Objectives:Cold-acclimated organisms accumulate low molecular weight organic solutes such as sugar alcohols and soluble sugars.This study aimed to compare the efficacy of five sugar alcohols and 14 soluble sugars in s...Objectives:Cold-acclimated organisms accumulate low molecular weight organic solutes such as sugar alcohols and soluble sugars.This study aimed to compare the efficacy of five sugar alcohols and 14 soluble sugars in stabilizing proteins under freezing,freeze-drying,and air-drying stresses.Materials and methods:Glucose-6-Phosphate Dehydrogenase(G6PD)was used as the model protein.G6PD solutions with or without sugar alcohols and or sugars were subjected to freezing,freeze-drying,and air-drying stresses.The recovery of G6PD activity was measured to evaluate the protective efficacy of these compounds.Results:Without stabilizers,freezing G6PD at-20℃ or-80℃ reduced enzyme activity by around 24%,while freeze-drying or air-drying reduced activity by 90%-95%.Among the five sugar alcohols tested,pinitol,quebrachitol and sorbitol stabilized G6PD,whereas mannitol and myo-inositol destabilized it.Among 14 soluble sugars,trehalose and raffinose showed slightly lower enzyme recovery after repeated freeze-thaw cycles at-20℃.Most soluble sugars(except arabinose and xylose)protected G6PD during freeze-drying,with di-,tri-,and oligosaccharides generally outperforming monosaccharides.During air-drying,lactose was ineffective,while arabinose,galactose,and xylose were detrimental.Conclusion:The study highlights the diverse mechanisms of sugar alcohols and sugars in protein stabilization under stress,offering insights for formulating stable protein-and cell-based drugs.展开更多
In the article“Silencing of the long non-coding RNA LINC00265 triggers autophagy and apoptosis in lung cancer by reducing protein stability of SIN3A oncogene”(Oncology Research.2024,Vol.32,No.7,pp.1185–1195.doi:10....In the article“Silencing of the long non-coding RNA LINC00265 triggers autophagy and apoptosis in lung cancer by reducing protein stability of SIN3A oncogene”(Oncology Research.2024,Vol.32,No.7,pp.1185–1195.doi:10.32604/or.2023.030771,https://www.techscience.com/or/v32n7/57163),an inadvertent error occurred during the compilation of Fig.3H.This needed corrections to ensure the accuracy and integrity of the data presented.展开更多
Subject Code:H16With the support by the National Natural Science Foundation of China,a collaborative study by the research groups led by Prof.Wang Chenji(王陈继)from Fudan University,Prof.Huang Haojie(黄浩杰)from Mayo...Subject Code:H16With the support by the National Natural Science Foundation of China,a collaborative study by the research groups led by Prof.Wang Chenji(王陈继)from Fudan University,Prof.Huang Haojie(黄浩杰)from Mayo Clinic and Sun Yinhao(孙颖浩)from the Second Military Medical University have展开更多
Protein A chromatography is a key technology in the industrial production of antibodies,and a variety of commercial protein A adsorbents are available in shelf.High stability and binding capacity of a protein A adsorb...Protein A chromatography is a key technology in the industrial production of antibodies,and a variety of commercial protein A adsorbents are available in shelf.High stability and binding capacity of a protein A adsorbent are two key issues for successful practice of protein A chromatography.Earlier versions of protein A adsorbents ever exhibited serious fragility to typical cleaning-in-place protocols(e.g.washing with sodium hydroxide solution),and suffered from low binding capacity,harsh elution,ligand leakage and other problems involved in industrial applications.During the last three decades,various techniques and approaches have been applied in the improvement of chemical stability and enhancement of binding capacity of protein A-based ligands and adsorbents for antibody purifications.This mini-review focuses on the technical explorations in protein A-based affinity adsorbents,especially protein A-based ligands,including the efforts to increase the chemical stability by site-directed mutations and to improve the binding capacity by ligand polymerization and site-directed immobilization.Moreover,the efforts to develop short peptide ligands based on the structure of protein A,including the biomimetic design strategies and the synthesis of peptide-mixed mode hybrid ligands are discussed.These peptide and peptidebased hybrid ligands exhibit high affinity and selectivity to antibodies,but noteworthy differences in the binding mechanism of antibody from protein A.As a result,bound antibody to the ligands could be effectively eluted under mild conditions.Perspectives for the development of the protein A-based peptide ligands have been extensively discussed,suggesting that the ligands represent a direction for technological development of antibody purification.展开更多
Objective To investigate whether α-hemoglobin stabilizing protein (AHSP), the α-globin-specific molecular chaperone, is regulated by erythroid transcription factor NF-E2. Methods We established the stable cell line ...Objective To investigate whether α-hemoglobin stabilizing protein (AHSP), the α-globin-specific molecular chaperone, is regulated by erythroid transcription factor NF-E2. Methods We established the stable cell line with NF-E2p45 (the larger subunit of NF-E2) short hairpin RNA to silence its expression. Western blot, real-time polymerase chain reaction, and chromatin immunoprecipitation (ChIP) analysis were performed to detect the expression of AHSP, the histone modifications at AHSP gene locus, and the binding of GATA-1 at the AHSP promoter with NF-E2p45 deficiency. ChIP was also carried out in dimethyl sulfoxide (DMSO)-induced DS19 cells and estrogen-induced G1E-ER4 cells to examine NF-E2 binding to the AHSP gene locus and its changes during cell erythroid differentiation. Finally, luciferase assay was applied in HeLa cells transfected with AHSP promoter fragments to examine AHSP promoter activity in the presence of exogenous NF-E2p45. Results We found that AHSP expression was highly dependent on NF-E2p45. NF-E2 bound to the regions across AHSP gene locus in vivo, and the transcription of AHSP was transactivated by exogenous NF-E2p45. In addition, we observed the decrease of H3K4 trimethylation and GATA-1 occupancy at the AHSP gene locus in NF-E2p45-deficient cells. Restoration of GATA-1 in G1E-ER4 cells in turn led to increased DNA binding of NF-E2p45. Conclusion NF-E2 may play an important role in AHSP gene regulation, providing new insights into the molecular mechanisms underlying the erythroid-specific expression of AHSP as well as new possibilities for β-thalassemia treatment.展开更多
Freeze-thaw cycles impact the amount of aggregation observed in antibodies and Fc-fusion proteins. Various formulation strategies are used to mitigate the amount of aggregation that occurs upon putting a protein solut...Freeze-thaw cycles impact the amount of aggregation observed in antibodies and Fc-fusion proteins. Various formulation strategies are used to mitigate the amount of aggregation that occurs upon putting a protein solution through a freeze-thaw cycle. Additionally, low pH solutions cause native antibodies to unfold, which are prone to aggregate upon pH neutralization, There is great interest in the mechanism that causes therapeutic proteins to aggregate since aggregate species can cause unwanted immunogenicity in patients, Herein, an increase in aggregation is reported when the pH is adjusted from pH 3 up to a pH ranging from pH 4 to pH 7 during the thaw process of a frozen antibody solution, Raising the pH during the thaw process caused a significant increase in the percent aggregation observed. Two antibodies and one Fc-fusion protein were evaluated during the pH jump thaw process and similar effects were observed. The results provide a new tool to study the kinetics of therapeutic protein ag- gregation upon pH increase,展开更多
Light is an environmental signaling,whereas Aux/IAA proteins and Auxin Response Factors(ARFs)are regulators of auxin signalling.Aux/IAA proteins are unstable,and their degradation dependents on 26S ubiquitin-proteasom...Light is an environmental signaling,whereas Aux/IAA proteins and Auxin Response Factors(ARFs)are regulators of auxin signalling.Aux/IAA proteins are unstable,and their degradation dependents on 26S ubiquitin-proteasome and is promoted by Auxin.Auxin binds directly to a SCF-type ubiquitin-protein ligase,TIR1,facilitates the interaction between Aux/IAA proteins and TIR1,and then the degradation of Aux/IAA proteins.A few studies have reported that some ARFs are also unstable proteins,and their degradation is also mediated by 26S proteasome.In this study,by using of antibodies recognizing endogenous ARF7 proteins,we found that protein stability of ARF7 was affected by light.By expressing MYC tagged ARF activators in protoplasts,we found that degradation of ARF7 was inhibited by 26 proteasome inhibitors.In addition,at least ARF5 and ARF19 were also unstable proteins,and degradation of ARF5 via 26S proteasome was further confirmed by using stable transformed plants overexpressing ARF5 with a GUS tag.展开更多
We review the use of nuclear magnetic resonance(NMR)spectroscopy to assess the exchange of amide protons for deuterons(HDX)in efforts to understand how high concentration of cosolutes,especially macromolecules,affect ...We review the use of nuclear magnetic resonance(NMR)spectroscopy to assess the exchange of amide protons for deuterons(HDX)in efforts to understand how high concentration of cosolutes,especially macromolecules,affect the equilibrium thermodynamics of protein stability.HDX NMR is the only method that can routinely provide such data at the level of individual amino acids.We begin by discussing the properties of the protein systems required to yield equilibrium thermodynamic data and then review publications using osmolytes,sugars,denaturants,synthetic polymers,proteins,cytoplasm and in cells.展开更多
To detect retrospectively the phenotype and stability of the E-protein gene in Japanese Encephalitis （JE） virus strain SA14-14-2 used in the live-attenuated JE vaccine prepears, the viral titer was titrated by plaqu...To detect retrospectively the phenotype and stability of the E-protein gene in Japanese Encephalitis （JE） virus strain SA14-14-2 used in the live-attenuated JE vaccine prepears, the viral titer was titrated by plaque formation in BHK-21 cell cultures, and the neuro-virulence of viruses was assayed in mice with body weight of 12-14 g by intracerebral inoculation. Meanwhile, the total RNA of virus gene was extracted and amplified by RT-PCR with the designed primers, and then it was purified and cloned to the expression vector pGEM-T. The recombinant plasmid was purified and sequenced. It was found that the loss of viral titer of vaccines stored in -20℃ for longer than 10 years was less than 0.5 Lg PFU/ml. No mice inoculated intracerebrally showed signs of illness or even death. The size of plagues of the vaccine virus remained to be small, and the E genes of primary virus seed SA14-14-2 and the vaccines prepared at different years （1987-2001） were unchanged, in- cluding the 8 critical amino acid sites which were different from the parent wild virus strain SA14 and the related neuro-virulence. These results indicate that the genotypic and biological characteristics of the attenuated JE virus strain SA14-14-2 and its vaccines sion noted. prepared are quite stable without any reversion noted.展开更多
Predicting free energy changes(DDG)is essential for enhancing our understanding of protein evolution and plays a pivotal role in protein engineering and pharmaceutical development.While traditional methods offer valua...Predicting free energy changes(DDG)is essential for enhancing our understanding of protein evolution and plays a pivotal role in protein engineering and pharmaceutical development.While traditional methods offer valuable insights,they are often constrained by computational speed and reliance on biased training datasets.These constraints become particularly evident when aiming for accurate DDG predictions across a diverse array of protein sequences.Herein,we introduce Pythia,a self-supervised graph neural network specifically designed for zero-shot DDG predictions.Our comparative benchmarks demonstrate that Pythia outperforms other self-supervised pretraining models and force field-based approaches while also exhibiting competitive performance with fully supervised models.Notably,Pythia shows strong correlations and achieves a remarkable increase in computational speed of up to 105-fold.We further validated Pythia’s performance in predicting the thermostabilizing mutations of limonene epoxide hydrolase,leading to higher experimental success rates.This exceptional efficiency has enabled us to explore 26 million high-quality protein structures,marking a significant advancement in our ability to navigate the protein sequence space and enhance our understanding of the relationships between protein genotype and phenotype.In addition,we established a web server at https://pythia.wulab.xyz to allow users to easily perform such predictions.展开更多
In the originally published version of this article,the designation of one of the corresponding authors was inadvertently omitted.The article correctly listed Dr.Lichuan Gu as a corresponding author,but failed to desi...In the originally published version of this article,the designation of one of the corresponding authors was inadvertently omitted.The article correctly listed Dr.Lichuan Gu as a corresponding author,but failed to designate Dr.Sujuan Xu as an additional corresponding author.The correct corresponding authors should be.展开更多
Aggregation-induced emission(AIE)materials offer promising perspectives in disease diagnosis and therapeutics given their unique optical and photochemical properties.A key step toward translational applications for AI...Aggregation-induced emission(AIE)materials offer promising perspectives in disease diagnosis and therapeutics given their unique optical and photochemical properties.A key step toward translational applications for AIE materials is to systematically and vigorously evaluate their biosafety and biocompatibility.While previous studies focus on cellular viability and toxicity,the impact of AIE materials on detailed stress responses manifesting cellular fitness has been less explored.Herein,this work provides the first piece of evidence to support amphiphilic functionalization of AIE nanoparticles minimizes the deterioration on proteome stability and cellular protein homeostasis(proteostasis).To this end,four scaffolds of AIE molecules were prepared,further functionalized into eight nanoparticles with two amphiphilic shells respectively,and characterized for their physicochemical properties.Thermal shift assay quantitatively demonstrates that AIE materials after amphiphilic functionalization into nanoparticles enhance proteome thermodynamic stability and ameliorate proteome aggregation propensity in cellular lysate,echoed by cell viability and fractionation experiments.Intriguingly,poor polydispersity index(PDI)of functionalized nanoparticles exaggerates their retention and aggregation in the cell.Comparative proteomic analysis uncovers that amphiphilic functionalization of AIE materials can minimize the deterioration of cellular protein homeostasis network.Finally,vigorous interrogation of functionalized AIE nanoparticles in mice model reveals the complexity of factors affecting the biocompatibility profiles in vivo,including materials’size,PDI,and treatment frequencies.Overall,amphiphilic functionalization of AIE materials into nanoparticles is necessary to maintain proteome stability and balance cellular protein homeostasis.展开更多
Cytokinin is a critical growth regulator for various aspects of plant growth and development. In Arabidopsis, cytokinin signaling is mediated by a two-component system-based phosphorelay that transmits a signal from t...Cytokinin is a critical growth regulator for various aspects of plant growth and development. In Arabidopsis, cytokinin signaling is mediated by a two-component system-based phosphorelay that transmits a signal from the receptors, through histidine phosphotransfer proteins, to the downstream response regulators （ARRs）. Of these ARRs, type-A ARR genes, whose transcription can be rapidly induced by cytokinin, act as negative regulators of eytokinin signaling. However, because of functional redundancy, the function of type-A ARR genes in plant growth and development is not well understood by analyzing loss-of-function mutants. In this study, we performed a comparative functional study on all ten type-A ARR genes by analyzing transgenic plants overexpressing these ARR genes fused to a MYC epitope tag. Overexpression of ARR genes results in a variety of cytokinin-associated phenotypes. Notably, overexpression of different ARR transgenes causes diverse phenotypes, even between phylogenetically closely-related gene pairs, such as within the ARR3-ARR4 and ARR5-ARR6 pairs. We found that the accumulation of a subset of ARR proteins （ARR3, ARR5, ARR7, ARR16 and ARR17; possibly ARR8 and ARR15） is increased by MG132, a specific proteasomal inhibitor, indicating that stability of these proteins is regulated by proteasomal degradation. Moreover, similar to that of previously characterized ARR5, ARR6 and ARR7, stability of ARR16 and ARR17, possibly including ARR8 and ARR15, is regulated by cytokinin. These results suggest that type-A ARR proteins are regulated by a combinatorial mechanism involving both the cytokinin and proteasome pathways, thereby executing distinctive functions in plant growth and development.展开更多
The adsorption dynamics of a model protein （the human insulin） onto graphene surfaces with different sizes was investigated by molecular dynamics simulations. During the adsorption, it has different effect on the st...The adsorption dynamics of a model protein （the human insulin） onto graphene surfaces with different sizes was investigated by molecular dynamics simulations. During the adsorption, it has different effect on the stability of the model protein in the fixed and non-fixed graphene systems. The tertiary structure of the protein was destroyed or partially destroyed, and graphene surfaces shows the selective protection for some α-helices in non-fixed Systems but not in fixed systems by reason of the flexibility of graphene. As indicated by the interaction energy curve and trajectory animation, the conformation and orientation selection of the protein were induced by the properties and the texture of graphene surfaces. The knowledge of protein adsorption on graphene surfaces would be helpful to better understand stability of protein on graphene surfaces and facilitate potential applications of graphene in biotechnology.展开更多
Adenylate cyclase(AC)is the key enzyme that catalyzes the formation of cAMP from ATP.In this study,we discovered two novel class Ⅲ ACs with a halophilic property from Thermobifida halotolerans DSM 44931(ThAC)and Halo...Adenylate cyclase(AC)is the key enzyme that catalyzes the formation of cAMP from ATP.In this study,we discovered two novel class Ⅲ ACs with a halophilic property from Thermobifida halotolerans DSM 44931(ThAC)and Haloactinopolyspora alba DSM 45211(HaAC),respectively.The recombinant ThAC and HaAC were expressed in Escherichia coli with molecular weights of 36.1 and 36.0 kDa respectively.The presence of 2500 and 2200 mmolL^(-1)1 NaCl significantly enhanced the enzyme activities of ThAC and HaAC,with 22-fold and 7.4-fold higher activities compared to those without NaCl,respectively.Several divalent metal ions were found to activate the recombinant ACs to different extents,and the optimal metal ion was Mg^(2+)for both ThAC and HaAC with concentrations of 80 mmol·L^(-1) and 40 mmol·L^(-1) respectively.Purified ThAC and HaAC had the optimal specific activities((4.59±0.35)×10^(4) and(7.76±0.52)×10^(4) U·mg^(-1))and catalytic efficiency(4.47 and 5.30 L·mmol^(-1)·s^(-1))at 45℃ and 40℃ respectively,while the optimum pH of both two recombinant ACs was 10.0.This is the first report of the halophilic Class III ACs,which could make new contributions to explore and study ACs for further associated investigations.展开更多
Rhizopus oryzae lipase(ROL)was immobilized on the surface of silica coated amino modified CoFe_(2)O_4 nanoparticles and applied for biodiesel production.The results indicated more affinity of the ROL toward its substr...Rhizopus oryzae lipase(ROL)was immobilized on the surface of silica coated amino modified CoFe_(2)O_4 nanoparticles and applied for biodiesel production.The results indicated more affinity of the ROL toward its substrate upon immobilization,as revealed by a lower Km value for the immobilized ROL compared to its free counterpart.Intrinsic fluorescence spectroscopy indicated a lower intensity for ROL immobilized on CoFe_(2)O_4 nanoparticles.Besides,immobilized ROL steady state anisotropy measurements presented lower values,which implied assembly of ROL molecules on magnetic nanoparticles upon immobilization as well as their restricted rotation upon covalent attachment.Thermal stability analysis revealed improved activity at higher temperatures for the immobilized enzyme compared to its free counterpart.Accordingly,Pace analysis to determine protein thermal stability revealed preservation of the protein conformation in the presence of increasing temperatures upon immobilization on nanoparticles.Finally,ROL immobilized on CoFe_(2)O_(4)nanoparticles exhibited improved efficiency of biodiesel production in agreement with thermal activity profile.Therefore,the authors suggest application of the lipase molecules immobilized on CoFe_(2)O_(4)nanoparticles for more efficient biodiesel production.展开更多
When a protein is encapsulated into poly( DL -lactide-co-glycolide)(PLGA) microspheres by means of the double-emulsion method,the harsh microspheres formation process including ultrasonification,exposure to an organic...When a protein is encapsulated into poly( DL -lactide-co-glycolide)(PLGA) microspheres by means of the double-emulsion method,the harsh microspheres formation process including ultrasonification,exposure to an organic solvent and a polymer may cause the denaturation of the protein. In this study,we investigated the enzymatic activity change and the effect of the excipients on the stability of recombinant human Cu,Zn-superoxide dismutase(rhCu,Zn-SOD) during the emulsification. The specific activity recovery was found to be concentration dependent and the excipients involved such as PEG 600 and Tween 20,and trehalose were shown to increase the stability of rhCu,Zn-SOD. The protein structural integrity within the microspheres was analyzed by FTIR. The structure of rhCu,Zn-SOD within PLGA microspheres containing trehalose was found to be similar to that of the native solid state,whereas the protein encapsulated during the preparation in the absence of any excipient changed due to the possible hydrophobic interaction with the polymer. The results suggest that a rational stability strategy for protein to be encapsulated into microspheres should aim at different processes.展开更多
Obesity and cancer are two interrelated conditions of high epidemiological need, with studies showing that obesity is responsible for nearly 25% of the relative contribution to cancer incidence. Given the connection b...Obesity and cancer are two interrelated conditions of high epidemiological need, with studies showing that obesity is responsible for nearly 25% of the relative contribution to cancer incidence. Given the connection between these conditions, a drug that can operate on both obesity and cancer is highly desirable. Such a drug is accomplishablethrough the development of potent anti-angiogenesis agents due to the shared underlying role of angiogenesis in the development of both diseases. Prior research has demonstrated a key role of type-2 methionine aminopeptidase(Met AP2) for angiogenesis, which has led to the development of numerous of novel inhibitors. Several irreversible Met AP2 inhibitors have entered clinical trials without great success. Though this lack of success could be attributed to off-target adverse effects, the underlying causes remain unclear. More promising reversible inhibitors have been recently developed with excellent pre-clinical results. However, due to insufficient knowledge of the biological functions of N-terminal protein processing, it is hard to predict whether these novel inhibitors would successfully pass clinical trials and thereby benefit cancer and obesity patients. Significantly more efforts are needed to advance our understanding of the regulation of methionine aminopeptidases and the processes by which they govern the function of proteins.展开更多
Typically,inherited metabolic diseases arise from point mutations in genes encoding metabolic enzymes. Although some of these mutations directly affect amino acid residues in the active sites of these enzymes,the majo...Typically,inherited metabolic diseases arise from point mutations in genes encoding metabolic enzymes. Although some of these mutations directly affect amino acid residues in the active sites of these enzymes,the majority do not. It is now well accepted that the majority of these disease-associated mutations exert their effects through alteration of protein stability,which causes a reduction in enzymatic activity. This finding suggests a way to predict the severity of newly discovered mutations. In silico prediction of the effects of amino acid sequence alterations on protein stability often correlates with disease severity. However,no stability prediction tool is perfect and,in general,better results are obtained if the predictions from a variety of tools are combined and then interpreted. In addition to predicted alterations to stability,the degree of conservation of a particular residue can also be a factor which needs to be taken into account: alterations to highly conserved residues are more likely to be associated with severe forms of the disease. The approach has been successfully applied in a variety of inherited metabolic diseases,but further improvements are necessary to enable robust translation into clinically useful tools.展开更多
Recombinant human bone morphogenetic protein-2(rhBMP-2)is the predominant growth factor that effectively induces osteogenic differentiation in orthopedic procedures.However,the bioactivity and stability of rhBMP-2 are...Recombinant human bone morphogenetic protein-2(rhBMP-2)is the predominant growth factor that effectively induces osteogenic differentiation in orthopedic procedures.However,the bioactivity and stability of rhBMP-2 are intrinsically associated with its sequence,structure,and storage conditions.In this study,we successfully determined the amino acid sequence and protein secondary structure model of non-glycosylated rhBMP-2 expressed by an E.coli expression system through X-ray crystal structure analysis.Furthermore,we observed that acidic storage conditions enhanced the proliferative and osteoinductive activity of rhBMP-2.Although the osteogenic activity of non-glycosylated rhBMP-2 is relatively weaker compared to glycosylated rhBMP-2;however,this discrepancy can be mitigated by incorporating exogenous chaperone molecules.Overall,such information is crucial for rationalizing the design of stabilization methods and enhancing the bioactivity of rhBMP-2,which may also be applicable to other growth factors.展开更多
基金supported by a research grant from the National University of Singapore to WQS(RP-3960366)a collaborative research grant from Sichuan Zhongke Organ Co.Ltd(Chengdu,China).
文摘Objectives:Cold-acclimated organisms accumulate low molecular weight organic solutes such as sugar alcohols and soluble sugars.This study aimed to compare the efficacy of five sugar alcohols and 14 soluble sugars in stabilizing proteins under freezing,freeze-drying,and air-drying stresses.Materials and methods:Glucose-6-Phosphate Dehydrogenase(G6PD)was used as the model protein.G6PD solutions with or without sugar alcohols and or sugars were subjected to freezing,freeze-drying,and air-drying stresses.The recovery of G6PD activity was measured to evaluate the protective efficacy of these compounds.Results:Without stabilizers,freezing G6PD at-20℃ or-80℃ reduced enzyme activity by around 24%,while freeze-drying or air-drying reduced activity by 90%-95%.Among the five sugar alcohols tested,pinitol,quebrachitol and sorbitol stabilized G6PD,whereas mannitol and myo-inositol destabilized it.Among 14 soluble sugars,trehalose and raffinose showed slightly lower enzyme recovery after repeated freeze-thaw cycles at-20℃.Most soluble sugars(except arabinose and xylose)protected G6PD during freeze-drying,with di-,tri-,and oligosaccharides generally outperforming monosaccharides.During air-drying,lactose was ineffective,while arabinose,galactose,and xylose were detrimental.Conclusion:The study highlights the diverse mechanisms of sugar alcohols and sugars in protein stabilization under stress,offering insights for formulating stable protein-and cell-based drugs.
文摘In the article“Silencing of the long non-coding RNA LINC00265 triggers autophagy and apoptosis in lung cancer by reducing protein stability of SIN3A oncogene”(Oncology Research.2024,Vol.32,No.7,pp.1185–1195.doi:10.32604/or.2023.030771,https://www.techscience.com/or/v32n7/57163),an inadvertent error occurred during the compilation of Fig.3H.This needed corrections to ensure the accuracy and integrity of the data presented.
文摘Subject Code:H16With the support by the National Natural Science Foundation of China,a collaborative study by the research groups led by Prof.Wang Chenji(王陈继)from Fudan University,Prof.Huang Haojie(黄浩杰)from Mayo Clinic and Sun Yinhao(孙颖浩)from the Second Military Medical University have
基金This work was supported by the National Natural Science Foundation of China(Nos.21476166 and 21878221)the Foundation for Innovative Research Groups of the National Natural Science Foundation of China(No.21621004).
文摘Protein A chromatography is a key technology in the industrial production of antibodies,and a variety of commercial protein A adsorbents are available in shelf.High stability and binding capacity of a protein A adsorbent are two key issues for successful practice of protein A chromatography.Earlier versions of protein A adsorbents ever exhibited serious fragility to typical cleaning-in-place protocols(e.g.washing with sodium hydroxide solution),and suffered from low binding capacity,harsh elution,ligand leakage and other problems involved in industrial applications.During the last three decades,various techniques and approaches have been applied in the improvement of chemical stability and enhancement of binding capacity of protein A-based ligands and adsorbents for antibody purifications.This mini-review focuses on the technical explorations in protein A-based affinity adsorbents,especially protein A-based ligands,including the efforts to increase the chemical stability by site-directed mutations and to improve the binding capacity by ligand polymerization and site-directed immobilization.Moreover,the efforts to develop short peptide ligands based on the structure of protein A,including the biomimetic design strategies and the synthesis of peptide-mixed mode hybrid ligands are discussed.These peptide and peptidebased hybrid ligands exhibit high affinity and selectivity to antibodies,but noteworthy differences in the binding mechanism of antibody from protein A.As a result,bound antibody to the ligands could be effectively eluted under mild conditions.Perspectives for the development of the protein A-based peptide ligands have been extensively discussed,suggesting that the ligands represent a direction for technological development of antibody purification.
基金Supported by National Natural Science Foundation of China (30130026, U0632005, 30721063)National Basic Research Program of China (973 Program) (2011CB964803)+1 种基金National Laboratory of Medical Molecular Biology grant (2060204)Beijing municipal government grant (YB20081002301)
文摘Objective To investigate whether α-hemoglobin stabilizing protein (AHSP), the α-globin-specific molecular chaperone, is regulated by erythroid transcription factor NF-E2. Methods We established the stable cell line with NF-E2p45 (the larger subunit of NF-E2) short hairpin RNA to silence its expression. Western blot, real-time polymerase chain reaction, and chromatin immunoprecipitation (ChIP) analysis were performed to detect the expression of AHSP, the histone modifications at AHSP gene locus, and the binding of GATA-1 at the AHSP promoter with NF-E2p45 deficiency. ChIP was also carried out in dimethyl sulfoxide (DMSO)-induced DS19 cells and estrogen-induced G1E-ER4 cells to examine NF-E2 binding to the AHSP gene locus and its changes during cell erythroid differentiation. Finally, luciferase assay was applied in HeLa cells transfected with AHSP promoter fragments to examine AHSP promoter activity in the presence of exogenous NF-E2p45. Results We found that AHSP expression was highly dependent on NF-E2p45. NF-E2 bound to the regions across AHSP gene locus in vivo, and the transcription of AHSP was transactivated by exogenous NF-E2p45. In addition, we observed the decrease of H3K4 trimethylation and GATA-1 occupancy at the AHSP gene locus in NF-E2p45-deficient cells. Restoration of GATA-1 in G1E-ER4 cells in turn led to increased DNA binding of NF-E2p45. Conclusion NF-E2 may play an important role in AHSP gene regulation, providing new insights into the molecular mechanisms underlying the erythroid-specific expression of AHSP as well as new possibilities for β-thalassemia treatment.
文摘Freeze-thaw cycles impact the amount of aggregation observed in antibodies and Fc-fusion proteins. Various formulation strategies are used to mitigate the amount of aggregation that occurs upon putting a protein solution through a freeze-thaw cycle. Additionally, low pH solutions cause native antibodies to unfold, which are prone to aggregate upon pH neutralization, There is great interest in the mechanism that causes therapeutic proteins to aggregate since aggregate species can cause unwanted immunogenicity in patients, Herein, an increase in aggregation is reported when the pH is adjusted from pH 3 up to a pH ranging from pH 4 to pH 7 during the thaw process of a frozen antibody solution, Raising the pH during the thaw process caused a significant increase in the percent aggregation observed. Two antibodies and one Fc-fusion protein were evaluated during the pH jump thaw process and similar effects were observed. The results provide a new tool to study the kinetics of therapeutic protein ag- gregation upon pH increase,
文摘Light is an environmental signaling,whereas Aux/IAA proteins and Auxin Response Factors(ARFs)are regulators of auxin signalling.Aux/IAA proteins are unstable,and their degradation dependents on 26S ubiquitin-proteasome and is promoted by Auxin.Auxin binds directly to a SCF-type ubiquitin-protein ligase,TIR1,facilitates the interaction between Aux/IAA proteins and TIR1,and then the degradation of Aux/IAA proteins.A few studies have reported that some ARFs are also unstable proteins,and their degradation is also mediated by 26S proteasome.In this study,by using of antibodies recognizing endogenous ARF7 proteins,we found that protein stability of ARF7 was affected by light.By expressing MYC tagged ARF activators in protoplasts,we found that degradation of ARF7 was inhibited by 26 proteasome inhibitors.In addition,at least ARF5 and ARF19 were also unstable proteins,and degradation of ARF5 via 26S proteasome was further confirmed by using stable transformed plants overexpressing ARF5 with a GUS tag.
文摘We review the use of nuclear magnetic resonance(NMR)spectroscopy to assess the exchange of amide protons for deuterons(HDX)in efforts to understand how high concentration of cosolutes,especially macromolecules,affect the equilibrium thermodynamics of protein stability.HDX NMR is the only method that can routinely provide such data at the level of individual amino acids.We begin by discussing the properties of the protein systems required to yield equilibrium thermodynamic data and then review publications using osmolytes,sugars,denaturants,synthetic polymers,proteins,cytoplasm and in cells.
文摘To detect retrospectively the phenotype and stability of the E-protein gene in Japanese Encephalitis （JE） virus strain SA14-14-2 used in the live-attenuated JE vaccine prepears, the viral titer was titrated by plaque formation in BHK-21 cell cultures, and the neuro-virulence of viruses was assayed in mice with body weight of 12-14 g by intracerebral inoculation. Meanwhile, the total RNA of virus gene was extracted and amplified by RT-PCR with the designed primers, and then it was purified and cloned to the expression vector pGEM-T. The recombinant plasmid was purified and sequenced. It was found that the loss of viral titer of vaccines stored in -20℃ for longer than 10 years was less than 0.5 Lg PFU/ml. No mice inoculated intracerebrally showed signs of illness or even death. The size of plagues of the vaccine virus remained to be small, and the E genes of primary virus seed SA14-14-2 and the vaccines prepared at different years （1987-2001） were unchanged, in- cluding the 8 critical amino acid sites which were different from the parent wild virus strain SA14 and the related neuro-virulence. These results indicate that the genotypic and biological characteristics of the attenuated JE virus strain SA14-14-2 and its vaccines sion noted. prepared are quite stable without any reversion noted.
基金supported by the National Key R&D Program of China(grant no.2023YFA0916000)the National Natural Science Foundation of China(32225002,32170033,and 32422001)+2 种基金the Key Research Program of Frontier Sciences(ZDBS-LYSM014)the Biological Resources Program(KFJ-BRP-009 and KFJ-BRP-017-58)from the Chinese Academy of Sciences,the Informatization Plan of Chinese Academy of Sciences(CAS-WX2021SF-0111)the Youth Innovation Promotion Association CAS(2022086).
文摘Predicting free energy changes(DDG)is essential for enhancing our understanding of protein evolution and plays a pivotal role in protein engineering and pharmaceutical development.While traditional methods offer valuable insights,they are often constrained by computational speed and reliance on biased training datasets.These constraints become particularly evident when aiming for accurate DDG predictions across a diverse array of protein sequences.Herein,we introduce Pythia,a self-supervised graph neural network specifically designed for zero-shot DDG predictions.Our comparative benchmarks demonstrate that Pythia outperforms other self-supervised pretraining models and force field-based approaches while also exhibiting competitive performance with fully supervised models.Notably,Pythia shows strong correlations and achieves a remarkable increase in computational speed of up to 105-fold.We further validated Pythia’s performance in predicting the thermostabilizing mutations of limonene epoxide hydrolase,leading to higher experimental success rates.This exceptional efficiency has enabled us to explore 26 million high-quality protein structures,marking a significant advancement in our ability to navigate the protein sequence space and enhance our understanding of the relationships between protein genotype and phenotype.In addition,we established a web server at https://pythia.wulab.xyz to allow users to easily perform such predictions.
文摘In the originally published version of this article,the designation of one of the corresponding authors was inadvertently omitted.The article correctly listed Dr.Lichuan Gu as a corresponding author,but failed to designate Dr.Sujuan Xu as an additional corresponding author.The correct corresponding authors should be.
基金National Natural Science Foundation of China,Grant/Award Numbers:22107100,21907091,21725506China Postdoctoral Science Foundation,Grant/Award Number:2019M661138+2 种基金LiaoNing Revitalization Talents Program from the Liaoning province of China,Grant/Award Number:XLYC1907048Dalian Innovation Fund,Grant/Award Number:2020JJ26GX027Youth Innovation Promotion Association,CAS,Grant/Award Number:2020184。
文摘Aggregation-induced emission(AIE)materials offer promising perspectives in disease diagnosis and therapeutics given their unique optical and photochemical properties.A key step toward translational applications for AIE materials is to systematically and vigorously evaluate their biosafety and biocompatibility.While previous studies focus on cellular viability and toxicity,the impact of AIE materials on detailed stress responses manifesting cellular fitness has been less explored.Herein,this work provides the first piece of evidence to support amphiphilic functionalization of AIE nanoparticles minimizes the deterioration on proteome stability and cellular protein homeostasis(proteostasis).To this end,four scaffolds of AIE molecules were prepared,further functionalized into eight nanoparticles with two amphiphilic shells respectively,and characterized for their physicochemical properties.Thermal shift assay quantitatively demonstrates that AIE materials after amphiphilic functionalization into nanoparticles enhance proteome thermodynamic stability and ameliorate proteome aggregation propensity in cellular lysate,echoed by cell viability and fractionation experiments.Intriguingly,poor polydispersity index(PDI)of functionalized nanoparticles exaggerates their retention and aggregation in the cell.Comparative proteomic analysis uncovers that amphiphilic functionalization of AIE materials can minimize the deterioration of cellular protein homeostasis network.Finally,vigorous interrogation of functionalized AIE nanoparticles in mice model reveals the complexity of factors affecting the biocompatibility profiles in vivo,including materials’size,PDI,and treatment frequencies.Overall,amphiphilic functionalization of AIE materials into nanoparticles is necessary to maintain proteome stability and balance cellular protein homeostasis.
文摘Cytokinin is a critical growth regulator for various aspects of plant growth and development. In Arabidopsis, cytokinin signaling is mediated by a two-component system-based phosphorelay that transmits a signal from the receptors, through histidine phosphotransfer proteins, to the downstream response regulators （ARRs）. Of these ARRs, type-A ARR genes, whose transcription can be rapidly induced by cytokinin, act as negative regulators of eytokinin signaling. However, because of functional redundancy, the function of type-A ARR genes in plant growth and development is not well understood by analyzing loss-of-function mutants. In this study, we performed a comparative functional study on all ten type-A ARR genes by analyzing transgenic plants overexpressing these ARR genes fused to a MYC epitope tag. Overexpression of ARR genes results in a variety of cytokinin-associated phenotypes. Notably, overexpression of different ARR transgenes causes diverse phenotypes, even between phylogenetically closely-related gene pairs, such as within the ARR3-ARR4 and ARR5-ARR6 pairs. We found that the accumulation of a subset of ARR proteins （ARR3, ARR5, ARR7, ARR16 and ARR17; possibly ARR8 and ARR15） is increased by MG132, a specific proteasomal inhibitor, indicating that stability of these proteins is regulated by proteasomal degradation. Moreover, similar to that of previously characterized ARR5, ARR6 and ARR7, stability of ARR16 and ARR17, possibly including ARR8 and ARR15, is regulated by cytokinin. These results suggest that type-A ARR proteins are regulated by a combinatorial mechanism involving both the cytokinin and proteasome pathways, thereby executing distinctive functions in plant growth and development.
基金V. ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China （No.60533050 and No.J0830413） and the Zhejiang Provincial Natural Science Foundation of China （No.R407042）.
文摘The adsorption dynamics of a model protein （the human insulin） onto graphene surfaces with different sizes was investigated by molecular dynamics simulations. During the adsorption, it has different effect on the stability of the model protein in the fixed and non-fixed graphene systems. The tertiary structure of the protein was destroyed or partially destroyed, and graphene surfaces shows the selective protection for some α-helices in non-fixed Systems but not in fixed systems by reason of the flexibility of graphene. As indicated by the interaction energy curve and trajectory animation, the conformation and orientation selection of the protein were induced by the properties and the texture of graphene surfaces. The knowledge of protein adsorption on graphene surfaces would be helpful to better understand stability of protein on graphene surfaces and facilitate potential applications of graphene in biotechnology.
基金supported by Jiangsu Province Natural Science Foundation for Distinguished Young Scholars(BK20190035)Jiangsu Government Scholarship for Overseas Studies(JS-2019-053)+6 种基金Key Research&Development plan of Jiangsu Province(BE2019001)the National Natural Science Foundation of China(2217080044 and 22008119)the Natural Science Foundation of Jiangsu Province(BK20202002)the National Key Research and Development Program of China(2021YFC2101204)the Program for Changjiang Scholars and Innovative Research Team in University(IRT_14R28)the Priority Academic Program Development of Jiangsu Higher Education Institutions(PAPD)the Jiangsu Synergetic Innovation Center for Advanced Bio-Manufacture.
文摘Adenylate cyclase(AC)is the key enzyme that catalyzes the formation of cAMP from ATP.In this study,we discovered two novel class Ⅲ ACs with a halophilic property from Thermobifida halotolerans DSM 44931(ThAC)and Haloactinopolyspora alba DSM 45211(HaAC),respectively.The recombinant ThAC and HaAC were expressed in Escherichia coli with molecular weights of 36.1 and 36.0 kDa respectively.The presence of 2500 and 2200 mmolL^(-1)1 NaCl significantly enhanced the enzyme activities of ThAC and HaAC,with 22-fold and 7.4-fold higher activities compared to those without NaCl,respectively.Several divalent metal ions were found to activate the recombinant ACs to different extents,and the optimal metal ion was Mg^(2+)for both ThAC and HaAC with concentrations of 80 mmol·L^(-1) and 40 mmol·L^(-1) respectively.Purified ThAC and HaAC had the optimal specific activities((4.59±0.35)×10^(4) and(7.76±0.52)×10^(4) U·mg^(-1))and catalytic efficiency(4.47 and 5.30 L·mmol^(-1)·s^(-1))at 45℃ and 40℃ respectively,while the optimum pH of both two recombinant ACs was 10.0.This is the first report of the halophilic Class III ACs,which could make new contributions to explore and study ACs for further associated investigations.
基金financially supported by Research Institute of Applied Science(RIAS)ACECRInstitute of Biochemistry and Biophysics(IBB)Iran National Science Foundation(INSF)。
文摘Rhizopus oryzae lipase(ROL)was immobilized on the surface of silica coated amino modified CoFe_(2)O_4 nanoparticles and applied for biodiesel production.The results indicated more affinity of the ROL toward its substrate upon immobilization,as revealed by a lower Km value for the immobilized ROL compared to its free counterpart.Intrinsic fluorescence spectroscopy indicated a lower intensity for ROL immobilized on CoFe_(2)O_4 nanoparticles.Besides,immobilized ROL steady state anisotropy measurements presented lower values,which implied assembly of ROL molecules on magnetic nanoparticles upon immobilization as well as their restricted rotation upon covalent attachment.Thermal stability analysis revealed improved activity at higher temperatures for the immobilized enzyme compared to its free counterpart.Accordingly,Pace analysis to determine protein thermal stability revealed preservation of the protein conformation in the presence of increasing temperatures upon immobilization on nanoparticles.Finally,ROL immobilized on CoFe_(2)O_(4)nanoparticles exhibited improved efficiency of biodiesel production in agreement with thermal activity profile.Therefore,the authors suggest application of the lipase molecules immobilized on CoFe_(2)O_(4)nanoparticles for more efficient biodiesel production.
文摘When a protein is encapsulated into poly( DL -lactide-co-glycolide)(PLGA) microspheres by means of the double-emulsion method,the harsh microspheres formation process including ultrasonification,exposure to an organic solvent and a polymer may cause the denaturation of the protein. In this study,we investigated the enzymatic activity change and the effect of the excipients on the stability of recombinant human Cu,Zn-superoxide dismutase(rhCu,Zn-SOD) during the emulsification. The specific activity recovery was found to be concentration dependent and the excipients involved such as PEG 600 and Tween 20,and trehalose were shown to increase the stability of rhCu,Zn-SOD. The protein structural integrity within the microspheres was analyzed by FTIR. The structure of rhCu,Zn-SOD within PLGA microspheres containing trehalose was found to be similar to that of the native solid state,whereas the protein encapsulated during the preparation in the absence of any excipient changed due to the possible hydrophobic interaction with the polymer. The results suggest that a rational stability strategy for protein to be encapsulated into microspheres should aim at different processes.
文摘Obesity and cancer are two interrelated conditions of high epidemiological need, with studies showing that obesity is responsible for nearly 25% of the relative contribution to cancer incidence. Given the connection between these conditions, a drug that can operate on both obesity and cancer is highly desirable. Such a drug is accomplishablethrough the development of potent anti-angiogenesis agents due to the shared underlying role of angiogenesis in the development of both diseases. Prior research has demonstrated a key role of type-2 methionine aminopeptidase(Met AP2) for angiogenesis, which has led to the development of numerous of novel inhibitors. Several irreversible Met AP2 inhibitors have entered clinical trials without great success. Though this lack of success could be attributed to off-target adverse effects, the underlying causes remain unclear. More promising reversible inhibitors have been recently developed with excellent pre-clinical results. However, due to insufficient knowledge of the biological functions of N-terminal protein processing, it is hard to predict whether these novel inhibitors would successfully pass clinical trials and thereby benefit cancer and obesity patients. Significantly more efforts are needed to advance our understanding of the regulation of methionine aminopeptidases and the processes by which they govern the function of proteins.
文摘Typically,inherited metabolic diseases arise from point mutations in genes encoding metabolic enzymes. Although some of these mutations directly affect amino acid residues in the active sites of these enzymes,the majority do not. It is now well accepted that the majority of these disease-associated mutations exert their effects through alteration of protein stability,which causes a reduction in enzymatic activity. This finding suggests a way to predict the severity of newly discovered mutations. In silico prediction of the effects of amino acid sequence alterations on protein stability often correlates with disease severity. However,no stability prediction tool is perfect and,in general,better results are obtained if the predictions from a variety of tools are combined and then interpreted. In addition to predicted alterations to stability,the degree of conservation of a particular residue can also be a factor which needs to be taken into account: alterations to highly conserved residues are more likely to be associated with severe forms of the disease. The approach has been successfully applied in a variety of inherited metabolic diseases,but further improvements are necessary to enable robust translation into clinically useful tools.
基金supported by the Basic Science Center Program of National Natural Science Foundation of China(No.T2288102)the Key Program of the National Natural Science Foundation of China(No.32230059)+4 种基金the National Natural Science Foundation of China(No.32101086)the National Postdoctoral Program for Innovative Talents(BX2021101)the China Postdoctoral Science Foundation Funded Project(2021M701192)Fundamental Research Funds for the Central Universities(JKD01221507)the Foundation of Frontiers Science Center for Materiobiology and Dynamic Chemistry(JKVD1211002).
文摘Recombinant human bone morphogenetic protein-2(rhBMP-2)is the predominant growth factor that effectively induces osteogenic differentiation in orthopedic procedures.However,the bioactivity and stability of rhBMP-2 are intrinsically associated with its sequence,structure,and storage conditions.In this study,we successfully determined the amino acid sequence and protein secondary structure model of non-glycosylated rhBMP-2 expressed by an E.coli expression system through X-ray crystal structure analysis.Furthermore,we observed that acidic storage conditions enhanced the proliferative and osteoinductive activity of rhBMP-2.Although the osteogenic activity of non-glycosylated rhBMP-2 is relatively weaker compared to glycosylated rhBMP-2;however,this discrepancy can be mitigated by incorporating exogenous chaperone molecules.Overall,such information is crucial for rationalizing the design of stabilization methods and enhancing the bioactivity of rhBMP-2,which may also be applicable to other growth factors.
