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1.
Int J Mol Sci ; 22(17)2021 Aug 24.
Artigo em Inglês | MEDLINE | ID: mdl-34502041

RESUMO

The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) outbreak in December 2019 has caused a global pandemic. The rapid mutation rate in the virus has created alarming situations worldwide and is being attributed to the false negativity in RT-PCR tests. It has also increased the chances of reinfection and immune escape. Recently various lineages namely, B.1.1.7 (Alpha), B.1.617.1 (Kappa), B.1.617.2 (Delta) and B.1.617.3 have caused rapid infection around the globe. To understand the biophysical perspective, we have performed molecular dynamic simulations of four different spikes (receptor binding domain)-hACE2 complexes, namely wildtype (WT), Alpha variant (N501Y spike mutant), Kappa (L452R, E484Q) and Delta (L452R, T478K), and compared their dynamics, binding energy and molecular interactions. Our results show that mutation has caused significant increase in the binding energy between the spike and hACE2 in Alpha and Kappa variants. In the case of Kappa and Delta variants, the mutations at L452R, T478K and E484Q increased the stability and intra-chain interactions in the spike protein, which may change the interaction ability of neutralizing antibodies to these spike variants. Further, we found that the Alpha variant had increased hydrogen interaction with Lys353 of hACE2 and more binding affinity in comparison to WT. The current study provides the biophysical basis for understanding the molecular mechanism and rationale behind the increase in the transmissivity and infectivity of the mutants compared to wild-type SARS-CoV-2.


Assuntos
Enzima de Conversão de Angiotensina 2/metabolismo , COVID-19/transmissão , SARS-CoV-2/patogenicidade , Glicoproteína da Espícula de Coronavírus/metabolismo , Enzima de Conversão de Angiotensina 2/ultraestrutura , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/metabolismo , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/metabolismo , COVID-19/virologia , Cristalografia por Raios X , Humanos , Simulação de Dinâmica Molecular , Mutação , Estabilidade Proteica , SARS-CoV-2/genética , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/imunologia , Glicoproteína da Espícula de Coronavírus/ultraestrutura , Termodinâmica
2.
Int J Mol Sci ; 22(16)2021 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-34445303

RESUMO

Macromolecular associates, such as membraneless organelles or lipid-protein assemblies, provide a hydrophobic environment, i.e., a liquid protein phase (LP), where folding preferences can be drastically altered. LP as well as the associated phase change from water (W) is an intriguing phenomenon related to numerous biological processes and also possesses potential in nanotechnological applications. However, the energetic effects of a hydrophobic yet water-containing environment on protein folding are poorly understood. Here, we focus on small ß-sheets, the key motifs of proteins, undergoing structural changes in liquid-liquid phase separation (LLPS) and also model the mechanism of energy-coupled unfolding, e.g., in proteases, during W → LP transition. Due to the importance of the accurate description for hydrogen bonding patterns, the employed models were studied by using quantum mechanical calculations. The results demonstrate that unfolding is energetically less favored in LP by ~0.3-0.5 kcal·mol-1 per residue in which the difference further increased by the presence of explicit structural water molecules, where the folded state was preferred by ~1.2-2.3 kcal·mol-1 per residue relative to that in W. Energetics at the LP/W interfaces was also addressed by theoretical isodesmic reactions. While the models predict folded state preference in LP, the unfolding from LP to W renders the process highly favorable since the unfolded end state has >1 kcal·mol-1 per residue excess stabilization.


Assuntos
Transição de Fase/efeitos dos fármacos , Conformação Proteica em Folha beta/efeitos dos fármacos , Água/farmacologia , Motivos de Aminoácidos/efeitos dos fármacos , Fracionamento Químico/métodos , Simulação por Computador , Interações Hidrofóbicas e Hidrofílicas/efeitos dos fármacos , Cinética , Substâncias Macromoleculares/química , Modelos Moleculares , Conformação Proteica/efeitos dos fármacos , Dobramento de Proteína/efeitos dos fármacos , Estabilidade Proteica/efeitos dos fármacos , Teoria Quântica , Viscosidade , Água/química
3.
Adv Protein Chem Struct Biol ; 127: 217-248, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34340768

RESUMO

Protein structure characterization is fundamental to understand protein properties, such as folding process and protein resistance to thermal stress, up to unveiling organism pathologies (e.g., prion disease). In this chapter, we provide an overview on how the spectral properties of the networks reconstructed from the Protein Contact Map (PCM) can be used to generate informative observables. As a specific case study, we apply two different network approaches to an example protein dataset, for the aim of discriminating protein folding state, and for the reconstruction of protein 3D structure.


Assuntos
Bases de Dados de Proteínas , Dobramento de Proteína , Mapas de Interação de Proteínas , Proteínas/química , Proteínas/metabolismo , Animais , Humanos , Domínios Proteicos , Estabilidade Proteica
4.
Proc Natl Acad Sci U S A ; 118(37)2021 09 14.
Artigo em Inglês | MEDLINE | ID: mdl-34452991

RESUMO

COVID-19, caused by severe acute respiratory coronavirus 2 (SARS-CoV-2), has presented a serious risk to global public health. The viral main protease Mpro (also called 3Clpro) encoded by NSP5 is an enzyme essential for viral replication. However, very few host proteins have been experimentally validated as targets of 3Clpro. Here, through bioinformatics analysis of 300 interferon stimulatory genes (ISGs) based on the prediction method NetCorona, we identify RNF20 (Ring Finger Protein 20) as a novel target of 3Clpro. We have also provided evidence that 3Clpro, but not the mutant 3ClproC145A without catalytic activity, cleaves RNF20 at a conserved Gln521 across species, which subsequently prevents SREBP1 from RNF20-mediated degradation and promotes SARS-CoV-2 replication. We show that RNA interference (RNAi)-mediated depletion of either RNF20 or RNF40 significantly enhances viral replication, indicating the antiviral role of RNF20/RNF40 complex against SARS-CoV-2. The involvement of SREBP1 in SARS-CoV-2 infection is evidenced by a decrease of viral replication in the cells with SREBP1 knockdown and inhibitor AM580. Taken together, our findings reveal RNF20 as a novel host target for SARS-CoV-2 main protease and indicate that 3Clpro inhibitors may treat COVID-19 through not only blocking viral polyprotein cleavage but also enhancing host antiviral response.


Assuntos
Proteases 3C de Coronavírus/metabolismo , Estabilidade Proteica , SARS-CoV-2/patogenicidade , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Replicação Viral , Animais , Antivirais/farmacologia , Linhagem Celular , Chlorocebus aethiops , Regulação da Expressão Gênica , Interferons/fisiologia , SARS-CoV-2/imunologia , Proteína de Ligação a Elemento Regulador de Esterol 1/antagonistas & inibidores , Células Vero
5.
Viruses ; 13(8)2021 08 12.
Artigo em Inglês | MEDLINE | ID: mdl-34452461

RESUMO

The COVID-19 pandemic, caused by SARS-CoV-2, has rapidly spread to more than 222 countries and has put global public health at high risk. The world urgently needs cost-effective and safe SARS-CoV-2 vaccines, antiviral, and therapeutic drugs to control it. In this study, we engineered the receptor binding domain (RBD) of the SARS-CoV-2 spike (S) protein and produced it in the plant Nicotiana benthamiana in a glycosylated and deglycosylated form. Expression levels of both glycosylated (gRBD) and deglycosylated (dRBD) RBD were greater than 45 mg/kg fresh weight. The purification yields were 22 mg of pure protein/kg of plant biomass for gRBD and 20 mg for dRBD, which would be sufficient for commercialization of these vaccine candidates. The purified plant-produced RBD protein was recognized by an S protein-specific monoclonal antibody, demonstrating specific reactivity of the antibody to the plant-produced RBD proteins. The SARS-CoV-2 RBD showed specific binding to angiotensin converting enzyme 2 (ACE2), the SARS-CoV-2 receptor. In mice, the plant-produced RBD antigens elicited high titers of antibodies with a potent virus-neutralizing activity. To our knowledge, this is the first report demonstrating that mice immunized with plant-produced deglycosylated RBD form elicited high titer of RBD-specific antibodies with potent neutralizing activity against SARS-CoV-2 infection. Thus, obtained data support that plant-produced glycosylated and in vivo deglycosylated RBD antigens, developed in this study, are promising vaccine candidates for the prevention of COVID-19.


Assuntos
Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/imunologia , Enzima de Conversão de Angiotensina 2/metabolismo , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Chlorocebus aethiops , Glicosilação , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Testes de Neutralização , Plantas Geneticamente Modificadas , Ligação Proteica , Domínios Proteicos , Engenharia de Proteínas , Estabilidade Proteica , Receptores de Coronavírus/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismo , Tabaco/genética , Tabaco/metabolismo , Células Vero
6.
Int J Mol Sci ; 22(15)2021 Jul 22.
Artigo em Inglês | MEDLINE | ID: mdl-34360583

RESUMO

High-density lipoproteins' (HDL) stability is a determinant of their residence times in plasma and consequently an important parameter that influences the beneficial properties of these lipoproteins. Since there are no accessible procedures for this purpose, here, we describe the methodological conditions to assess the stability of the HDL based on the redshift of the fluorescence spectrum of tryptophans contained in the structure of HDL-apolipoproteins during incubation with urea 8M. Along the HDL denaturation kinetics, the main variations of fluorescence were observed at the wavelengths of 330, 344, and 365 nm at room temperature. Therefore, HDL denaturation was estimated using the tryptophan (Trp)-ratio of fluorescence intensity (rfi) at such wavelengths. By setting 100% of the measurable denaturation at 26 h, HDL reached 50% after 8 h of incubation with urea. Then, for further analyses we determined the percentage of HDL denaturation at 8 h as an estimation of the stability of these lipoproteins. To explore the potential usefulness of this test, we analyzed the stability of HDL isolated from the plasma of 24 patients diagnosed with acute coronary syndrome (ACS). These HDL presented significantly higher percentages of denaturation (64.9% (58.7-78.4)) than HDLs of healthy individuals (23.3% (20.3-27.0)). These results indicate that HDL in ACS are less stable than in control subjects. Moreover, the percentage of denaturation of HDL correlated with body mass index and aspartate transaminase plasma activity. Furthermore, apo-I, HDL-cholesterol, HDL-triglycerides, and apo A-I-to-triglycerides ratio correlated with the percentage of HDL denaturation, suggesting that the lipoprotein composition is a main determinant of HDL stability. Finally, the percentage of HDL denaturation is the parameter that predicted the presence of ACS as determined by a machine learning procedure and logistic regression analysis. In conclusion, we established the methodological conditions to assess the stability of HDL by a fluorescence-based method that merits exploration in prospective studies for evaluating the coronary artery disease risk.


Assuntos
Síndrome Coronariana Aguda/patologia , Fluorescência , Lipoproteínas HDL/química , Lipoproteínas HDL/metabolismo , Triptofano/química , Síndrome Coronariana Aguda/metabolismo , Estudos de Casos e Controles , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Desnaturação Proteica , Estabilidade Proteica
7.
Chem Biol Interact ; 347: 109604, 2021 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-34352275

RESUMO

Angiotensin-converting enzyme (ACE, EC 3.4.15.1) synthesized by endothelial cells and responsible for the regulation of blood pressure was purified from the bovine lung with affinity chromatography method. The purification rate of the ACE of the bovine lung was calculated as 1748- fold. Optimum pH and optimum temperature for the purified ACE were found to be 7.6 and 35-40 °C, respectively. The purity and molecular weight of the ACE were designated with SDS-PAGE. The ACE was found to have three subunits with molecular weights of 57 kDa, 66 kDa, and 190 kDa. Then, the total molecular weight of the ACE was designated as 303 kDa with gel filtration chromatography. The effects of ACE inhibitors captopril, fosinopril, lisinopril, and beta-blockers propranolol, atenolol, and diuretic triamterene on ACE activity were studied. ACE inhibitors lisinopril, captopril, fosinopril, and diuretic triamterene demonstrated an inhibition effect on ACE activity. Beta-blockers indicated no effect on ACE. IC50 values of captopril, fosinopril, lisinopril, and triamterene from the graphical equation were calculated as 0.835 nM, 1.159 µM, 4.085 nM, and 227 µM, respectively. The inhibition type and Ki values of these compounds were determined from Lineweaver-Burk plots. Captopril, fosinopril, lisinopril, and triamterene demonstrated a non-competitive inhibition effect on ACE activity. Ki constants were found as 1.057 nM, 1.675 µM, 6.449 nM, and 419.5 µM, respectively. Captopril indicated the highest inhibitor effect with an IC50 value of 0.835 nM.


Assuntos
Peptidil Dipeptidase A/isolamento & purificação , Inibidores da Enzima Conversora de Angiotensina/química , Animais , Captopril/química , Bovinos , Cromatografia de Afinidade , Fosinopril/química , Concentração de Íons de Hidrogênio , Cinética , Lisinopril/química , Pulmão/química , Peptidil Dipeptidase A/química , Estabilidade Proteica , Temperatura , Triantereno/química
8.
Molecules ; 26(15)2021 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-34361782

RESUMO

Thermal treatments of milk induce changes in the properties of milk whey proteins. The aim of this study was to investigate the specific changes related to nutrients in the whey proteins of dairy cow milk after pasteurization at 85 °C for 15 s or ultra-high temperature (UHT) at 135 °C for 15 s. A total of 223 whey proteins were confidently identified and quantified by TMT-based global discovery proteomics in this study. We found that UHT thermal treatment resulted in an increased abundance of 17 proteins, which appeared to show heat insensitivity. In contrast, 15 heat-sensitive proteins were decreased in abundance after UHT thermal treatment. Some of the heat-sensitive proteins were connected with the biological immune functionality, suggesting that UHT thermal treatment results in a partial loss of immune function in the whey proteins of dairy cow milk. The information reported here will considerably expand our knowledge about the degree of heat sensitivity in the whey proteins of dairy cow milk in response to different thermal treatments and offer a knowledge-based reference to aid in choosing dairy products. It is worth noting that the whey proteins (lactoperoxidase and lactoperoxidase) in milk that were significantly decreased by high heat treatment in a previous study (142 °C) showed no significant difference in the present study (135 °C). These results may imply that an appropriately reduced heating intensity of UHT retains the immunoactive proteins to the maximum extent possible.


Assuntos
Leite/química , Pasteurização/métodos , Proteínas do Soro do Leite/química , Soro do Leite/química , Animais , Feminino , Temperatura Alta , Leite/imunologia , Anotação de Sequência Molecular , Estabilidade Proteica , Proteômica/métodos , Soro do Leite/imunologia , Proteínas do Soro do Leite/classificação , Proteínas do Soro do Leite/imunologia , Proteínas do Soro do Leite/isolamento & purificação
9.
J Phys Chem Lett ; 12(32): 7659-7664, 2021 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-34351767

RESUMO

From stem cell freeze-drying to organ storage, considerable recent efforts have been directed toward the development of new preservation technologies. A prominent protein stabilizing strategy involves vitrification in glassy matrices, most notably those formed of sugars such as the biologically relevant preservative trehalose. Here, we compare the folding thermodynamics of a model miniprotein in solution and in the glassy state of the sugars trehalose and glucose. Using synchrotron radiation circular dichroism (SRCD), we find that the same native structure persists in solution and glass. However, upon transition to the glass, a completely different, conformationally restricted unfolded state replaces the disordered denatured state found in solution, potentially inhibiting misfolding. Concomitantly, a large exothermic contribution is observed in glass, exposing the stabilizing effect of interactions with the sugar matrix on the native state. Our results shed light on the mechanism of protein stabilization in sugar glass and should aid in future preservation technologies.


Assuntos
Conformação Proteica/efeitos dos fármacos , Estabilidade Proteica/efeitos dos fármacos , Proteínas/metabolismo , Trealose/química , Sequência de Aminoácidos , Dobramento de Proteína/efeitos dos fármacos , Proteínas/química , Termodinâmica , Vitrificação
10.
Cells ; 10(7)2021 07 13.
Artigo em Inglês | MEDLINE | ID: mdl-34359938

RESUMO

Transthyretin (TTR) is a tetrameric protein transporting hormones in the plasma and brain, which has many other activities that have not been fully acknowledged. TTR is a positive indicator of nutrition status and is negatively correlated with inflammation. TTR is a neuroprotective and oxidative-stress-suppressing factor. The TTR structure is destabilized by mutations, oxidative modifications, aging, proteolysis, and metal cations, including Ca2+. Destabilized TTR molecules form amyloid deposits, resulting in senile and familial amyloidopathies. This review links structural stability of TTR with the environmental factors, particularly oxidative stress and Ca2+, and the processes involved in the pathogenesis of TTR-related diseases. The roles of TTR in biomineralization, calcification, and osteoarticular and cardiovascular diseases are broadly discussed. The association of TTR-related diseases and vascular and ligament tissue calcification with TTR levels and TTR structure is presented. It is indicated that unaggregated TTR and TTR amyloid are bound by vicious cycles, and that TTR may have an as yet undetermined role(s) at the crossroads of calcification, blood coagulation, and immune response.


Assuntos
Artrite/metabolismo , Doenças Cardiovasculares/metabolismo , Osteoporose/metabolismo , Pré-Albumina/metabolismo , Amiloide/química , Amiloide/metabolismo , Amiloidose/metabolismo , Animais , Humanos , Estresse Oxidativo , Pré-Albumina/química , Conformação Proteica , Estabilidade Proteica
11.
Int J Mol Sci ; 22(15)2021 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-34361043

RESUMO

Intravesicular pH plays a crucial role in melanosome maturation and function. Melanosomal pH changes during maturation from very acidic in the early stages to neutral in late stages. Neutral pH is critical for providing optimal conditions for the rate-limiting, pH-sensitive melanin-synthesizing enzyme tyrosinase (TYR). This dramatic change in pH is thought to result from the activity of several proteins that control melanosomal pH. Here, we computationally investigated the pH-dependent stability of several melanosomal membrane proteins and compared them to the pH dependence of the stability of TYR. We confirmed that the pH optimum of TYR is neutral, and we also found that proteins that are negative regulators of melanosomal pH are predicted to function optimally at neutral pH. In contrast, positive pH regulators were predicted to have an acidic pH optimum. We propose a competitive mechanism among positive and negative regulators that results in pH equilibrium. Our findings are consistent with previous work that demonstrated a correlation between the pH optima of stability and activity, and they are consistent with the expected activity of positive and negative regulators of melanosomal pH. Furthermore, our data suggest that disease-causing variants impact the pH dependence of melanosomal proteins; this is particularly prominent for the OCA2 protein. In conclusion, melanosomal pH appears to affect the activity of multiple melanosomal proteins.


Assuntos
Antígenos de Neoplasias/química , ATPases Transportadoras de Cobre/química , Melanossomas/metabolismo , Proteínas de Membrana Transportadoras/química , Simulação de Dinâmica Molecular , Monofenol Mono-Oxigenase/química , Prótons , Antígenos de Neoplasias/metabolismo , ATPases Transportadoras de Cobre/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Melanossomas/química , Proteínas de Membrana Transportadoras/metabolismo , Monofenol Mono-Oxigenase/metabolismo , Estabilidade Proteica
12.
Biol Aujourdhui ; 215(1-2): 25-43, 2021.
Artigo em Francês | MEDLINE | ID: mdl-34397373

RESUMO

Targeted protein degradation (TPD), discovered twenty years ago through the PROTAC technology, is rapidly developing thanks to the implication of many scientists from industry and academia. PROTAC chimeras are heterobifunctional molecules able to link simultaneously a protein to be degraded and an E3 ubiquitin ligase. This allows the protein ubiquitination and its degradation by 26S proteasome. PROTACs have evolved from small peptide molecules to small non-peptide and orally available molecules. It was shown that PROTACs are capable to degrade proteins considered as "undruggable" i.e. devoid of well-defined pockets and deep grooves possibly occupied by small molecules. Among these "hard to drug" proteins, several can be degraded by PROTACs: scaffold proteins, BAF complex, transcription factors, Ras family proteins. Two PROTACs are clinically tested for breast (ARV471) and prostate (ARV110) cancers. The protein degradation by proteasome is also induced by other types of molecules: molecular glues, hydrophobic tagging (HyT), HaloPROTACs and homo-PROTACs. Other cellular constituents are eligible to induced degradation: RNA-PROTACs for RNA binding proteins and RIBOTACs for degradation of RNA itself (SARS-CoV-2 RNA). TPD has recently moved beyond the proteasome with LYTACs (lysosome targeting chimeras) and MADTACs (macroautophagy degradation targeting chimeras). Several techniques such as screening platforms together with mathematical modeling and computational design are now used to improve the discovery of new efficient PROTACs.


Assuntos
COVID-19/tratamento farmacológico , Desenho de Fármacos , Terapia de Alvo Molecular/métodos , Proteólise , Proteínas Recombinantes de Fusão/farmacologia , SARS-CoV-2/efeitos dos fármacos , Antineoplásicos/química , Antineoplásicos/farmacologia , Autofagia , Catálise , Humanos , Lisossomos/metabolismo , Proteínas de Neoplasias/antagonistas & inibidores , Neoplasias/tratamento farmacológico , Complexo de Endopeptidases do Proteassoma/metabolismo , Conformação Proteica , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Estabilidade Proteica , Proteólise/efeitos dos fármacos , RNA/efeitos dos fármacos , Proteínas de Ligação a RNA/antagonistas & inibidores , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/farmacocinética , Relação Estrutura-Atividade , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação
13.
Nat Commun ; 12(1): 5068, 2021 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-34417460

RESUMO

p53 regulates several signaling pathways to maintain the metabolic homeostasis of cells and modulates the cellular response to stress. Deficiency or excess of nutrients causes cellular metabolic stress, and we hypothesized that p53 could be linked to glucose maintenance. We show here that upon starvation hepatic p53 is stabilized by O-GlcNAcylation and plays an essential role in the physiological regulation of glucose homeostasis. More specifically, p53 binds to PCK1 promoter and regulates its transcriptional activation, thereby controlling hepatic glucose production. Mice lacking p53 in the liver show a reduced gluconeogenic response during calorie restriction. Glucagon, adrenaline and glucocorticoids augment protein levels of p53, and administration of these hormones to p53 deficient human hepatocytes and to liver-specific p53 deficient mice fails to increase glucose levels. Moreover, insulin decreases p53 levels, and over-expression of p53 impairs insulin sensitivity. Finally, protein levels of p53, as well as genes responsible of O-GlcNAcylation are elevated in the liver of type 2 diabetic patients and positively correlate with glucose and HOMA-IR. Overall these results indicate that the O-GlcNAcylation of p53 plays an unsuspected key role regulating in vivo glucose homeostasis.


Assuntos
Acetilglucosamina/metabolismo , Glucose/metabolismo , Fígado/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Animais , Sequência de Bases , Restrição Calórica , Linhagem Celular , Colforsina/farmacologia , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/metabolismo , Epinefrina/metabolismo , Glucagon/metabolismo , Glucocorticoides/metabolismo , Gluconeogênese/efeitos dos fármacos , Glicosilação , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Hidrocortisona/metabolismo , Hiperglicemia/complicações , Hiperglicemia/metabolismo , Resistência à Insulina , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Fígado/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Obesidade/complicações , Obesidade/metabolismo , Fosfoenolpiruvato Carboxiquinase (GTP)/metabolismo , Regiões Promotoras Genéticas/genética , Ligação Proteica/efeitos dos fármacos , Estabilidade Proteica/efeitos dos fármacos , Ácido Pirúvico/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transcrição Genética/efeitos dos fármacos , Proteína Supressora de Tumor p53/genética
14.
Int J Mol Sci ; 22(16)2021 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-34445414

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) affects the COVID-19 pandemic in the world. The spike protein of the various proteins encoded in SARS-CoV-2 binds to human ACE2, fuses, and enters human cells in the respiratory system. Spike protein, however, is highly variable, and many variants were identified continuously. In this study, Korean mutants for spike protein (D614G and D614A-C terminal domain, L455F and F456L-RBD, and Q787H-S2 domain) were investigated in patients. Because RBD in spike protein is related to direct interaction with ACE2, almost all researches were focused on the RBD region or ACE2-free whole domain region. The 3D structure for spike protein complexed with ACE2 was recently released. The stability analysis through RBD distance among each spike protein chain and the binding free energy calculation between spike protein and ACE2 were performed using MD simulation depending on mutant types in 1-, 2-, and 3-open-complex forms. D614G mutant of CT2 domain, showing to be the most prevalent in the global pandemic, showed higher stability in all open-complex forms than the wild type and other mutants. We hope this study will provide an insight into the importance of conformational fluctuation in the whole domain, although RBD is involved in the direct interaction with ACE2.


Assuntos
COVID-19/virologia , SARS-CoV-2/química , SARS-CoV-2/metabolismo , Enzima de Conversão de Angiotensina 2/química , Enzima de Conversão de Angiotensina 2/metabolismo , Sítios de Ligação , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Mutação , Conformação Proteica , Domínios Proteicos , Estabilidade Proteica , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/química
15.
Biochim Biophys Acta Proteins Proteom ; 1869(10): 140685, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34216797

RESUMO

Selenoprotein W is widespread among pro- and eukaryotic organisms. It possesses antioxidant activity and plays pivotal roles in mammalian embryonic development and cellular functions. A very simple, prototypical selenoprotein W is SelW1 from Chlamydomonas. The U14C mutant of SelW1 was isolated and biophysically characterized. It contains an intramolecular disulfide bond and is thermally stable up to 70 °C. NMR resonance assignment of reduced and oxidized SelW1 showed that SelW1 adopts a thioredoxin fold. Interestingly, both forms show two additional sets of resonance for amino acid residues near the termini and have basically identical dynamic behavior. Since SelW1 from Chlamydomonas resembles the ancestor of mammalian selenoproteins in certain aspects, this study lays the basis for future characterization of SelW1 function and possible interaction partners.


Assuntos
Chlamydomonas reinhardtii/metabolismo , Mutação , Selenoproteína W/química , Selenoproteína W/metabolismo , Proteínas de Algas/química , Proteínas de Algas/genética , Proteínas de Algas/metabolismo , Chlamydomonas reinhardtii/química , Chlamydomonas reinhardtii/genética , Dissulfetos/química , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Oxirredução , Estabilidade Proteica , Estrutura Secundária de Proteína , Selenoproteína W/genética , Termodinâmica
16.
Fish Shellfish Immunol ; 116: 84-90, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34214656

RESUMO

Viral hemorrhagic septicemia virus (VHSV) causes severe mortality among more than 90 fish species. The 11 kb viral genome encodes six proteins including nonvirion protein (NV). In previous study, we reported that NV gene variations of VHSV decrease cellular energy metabolism. Among several NV mutant proteins, NV-S56L showed the highest cellular energy deprivation. Based on this finding, we further examined a molecular mechanism of one amino acid (S56L) change on differential cellular dysregulation. In the fish cells, the NV-S56L protein showed an increased level of cellular expression than normal and other mutant NV proteins without change of mRNA expression. Using cycloheximide treatment for exclude de novo NV protein expression, NV-S56L had an extensive half-life of intracellular protein. The proteasome inhibitor, MG-132, treatment recovered the all NV protein levels. The ubiquitination of NV was increased in the treatment of MG132 via inhibition of the ubiquitin/proteasome system process. Finally, increased protein stability of NV-S56L led to downregulation of NF-κB response immune gene expression. These results indicate that the prolonged protein stabilization of NV protein variant (NV-S56L) increases its pathological duration and might eventually lead to high virulence activity in the host fish cell.


Assuntos
Septicemia Hemorrágica Viral , Novirhabdovirus/genética , Proteínas Virais/genética , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , Peixes , Expressão Gênica/imunologia , Septicemia Hemorrágica Viral/genética , Septicemia Hemorrágica Viral/imunologia , Estabilidade Proteica
17.
Langmuir ; 37(28): 8474-8485, 2021 07 20.
Artigo em Inglês | MEDLINE | ID: mdl-34236863

RESUMO

Protein stability and performance in various natural and artificial systems incorporating many other macromolecules for therapeutic, diagnostic, sensor, and biotechnological applications attract increasing interest with the expansion of these technologies. Here we address the catalytic activity of lysozyme protein (LYZ) in the presence of a polyethylene glycol (PEG) crowder in a broad range of concentrations and temperatures in aqueous solutions of two different molecular mass PEG samples (Mw = 3350 and 10000 g/mol). The phase behavior of PEG-protein solutions is examined by using dynamic light scattering (DLS) and small-angle X-ray scattering (SAXS), while the enzyme denaturing is monitored by using an activity assay (AS) and circular dichroism (CD) spectroscopy. Molecular dynamic (MD) simulations are used to illustrate the effect of PEG concentration on protein stability at high temperatures. The results demonstrate that LYZ residual activity after 1 h incubation at 80 °C is improved from 15% up to 55% with the addition of PEG. The improvement is attributed to two underlying mechanisms. (i) Primarily, the stabilizing effect is due to the suppression of the enzyme aggregation because of the stronger PEG-protein interactions caused by the increased hydrophobicity of PEG and lysozyme at elevated temperatures. (ii) The MD simulations showed that the addition of PEG to some degree stabilizes the secondary structures of the enzyme by delaying unfolding at elevated temperatures. The more pronounced effect is observed with an increase in PEG concentration. This trend is consistent with CD and AS experimental results, where the thermal stability is strengthened with increasing of PEG concentration and molecular mass. The results show that the highest stabilizing effect is approached at the critical overlap concentration of PEG.


Assuntos
Polietilenoglicóis , Estabilidade Enzimática , Peso Molecular , Estabilidade Proteica , Espalhamento a Baixo Ângulo , Difração de Raios X
18.
BMC Bioinformatics ; 22(Suppl 7): 345, 2021 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-34225665

RESUMO

BACKGROUND: Despite decades on developing dedicated Web tools, it is still difficult to predict correctly the changes of the thermodynamic stability of proteins caused by mutations. Here, we assessed the reliability of five recently developed Web tools, in order to evaluate the progresses in the field. RESULTS: The results show that, although there are improvements in the field, the assessed predictors are still far from ideal. Prevailing problems include the bias towards destabilizing mutations, and, in general, the results are unreliable when the mutation causes a ΔΔG within the interval ± 0.5 kcal/mol. We found that using several predictors and combining their results into a consensus is a rough, but effective way to increase reliability of the predictions. CONCLUSIONS: We suggest all developers to consider in their future tools the usage of balanced data sets for training of predictors, and all users to combine the results of multiple tools to increase the chances of having correct predictions about the effect of mutations on the thermodynamic stability of a protein.


Assuntos
Proteínas , Mutação , Estabilidade Proteica , Proteínas/genética , Reprodutibilidade dos Testes , Termodinâmica
19.
Nat Commun ; 12(1): 4536, 2021 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-34315872

RESUMO

Despite the substantial impact of post-translational modifications on programmed cell death 1 ligand 1 (PD-L1), its importance in therapeutic resistance in pancreatic cancer remains poorly defined. Here, we demonstrate that never in mitosis gene A-related kinase 2 (NEK2) phosphorylates PD-L1 to maintain its stability, causing PD-L1-targeted pancreatic cancer immunotherapy to have poor efficacy. We identify NEK2 as a prognostic factor in immunologically "hot" pancreatic cancer, involved in the onset and development of pancreatic tumors in an immune-dependent manner. NEK2 deficiency results in the suppression of PD-L1 expression and enhancement of lymphocyte infiltration. A NEK binding motif (F/LXXS/T) is identified in the glycosylation-rich region of PD-L1. NEK2 interacts with PD-L1, phosphorylating the T194/T210 residues and preventing ubiquitin-proteasome pathway-mediated degradation of PD-L1 in ER lumen. NEK2 inhibition thereby sensitizes PD-L1 blockade, synergically enhancing the anti-pancreatic cancer immune response. Together, the present study proposes a promising strategy for improving the effectiveness of pancreatic cancer immunotherapy.


Assuntos
Antígeno B7-H1/metabolismo , Imunidade , Quinases Relacionadas a NIMA/antagonistas & inibidores , Neoplasias Pancreáticas/imunologia , Adenocarcinoma/genética , Adenocarcinoma/imunologia , Adenocarcinoma/patologia , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/imunologia , Carcinoma Ductal Pancreático/patologia , Linhagem Celular Tumoral , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Camundongos Nus , Pessoa de Meia-Idade , Modelos Biológicos , Quinases Relacionadas a NIMA/deficiência , Quinases Relacionadas a NIMA/genética , Quinases Relacionadas a NIMA/metabolismo , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Fosforilação , Fosfosserina/metabolismo , Prognóstico , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica , Estabilidade Proteica , Proteólise , Ubiquitinação
20.
Nat Commun ; 12(1): 4551, 2021 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-34315879

RESUMO

Cornelia de Lange syndrome (CdLS) is a rare disease affecting multiple organs and systems during development. Mutations in the cohesin loader, NIPBL/Scc2, were first described and are the most frequent in clinically diagnosed CdLS patients. The molecular mechanisms driving CdLS phenotypes are not understood. In addition to its canonical role in sister chromatid cohesion, cohesin is implicated in the spatial organization of the genome. Here, we investigate the transcriptome of CdLS patient-derived primary fibroblasts and observe the downregulation of genes involved in development and system skeletal organization, providing a link to the developmental alterations and limb abnormalities characteristic of CdLS patients. Genome-wide distribution studies demonstrate a global reduction of NIPBL at the NIPBL-associated high GC content regions in CdLS-derived cells. In addition, cohesin accumulates at NIPBL-occupied sites at CpG islands potentially due to reduced cohesin translocation along chromosomes, and fewer cohesin peaks colocalize with CTCF.


Assuntos
Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Síndrome de Cornélia de Lange/genética , Genoma Humano , Transcriptoma/genética , Diferenciação Celular/genética , Cromatina/metabolismo , Fibroblastos/metabolismo , Fibroblastos/patologia , Humanos , Estabilidade Proteica
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