , Simulação por Computador , Humanos , Modelos Moleculares , Mutação , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , SARS-CoV-2/química , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Eletricidade Estática
20.
RNA ; 24(11): 1457-1465, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30093489

RESUMO

Structural information about protein-RNA complexes supports the understanding of crucial recognition processes in the cell, and it can allow the development of high affinity ligands to interfere with these processes. In this respect, the identification of amino acid hotspots is particularly important. In contrast to protein-protein interactions, in silico approaches for protein-RNA interactions lag behind in their development. Herein, we report an analysis of available protein-RNA structures. We assembled a data set of 322 crystal and NMR structures and analyzed them regarding interface properties. In addition, we describe a computational alanine-scanning approach which provides interaction scores for interface amino acids, allowing the identification of potential hotspots in protein-RNA interfaces. We have made the computational approach available as an online tool, which allows interaction scores to be calculated for any structure of a protein-RNA complex by uploading atomic coordinates to the PRI HotScore web server (https://pri-hotscore.labs.vu.nl).


Assuntos
Proteínas de Ligação a RNA/química , RNA/química , Alanina/química , Aminoácidos/química , Sítios de Ligação , Espectroscopia de Ressonância Magnética , Conformação de Ácido Nucleico , Ligação Proteica , Conformação Proteica , Mapeamento de Interação de Proteínas , RNA/metabolismo , Proteínas de Ligação a RNA/metabolismo , Relação Estrutura-Atividade
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