In Bacillus thuringenesis(Bt) transgenic cotton, the cotton boll has the lowest insecticidal protein content when compared to the other organs. The present study investigated the effects of amino acid spray applicatio...In Bacillus thuringenesis(Bt) transgenic cotton, the cotton boll has the lowest insecticidal protein content when compared to the other organs. The present study investigated the effects of amino acid spray application at the peak flowering stage on the cotton boll Bt toxin concentration and yield formation. Boll protein synthesis and carbohydrate conversion were also studied to reveal the fundamental mechanism. Three treatments(i.e., CK, the untreated control;LA1, five amino acids;LA2, 21 amino acids) were applied to two Bt cultivars of G. hirsutum(i.e., the hybrid Sikang 3 and the conventional Sikang 1) in the cotton-growing seasons during 2017 and 2018. Amino acid spray application at the peak flowering stage resulted in an increase of 5.2–16.4% in the boll Bt protein concentration and an increase of 5.5–11.3%in the seed cotton yield, but there was no difference between the two amino acid treatments. In addition, amino acid applications led to increases in the amino acid content, soluble protein content, glutamate pyruvate transaminase(GPT)activity, glutamate oxaloacetate transaminase(GOT) activity, glucose content, fructose content and soluble acid invertase(SAI) activity. This study also found that Bt protein content, enhanced boll number and the weight of opened bolls were closely related to carbon and nitrogen metabolism. The Bt protein content had significant linear positive correlations with amino acid and soluble protein contents. Enhanced boll number had significant linear positive correlations with the GPT and GOT activities from 15–25 days after flowering(DAF). The weight of opened bolls from 55–65 DAF had a significant linear positive correlation with the SAI activity. These results indicate that the enhancement of boll protein synthesis and carbohydrate conversion by amino acid application resulted in a simultaneous increase in the boll Bt protein concentration and cotton lint yield.展开更多
Protein synthesis is essential for cells to perform life metabolic processes.Pathological alterations of protein content can lead to particular diseases.Cells have an intrinsic array of mechanisms and pathways that ar...Protein synthesis is essential for cells to perform life metabolic processes.Pathological alterations of protein content can lead to particular diseases.Cells have an intrinsic array of mechanisms and pathways that are activated when protein misfolding,accumulation,aggregation or mislocalization occur.Some of them(like the unfolded protein response)represent complex interactions between endoplasmic reticulum sensors and elongation factors that tend to increase expression of chaperone proteins and/or repress translation in order to restore protein homeostasis(also known as proteostasis).This is even more important in neurons,as they are very susceptible to harmful effects associated with protein overload and proteostatic mechanisms are less effective with age.Several neurodegenerative pathologies such as Alzheimer’s,Parkinson’s,and Huntington’s diseases,amyotrophic lateral sclerosis and frontotemporal dementia exhibit a particular molecular signature of distinct,unbalanced protein overload.In amyotrophic lateral sclerosis and frontotemporal dementia,the majority of cases present intracellular inclusions of ubiquitinated transactive response DNA-binding protein of 43 kDa(TDP-43).TDP-43 is an RNA binding protein that participates in RNA metabolism,among other functions.Dysregulation of TDP-43(e.g.aggregation and mislocalization)can dramatically affect neurons,and this has been linked to disease development.Expression of amyotrophic lateral sclerosis/frontotemporal dementia TDP-43-related mutations in cellular and animal models has been shown to recapitulate key features of the amyotrophic lateral sclerosis/frontotemporal dementia disease spectrum.These variants can be causative of degeneration onset and progression.Most neurodegenerative diseases(including amyotrophic lateral sclerosis and frontotemporal dementia)have no cure at the moment;however,modulating translation has recently emerged as an attractive approach that can be performed at several steps(i.e.regulating activation of initiation and elongation factors,inhibiting unfolded protein response activation or inducing chaperone expression and activity).This review focuses on the features of protein imbalance in neurodegenerative disorders and the relevance of developing therapeutical compounds aiming at restoring proteostasis.We strive to highlight the importance of research on drugs that,not only restore protein imbalance without compromising translational activity of cells,but are also as safe as possible for the patients.展开更多
Selenium has been recognized as an essential nutrient in animals since the 1950s. Demonstration of the role of dietary selenium in protection from oxidative stress foIlowed in the early 1970s, and was largely attribut...Selenium has been recognized as an essential nutrient in animals since the 1950s. Demonstration of the role of dietary selenium in protection from oxidative stress foIlowed in the early 1970s, and was largely attributed to its presence as an integral part of cellular glutathione peroxidase. However, the functions of this enzyme did not explain many of the other effects of selenium deficiency. The identification of other mammalian selenoproteins during the last few years has provided new insights into the functions of this trace nutrient. The discovery that type 1 deiodinase (D1) is a selenoenzyme, in addition to unveiling an essential role for selenium in thyroid hormone action, has had more far-reaching implications. Studies of this protein opened the door for investigation of the requirements for eukaryotic selenoprotein synthesis,and the features that distinguish this pathway from the corresponding prokaryotic pathway.Selenium is present in a number of prokaryotic and eukaryotic proteins in the form of the unusual amino acid, selenocysteine. Incorporation of selenocysteine into these proteins requires a novel translation step in which UGA specifies selenocysteine insertion. Since UGA codons are typically recognized as translation stop signals, an intriguing question is raised: How does a cell recognize and distinguish a UGA selenocysteine codon from a UGA stop codon? In this review, we will focus on what is known about selenocysteine incorporation in eukaryotes, briefly summarizing initial studies and discussing a few recent advances in our understanding of this unique 'recoding' process展开更多
The lactating mammary gland is a prodigious protein-producing factory, but the milk protein synthesis mechanisms are not well understood. The major objective of this paper was to elucidate which genes and pathways wer...The lactating mammary gland is a prodigious protein-producing factory, but the milk protein synthesis mechanisms are not well understood. The major objective of this paper was to elucidate which genes and pathways were involved in the regulation of milk protein synthesis in the dairy goat mammary gland. Total 36 primiparous Guanzhong dairy goats were allotted in 12 groups according to their mammary development stages: days 90 and 150 of virgin, days 30, 90, and 150 of pregnancy, days 1, 10, 35, and 60 of lactation and days 3, 7, and 21 of involution (three animals per group). Mammary tissue RNA was isolated for quantitative real- time RT-PCR of four casein genes alpha-s 1 casein (CSN 1S 1 ), alpha-s2 casein (CSN 1 S2), beta-casein (CSN2) and casein kappa (CSN3), four whey protein genes lactoglobulin (LGB), laetalbumin (LALBA), laetofarrin (LTF), and Whey acidic protein (WAP) and the genes which were potentially to regulate dairy goat milk protein synthesis at the level of transcription or translation [prolactin receptor (PRLR), AKT1, signal transducers and activators of transcription 5 (STAT5), E74-Like Factor 5 (ELF5), eukaryotic translation initiation factor 4E binding protein 1 (EIF4E-BP1), S6kinase (S6K) and caveolin 1]. The results showed that all genes were up-regulated in lactation period. The expressions of PRLR, AKT1, STAT5, ELF5, and S6K were similar to mRNA expressions of milk proteins. Our results indicated that milk protein synthesis in dairy goat mammary gland was possibly regulated by these genes.展开更多
Objective To investigate the influence of sodium fluoride(NaF)on alkaline phosphatase(ALP)activity and bone gla protein(BGP)synthesis in yellow ligament cells from different surgical simples in vitro.Methods The human...Objective To investigate the influence of sodium fluoride(NaF)on alkaline phosphatase(ALP)activity and bone gla protein(BGP)synthesis in yellow ligament cells from different surgical simples in vitro.Methods The human ligament cells展开更多
The central dogma of modern biology underscores the pivotal roles proteins play in diverse biological processes,the study of which necessitates advanced methods to produce proteins with precision and versatility.Chemi...The central dogma of modern biology underscores the pivotal roles proteins play in diverse biological processes,the study of which necessitates advanced methods to produce proteins with precision and versatility.Chemical protein synthesis,a powerful approach utilizing chemical reactions for the de novo construction of structurally accurate proteins,has emerged as a transformative tool for studying proteins and generating protein derivatives/mimics inaccessible by natural biological machinery,including post-translationally modified proteins,proteins comprised of unnatural amino acids,as well as mirror-image proteins.This review summarizes recent strides in synthetic method developments for chemical protein synthesis,including innovative techniques in solid-phase peptide synthesis,the challenges presented by difficult sequences in either synthesis or folding and the exploration of novel ligation reactions using both chemical and enzymatic methods.Furthermore,the review also delves into newly developed protocols for site-selective protein modifications and the generation of stapled or macrocyclized peptides/miniproteins,highlighting the power of chemical methods to make structurally diverse proteins.Recent applications of synthetic proteins in investigating post-translational modifications(phosphorylation,lipidation,glycosylation,ubiquitination,etc.),mirror-image biological processes and drug development are further discussed.Together,these topics provide a comprehensive overview of the current landscape of chemical protein synthesis.展开更多
The 2-hydroxy-4-methoxybenzyl(Hmb)backbone modification can prevent amide bond-mediated sidereactions(e.g.,aspartimide formation,peptide aggregation)by installing the removable Hmb group into a peptide bond,thus impro...The 2-hydroxy-4-methoxybenzyl(Hmb)backbone modification can prevent amide bond-mediated sidereactions(e.g.,aspartimide formation,peptide aggregation)by installing the removable Hmb group into a peptide bond,thus improving the synthesis of long and challenging peptides and proteins.However,its use is largely precluded by the limited Hmb’s installation sites.In this report,an improved installation of Hmb(iHmb)method was developed to achieve the flexible installation and the convenient removal of Hmb.The iHmb method involves two critical steps:(1)oxidative diazotization of the readily installed 2-hydroxy-4-methoxy-5-amino-benzyl(Hmab)to give 2-hydroxy-4-methoxy-5-diazonium-benzyl(Hmdab)by combining soamyl nitrite(IAN)/HBF_(4),and(2)reductive elimination of Hmdab to give the desired Hmb by 1,2-ethanedithiol(EDT).The iHmb method enables the installation of Hmb at any primary amino acid including the highly sterically hindered amino acids(e.g.,valine and isoleucine).The practicality and utility of the iHmb method was demonstrated by one-shot solid-phase synthesis of a challenging aspartimide-prone peptide,the mirror-image version of a hydrophobic peptide and a long-chain peptide up to 76-residue.Furthermore,the iHmb method can be utilized to facilitate chemical protein ligation,as exemplified by the synthesis of the single-spanning membrane protein sarcolipin.The iHmb method expands the toolkit for peptide synthesis and ligation and facilitates the preparation of peptides/proteins.展开更多
Since starch digestion in the small intestine provides more energy than digestion in the rumen of ru-minants,reducing dietary rumen degradable starch(RDS)content is beneficial for improving energy utilization of starc...Since starch digestion in the small intestine provides more energy than digestion in the rumen of ru-minants,reducing dietary rumen degradable starch(RDS)content is beneficial for improving energy utilization of starch in ruminants.The present study tested whether the reduction of rumen degradable starch by restricting dietary corn processing for growing goats could improve growth performance,and further investigated the possible underlying mechanism.In this study,twenty-four 12-wk-old goats were selected and randomly allocated to receive either a high RDS diet(HRDS,crushed corn-based concen-trate,the mean of particle sizes of corn grain=1.64 mm,n=12)or a low RDS diet(LRDS,non-processed corn-based concentrate,the mean of particle sizes of corn grain>8 mm,n=12).Growth performance,carcass traits,plasma biochemical indices,gene expression of glucose and amino acid transporters,and protein expression of the AMPK-mTOR pathway were measured.Compared to the HRDS,LRDS tended to increase the average daily gain(ADG,P=0.054)and decreased the feed-to-gain ratio(F/G,P<0.05).Furthermore,LRDS increased the net lean tissue rate(P<0.01),protein content(P<0.05)and total free amino acids(P<0.05)in the biceps femoris(BF)muscle of goats.LRDS increased the glucose concen-tration(P<0.01),but reduced total amino acid concentration(P<0.05)and tended to reduce blood urea nitrogen(BUN)concentration(P=0.062)in plasma of goats.The mRNA expression of insulin receptors(INSR),glucose transporter 4(GLUT4),L-type amino acid transporter 1(LAT1)and 4F2 heavy chain(4F2hc)in BF muscle,and sodium-glucose cotransporters 1(SGLT1)and glucose transporter 2(GLUT2)in the small intestine were significantly increased(P<0.05)in LRDS goats.LRDS also led to marked activation of p70-S6 kinase(S6K)(P<0.05),but lower activation of AMP-activated protein kinase(AMPK)(P<0.05)and eukaryotic initiation factor 2a(P<0.01).Our findings suggested that reducing the content of dietary RDS enhanced postruminal starch digestion and increased plasma glucose,thereby improving amino acid utilization and promoting protein synthesis in the skeletal muscle of goats via the AMPK-mTOR pathway.These changes may contribute to improvement in growth performance and carcass traits in LRDS goats.展开更多
Tyrosine sulfation is an important post-translational modification that enhances the inhibitory activity of hirudin.Herein,we developed a facile synthetic strategy to afford the sulfated hirudins with up to three modi...Tyrosine sulfation is an important post-translational modification that enhances the inhibitory activity of hirudin.Herein,we developed a facile synthetic strategy to afford the sulfated hirudins with up to three modifications and in multi-milligram scales,after a single HPLC purification step.Through these synthetic proteins,a novel type of modulation mechanism exhibited by tyrosine sulfation was proposed,which would help to delineate the structure-function relationships in other sulfated proteins and more importantly,to serve as a basis for the development of related antithrombotic agents.展开更多
Advances in metabolic engineering and synthetic biology have facilitated the manufacturing of many valuable-added compounds and commodity chemicals using microbial cell factories in the past decade.However,due to comp...Advances in metabolic engineering and synthetic biology have facilitated the manufacturing of many valuable-added compounds and commodity chemicals using microbial cell factories in the past decade.However,due to complexity of cellular metabolism,the optimization of metabolic pathways for maximal production represents a grand challenge and an unavoidable barrier for metabolic engineering.Recently,cell-free protein synthesis system(CFPS)has been emerging as an enabling alternative to address challenges in biomanufacturing.This review summarizes the recent progresses of CFPS in rapid prototyping of biosynthetic pathways and genetic circuits(biosensors)to speed up design-build-test(DBT)cycles of metabolic engineering and synthetic biology.展开更多
Cell-free protein synthesis(CFPS)systems from crude lysates have benefitted from modifications to their enzyme composition.For example,functionally deleting enzymes in the source strain that are deleterious to CFPS ca...Cell-free protein synthesis(CFPS)systems from crude lysates have benefitted from modifications to their enzyme composition.For example,functionally deleting enzymes in the source strain that are deleterious to CFPS can improve protein synthesis yields.However,making such modifications can take substantial time.As a proof-of-concept to accelerate prototyping capabilities,we assessed the feasibility of using the yeast knockout collection to identify negative effectors in a Saccharomyces cerevisiae CFPS platform.We analyzed extracts made from six deletion strains that targeted the single deletion of potentially negative effectors(e.g.,nucleases).We found a statistically significant increase in luciferase yields upon loss of function of GCN3,PEP4,PPT1,NGL3,and XRN1 with a maximum increase of over 6-fold as compared to the wild type.Our work has implications for yeast CFPS and for rapidly prototyping strains to enable cell-free synthetic biology applications.展开更多
D-peptides are recognized as a new class of synthetic chemical drugs and they possess many interesting advantages such as high enzymatic stability,improved oral bioavailability,as well as high binding affinity and spe...D-peptides are recognized as a new class of synthetic chemical drugs and they possess many interesting advantages such as high enzymatic stability,improved oral bioavailability,as well as high binding affinity and specificity.Recently,D-peptide drugs have been attracting increasing attention in both academic and industrial researches over recent years.One D-peptide etelcalcetide has even entered the market that targets the calcium(Ca2+)-sensing receptor(CaSR) to fight secondary hyperparathyroidism.Effective discovery and optimization of D-peptide ligands that can bind to various disease-related targets with high specificity and potency is of great importance for the development of D-peptide drugs.This review surveys the recent method development in this area especially the chemical protein synthesis-assisted high-throughput screening strategies for D-peptide ligands and their application in drug discovery.展开更多
Cell-free systems contain many proteins and metabolites required for complex functions such as transcription and translation or multi-step metabolic conversions.Research into expanding the delivery of these systems by...Cell-free systems contain many proteins and metabolites required for complex functions such as transcription and translation or multi-step metabolic conversions.Research into expanding the delivery of these systems by drying or by embedding into other materials is enabling new applications in sensing,point-of-need manufacturing,and responsive materials.Meanwhile,silk fibroin from the silk worm,Bombyx mori,has received attention as a protective additive for dried enzyme formulations and as a material to build biocompatible hydrogels for controlled localization or delivery of biomolecular cargoes.In this work,we explore the effects of silk fibroin as an additive in cell-free protein synthesis(CFPS)reactions.Impacts of silk fibroin on CFPS activity and stability after drying,as well as the potential for incorporation of CFPS into hydrogels of crosslinked silk fibroin are assessed.We find that simple addition of silk fibroin increased productivity of the CFPS reactions by up to 42%,which we attribute to macromolecular crowding effects.However,we did not find evidence that silk fibroin provides a protective effects after drying as previously described for purified enzymes.Further,the enzymatic crosslinking transformations of silk fibroin typically used to form hydrogels are inhibited in the presence of the CFPS reaction mixture.Crosslinking attempts did not impact CFPS activity,but did yield localized protein aggregates rather than a hydrogel.We discuss the mechanisms at play in these results and how the silk fibroin-CFPS system might be improved for the design of cell-free devices.展开更多
Gases are the vital nutrition of all organisms as the precursor of metabolism pathways.As a potential biological process,protein synthesis is inevitably regulated by gas transport and utilization.However,the effect of...Gases are the vital nutrition of all organisms as the precursor of metabolism pathways.As a potential biological process,protein synthesis is inevitably regulated by gas transport and utilization.However,the effect of carbon dioxide(CO_(2))present in many metabolic pathways on protein synthesis has not been studied well.In this work,carbon dioxide combined with oxygen was employed for cell-free protein synthesis(CFPS)in the tube-in-tube reactor with precise control of gas concentration.In this in vitro system,gases could directly affect the protein synthesis process without transmembrane transport.Varied concentrations of carbon dioxide(0-1%)and constant oxygen concentration(21%)were employed for CFPS to assess the effects.The cell-free reactions with 0.3%CO_(2) and 21%O_(2) showed the highest protein yields.The combined effect of CO_(2) and O_(2) also resulted in relatively high protein expression under high oxygen conditions(0.3%CO_(2) and 100%O_(2)).Moreover,metabolomics assays were performed to gain insight into metabolic changes,which showed that CO_(2) slightly improved energy metabolism and redox balance.In particular,the extra supplied CO_(2) activated the decarboxylating reactions and removed toxic metabolites to recover the protein synthesis activity.The exploration of CO_(2) on protein synthesis could provide guiding implications for basic studies and biomanufacturing.展开更多
In the present experiments the changes in levels of ribosome,polysome and 3H-leucine incorporation rate in liver post-mitochondrial supernatant (PM-supernatant) were investigated in Sedericient and Se-supplented rats....In the present experiments the changes in levels of ribosome,polysome and 3H-leucine incorporation rate in liver post-mitochondrial supernatant (PM-supernatant) were investigated in Sedericient and Se-supplented rats.The results demonstrated that the amounts of ribosome and polysome as well as the ratio of polysome to ribosome in liver PM-supernatant from the Se-deficient rats were all remarkahly decreased.In the meantime,the rate of protein synthesis expressed as radioactivity or 3H-leucine incorporated into protein in the PM-supernatant system also decreased significantly.The results suggest that the decreases of ribosomes and proportion of ribosomal aggregates in PM-supernatant may be responsible for the decrease of the protein synthesis activity in liver of the Se-deficient animals.展开更多
The interferon-inducible-stranded-RNA-depedet protein kinase PKR has been implicated in both the antiviral aand cell growth-regulatory effects of the interferons.Over-expression of the wild-type form of this protein i...The interferon-inducible-stranded-RNA-depedet protein kinase PKR has been implicated in both the antiviral aand cell growth-regulatory effects of the interferons.Over-expression of the wild-type form of this protein inhibits cell proliferation,whereas over-expression of inactive mutant forms transforms cells to a tumouri-genie phenotype.It has been suggested that mutant PKR exerts a dominant negative effect on the activity of the wild-type protein kinase.We have investigated this possibility using the rabbbit reticulocyte cell-free translation system in which protein synthesis is inhibited by dsRNA due to activation of PKR and phosphorylation of initiation factor elF-2. Addition of a highly purified inactive PKR mutant,synthesised in a baculovirus-infected insect cell system, rescues protein synthesis from inhibition by the low concentrations of dsRNA in a dose-dependent manner. The PKR mutant has no effect on protein synthesis in the absence of dsRNA of in the presence of another inhibitory protein kinase,the haem-controlled repressor.Inhibition of translation can be re-established in the presence of the mutant PKR by adding a higher concentration of dsRNA.These results suggest that inactive mutant PKR does exert a dominant negative effect on wild-type PKR and that this may be due to competition for dsRNA binding.展开更多
An“endotoxin-free”E.coli-based cell-free protein synthesis system has been reported to produce therapeutic proteins rapidly and on-demand.However,preparation of the most complex CFPS reagent–the cell extract–remai...An“endotoxin-free”E.coli-based cell-free protein synthesis system has been reported to produce therapeutic proteins rapidly and on-demand.However,preparation of the most complex CFPS reagent–the cell extract–remains time-consuming and labor-intensive because of the relatively slow growth kinetics of the endotoxin-free ClearColiTMBL21(DE3)strain.Here we report a streamlined procedure for preparing E.coli cell extract from ClearColi™using auto-induction media.In this work,the term auto-induction describes cell culture media which eliminates the need for manual induction of protein expression.Culturing Clearcoli™cells in autoinduction media significantly reduces the hands-on time required during extract preparation,and the resulting“endotoxinfree”cell extract maintained the same cell-free protein synthesis capability as extract produced with traditional induction as demonstrated by the high-yield expression of crisantaspase,an FDA approved leukemia therapeutic.It is anticipated that this work will lower the barrier for researchers to enter the field and use this technology as the method to produce endotoxin-free E.coli-based extract for CFPS.展开更多
Aminoacyl-tRNA synthetases (aaRSs) are a family of ancient enzymes that are responsible for catalysis of the attachment of amino acids to their cognate tRNAs in the first step
In this study the effect of human recombinant interferon gamma hrIFN-γ)on hCG secretion of human first trimester trophoblast and protein synthesis of decidual tissue was investigated in vitro.The results indicated th...In this study the effect of human recombinant interferon gamma hrIFN-γ)on hCG secretion of human first trimester trophoblast and protein synthesis of decidual tissue was investigated in vitro.The results indicated that hrIFN-γat the doses of 250 U/ml medium and 2500 U/ml medium decreased hCG secretion of trophoblast obviously(P<0.05, P<0.01)in a dose dependent manner.The effect of hrIFN-γon protein synthesis at the doses of 10 U to 1,000 U/ml medium inhibited the 3H leucine incorporation obviously.The cpm values between control and experimental groups were significantly different(P<0. 05) in a dosedependent manner.Furthermore its inhibitory effect is in a dose-dependent manner and was neutralized by IFN-γantiserum.The IFN-a at the doses used did not find any effect on protein synthesis in decidual tissue.展开更多
As a key coordinator of metabolism,AMP-activated protein kinase(AMPK)is vitally involved in skeletal muscle maintenance.AMPK exerts its cellular effects through its function as a serine/threonine protein kinase by reg...As a key coordinator of metabolism,AMP-activated protein kinase(AMPK)is vitally involved in skeletal muscle maintenance.AMPK exerts its cellular effects through its function as a serine/threonine protein kinase by regulating many downstream targets and plays important roles in the development and growth of skeletal muscle.AMPK is activated by phosphorylation and exerts its function as a kinase in many processes,including synthesis and degradation of proteins,mitochondrial biogenesis,glucose uptake,and fatty acid and cholesterol metabolism.Skeletal muscle atrophy is a result of various diseases or disorders and is characterized by a decrease in muscle mass.The pathogenesis and therapeutic strategies of skeletal muscle atrophy are still under investigation.In this review,we discuss the role of AMPK in skeletal muscle metabolism and atrophy.We also discuss targeting AMPK for skeletal muscle treatment,including exercise,AMPK activators including 5-amino-4-imidazolecarboxamide ribonucleoside and metformin,and low-level lasers.These studies show the important roles of AMPK in regulating muscle metabolism and function;thus,the treatment of skeletal muscle atrophy needs to take into account the roles of AMPK.展开更多
基金supported by the National Natural Science Foundation of China (31901462 and 31671613)the Natural Science Foundation of Jiangsu Province,China (BK20191439)+1 种基金the Postgraduate Research&Practice Innovation Program of Jiangsu Province,China(KYCX22_3508)the Priority Academic Program Development of Jiangsu Higher Education Institutions,China (PAPD)。
文摘In Bacillus thuringenesis(Bt) transgenic cotton, the cotton boll has the lowest insecticidal protein content when compared to the other organs. The present study investigated the effects of amino acid spray application at the peak flowering stage on the cotton boll Bt toxin concentration and yield formation. Boll protein synthesis and carbohydrate conversion were also studied to reveal the fundamental mechanism. Three treatments(i.e., CK, the untreated control;LA1, five amino acids;LA2, 21 amino acids) were applied to two Bt cultivars of G. hirsutum(i.e., the hybrid Sikang 3 and the conventional Sikang 1) in the cotton-growing seasons during 2017 and 2018. Amino acid spray application at the peak flowering stage resulted in an increase of 5.2–16.4% in the boll Bt protein concentration and an increase of 5.5–11.3%in the seed cotton yield, but there was no difference between the two amino acid treatments. In addition, amino acid applications led to increases in the amino acid content, soluble protein content, glutamate pyruvate transaminase(GPT)activity, glutamate oxaloacetate transaminase(GOT) activity, glucose content, fructose content and soluble acid invertase(SAI) activity. This study also found that Bt protein content, enhanced boll number and the weight of opened bolls were closely related to carbon and nitrogen metabolism. The Bt protein content had significant linear positive correlations with amino acid and soluble protein contents. Enhanced boll number had significant linear positive correlations with the GPT and GOT activities from 15–25 days after flowering(DAF). The weight of opened bolls from 55–65 DAF had a significant linear positive correlation with the SAI activity. These results indicate that the enhancement of boll protein synthesis and carbohydrate conversion by amino acid application resulted in a simultaneous increase in the boll Bt protein concentration and cotton lint yield.
基金supported by research grants to LMI from University of Buenos Aires(UBACyT)the Agencia Nacional de Promoción Científica y Tecnológica(ANPCyT)under grants PICT 2015-0975 and PICT 2017-2140。
文摘Protein synthesis is essential for cells to perform life metabolic processes.Pathological alterations of protein content can lead to particular diseases.Cells have an intrinsic array of mechanisms and pathways that are activated when protein misfolding,accumulation,aggregation or mislocalization occur.Some of them(like the unfolded protein response)represent complex interactions between endoplasmic reticulum sensors and elongation factors that tend to increase expression of chaperone proteins and/or repress translation in order to restore protein homeostasis(also known as proteostasis).This is even more important in neurons,as they are very susceptible to harmful effects associated with protein overload and proteostatic mechanisms are less effective with age.Several neurodegenerative pathologies such as Alzheimer’s,Parkinson’s,and Huntington’s diseases,amyotrophic lateral sclerosis and frontotemporal dementia exhibit a particular molecular signature of distinct,unbalanced protein overload.In amyotrophic lateral sclerosis and frontotemporal dementia,the majority of cases present intracellular inclusions of ubiquitinated transactive response DNA-binding protein of 43 kDa(TDP-43).TDP-43 is an RNA binding protein that participates in RNA metabolism,among other functions.Dysregulation of TDP-43(e.g.aggregation and mislocalization)can dramatically affect neurons,and this has been linked to disease development.Expression of amyotrophic lateral sclerosis/frontotemporal dementia TDP-43-related mutations in cellular and animal models has been shown to recapitulate key features of the amyotrophic lateral sclerosis/frontotemporal dementia disease spectrum.These variants can be causative of degeneration onset and progression.Most neurodegenerative diseases(including amyotrophic lateral sclerosis and frontotemporal dementia)have no cure at the moment;however,modulating translation has recently emerged as an attractive approach that can be performed at several steps(i.e.regulating activation of initiation and elongation factors,inhibiting unfolded protein response activation or inducing chaperone expression and activity).This review focuses on the features of protein imbalance in neurodegenerative disorders and the relevance of developing therapeutical compounds aiming at restoring proteostasis.We strive to highlight the importance of research on drugs that,not only restore protein imbalance without compromising translational activity of cells,but are also as safe as possible for the patients.
文摘Selenium has been recognized as an essential nutrient in animals since the 1950s. Demonstration of the role of dietary selenium in protection from oxidative stress foIlowed in the early 1970s, and was largely attributed to its presence as an integral part of cellular glutathione peroxidase. However, the functions of this enzyme did not explain many of the other effects of selenium deficiency. The identification of other mammalian selenoproteins during the last few years has provided new insights into the functions of this trace nutrient. The discovery that type 1 deiodinase (D1) is a selenoenzyme, in addition to unveiling an essential role for selenium in thyroid hormone action, has had more far-reaching implications. Studies of this protein opened the door for investigation of the requirements for eukaryotic selenoprotein synthesis,and the features that distinguish this pathway from the corresponding prokaryotic pathway.Selenium is present in a number of prokaryotic and eukaryotic proteins in the form of the unusual amino acid, selenocysteine. Incorporation of selenocysteine into these proteins requires a novel translation step in which UGA specifies selenocysteine insertion. Since UGA codons are typically recognized as translation stop signals, an intriguing question is raised: How does a cell recognize and distinguish a UGA selenocysteine codon from a UGA stop codon? In this review, we will focus on what is known about selenocysteine incorporation in eukaryotes, briefly summarizing initial studies and discussing a few recent advances in our understanding of this unique 'recoding' process
基金Supported by the National Basic Research Program of China(973 Program,2011CB100804)the National Natural Science Foundation of China(31101784)Funds for Young Researchers from Northeast Agricultural University(14QC43)
文摘The lactating mammary gland is a prodigious protein-producing factory, but the milk protein synthesis mechanisms are not well understood. The major objective of this paper was to elucidate which genes and pathways were involved in the regulation of milk protein synthesis in the dairy goat mammary gland. Total 36 primiparous Guanzhong dairy goats were allotted in 12 groups according to their mammary development stages: days 90 and 150 of virgin, days 30, 90, and 150 of pregnancy, days 1, 10, 35, and 60 of lactation and days 3, 7, and 21 of involution (three animals per group). Mammary tissue RNA was isolated for quantitative real- time RT-PCR of four casein genes alpha-s 1 casein (CSN 1S 1 ), alpha-s2 casein (CSN 1 S2), beta-casein (CSN2) and casein kappa (CSN3), four whey protein genes lactoglobulin (LGB), laetalbumin (LALBA), laetofarrin (LTF), and Whey acidic protein (WAP) and the genes which were potentially to regulate dairy goat milk protein synthesis at the level of transcription or translation [prolactin receptor (PRLR), AKT1, signal transducers and activators of transcription 5 (STAT5), E74-Like Factor 5 (ELF5), eukaryotic translation initiation factor 4E binding protein 1 (EIF4E-BP1), S6kinase (S6K) and caveolin 1]. The results showed that all genes were up-regulated in lactation period. The expressions of PRLR, AKT1, STAT5, ELF5, and S6K were similar to mRNA expressions of milk proteins. Our results indicated that milk protein synthesis in dairy goat mammary gland was possibly regulated by these genes.
文摘Objective To investigate the influence of sodium fluoride(NaF)on alkaline phosphatase(ALP)activity and bone gla protein(BGP)synthesis in yellow ligament cells from different surgical simples in vitro.Methods The human ligament cells
基金supported by the National Key R&D Program of China(2022YFC3401500)the National Natural Science Foundation of China(22137005,92253302,22227810 to Lei Liu,22177004,92153301,22321005 to Suwei Dong,22277020 to Yiming Li,22022703,22177108,22377118 to Ji-Shen Zheng,92353302,22177059 to Yongxiang Chen,22177035 to Jun Guo,22277029,22077036 to Chunmao He,22077078 to Honggang Hu92353302,92053108 to Yanmei Li,22277015 to Junfeng Zhao)。
文摘The central dogma of modern biology underscores the pivotal roles proteins play in diverse biological processes,the study of which necessitates advanced methods to produce proteins with precision and versatility.Chemical protein synthesis,a powerful approach utilizing chemical reactions for the de novo construction of structurally accurate proteins,has emerged as a transformative tool for studying proteins and generating protein derivatives/mimics inaccessible by natural biological machinery,including post-translationally modified proteins,proteins comprised of unnatural amino acids,as well as mirror-image proteins.This review summarizes recent strides in synthetic method developments for chemical protein synthesis,including innovative techniques in solid-phase peptide synthesis,the challenges presented by difficult sequences in either synthesis or folding and the exploration of novel ligation reactions using both chemical and enzymatic methods.Furthermore,the review also delves into newly developed protocols for site-selective protein modifications and the generation of stapled or macrocyclized peptides/miniproteins,highlighting the power of chemical methods to make structurally diverse proteins.Recent applications of synthetic proteins in investigating post-translational modifications(phosphorylation,lipidation,glycosylation,ubiquitination,etc.),mirror-image biological processes and drug development are further discussed.Together,these topics provide a comprehensive overview of the current landscape of chemical protein synthesis.
基金supported by the National Key Research and Development Program of China(No.2019YFA0706900)the National Natural Science Foundation of China(Nos.22022703 and 22177108)the Collaborative Innovation Program of Hefei Science Center,CAS(No.2022HSC-CIP013).
文摘The 2-hydroxy-4-methoxybenzyl(Hmb)backbone modification can prevent amide bond-mediated sidereactions(e.g.,aspartimide formation,peptide aggregation)by installing the removable Hmb group into a peptide bond,thus improving the synthesis of long and challenging peptides and proteins.However,its use is largely precluded by the limited Hmb’s installation sites.In this report,an improved installation of Hmb(iHmb)method was developed to achieve the flexible installation and the convenient removal of Hmb.The iHmb method involves two critical steps:(1)oxidative diazotization of the readily installed 2-hydroxy-4-methoxy-5-amino-benzyl(Hmab)to give 2-hydroxy-4-methoxy-5-diazonium-benzyl(Hmdab)by combining soamyl nitrite(IAN)/HBF_(4),and(2)reductive elimination of Hmdab to give the desired Hmb by 1,2-ethanedithiol(EDT).The iHmb method enables the installation of Hmb at any primary amino acid including the highly sterically hindered amino acids(e.g.,valine and isoleucine).The practicality and utility of the iHmb method was demonstrated by one-shot solid-phase synthesis of a challenging aspartimide-prone peptide,the mirror-image version of a hydrophobic peptide and a long-chain peptide up to 76-residue.Furthermore,the iHmb method can be utilized to facilitate chemical protein ligation,as exemplified by the synthesis of the single-spanning membrane protein sarcolipin.The iHmb method expands the toolkit for peptide synthesis and ligation and facilitates the preparation of peptides/proteins.
基金supported by the National Key Research and Development Program of China(grant number 2017YFD0500500).
文摘Since starch digestion in the small intestine provides more energy than digestion in the rumen of ru-minants,reducing dietary rumen degradable starch(RDS)content is beneficial for improving energy utilization of starch in ruminants.The present study tested whether the reduction of rumen degradable starch by restricting dietary corn processing for growing goats could improve growth performance,and further investigated the possible underlying mechanism.In this study,twenty-four 12-wk-old goats were selected and randomly allocated to receive either a high RDS diet(HRDS,crushed corn-based concen-trate,the mean of particle sizes of corn grain=1.64 mm,n=12)or a low RDS diet(LRDS,non-processed corn-based concentrate,the mean of particle sizes of corn grain>8 mm,n=12).Growth performance,carcass traits,plasma biochemical indices,gene expression of glucose and amino acid transporters,and protein expression of the AMPK-mTOR pathway were measured.Compared to the HRDS,LRDS tended to increase the average daily gain(ADG,P=0.054)and decreased the feed-to-gain ratio(F/G,P<0.05).Furthermore,LRDS increased the net lean tissue rate(P<0.01),protein content(P<0.05)and total free amino acids(P<0.05)in the biceps femoris(BF)muscle of goats.LRDS increased the glucose concen-tration(P<0.01),but reduced total amino acid concentration(P<0.05)and tended to reduce blood urea nitrogen(BUN)concentration(P=0.062)in plasma of goats.The mRNA expression of insulin receptors(INSR),glucose transporter 4(GLUT4),L-type amino acid transporter 1(LAT1)and 4F2 heavy chain(4F2hc)in BF muscle,and sodium-glucose cotransporters 1(SGLT1)and glucose transporter 2(GLUT2)in the small intestine were significantly increased(P<0.05)in LRDS goats.LRDS also led to marked activation of p70-S6 kinase(S6K)(P<0.05),but lower activation of AMP-activated protein kinase(AMPK)(P<0.05)and eukaryotic initiation factor 2a(P<0.01).Our findings suggested that reducing the content of dietary RDS enhanced postruminal starch digestion and increased plasma glucose,thereby improving amino acid utilization and promoting protein synthesis in the skeletal muscle of goats via the AMPK-mTOR pathway.These changes may contribute to improvement in growth performance and carcass traits in LRDS goats.
基金The financial support from the National Natural Science Foundation of China(Nos.91853117 and 22077036)the Natural Science Foundation of Guangdong Province(No.2020A1515010766)are greatly acknowledged。
文摘Tyrosine sulfation is an important post-translational modification that enhances the inhibitory activity of hirudin.Herein,we developed a facile synthetic strategy to afford the sulfated hirudins with up to three modifications and in multi-milligram scales,after a single HPLC purification step.Through these synthetic proteins,a novel type of modulation mechanism exhibited by tyrosine sulfation was proposed,which would help to delineate the structure-function relationships in other sulfated proteins and more importantly,to serve as a basis for the development of related antithrombotic agents.
基金This work was financially supported by the National Natural Science Foundation of China(Grant No.21606205,21576232&21506185)the Fundamental Research Funds for the Central Universities,and the Startup Fund from Zhejiang University.
文摘Advances in metabolic engineering and synthetic biology have facilitated the manufacturing of many valuable-added compounds and commodity chemicals using microbial cell factories in the past decade.However,due to complexity of cellular metabolism,the optimization of metabolic pathways for maximal production represents a grand challenge and an unavoidable barrier for metabolic engineering.Recently,cell-free protein synthesis system(CFPS)has been emerging as an enabling alternative to address challenges in biomanufacturing.This review summarizes the recent progresses of CFPS in rapid prototyping of biosynthetic pathways and genetic circuits(biosensors)to speed up design-build-test(DBT)cycles of metabolic engineering and synthetic biology.
基金YKO collection strains were generously provided by the Northwestern High Throughput Core.We acknowledge Northwestern University and the DARPA Biomedicines on Demand program(N66001-13-C-4024)for support.J.A.S.was supported by the National Science Foundation Graduate Research Fellowship,grant number DGE-1324585.
文摘Cell-free protein synthesis(CFPS)systems from crude lysates have benefitted from modifications to their enzyme composition.For example,functionally deleting enzymes in the source strain that are deleterious to CFPS can improve protein synthesis yields.However,making such modifications can take substantial time.As a proof-of-concept to accelerate prototyping capabilities,we assessed the feasibility of using the yeast knockout collection to identify negative effectors in a Saccharomyces cerevisiae CFPS platform.We analyzed extracts made from six deletion strains that targeted the single deletion of potentially negative effectors(e.g.,nucleases).We found a statistically significant increase in luciferase yields upon loss of function of GCN3,PEP4,PPT1,NGL3,and XRN1 with a maximum increase of over 6-fold as compared to the wild type.Our work has implications for yeast CFPS and for rapidly prototyping strains to enable cell-free synthetic biology applications.
基金supported by the National Key R&D Program of China(No.2019YFA0706902)National Natural Science Foundation of China(Nos.U1732161 and 91753120)Science and Technological Fund of Anhui Province for Outstanding Youth(No.1808085J04)。
文摘D-peptides are recognized as a new class of synthetic chemical drugs and they possess many interesting advantages such as high enzymatic stability,improved oral bioavailability,as well as high binding affinity and specificity.Recently,D-peptide drugs have been attracting increasing attention in both academic and industrial researches over recent years.One D-peptide etelcalcetide has even entered the market that targets the calcium(Ca2+)-sensing receptor(CaSR) to fight secondary hyperparathyroidism.Effective discovery and optimization of D-peptide ligands that can bind to various disease-related targets with high specificity and potency is of great importance for the development of D-peptide drugs.This review surveys the recent method development in this area especially the chemical protein synthesis-assisted high-throughput screening strategies for D-peptide ligands and their application in drug discovery.
基金our funding sources:US Office of the Secretary of Defense Applied Research for the Advancement of S&T Priorities program Synthetic Biology for Military Environments and the US Army Combat Capabilities Development Command Chemical Biological Center Section 2363 Biological Engineering for Applied Materials Solutions program.This work was performed while Marilyn Lee held an NRC Research Associateship award at US Army CCDC CBC.
文摘Cell-free systems contain many proteins and metabolites required for complex functions such as transcription and translation or multi-step metabolic conversions.Research into expanding the delivery of these systems by drying or by embedding into other materials is enabling new applications in sensing,point-of-need manufacturing,and responsive materials.Meanwhile,silk fibroin from the silk worm,Bombyx mori,has received attention as a protective additive for dried enzyme formulations and as a material to build biocompatible hydrogels for controlled localization or delivery of biomolecular cargoes.In this work,we explore the effects of silk fibroin as an additive in cell-free protein synthesis(CFPS)reactions.Impacts of silk fibroin on CFPS activity and stability after drying,as well as the potential for incorporation of CFPS into hydrogels of crosslinked silk fibroin are assessed.We find that simple addition of silk fibroin increased productivity of the CFPS reactions by up to 42%,which we attribute to macromolecular crowding effects.However,we did not find evidence that silk fibroin provides a protective effects after drying as previously described for purified enzymes.Further,the enzymatic crosslinking transformations of silk fibroin typically used to form hydrogels are inhibited in the presence of the CFPS reaction mixture.Crosslinking attempts did not impact CFPS activity,but did yield localized protein aggregates rather than a hydrogel.We discuss the mechanisms at play in these results and how the silk fibroin-CFPS system might be improved for the design of cell-free devices.
基金supported by the National Natural Science Foundation of China(Grant No.21878173)National Key R&D Program of China(Grant No.2018YFA0901700)a grant from the Institute Guo Qiang,Tsinghua University(2019GQG1016).
文摘Gases are the vital nutrition of all organisms as the precursor of metabolism pathways.As a potential biological process,protein synthesis is inevitably regulated by gas transport and utilization.However,the effect of carbon dioxide(CO_(2))present in many metabolic pathways on protein synthesis has not been studied well.In this work,carbon dioxide combined with oxygen was employed for cell-free protein synthesis(CFPS)in the tube-in-tube reactor with precise control of gas concentration.In this in vitro system,gases could directly affect the protein synthesis process without transmembrane transport.Varied concentrations of carbon dioxide(0-1%)and constant oxygen concentration(21%)were employed for CFPS to assess the effects.The cell-free reactions with 0.3%CO_(2) and 21%O_(2) showed the highest protein yields.The combined effect of CO_(2) and O_(2) also resulted in relatively high protein expression under high oxygen conditions(0.3%CO_(2) and 100%O_(2)).Moreover,metabolomics assays were performed to gain insight into metabolic changes,which showed that CO_(2) slightly improved energy metabolism and redox balance.In particular,the extra supplied CO_(2) activated the decarboxylating reactions and removed toxic metabolites to recover the protein synthesis activity.The exploration of CO_(2) on protein synthesis could provide guiding implications for basic studies and biomanufacturing.
文摘In the present experiments the changes in levels of ribosome,polysome and 3H-leucine incorporation rate in liver post-mitochondrial supernatant (PM-supernatant) were investigated in Sedericient and Se-supplented rats.The results demonstrated that the amounts of ribosome and polysome as well as the ratio of polysome to ribosome in liver PM-supernatant from the Se-deficient rats were all remarkahly decreased.In the meantime,the rate of protein synthesis expressed as radioactivity or 3H-leucine incorporated into protein in the PM-supernatant system also decreased significantly.The results suggest that the decreases of ribosomes and proportion of ribosomal aggregates in PM-supernatant may be responsible for the decrease of the protein synthesis activity in liver of the Se-deficient animals.
文摘The interferon-inducible-stranded-RNA-depedet protein kinase PKR has been implicated in both the antiviral aand cell growth-regulatory effects of the interferons.Over-expression of the wild-type form of this protein inhibits cell proliferation,whereas over-expression of inactive mutant forms transforms cells to a tumouri-genie phenotype.It has been suggested that mutant PKR exerts a dominant negative effect on the activity of the wild-type protein kinase.We have investigated this possibility using the rabbbit reticulocyte cell-free translation system in which protein synthesis is inhibited by dsRNA due to activation of PKR and phosphorylation of initiation factor elF-2. Addition of a highly purified inactive PKR mutant,synthesised in a baculovirus-infected insect cell system, rescues protein synthesis from inhibition by the low concentrations of dsRNA in a dose-dependent manner. The PKR mutant has no effect on protein synthesis in the absence of dsRNA of in the presence of another inhibitory protein kinase,the haem-controlled repressor.Inhibition of translation can be re-established in the presence of the mutant PKR by adding a higher concentration of dsRNA.These results suggest that inactive mutant PKR does exert a dominant negative effect on wild-type PKR and that this may be due to competition for dsRNA binding.
基金This work was supported by the National Science Foundation CBET Division CAREER Award(grant number:1254148)the Utah NASA Space Grant Consortiumand by the Simmons Center for Cancer Research at Brigham Young University.
文摘An“endotoxin-free”E.coli-based cell-free protein synthesis system has been reported to produce therapeutic proteins rapidly and on-demand.However,preparation of the most complex CFPS reagent–the cell extract–remains time-consuming and labor-intensive because of the relatively slow growth kinetics of the endotoxin-free ClearColiTMBL21(DE3)strain.Here we report a streamlined procedure for preparing E.coli cell extract from ClearColi™using auto-induction media.In this work,the term auto-induction describes cell culture media which eliminates the need for manual induction of protein expression.Culturing Clearcoli™cells in autoinduction media significantly reduces the hands-on time required during extract preparation,and the resulting“endotoxinfree”cell extract maintained the same cell-free protein synthesis capability as extract produced with traditional induction as demonstrated by the high-yield expression of crisantaspase,an FDA approved leukemia therapeutic.It is anticipated that this work will lower the barrier for researchers to enter the field and use this technology as the method to produce endotoxin-free E.coli-based extract for CFPS.
文摘Aminoacyl-tRNA synthetases (aaRSs) are a family of ancient enzymes that are responsible for catalysis of the attachment of amino acids to their cognate tRNAs in the first step
文摘In this study the effect of human recombinant interferon gamma hrIFN-γ)on hCG secretion of human first trimester trophoblast and protein synthesis of decidual tissue was investigated in vitro.The results indicated that hrIFN-γat the doses of 250 U/ml medium and 2500 U/ml medium decreased hCG secretion of trophoblast obviously(P<0.05, P<0.01)in a dose dependent manner.The effect of hrIFN-γon protein synthesis at the doses of 10 U to 1,000 U/ml medium inhibited the 3H leucine incorporation obviously.The cpm values between control and experimental groups were significantly different(P<0. 05) in a dosedependent manner.Furthermore its inhibitory effect is in a dose-dependent manner and was neutralized by IFN-γantiserum.The IFN-a at the doses used did not find any effect on protein synthesis in decidual tissue.
基金supported by the Natural Science Foundation of China(Grant No.32071517,82072106)the Natural Science Basic Research Plan in Shaanxi Province of China(Grant No.2020JM-100).
文摘As a key coordinator of metabolism,AMP-activated protein kinase(AMPK)is vitally involved in skeletal muscle maintenance.AMPK exerts its cellular effects through its function as a serine/threonine protein kinase by regulating many downstream targets and plays important roles in the development and growth of skeletal muscle.AMPK is activated by phosphorylation and exerts its function as a kinase in many processes,including synthesis and degradation of proteins,mitochondrial biogenesis,glucose uptake,and fatty acid and cholesterol metabolism.Skeletal muscle atrophy is a result of various diseases or disorders and is characterized by a decrease in muscle mass.The pathogenesis and therapeutic strategies of skeletal muscle atrophy are still under investigation.In this review,we discuss the role of AMPK in skeletal muscle metabolism and atrophy.We also discuss targeting AMPK for skeletal muscle treatment,including exercise,AMPK activators including 5-amino-4-imidazolecarboxamide ribonucleoside and metformin,and low-level lasers.These studies show the important roles of AMPK in regulating muscle metabolism and function;thus,the treatment of skeletal muscle atrophy needs to take into account the roles of AMPK.