Talin-1 head(hereinafter referred to as TH)is the head structure of talin protein,which contains a four-point-one-protein/ezrin/radixin/moesin(FERM)domain.Its F1 domain contains an unstructured loop of 30 amino acids(...Talin-1 head(hereinafter referred to as TH)is the head structure of talin protein,which contains a four-point-one-protein/ezrin/radixin/moesin(FERM)domain.Its F1 domain contains an unstructured loop of 30 amino acids(139-168),which does not interact with other domains.Because TH doesn’t get the crystal structure and whether the unstructured loop has obvious influence on the TH secondary structure,therefore,the truncated talin-1 headΔ139-168(hereinafter referred to as THΔ)was constructed and its structure and the impact of stability after truncation were analyzed.Molecular biology and structural biology methods were used to construct prokaryotic expression vectors of TH and THΔ,explore and optimize the expression conditions of recombinants,and they were purified by affinity chromatography and FPLC gel filtration chromatography.Finally,a large number of stable,high-purity protein samples were prepared successfully.The physicochemical properties and structural stability of the proteins were investigated by dynamic light scattering and circular dichroism.The results showed that the THΔsecondary structure of the truncated body did not change significantly,the structural stability was enhanced and the resistance to guanidine hydrochloride and high temperature was stronger.展开更多
Biomarke rs are required for the early detection,prognosis prediction,and monitoring of amyotrophic lateral sclerosis,a progressive disease.Proteomics is an unbiased and quantitative method that can be used to detect ...Biomarke rs are required for the early detection,prognosis prediction,and monitoring of amyotrophic lateral sclerosis,a progressive disease.Proteomics is an unbiased and quantitative method that can be used to detect neurochemical signatures to aid in the identification of candidate biomarke rs.In this study,we used a label-free quantitative proteomics approach to screen for substantially differentially regulated proteins in ten patients with sporadic amyotrophic lateral scle rosis compared with five healthy controls.Su bstantial upregulation of serum proteins related to multiple functional clusters was observed in patients with spo radic amyotrophic lateral sclerosis.Potential biomarke rs were selected based on functionality and expression specificity.To validate the proteomics profiles,blood samples from an additional cohort comprising 100 patients with sporadic amyotrophic lateral sclerosis and 100 healthy controls were subjected to enzyme-linked immunosorbent assay.Eight substantially upregulated serum proteins in patients with spora dic amyotrophic lateral sclerosis were selected,of which the cathelicidin-related antimicrobial peptide demonstrated the best discriminative ability between patients with sporadic amyotrophic lateral sclerosis and healthy controls(area under the curve[AUC]=0.713,P<0.0001).To further enhance diagnostic accuracy,a multi-protein combined discriminant algorithm was developed incorporating five proteins(hemoglobin beta,cathelicidin-related antimicrobial peptide,talin-1,zyxin,and translationally-controlled tumor protein).The algo rithm achieved an AUC of 0.811 and a P-value of<0.0001,resulting in 79%sensitivity and 71%specificity for the diagnosis of sporadic amyotrophic lateral scle rosis.Subsequently,the ability of candidate biomarkers to discriminate between early-stage amyotrophic lateral sclerosis patients and controls,as well as patients with different disease severities,was examined.A two-protein panel comprising talin-1 and translationally-controlled tumor protein effectively distinguished early-stage amyotrophic lateral sclerosis patients from controls(AUC=0.766,P<0.0001).Moreove r,the expression of three proteins(FK506 binding protein 1A,cathelicidin-related antimicrobial peptide,and hemoglobin beta-1)was found to increase with disease progression.The proteomic signatures developed in this study may help facilitate early diagnosis and monitor the progression of sporadic amyotrophic lateral sclerosis when used in co mbination with curre nt clinical-based parameters.展开更多
基金Supported by the Scientific Research Personnel of Northeast Agricultural University(518001)。
文摘Talin-1 head(hereinafter referred to as TH)is the head structure of talin protein,which contains a four-point-one-protein/ezrin/radixin/moesin(FERM)domain.Its F1 domain contains an unstructured loop of 30 amino acids(139-168),which does not interact with other domains.Because TH doesn’t get the crystal structure and whether the unstructured loop has obvious influence on the TH secondary structure,therefore,the truncated talin-1 headΔ139-168(hereinafter referred to as THΔ)was constructed and its structure and the impact of stability after truncation were analyzed.Molecular biology and structural biology methods were used to construct prokaryotic expression vectors of TH and THΔ,explore and optimize the expression conditions of recombinants,and they were purified by affinity chromatography and FPLC gel filtration chromatography.Finally,a large number of stable,high-purity protein samples were prepared successfully.The physicochemical properties and structural stability of the proteins were investigated by dynamic light scattering and circular dichroism.The results showed that the THΔsecondary structure of the truncated body did not change significantly,the structural stability was enhanced and the resistance to guanidine hydrochloride and high temperature was stronger.
基金supported by the grants from Shanghai Shuguang Plan Project,No.18SG15(to SC)Shanghai Outstanding Young Scholars Project+2 种基金Shanghai Talent Development Project,No.2019044(to SC)Medical-engineering cross fund of Shanghai Jiao Tong University,No.YG2022QN009(to QZ)the National Natural Science Foundation of China,No.82201558(to QZ)。
文摘Biomarke rs are required for the early detection,prognosis prediction,and monitoring of amyotrophic lateral sclerosis,a progressive disease.Proteomics is an unbiased and quantitative method that can be used to detect neurochemical signatures to aid in the identification of candidate biomarke rs.In this study,we used a label-free quantitative proteomics approach to screen for substantially differentially regulated proteins in ten patients with sporadic amyotrophic lateral scle rosis compared with five healthy controls.Su bstantial upregulation of serum proteins related to multiple functional clusters was observed in patients with spo radic amyotrophic lateral sclerosis.Potential biomarke rs were selected based on functionality and expression specificity.To validate the proteomics profiles,blood samples from an additional cohort comprising 100 patients with sporadic amyotrophic lateral sclerosis and 100 healthy controls were subjected to enzyme-linked immunosorbent assay.Eight substantially upregulated serum proteins in patients with spora dic amyotrophic lateral sclerosis were selected,of which the cathelicidin-related antimicrobial peptide demonstrated the best discriminative ability between patients with sporadic amyotrophic lateral sclerosis and healthy controls(area under the curve[AUC]=0.713,P<0.0001).To further enhance diagnostic accuracy,a multi-protein combined discriminant algorithm was developed incorporating five proteins(hemoglobin beta,cathelicidin-related antimicrobial peptide,talin-1,zyxin,and translationally-controlled tumor protein).The algo rithm achieved an AUC of 0.811 and a P-value of<0.0001,resulting in 79%sensitivity and 71%specificity for the diagnosis of sporadic amyotrophic lateral scle rosis.Subsequently,the ability of candidate biomarkers to discriminate between early-stage amyotrophic lateral sclerosis patients and controls,as well as patients with different disease severities,was examined.A two-protein panel comprising talin-1 and translationally-controlled tumor protein effectively distinguished early-stage amyotrophic lateral sclerosis patients from controls(AUC=0.766,P<0.0001).Moreove r,the expression of three proteins(FK506 binding protein 1A,cathelicidin-related antimicrobial peptide,and hemoglobin beta-1)was found to increase with disease progression.The proteomic signatures developed in this study may help facilitate early diagnosis and monitor the progression of sporadic amyotrophic lateral sclerosis when used in co mbination with curre nt clinical-based parameters.
