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1.
Food Chem ; 398: 133874, 2023 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-35964567

RESUMO

The influence of ultrasound-assisted immersion freezing (UF), immersion freezing (IF), and air freezing (AF) on the protein oxidation, structure, and thermal stability of chicken breast during frozen storage was evaluated in this study. Compared to IF and AF samples, the UF samples had a lower carbonyl content, dityrosine content, and surface hydrophobicity of myofibrillar protein (MP) (P < 0.05), as well as a higher free amino group content and total and reactive sulfhydryl content (P < 0.05). Moreover, UF significantly delayed the deterioration of protein secondary and tertiary structures and the decrease in protein thermal stability during frozen storage (P < 0.05). Additionally, the UF samples at 180 days had similar protein structures and quality characteristics to the IF samples at 90 days or the AF samples at 60 days. Overall, UF treatment can effectively retard protein oxidation, protein structure deterioration, and protein thermal stability loss caused by frozen storage.


Assuntos
Galinhas , Proteínas , Animais , Congelamento , Interações Hidrofóbicas e Hidrofílicas , Estabilidade Proteica
2.
Sci Rep ; 12(1): 14923, 2022 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-36056157

RESUMO

TAR DNA-binding protein 43 kDa (TDP-43), a nuclear protein, plays an important role in the molecular pathogenesis of amyotrophic lateral sclerosis (ALS). The long-disordered C-terminal region (CTR) of TDP-43 is known to be aggregation-prone and a hotspot for ALS mutations, so elucidation of the physiological function of CTR will provide insights into the pathogenesis of ALS. The CTR has two Gly, aromatic, and Ser-rich (GaroS) segments and an amyloidogenic core divided into a hydrophobic patch (HP) and a Gln/Asn (Q/N)-rich segment. Although TDP-43 lacking the CTR is known to be unstable, as observed in knock-in mice, it is unclear which of these segments contributes to the stability of TDP-43. Here, we generated 12 mouse lines lacking the various sub-regions of CTR by genome editing and compared the embryonic lethality of homozygotes, and protein and mRNA expression levels of TDP-43. We demonstrated the functional diversity of the four segments of CTR, finding that the presence of the Q/N-rich segment greatly restored the protein stability of TDP-43. In addition, we found that the second GaroS deletion did not affect protein stability and mouse development.


Assuntos
Proteínas de Ligação a DNA/química , Estabilidade Proteica , Esclerose Amiotrófica Lateral/metabolismo , Animais , Proteínas de Ligação a DNA/metabolismo , Camundongos , Mutação
3.
Int J Mol Sci ; 23(17)2022 Sep 04.
Artigo em Inglês | MEDLINE | ID: mdl-36077536

RESUMO

Cytochrome P450 oxidoreductase (POR) is the redox partner of steroid and drug-metabolising cytochromes P450 located in the endoplasmic reticulum. Mutations in POR cause a broad range of metabolic disorders. The POR variant rs17853284 (P228L), identified by genome sequencing, has been linked to lower testosterone levels and reduced P450 activities. We expressed the POR wild type and the P228L variant in bacteria, purified the proteins, and performed protein stability and catalytic functional studies. Variant P228L affected the stability of the protein as evidenced by lower unfolding temperatures and higher sensitivity to urea denaturation. A significant decline in the rate of electron transfer to cytochrome c and thiazolyl blue tetrazolium (MTT) was observed with POR P228L, while activities of CYP3A4 were reduced by 25% and activities of CYP3A5 and CYP2C9 were reduced by more than 40% compared with WT POR. The 17,20 lyase activity of CYP17A1, responsible for the production of the main androgen precursor dehydroepiandrosterone, was reduced to 27% of WT in the presence of the P228L variant of POR. Based on in silico and in vitro studies, we predict that the change of proline to leucine may change the rigidity of the protein, causing conformational changes in POR, leading to altered electron transfer to redox partners. A single amino acid change can affect protein stability and cause a severe reduction in POR activity. Molecular characterisation of individual POR mutations is crucial for a better understanding of the impact on different redox partners of POR.


Assuntos
NADPH-Ferri-Hemoproteína Redutase , Esteroides , Mutação , NADPH-Ferri-Hemoproteína Redutase/genética , NADPH-Ferri-Hemoproteína Redutase/metabolismo , Estabilidade Proteica , Testosterona
4.
Molecules ; 27(16)2022 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-36014329

RESUMO

Due to the growing demand for patient-friendly subcutaneous dosage forms, the ability to increasing protein solubility and stability in formulations to deliver on the required high protein concentrations is crucial. A common approach to ensure protein solubility and stability in high concentration protein formulations is the addition of excipients such as sugars, amino acids, surfactants, approved by the Food and Drug Administration. In a best-case scenario, these excipients fulfil multiple demands simultaneously, such as increasing long-term stability of the formulation, reducing protein adsorption on surfaces/interfaces, and stabilizing the protein against thermal or mechanical stress. 2-Hydroxylpropyl-ß-cyclodextrin (derivative of ß-cyclodextrin) holds this potential, but has not yet been sufficiently investigated for use in protein formulations. Within this work, we have systematically investigated the relevant molecular interactions to identify the potential of Kleptose®HPB (2-hydroxylpropyl-ß-cyclodextrin from Roquette Freres, Lestrem, France) as "multirole" excipient within liquid protein formulations. Based on our results three factors determine the influence of Kleptose®HPB on protein formulation stability: (1) concentration of Kleptose®HPB, (2) protein type and protein concentration, and (3) quality of the protein formulation. Our results not only contribute to the understanding of the relevant interactions but also enable the target-oriented use of Kleptose®HPB within formulation design.


Assuntos
Excipientes , beta-Ciclodextrinas , Estabilidade de Medicamentos , Excipientes/química , Humanos , Estabilidade Proteica , Proteínas/química , Solubilidade , beta-Ciclodextrinas/química
5.
BMC Bioinformatics ; 23(Suppl 10): 352, 2022 Aug 23.
Artigo em Inglês | MEDLINE | ID: mdl-35999491

RESUMO

BACKGROUND: The stability of protein sequence structure plays an important role in the prevention and treatment of diseases. RESULTS: In this paper, particle swarm optimization and tabu search are combined to propose a new method for protein structure prediction. The experimental results show that: for four groups of artificial protein sequences with different lengths, this method obtains the lowest potential energy value and stable structure prediction results, and the effect is obviously better than the other two comparison methods. Taking the first group of protein sequences as an example, our method improves the prediction of minimum potential energy by 127% and 7% respectively. CONCLUSIONS: Therefore, the method proposed in this paper is more suitable for the prediction of protein structural stability.


Assuntos
Algoritmos , Proteínas , Sequência de Aminoácidos , Estabilidade Proteica , Proteínas/química
6.
Biomed Res Int ; 2022: 7736485, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35978633

RESUMO

Leptin is a polypeptide hormone produced in the adipose tissue and governs many processes in the body. Recently, polymorphisms in the LEP gene revealed a significant change in body weight regulation, energy balance, food intake, and reproductive hormone secretion. This study considers its crucial role in the regulation of the economically important traits of sheep. Several computational tools, including SIFT, Predict SNP2, SNAP2, and PROVEAN, have been used to screen out the deleterious nsSNPs. Following the screening of 11 nsSNPs in the sheep genome, 5 nsSNPs, T86M (C → T), D98N (G → A), N136T (A → C), R142Q (G → A), and P157Q (C → A), were predicted to have a significant deleterious effect on the LEP protein function, leading to phenotypic difference. The analysis of proteins' stability change due to amino acid substitution using the I-stable, SDM, and DynaMut consistently confirmed that three nsSNPs (T86M (C → T), D98N (G → A), and P157Q (C → A)) increased protein stability. It is suggested that these three nsSNPs may enhance the evolvability of LEP protein, which is vital for the evolutionary adaptation of sheep. Our findings demonstrate that the five nsSNPs reported in this study might be responsible for sheep's structural and functional modifications of LEP protein. This is the first comprehensive report on the sheep LEP gene. It narrow downs the candidate nsSNPs for in vitro experiments to facilitate the development of reliable molecular markers for associated traits.


Assuntos
Biologia Computacional , Polimorfismo de Nucleotídeo Único , Substituição de Aminoácidos , Animais , Simulação por Computador , Polimorfismo de Nucleotídeo Único/genética , Estabilidade Proteica , Ovinos/genética
7.
Proc Natl Acad Sci U S A ; 119(33): e2207275119, 2022 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-35939685

RESUMO

The circadian clock is a timekeeping, homeostatic system that temporally coordinates all major cellular processes. The function of the circadian clock is compensated in the face of variable environmental conditions ranging from normal to stress-inducing conditions. Salinity is a critical environmental factor affecting plant growth, and plants have evolved the SALT OVERLY SENSITIVE (SOS) pathway to acquire halotolerance. However, the regulatory systems for clock compensation under salinity are unclear. Here, we show that the plasma membrane Na+/H+ antiporter SOS1 specifically functions as a salt-specific circadian clock regulator via GIGANTEA (GI) in Arabidopsis thaliana. SOS1 directly interacts with GI in a salt-dependent manner and stabilizes this protein to sustain a proper clock period under salinity conditions. SOS1 function in circadian clock regulation requires the salt-mediated secondary messengers cytosolic free calcium and reactive oxygen species, pointing to a distinct regulatory role for SOS1 in addition to its function as a transporter to maintain Na+ homeostasis. Our results demonstrate that SOS1 maintains homeostasis of the salt response under high or daily fluctuating salt levels. These findings highlight the genetic capacity of the circadian clock to maintain timekeeping activity over a broad range of salinity levels.


Assuntos
Proteínas de Arabidopsis , Arabidopsis , Ritmo Circadiano , Estresse Salino , Trocadores de Sódio-Hidrogênio , Arabidopsis/genética , Arabidopsis/fisiologia , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Estabilidade Proteica , Trocadores de Sódio-Hidrogênio/genética , Trocadores de Sódio-Hidrogênio/metabolismo
8.
Biochem Biophys Res Commun ; 627: 97-102, 2022 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-36030658

RESUMO

A nuclear serine/threonine kinase homeodomain-interacting protein kinase 2 (HIPK2) is a critical regulator of development and DNA damage response. HIPK2 can induce apoptosis under cellular stress conditions and thus its protein level is maintained low by constant proteasomal degradation. In the present study, we present evidence that TNF receptor-associated factor 2 (TRAF2) regulates the protein stability of HIPK2. Overexpression of TRAF2 decreased while its knockdown increased the HIPK2 protein level. The TRAF2-mediated decrease in HIPK2 protein expression was blocked by proteasomal inhibitor. In addition, TRAF2 decreased the protein half-life of HIPK2. We found that HIPK2 and TRAF2 co-immunoprecipitated. Interestingly, the co-immunoprecipitation was reduced while HIPK2 protein level increased following TNFα treatment, suggesting TNFα induced dissociation of TRAF2 from HIPK2 to accumulate HIPK2. Inhibition of HIPK2 partially suppressed TNFα-induced cell death, indicating that the accumulated HIPK2 may contribute to the TNFα-induced cell death. Our results suggest that TRAF2 can regulate proapoptotic function of HIPK2 by promoting proteasomal degradation.


Assuntos
Proteínas Serina-Treonina Quinases , Fator de Necrose Tumoral alfa , Apoptose , Proteínas Serina-Treonina Quinases/genética , Estabilidade Proteica , Fator 2 Associado a Receptor de TNF/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Ubiquitina-Proteína Ligases/metabolismo
9.
J Chem Inf Model ; 62(17): 4270-4282, 2022 Sep 12.
Artigo em Inglês | MEDLINE | ID: mdl-35973091

RESUMO

An essential step in engineering proteins and understanding disease-causing missense mutations is to accurately model protein stability changes when such mutations occur. Here, we developed a new sequence-based predictor for the protein stability (PROST) change (Gibb's free energy change, ΔΔG) upon a single-point missense mutation. PROST extracts multiple descriptors from the most promising sequence-based predictors, such as BoostDDG, SAAFEC-SEQ, and DDGun. RPOST also extracts descriptors from iFeature and AlphaFold2. The extracted descriptors include sequence-based features, physicochemical properties, evolutionary information, evolutionary-based physicochemical properties, and predicted structural features. The PROST predictor is a weighted average ensemble model based on extreme gradient boosting (XGBoost) decision trees and an extra-trees regressor; PROST is trained on both direct and hypothetical reverse mutations using the S5294 (S2647 direct mutations + S2647 inverse mutations). The parameters for the PROST model are optimized using grid searching with 5-fold cross-validation, and feature importance analysis unveils the most relevant features. The performance of PROST is evaluated in a blinded manner, employing nine distinct data sets and existing state-of-the-art sequence-based and structure-based predictors. This method consistently performs well on frataxin, S217, S349, Ssym, S669, Myoglobin, and CAGI5 data sets in blind tests and similarly to the state-of-the-art predictors for p53 and S276 data sets. When the performance of PROST is compared with the latest predictors such as BoostDDG, SAAFEC-SEQ, ACDC-NN-seq, and DDGun, PROST dominates these predictors. A case study of mutation scanning of the frataxin protein for nine wild-type residues demonstrates the utility of PROST. Taken together, these findings indicate that PROST is a well-suited predictor when no protein structural information is available. The source code of PROST, data sets, examples, and pretrained models along with how to use PROST are available at https://github.com/ShahidIqb/PROST and https://prost.erc.monash.edu/seq.


Assuntos
Mutação de Sentido Incorreto , Transferência Intratubária do Zigoto , Estabilidade Proteica , Proteínas/química , Software
10.
J Phys Chem Lett ; 13(33): 7741-7748, 2022 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-35969173

RESUMO

Extraordinarily stable protein and peptide structures are critically demanded in many applications. Typical approaches to enhance protein and peptide stability are strengthening certain interactions. Here, we develop a very different approach: stabilizing peptide structures through side-chain-locked knots. More specifically, a peptide core consists of a knot, which is prevented from unknotting and unfolding by large side chains of amino acids at knot boundaries. These side chains impose free energy barriers for unknotting. The free energy barriers are quantified using all-atom and coarse-grained simulations. The barriers become infinitely high for large side chains and tight knot cores, resulting in stable peptide structures, which never unfold unless one chemical bond is broken. The extraordinary stability is essentially kinetic stability. Our new approach lifts the thermodynamic restriction in designing peptide structures, provides extra freedom in selecting sequence and structural motifs that are thermodynamically unstable, and should expand the functionality of peptides. This work also provides a bottom-up understanding of how knotting enhances protein stability.


Assuntos
Peptídeos , Proteínas , Aminoácidos/química , Peptídeos/química , Estabilidade Proteica , Proteínas/química , Termodinâmica
11.
J Phys Chem Lett ; 13(34): 7980-7986, 2022 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-35984361

RESUMO

Using a combination of molecular dynamics simulation, dialysis experiments, and electronic circular dichroism measurements, we studied the solvation thermodynamics of proteins in two osmolyte solutions, trimethylamine N-oxide (TMAO) and betaine. We showed that existing force fields are unable to capture the solvation properties of the proteins lysozyme and ribonuclease T1 and that the inaccurate parametrization of protein-osmolyte interactions in these force fields promoted an unphysical strong thermal denaturation of the trpcage protein. We developed a novel force field for betaine (the KBB force field) which reproduces the experimental solution Kirkwood-Buff integrals and density. We further introduced appropriate scaling to protein-osmolyte interactions in both the betaine and TMAO force fields which led to successful reproduction of experimental protein-osmolyte preferential binding coefficients for lysozyme and ribonuclease T1 and prevention of the unphysical denaturation of trpcage in osmolyte solutions. Correct parametrization of protein-TMAO interactions also led to the stabilization of the collapsed conformations of a disordered elastin-like peptide, while the uncorrected parameters destabilized the collapsed structures. Our results establish that the thermodynamic stability of proteins in both betaine and TMAO solutions is governed by osmolyte exclusion from proteins.


Assuntos
Betaína , Muramidase , Metilaminas/química , Muramidase/metabolismo , Estabilidade Proteica , Ribonuclease T1/metabolismo , Soluções , Termodinâmica , Água/química
12.
J Chem Phys ; 157(5): 055101, 2022 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-35933209

RESUMO

The formation of the fibrillar structure of amyloid proteins/peptides is believed to be associated with neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis. Since the rate of aggregation can influence neurotoxicity, finding the key factors that control this rate is of paramount importance. It was recently found that the rate of protein aggregation is related to the mechanical stability of the fibrillar structure such that the higher the mechanical stability, the faster the fibril is formed. However, this conclusion was supported by a limited dataset. In this work, we expand the previous study to a larger dataset, including the wild type of Aß42 peptide and its 20 mutants, the aggregation rate of which was measured experimentally. By using all-atom steered molecular dynamics (SMD) simulations, we can assess the mechanical stability of the fibril structure, which is characterized by the rupture force, pulling work, and unbinding free energy barrier. Our result confirms that mechanical stability is indeed related to the aggregation rate. Since the estimation of the aggregation rate using all-atom simulations is almost forbidden by the current computational capabilities, our result is useful for predicting it based on information obtained from fast SMD simulations for fibrils.


Assuntos
Doença de Alzheimer , Agregados Proteicos , Doença de Alzheimer/metabolismo , Amiloide/química , Peptídeos beta-Amiloides/química , Humanos , Fenômenos Mecânicos , Simulação de Dinâmica Molecular , Fragmentos de Peptídeos/química , Estabilidade Proteica
13.
Nature ; 609(7925): 151-158, 2022 09.
Artigo em Inglês | MEDLINE | ID: mdl-35978186

RESUMO

Compelling evidence shows that brown and beige adipose tissue are protective against metabolic diseases1,2. PR domain-containing 16 (PRDM16) is a dominant activator of the biogenesis of beige adipocytes by forming a complex with transcriptional and epigenetic factors and is therefore an attractive target for improving metabolic health3-8. However, a lack of knowledge surrounding the regulation of PRDM16 protein expression hampered us from selectively targeting this transcriptional pathway. Here we identify CUL2-APPBP2 as the ubiquitin E3 ligase that determines PRDM16 protein stability by catalysing its polyubiquitination. Inhibition of CUL2-APPBP2 sufficiently extended the half-life of PRDM16 protein and promoted beige adipocyte biogenesis. By contrast, elevated CUL2-APPBP2 expression was found in aged adipose tissues and repressed adipocyte thermogenesis by degrading PRDM16 protein. Importantly, extended PRDM16 protein stability by adipocyte-specific deletion of CUL2-APPBP2 counteracted diet-induced obesity, glucose intolerance, insulin resistance and dyslipidaemia in mice. These results offer a cell-autonomous route to selectively activate the PRDM16 pathway in adipose tissues.


Assuntos
Tecido Adiposo Bege , Proteínas de Ligação a DNA , Fatores de Transcrição , Adipócitos Bege/metabolismo , Tecido Adiposo Bege/metabolismo , Tecido Adiposo Marrom/metabolismo , Animais , Proteínas Culina , Proteínas de Ligação a DNA/metabolismo , Dislipidemias , Intolerância à Glucose , Resistência à Insulina , Camundongos , Obesidade , Estabilidade Proteica , Termogênese/fisiologia , Fatores de Transcrição/metabolismo , Ubiquitinação
14.
Genes (Basel) ; 13(8)2022 08 08.
Artigo em Inglês | MEDLINE | ID: mdl-36011324

RESUMO

Myeloperoxidase (MPO) is a heme peroxidase with microbicidal properties. MPO plays a role in the host's innate immunity by producing reactive oxygen species inside the cell against foreign organisms. However, there is little functional evidence linking missense mutations to human diseases. We utilized in silico saturation mutagenesis to generate and analyze the effects of 10,811 potential missense mutations on MPO stability. Our results showed that ~71% of the potential missense mutations destabilize MPO, and ~8% stabilize the MPO protein. We showed that G402W, G402Y, G361W, G402F, and G655Y would have the highest destabilizing effect on MPO. Meanwhile, D264L, G501M, D264H, D264M, and G501L have the highest stabilization effect on the MPO protein. Our computational tool prediction showed the destabilizing effects in 13 out of 14 MPO missense mutations that cause diseases in humans. We also analyzed putative post-translational modification (PTM) sites on the MPO protein and mapped the PTM sites to disease-associated missense mutations for further analysis. Our analysis showed that R327H associated with frontotemporal dementia and R548W causing generalized pustular psoriasis are near these PTM sites. Our results will aid further research into MPO as a biomarker for human complex diseases and a candidate for drug target discovery.


Assuntos
Mutação de Sentido Incorreto , Peroxidase/genética , Humanos , Mutagênese , Neutrófilos/metabolismo , Peroxidase/metabolismo , Estabilidade Proteica
15.
Molecules ; 27(14)2022 Jul 12.
Artigo em Inglês | MEDLINE | ID: mdl-35889332

RESUMO

Despite the recent advances in the prediction of protein structures by deep neutral networks, the elucidation of protein-folding mechanisms remains challenging. A promising theory for describing protein folding is a coarse-grained statistical mechanical model called the Wako-Saitô-Muñoz-Eaton (WSME) model. The model can calculate the free-energy landscapes of proteins based on a three-dimensional structure with low computational complexity, thereby providing a comprehensive understanding of the folding pathways and the structure and stability of the intermediates and transition states involved in the folding reaction. In this review, we summarize previous and recent studies on protein folding and dynamics performed using the WSME model and discuss future challenges and prospects. The WSME model successfully predicted the folding mechanisms of small single-domain proteins and the effects of amino-acid substitutions on protein stability and folding in a manner that was consistent with experimental results. Furthermore, extended versions of the WSME model were applied to predict the folding mechanisms of multi-domain proteins and the conformational changes associated with protein function. Thus, the WSME model may contribute significantly to solving the protein-folding problem and is expected to be useful for predicting protein folding, stability, and dynamics in basic research and in industrial and medical applications.


Assuntos
Dobramento de Proteína , Proteínas , Cinética , Modelos Moleculares , Estabilidade Proteica , Proteínas/química , Termodinâmica
16.
Proc Natl Acad Sci U S A ; 119(30): e2114119119, 2022 07 26.
Artigo em Inglês | MEDLINE | ID: mdl-35867819

RESUMO

Alphaviruses can cause severe human arthritis and encephalitis. During virus infection, structural changes of viral glycoproteins in the acidified endosome trigger virus-host membrane fusion for delivery of the capsid core and RNA genome into the cytosol to initiate virus translation and replication. However, mechanisms by which E1 and E2 glycoproteins rearrange in this process remain unknown. Here, we investigate prefusion cryoelectron microscopy (cryo-EM) structures of eastern equine encephalitis virus (EEEV) under acidic conditions. With models fitted into the low-pH cryo-EM maps, we suggest that E2 dissociates from E1, accompanied by a rotation (∼60°) of the E2-B domain (E2-B) to expose E1 fusion loops. Cryo-EM reconstructions of EEEV bound to a protective antibody at acidic and neutral pH suggest that stabilization of E2-B prevents dissociation of E2 from E1. These findings reveal conformational changes of the glycoprotein spikes in the acidified host endosome. Stabilization of E2-B may provide a strategy for antiviral agent development.


Assuntos
Vírus da Encefalite Equina do Leste , Proteínas do Envelope Viral , Antivirais/química , Antivirais/farmacologia , Microscopia Crioeletrônica , Vírus da Encefalite Equina do Leste/química , Concentração de Íons de Hidrogênio , Conformação Proteica , Estabilidade Proteica/efeitos dos fármacos , Proteínas do Envelope Viral/química
17.
Biomolecules ; 12(7)2022 07 10.
Artigo em Inglês | MEDLINE | ID: mdl-35883520

RESUMO

In this study, we combined all-atom MD simulations, the ensemble-based mutational scanning of protein stability and binding, and perturbation-based network profiling of allosteric interactions in the SARS-CoV-2 spike complexes with a panel of cross-reactive and ultra-potent single antibodies (B1-182.1 and A23-58.1) as well as antibody combinations (A19-61.1/B1-182.1 and A19-46.1/B1-182.1). Using this approach, we quantify the local and global effects of mutations in the complexes, identify protein stability centers, characterize binding energy hotspots, and predict the allosteric control points of long-range interactions and communications. Conformational dynamics and distance fluctuation analysis revealed the antibody-specific signatures of protein stability and flexibility of the spike complexes that can affect the pattern of mutational escape. A network-based perturbation approach for mutational profiling of allosteric residue potentials revealed how antibody binding can modulate allosteric interactions and identified allosteric control points that can form vulnerable sites for mutational escape. The results show that the protein stability and binding energetics of the SARS-CoV-2 spike complexes with the panel of ultrapotent antibodies are tolerant to the effect of Omicron mutations, which may be related to their neutralization efficiency. By employing an integrated analysis of conformational dynamics, binding energetics, and allosteric interactions, we found that the antibodies that neutralize the Omicron spike variant mediate the dominant binding energy hotpots in the conserved stability centers and allosteric control points in which mutations may be restricted by the requirements of the protein folding stability and binding to the host receptor. This study suggested a mechanism in which the patterns of escape mutants for the ultrapotent antibodies may not be solely determined by the binding interaction changes but are associated with the balance and tradeoffs of multiple local and global factors, including protein stability, binding affinity, and long-range interactions.


Assuntos
COVID-19 , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus/genética , COVID-19/genética , Humanos , Conformação Molecular , Mutação , Ligação Proteica , Estabilidade Proteica , SARS-CoV-2/genética
18.
Clin Transl Med ; 12(7): e871, 2022 07.
Artigo em Inglês | MEDLINE | ID: mdl-35853101

RESUMO

The stability of a protein, as well as its function and versatility, can be enhanced through oligomerization. KITENIN (KAI1 C-terminal interacting tetraspanin) is known to promote the malignant progression of colorectal cancer (CRC). How KITENIN maintains its structural integrity and stability are largely unknown, however. Here we investigated the mechanisms regulating the stability of KITENIN with the aim of developing therapeutics blocking its oncogenic functions. We found that KITENIN formed a homo-oligomeric complex and that the intracellular C-terminal domain (KITENIN-CTD) was needed for this oligomerization. Expression of the KITENIN-CTD alone interfered with the formation of the KITENIN homodimer, and the amino acid sequence from 463 to 471 within the KITENIN-CTD was the most effective. This sequence coupled with a cell-penetrating peptide was named a KITENIN dimerization-interfering peptide (KDIP). We next studied the mechanisms by which KDIP affected the stability of KITENIN. The KITENIN-interacting protein myosin-X (Myo10), which has oncogenic activity in several cancers, functioned as an effector to stabilize the KITENIN homodimer in the cis formation. Treatment with KDIP resulted in the disintegration of the homodimer via downregulation of Myo10, which led to increased binding of RACK1 to the exposed RACK1-interacting motif (463-471 aa), and subsequent autophagy-dependent degradation of KITENIN and reduced CRC cell invasion. Intravenous injection of KDIP significantly reduced the tumour burden in a syngeneic mouse tumour model and colorectal liver metastasis in an intrasplenic hepatic metastasis model. Collectively, our present results provide a new cancer therapeutic peptide for blocking colorectal liver metastasis, which acts by inducing the downregulation of Myo10 and specifically targeting the stability of the oncogenic KITENIN protein.


Assuntos
Neoplasias Colorretais , Proteínas de Membrana , Peptídeos , Animais , Proteínas de Transporte/metabolismo , Linhagem Celular Tumoral , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Dimerização , Neoplasias Hepáticas Experimentais/metabolismo , Neoplasias Hepáticas Experimentais/secundário , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Camundongos , Miosinas/química , Miosinas/metabolismo , Proteínas Oncogênicas/química , Proteínas Oncogênicas/metabolismo , Peptídeos/farmacologia , Estabilidade Proteica/efeitos dos fármacos
19.
Int J Pharm ; 624: 121974, 2022 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-35787458

RESUMO

The high propensity of mannitol to crystallize in frozen solutions along with its high eutectic temperature enabling higher primary drying temperatures makes it a good bulking agent. In protein formulations, addition of a sugar (sucrose) that has the ability to remain amorphous throughout processing as well as storage is imperative to retain the protein in its native state. It is well known that in the presence of amorphous excipients and protein, mannitol can crystallize as a mixture of anhydrous polymorphs - α-, ß- and δ-forms and a hemihydrate form [mannitol hemihydrate (MHH); C6H14O6·0.5H2O]. The conditions of formation of MHH due to processing and formulation variables are well established in the literature. However, MHH's dehydration kinetics on storage and its impact on the stability of a protein has not been systematically evaluated. The overall objective was to identify conditions (temperature and humidity) at which MHH can dehydrate on storage and the consequences of the release of associated water on sucrose phase behavior and protein stability. In a mannitol-sucrose-protein lyophile, the purpose of this study was (i) to investigate the dehydration behavior of MHH (ii) to determine the influence of dehydration on sucrose crystallization and (iii) the effect of moisture released due to MHH dehydration on model protein (Bovine serum albumin, BSA or Human serum albumin, HSA) aggregation. MHH dehydration and sucrose crystallization was observed in cases where the relative humidity was ≥ 55% (open vials). A relative humidity of ≤ 33% RH prevented MHH dehydration while retaining sucrose amorphous. No protein aggregation was observed irrespective of presence of MHH or its dehydration.


Assuntos
Manitol , Sacarose , Varredura Diferencial de Calorimetria , Desidratação , Excipientes/química , Liofilização , Humanos , Manitol/química , Estabilidade Proteica , Soroalbumina Bovina , Sacarose/química
20.
Phys Chem Chem Phys ; 24(29): 17723-17743, 2022 Jul 27.
Artigo em Inglês | MEDLINE | ID: mdl-35839100

RESUMO

Dissecting the regulatory principles underlying function and activity of the SARS-CoV-2 spike protein at the atomic level is of paramount importance for understanding the mechanisms of virus transmissibility and immune escape. In this work, we introduce a hierarchical computational approach for atomistic modeling of allosteric mechanisms in the SARS-CoV-2 Omicron spike proteins and present evidence of a frustration-based allostery as an important energetic driver of the conformational changes and spike activation. By examining conformational landscapes and the residue interaction networks in the SARS-CoV-2 Omicron spike protein structures, we have shown that the Omicron mutational sites are dynamically coupled and form a central engine of the allosterically regulated spike machinery that regulates the balance and tradeoffs between conformational plasticity, protein stability, and functional adaptability. We have found that the Omicron mutational sites at the inter-protomer regions form regulatory hotspot clusters that control functional transitions between the closed and open states. Through perturbation-based modeling of allosteric interaction networks and diffusion analysis of communications in the closed and open spike states, we have quantified the allosterically regulated activation mechanism and uncover specific regulatory roles of the Omicron mutations. Atomistic reconstruction of allosteric communication pathways and kinetic modeling using Markov transient analysis reveal that the Omicron mutations form the inter-protomer electrostatic bridges that operate as a network of coupled regulatory switches that could control global conformational changes and signal transmission in the spike protein. The results of this study have revealed distinct and yet complementary roles of the Omicron mutation sites as a network of hotspots that enable allosteric modulation of structural stability and conformational changes which are central for spike activation and virus transmissibility.


Assuntos
COVID-19 , Glicoproteína da Espícula de Coronavírus , Regulação Alostérica , Humanos , Simulação de Dinâmica Molecular , Mutação , Conformação Proteica , Estabilidade Proteica , Subunidades Proteicas , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/metabolismo
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