The E3 ubiquitin ligase,carboxyl terminus of heat shock protein 70(Hsp70)interacting protein(CHIP),also functions as a co-chaperone and plays a crucial role in the protein quality control system.In this study,we aimed...The E3 ubiquitin ligase,carboxyl terminus of heat shock protein 70(Hsp70)interacting protein(CHIP),also functions as a co-chaperone and plays a crucial role in the protein quality control system.In this study,we aimed to investigate the neuroprotective effect of overexpressed CHIP on Alzheimer’s disease.We used an adeno-associated virus vector that can cross the blood-brain barrier to mediate CHIP overexpression in APP/PS1 mouse brain.CHIP overexpression significantly ameliorated the performance of APP/PS1 mice in the Morris water maze and nest building tests,reduced amyloid-βplaques,and decreased the expression of both amyloid-βand phosphorylated tau.CHIP also alleviated the concentration of microglia and astrocytes around plaques.In APP/PS1 mice of a younger age,CHIP overexpression promoted an increase in ADAM10 expression and inhibitedβ-site APP cleaving enzyme 1,insulin degrading enzyme,and neprilysin expression.Levels of HSP70 and HSP40,which have functional relevance to CHIP,were also increased.Single nuclei transcriptome sequencing in the hippocampus of CHIP overexpressed mice showed that the lysosomal pathway and oligodendrocyte-related biological processes were up-regulated,which may also reflect a potential mechanism for the neuroprotective effect of CHIP.Our research shows that CHIP effectively reduces the behavior and pathological manifestations of APP/PS1 mice.Indeed,overexpression of CHIP could be a beneficial approach for the treatment of Alzheimer’s disease.展开更多
HCLS1-associated protein X-1(HAX1)is a multifunctional mitochondrial protein involved in the regulation of apoptosis,a crucial process of programmed cell death,and mRNA processing.Despite its significance,limited stru...HCLS1-associated protein X-1(HAX1)is a multifunctional mitochondrial protein involved in the regulation of apoptosis,a crucial process of programmed cell death,and mRNA processing.Despite its significance,limited structural data is available for HAX1,hindering a comprehensive understanding of its biological function.Notably,the caseinolytic mitochondrial matrix peptidase chaperone subunit B(CLPB)has been identified as an interacting partner of HAX1,yet the biophysical properties and binding affinity governing their interaction remain poorly defined.In this study,we present a thorough biophysical characterization of full-length human HAX1 and CLPB,accomplished through recombinant expression and purification.By employing size exclusion chromatography,dynamic light scattering,and circular dichroism spectroscopy,we successfully established their biophysical properties,revealing contrasting structural features,with CLPB displaying a-helical content and HAX1 exhibiting a disordered nature.Moreover,we employed solutionstate nuclear magnetic resonance(NMR)spectroscopy to probe their binding affinity.Our findings demonstrate the formation of stable multimeric complexes between HAX1 and CLPB,and we quantified a dissociation constant in the low range of micro-molar for their high affinity interaction.These results lay the foundation for further in-depth investigations into the dynamics and energetics governing the HAX1-CLPB interaction,ultimately contributing to a comprehensive understanding of their functional mechanisms.展开更多
Elucidation of ligand-protein interactions provides new insights into the physiological functions and mechanisms of ligand molecules,enabling new ideas for the treatment of diseases,and drug discovery and development....Elucidation of ligand-protein interactions provides new insights into the physiological functions and mechanisms of ligand molecules,enabling new ideas for the treatment of diseases,and drug discovery and development.Most ligand-protein binding occurs only in specific regions of proteins.The identification of protein targets and binding regions is crucial for drug discovery and development,as well as for the in-depth study of drug-protein conformational relationships[1].展开更多
Protein interacting with C kinase 1(PICK1)interacts with a variety of membrane proteins and receptors involved in nervous system diseases and multiple cancers.However,the role of PICK1 in gastric cancer remains unclea...Protein interacting with C kinase 1(PICK1)interacts with a variety of membrane proteins and receptors involved in nervous system diseases and multiple cancers.However,the role of PICK1 in gastric cancer remains unclear.In the present work,we explored the expression and interactions of PICK 1 with Toll-like receptor 4(TLR4)in gastric cancer.Clinical data analysis showed that PICK1 expression decreases and is predictive of worse outcomes in patients with gastric cancer.High PICK1 levels attenuate the proliferation and migration of gastric cancer cells,which is dependent on the TLR4/myeloid differentiation primary response 88(MyD88)signaling pathway.Furthermore,in vitro experiments demonstrated that PICK1 affects the trafficking and degradation of TLR4 and promotes TLR4 degradation via autophagy in gastric cancer cells.Molecular dynamics simulations highlighted the binding strength and stability of the TLR4-PICK1 complex.Our study provides new insights into the cellular and pathological functions of PICK1 in gastric cancer.展开更多
The yeast two\|hybrid system is a molecular genetic approach for protein interaction and it is widely used to screen for proteins that interact with a protein of interest in recent years.This process includes,construc...The yeast two\|hybrid system is a molecular genetic approach for protein interaction and it is widely used to screen for proteins that interact with a protein of interest in recent years.This process includes,construction and testing of the bait plasmid,screening a plasmid library for interacting fusion protein,elimination of false positives and delection analysis of true positives.This procedure is designed to allow investigators to identify proteins and their encoding cDNAs that have a biologically significant interaction with a protein of interest.More and more studies have demonstrated that the two\|hybrid system is a powerful and sensitive technique for the identification of genes that code for proteins that interact in a biologically significant fashion with a protein of interest in higher plants.This method has been used to identify new interaction protein in many laboratories.The recently reported yeast tri\|brid system,should allow the investigation of more complex protein\|protein interactions.The aim of this review is to outline the recent progress made in protein interactions by using yeast two\|hybrid system.展开更多
Tissue and systemic inflammation have been the main culprit behind the cellular response to multiple insults and maintaining homeostasis.Obesity is an independent disease state that has been reported as a common risk ...Tissue and systemic inflammation have been the main culprit behind the cellular response to multiple insults and maintaining homeostasis.Obesity is an independent disease state that has been reported as a common risk factor for multiple metabolic and microvascular diseases including nonalcoholic fatty liver disease(NAFLD),retinopathy,critical limb ischemia,and impaired angiogenesis.Sterile inflammation driven by high-fat diet,increased formation of reactive oxygen species,alteration of intracellular calcium level and associated release of inflammatory mediators,are the main common underlying forces in the pathophysiology of NAFLD,ischemic retinopathy,stroke,and aging brain.This work aims to examine the contribution of the pro-oxidative and pro-inflammatory thioredoxin interacting protein(TXNIP)to the expression and activation of NLRP3-inflammasome resulting in initiation or exacerbation of sterile inflammation in these disease states.Finally,the potential for TXNIP as a therapeutic target and whether TXNIP expression can be modulated using natural antioxidants or repurposing other drugs will be discussed.展开更多
Receptors interaction protein 2(RIP2)is a specific adaptor molecule in the downstream of NOD2.The role of RIP2 during foot-and-mouth disease virus(FMDV)infection remains unknown.Here,our results showed that RIP2 inhib...Receptors interaction protein 2(RIP2)is a specific adaptor molecule in the downstream of NOD2.The role of RIP2 during foot-and-mouth disease virus(FMDV)infection remains unknown.Here,our results showed that RIP2 inhibited FMDV replication and played an important role in the activation of IFN-βand NF-κB signal pathways during FMDV infection.FMDV infection triggered RIP2 transcription,while it reduced the expression of RIP2 protein.Detailed analysis showed that FMDV 2B,2C,3C^(pro),and L^(pro) proteins were responsible for inducing the reduction of RIP2 protein.3C^(pro) and L^(pro) are viral proteinases that can induce the cleavage or reduction of many host proteins and block host protein synthesis.The carboxyl terminal 105-C114 and 135-C144 regions of 2B were essential for reduction of RIP2.Our results also showed that the N terminal 1-61 region of 2C were essential for the reduction of RIP2.The 2C-induced reduction of RIP2 was dependent on inducing the reduction of poly(A)-binding protein 1(PABPC1).The interaction between RIP2 and 2C was observed in the context of viral infection,and the residues 1-61 were required for the interaction.These data clarify novel mechanisms of reduction of RIP2 mediated by FMDV.展开更多
AIM:To understand the complex reaction of gastric inflammation induced by Helicobacter pylori(H pylori) in a systematic manner using a protein interaction network. METHODS:The expression of genes significantly changed...AIM:To understand the complex reaction of gastric inflammation induced by Helicobacter pylori(H pylori) in a systematic manner using a protein interaction network. METHODS:The expression of genes significantly changed on microarray during H pylori infection was scanned from the web literary database and translated into proteins.A network of protein interactions was constructed by searching the primary interactions of selected proteins.The constructed network was mathematically analyzed and its biological function was examined.In addition,the nodes on the network were checked to determine if they had any further functional importance or relation to other proteins by extending them. RESULTS:The scale-free network showing the relationship between inflammation and carcinogenesis was constructed.Mathematical analysis showed hub and bottleneck proteins,and these proteins were mostly related to immune response.The network contained pathways and proteins related to H pylori infection,such as the JAK-STAT pathway triggered by interleukins.Activation of nuclear factor (NF)-κB,TLR4,and other proteins known to function as core proteins of immune response were also found. These immune-related proteins interacted on the network with pathways and proteins related to the cell cycle,cell maintenance and proliferation,andtranscription regulators such as BRCA1,FOS,REL,and zinc finger proteins.The extension of nodes showed interactions of the immune proteins with cancer- related proteins.One extended network,the core network,a summarized form of the extended network, and cell pathway model were constructed. CONCLUSION:Immune-related proteins activated by H pylori infection interact with proto-oncogene proteins.The hub and bottleneck proteins are potential drug targets for gastric inflammation and cancer.展开更多
The B-box(BBX)family of proteins consists of zinc-finger transcription factors with one or two highly conserved B-box motifs at their N-termini.BBX proteins play crucial roles in various aspects of plant growth and de...The B-box(BBX)family of proteins consists of zinc-finger transcription factors with one or two highly conserved B-box motifs at their N-termini.BBX proteins play crucial roles in various aspects of plant growth and development,including seedling photomorphogenesis,shade avoidance,flowering time,and biotic and abiotic stress responses.Previous studies have identified many different BBXs from several plant species,although the BBX family members in maize are largely unknown.Genome-wide identification and comprehensive analysis of maize BBX(ZmBBX)expression and interaction networks would therefore provide valuable information for understanding their functions.In this study,36 maize BBXs in three major clades were identified.The ZmBBXs within a given clade were found to share similar domains,motifs,and genomic structures.Gene duplication analyses revealed that the expansion of BBX proteins in maize has mainly occurred by segmental duplication.The expression levels of ZmBBXs were analyzed in various organs and tissues,and under different abiotic stress conditions.Protein–protein interaction networks of ZmBBXs were established using bioinformatic tools and verified by bimolecular fluorescence complementation(BiFC)assays.Our findings can facilitate a greater understanding of the complexity of the ZmBBX family and provide novel clues for unravelling ZmBBX protein functions.展开更多
Proteasome dysfunction has been repeatedly reported in alcoholic liver disease. Ethanol metabolism endproducts affect the structure of the proteasome, and, therefore, change the proteasome interaction with its regulat...Proteasome dysfunction has been repeatedly reported in alcoholic liver disease. Ethanol metabolism endproducts affect the structure of the proteasome, and, therefore, change the proteasome interaction with its regulatory complexes 19S and PA28, as well as its interacting proteins. Chronic ethanol feeding alters the ubiquitin-proteasome activity by altering the interaction between the 19S and the 20S proteasome interaction. The degradation of oxidized and damaged proteins is thus decreased and leads to accumulation of insoluble protein aggregates, such as Mallory-Denk bodies. Ethanol also affects the immunoproteasome formation. PA28a/b interactions with the 20S proteasome are decreased in the proteasome fraction isolated from the liver of rats fed ethanol chronically, thus affecting the cellular antigen presentation and defense against pathogenic agents. Recently, it has been shown that ethanol also affects the proteasome interacting proteins (PIPs). Interaction of the proteasome with Ecm29 and with deubiquitinating enzymes Rpn11, UCH37, and Usp14 has been found to decrease. However, the two UBL-ubiquitin-associated domain (UBA) PIPs p62 and valosin-containing protein are upregulated when the proteasome is inhibited. The increase of these UBL-UBA proteins, as well as the increase in Hsp70 and Hsp25 levels, compensated for the proteasome failure and helped in the unfolding/docking of misfolded proteins. Chronic alcohol feeding to rats causes a significant inhibition of the proteasome pathway and this inhibition results from a decreases of the interaction between the 20S proteasome and the regulatory complexes, PIPs, and the ubiquitin system components.展开更多
AIM： To investigate the biological function of p7 protein and to look for proteins interacting with p7 protein in hepatocytes. METHODS： We constructed p7 protein bait plasmid by cloning the gene of p7 protein into p...AIM： To investigate the biological function of p7 protein and to look for proteins interacting with p7 protein in hepatocytes. METHODS： We constructed p7 protein bait plasmid by cloning the gene of p7 protein into pGBKTT, then transformed it into yeast AH109 （a type）. The transformed yeast was mated with yeast Y187 （α type） containing liver cDNA library plasmid, pACT2 in 2×YPDA medium. Diploid yeast was plated on synthetic dropout nutrient medium （SD/- Trp-Leu-His-Ade） containing x-α-gal for selection and screening. After extracting and sequencing of plasmids from blue colonies, we performed sequence analysis by bioinformatics. RESULTS： Fifty colonies were selected and sequenced. Among them, one colony was Homo sapiens signal sequence receptor, seven colonies were Homo sapiens H19, seven colonies were immunoglobulin superfamily containing leucine-rich repeat, three colonies were spermatid peri-nuclear RNA binding proteins, two colonies were membrane-spanning 4-domains, 24 colonies were cancer-associated antigens, four colonies were nudeoporin 214 ku and two colonies were CLL-associated antigens. CONCLUSION： The successful cloning of gene of protein interacting with p7 protein paves a way for the study of the physiological function of p7 protein and its assodated protein.展开更多
The Ca^(2+)/CaM signal transduction pathway helps plants adapt to environmental stress. However, our knowledge on the functional proteins of C^(2+)/CaM pathway in peanut(Arachis hypogeae L.) remains limited. In the pr...The Ca^(2+)/CaM signal transduction pathway helps plants adapt to environmental stress. However, our knowledge on the functional proteins of C^(2+)/CaM pathway in peanut(Arachis hypogeae L.) remains limited. In the present study, a novel calmodulin 4(CaM4)-binding protein S-adenosyl-methionine synthetase 1(SAMS1) in peanut was identified using a yeast two-hybrid assay. Expression of AhSAMS1was induced by Ca^(2+), ABA, and salt stress. To elucidate the function of AhSAMS1, physiological and phenotypic analyses were performed with wild-type and transgenic materials. Overexpression of AhSAMS1increased spermidine and spermidine synthesis while decreased the contents of ethylene, thereby eliminating excessive reactive oxygen species(ROS) in transgenic lines under salt stress. AhSAMS1 reduced uptake of Na+and leakage of K+from mesophyll cells, and was less sensitive to salt stress during early seedling growth, in agreement with the induction of SOS and NHX genes Transcriptomics combined with epigenetic regulation uncovered relationships between differentially expressed genes and differentially methylated regions, which raised the salt tolerance and plants growth. Our findings support a model in which the role of AhSAMS1 in the ROS-dependent regulation of ion homeostasis was enhanced by Ca^(2+)/CaM while AhSAMS1-induced methylation was regulated by CaM, thus providing a new strategy for increasing the tolerance of plants to salt stress.展开更多
To screen for novel binding proteins interacting with high-risk HPV 18 E6 oncogene, the strain AH 109 was transformed with pGBKT7-HPV 18 E6 plasmid, and subsequent transference was utilized to screen for interacting p...To screen for novel binding proteins interacting with high-risk HPV 18 E6 oncogene, the strain AH 109 was transformed with pGBKT7-HPV 18 E6 plasmid, and subsequent transference was utilized to screen for interacting proteins with HPV 18 E6 in human Hela cDNA library. HPV 18 E6 mRNA was expressed in yeast and there was no self-activation and toxicity in AH109. Seven proteins that interacted with HPV18 E6, including transmembrane protein 87B, phosphonoformate immuno-associated protein 5, vimentin, KM-HN-1 protein, dedicator of cytokinesis 7, vaccinia related kinase 2 and a hypothetical protein, were identified. It was suggested that yeast two-hybrid system is an efficient for screening interacting proteins. The high-risk HPV 18 E6 oncogene may interact with the proteins, which may be associated with signal transduction and transcriptional control, epithelial cell invasion and migration, as well as humoral and cellular immune etc. This investigation provides functional clues for further exploration of potential oncogenesis targets for cancer biotherapy.展开更多
Interactions are the essence of all biomolecules because they cannot fulfill their roles without interacting with other molecules. Hence, mapping the interactions of biomolecules can be useful for understanding their ...Interactions are the essence of all biomolecules because they cannot fulfill their roles without interacting with other molecules. Hence, mapping the interactions of biomolecules can be useful for understanding their roles and functions. Furthermore, the development of molecular based systems biology requires an understanding of the biomolecular interactions. In recent years, the mapping of protein-protein interactions in different species has been reported, but few reports have focused on the large-scale mapping of protein-protein interactions in human. Here, we review the developments in protein interaction mapping and we discuss issues and strategies for the mapping of the human protein interactome.展开更多
The mechanism and interaction among Rb/p16, Rb/E2F1 and HDAC1 proteins in gallbladder carcinoma were investigated. By using the immunoprecipitation method, the interactions among Rb, p16, E2F1, HDAC1 proteins in gallb...The mechanism and interaction among Rb/p16, Rb/E2F1 and HDAC1 proteins in gallbladder carcinoma were investigated. By using the immunoprecipitation method, the interactions among Rb, p16, E2F1, HDAC1 proteins in gallbladder carcinoma cell line （Mz-ChA-1） were studied. It was found that there were Rb and E2F1 proteins in the precipitates with anti-HDAC1, and there were HDAC1 and E2F1 proteins in the precipitate with anti-Rb. It was concluded that there are specific interactions among Rb, HDAC1 and E2F1 proteins in gallbladder carcinoma, indicating the existence of the direct Rb/E2F1/HDAC1 signal transduction pathway. There is no direct relationship between p16 proteins with Rb, HDAC1, and E2F1 proteins.展开更多
Avian influenza virus(AIV)nonstructural 1(NS1)gene was amplified by real-time polymerse chain reac tion(RT-PCR)and inserted into pET28a,then transformed into E.coli BL21(DE3)competent cell.With the induction of isopro...Avian influenza virus(AIV)nonstructural 1(NS1)gene was amplified by real-time polymerse chain reac tion(RT-PCR)and inserted into pET28a,then transformed into E.coli BL21(DE3)competent cell.With the induction of isopropyl-β-D-thiogalactoside(IPTG)and the purification of Ni-NTA column,we finally obtained purified NS1 protein.T7-phage display system was used to screen the proteins that interacted with NS1 from lung cell cDNA li brary.The selected positive clones were identified by DNA sequencing and analyzed by BLAST program in Gene Bank.Two proteins were obtained as NS1 binding proteins,Homo sapiens nucleolar and coiled-body phosphoprotein 1(NOLC1)and Homo sapiens similar to colon cancer-associated antigen.By co-immunoprecipitation and other me thods,Homo sapiens NOLC1 was found to interact with the NS1 protein,the results would provide the basis for fur ther studying biological function of NS1 protein.展开更多
Regulation of the number of aetivin receptors that are present in the cell membrane plays a key role in the modulation of cellular responses to activin. In order to find the regulators, a novel protein ARIPzip, intera...Regulation of the number of aetivin receptors that are present in the cell membrane plays a key role in the modulation of cellular responses to activin. In order to find the regulators, a novel protein ARIPzip, interacting with activin type II receptors, was searched and identified by using yeast two-hybrid screening. ARIPzip is a splicing variant of ARIP2. This has been discussed previously. ARIPzip can specifically interact with ActR Ⅱ A, and is widely distributed in mouse tissues. Overexpression of ARIPzip can cause the activin-induced transcriptional activities to increase in a dose-dependent manner while the overexpression of ARIV2 can decrease these activities. These data suggest that the C-terminal rezions of ARIP2 and ARIPzip are involved in the regulation of activin signaling.展开更多
Zebrafish △113p53, an N-terminal truncated p53 isoform, is a p53-target gene that antagonises p53-mediated apoptotic activity. Interestingly, △113p53 does not act on p53 in a dominant-negative manner, but rather int...Zebrafish △113p53, an N-terminal truncated p53 isoform, is a p53-target gene that antagonises p53-mediated apoptotic activity. Interestingly, △113p53 does not act on p53 in a dominant-negative manner, but rather interferes with the p53 function by differentially modulating p53-target gene expression to protect cells from apoptosis. Previous studies showed that over-expressed △113p53 and p53 proteins formed a complex. However, it is not known whether endogenous p53 and △113p53 proteins also interact with each other, and if this interaction is required for △113p53 to inhibit the apoptotic activity of full-length p53. In this study, we used two available zebrafish p53 antibodies to address these questions. One, Zfp53-N, only recognises full-length p53, whereas the other, Zfp53-A7C10, detects both full-length p53 and △113p53. Using Zfp53-N for immunoprecipitation and Zfp53-A7C 10 for detection, we demonstrated that endogenous △113p53 and full-length p53 induced by a DNA-damaging drug formed a complex in vivo. Furthermore, of the six △113p53 mutants we generated with different point mutations in the oligomerisation domain, two failed to interact with p53 and lost the ability to modulate p53-target gene expression and inhibit p53-induced cell apoptosis. However, those △113p53 mutants that could interact with p53 retained the ability to antagonise the apoptotic activity of p53. Therefore, our data demonstrated that protein--protein interaction between △113p53 and p53 is essential for the anti-apoptotic function of △113p53. In addition, the two △113p53 mutants that failed to interact with p53 are also useful for the study of the mechanisms of other functions of △113p53.展开更多
Hot pepper(Capsicum annuum var.conoides)is a significant vegetable that is widely cultivated around the world.Currently,global climate change has caused frequent severe weather events,and waterlogging stress harms the...Hot pepper(Capsicum annuum var.conoides)is a significant vegetable that is widely cultivated around the world.Currently,global climate change has caused frequent severe weather events,and waterlogging stress harms the pepper industry by affecting the planting period,growth conditions,and disease susceptibility.The gene CaABI3/VP1-1 could improve pepper waterlogging tolerance.In order to explore the upstream regulatory mechanism of CaABI3/VP1-1,a high-quality standardized yeast hybrid library was successfully constructed for yeast one-,two-,and threehybrid screening using pepper‘ZHC2’as the experimental material,with a library recombinant efficiency of up to 100%.The length of inserted fragments varied from 650 to 5000 bp,the library titer was 5.18×10^(6)colony-forming units(CFU)·mL-1,and the library capacity was 1.04×10^(7)CFU of cDNA inserts.The recombinant bait plasmid was used to successfully identify 78 different proteins through the yeast one-hybrid system,including one transcription factor within the ethylene-responsive factor family and the other within the growth-regulating factor family.The interaction happened between LOC124895848 and CaABI3/VP1-1 promoter by point-to-point yeast one-hybrid experiment.The expression level of the 12 selected protein-coding genes was then evaluated by quantitative real-time polymerase chain reaction.Results indicated the protein coding genes showed different responses to waterlogging stress and that the activity of the CaABI3/VP1-1 promoter could be inhibited or activated by up-regulating or down-regulating gene expression,respectively.The identification of these proteins interacting with the promoter provides a new perspective for understanding the gene regulatory network of hot pepper operating under waterlogging stress and provides theoretical support for further analysis of the complex regulatory relationship between transcription factors and promoters.展开更多
Cytoskeletal microtubule rearrangement and movement are crucial in the repair of spinal cord injury.Spastin plays an important role in the regulation of microtubule severing.Both spastin and collapsin response mediato...Cytoskeletal microtubule rearrangement and movement are crucial in the repair of spinal cord injury.Spastin plays an important role in the regulation of microtubule severing.Both spastin and collapsin response mediator proteins can regulate neurite growth and branching;however,whether spastin interacts with collapsin response mediator protein 3(CRMP3)during this process remains unclear,as is the mechanism by which CRMP3 participates in the repair of spinal cord injury.In this study,we used a proteomics approach to identify key proteins associated with spinal cord injury repair.We then employed liquid chromatography-mass spectrometry to identify proteins that were able to interact with glutathione S-transferase-spastin.Then,co-immunoprecipitation and staining approaches were used to evaluate potential interactions between spastin and CRMP3.Finally,we co-transfected primary hippocampal neurons with CRMP3 and spastin to evaluate their role in neurite outgrowth.Mass spectrometry identified the role of CRMP3 in the spinal cord injury repair process.Liquid chromatography-mass spectrometry pulldown assays identified three CRMP3 peptides that were able to interact with spastin.CRMP3 and spastin were co-expressed in the spinal cord and were able to interact with one another in vitro and in vivo.Lastly,CRMP3 overexpression was able to enhance the ability of spastin to promote neurite growth and branching.Therefore,our results confirm that spastin and CRMP3 play roles in spinal cord injury repair by regulating neurite growth and branching.These proteins may therefore be novel targets for spinal cord injury repair.The Institutional Animal Care and Use Committee of Jinan University,China approved this study(approval No.IACUS-20181008-03)on October 8,2018.展开更多
基金supported by the National Natural Science Foundation of China,Nos.91849115 and U1904207(to YX),81974211 and 82171247(to CS)Non-profit Central Research Institute Fund of Chinese Academy of Medical Sciences,No.2020-PT310-01(to YX).
文摘The E3 ubiquitin ligase,carboxyl terminus of heat shock protein 70(Hsp70)interacting protein(CHIP),also functions as a co-chaperone and plays a crucial role in the protein quality control system.In this study,we aimed to investigate the neuroprotective effect of overexpressed CHIP on Alzheimer’s disease.We used an adeno-associated virus vector that can cross the blood-brain barrier to mediate CHIP overexpression in APP/PS1 mouse brain.CHIP overexpression significantly ameliorated the performance of APP/PS1 mice in the Morris water maze and nest building tests,reduced amyloid-βplaques,and decreased the expression of both amyloid-βand phosphorylated tau.CHIP also alleviated the concentration of microglia and astrocytes around plaques.In APP/PS1 mice of a younger age,CHIP overexpression promoted an increase in ADAM10 expression and inhibitedβ-site APP cleaving enzyme 1,insulin degrading enzyme,and neprilysin expression.Levels of HSP70 and HSP40,which have functional relevance to CHIP,were also increased.Single nuclei transcriptome sequencing in the hippocampus of CHIP overexpressed mice showed that the lysosomal pathway and oligodendrocyte-related biological processes were up-regulated,which may also reflect a potential mechanism for the neuroprotective effect of CHIP.Our research shows that CHIP effectively reduces the behavior and pathological manifestations of APP/PS1 mice.Indeed,overexpression of CHIP could be a beneficial approach for the treatment of Alzheimer’s disease.
基金supported by grants from the Special Foundation of President of the Chinese Academy of Sciences(Grant No.,YZJJ2020QN27,YZJJ2021QN33)Anhui Provincial Natural Science Foundation(Grant No.,2108085MC79).
文摘HCLS1-associated protein X-1(HAX1)is a multifunctional mitochondrial protein involved in the regulation of apoptosis,a crucial process of programmed cell death,and mRNA processing.Despite its significance,limited structural data is available for HAX1,hindering a comprehensive understanding of its biological function.Notably,the caseinolytic mitochondrial matrix peptidase chaperone subunit B(CLPB)has been identified as an interacting partner of HAX1,yet the biophysical properties and binding affinity governing their interaction remain poorly defined.In this study,we present a thorough biophysical characterization of full-length human HAX1 and CLPB,accomplished through recombinant expression and purification.By employing size exclusion chromatography,dynamic light scattering,and circular dichroism spectroscopy,we successfully established their biophysical properties,revealing contrasting structural features,with CLPB displaying a-helical content and HAX1 exhibiting a disordered nature.Moreover,we employed solutionstate nuclear magnetic resonance(NMR)spectroscopy to probe their binding affinity.Our findings demonstrate the formation of stable multimeric complexes between HAX1 and CLPB,and we quantified a dissociation constant in the low range of micro-molar for their high affinity interaction.These results lay the foundation for further in-depth investigations into the dynamics and energetics governing the HAX1-CLPB interaction,ultimately contributing to a comprehensive understanding of their functional mechanisms.
文摘Elucidation of ligand-protein interactions provides new insights into the physiological functions and mechanisms of ligand molecules,enabling new ideas for the treatment of diseases,and drug discovery and development.Most ligand-protein binding occurs only in specific regions of proteins.The identification of protein targets and binding regions is crucial for drug discovery and development,as well as for the in-depth study of drug-protein conformational relationships[1].
基金supported by the National Natural Science Foundation of China(No.82172363)the Traditional Chinese Medicine Scientific Research Project of Zhejiang Province(No.2023ZL010)the Zhejiang Medical and Health Science and Technology Project(Nos.2022KY529 and 2024KY636),China.
文摘Protein interacting with C kinase 1(PICK1)interacts with a variety of membrane proteins and receptors involved in nervous system diseases and multiple cancers.However,the role of PICK1 in gastric cancer remains unclear.In the present work,we explored the expression and interactions of PICK 1 with Toll-like receptor 4(TLR4)in gastric cancer.Clinical data analysis showed that PICK1 expression decreases and is predictive of worse outcomes in patients with gastric cancer.High PICK1 levels attenuate the proliferation and migration of gastric cancer cells,which is dependent on the TLR4/myeloid differentiation primary response 88(MyD88)signaling pathway.Furthermore,in vitro experiments demonstrated that PICK1 affects the trafficking and degradation of TLR4 and promotes TLR4 degradation via autophagy in gastric cancer cells.Molecular dynamics simulations highlighted the binding strength and stability of the TLR4-PICK1 complex.Our study provides new insights into the cellular and pathological functions of PICK1 in gastric cancer.
文摘The yeast two\|hybrid system is a molecular genetic approach for protein interaction and it is widely used to screen for proteins that interact with a protein of interest in recent years.This process includes,construction and testing of the bait plasmid,screening a plasmid library for interacting fusion protein,elimination of false positives and delection analysis of true positives.This procedure is designed to allow investigators to identify proteins and their encoding cDNAs that have a biologically significant interaction with a protein of interest.More and more studies have demonstrated that the two\|hybrid system is a powerful and sensitive technique for the identification of genes that code for proteins that interact in a biologically significant fashion with a protein of interest in higher plants.This method has been used to identify new interaction protein in many laboratories.The recently reported yeast tri\|brid system,should allow the investigation of more complex protein\|protein interactions.The aim of this review is to outline the recent progress made in protein interactions by using yeast two\|hybrid system.
文摘Tissue and systemic inflammation have been the main culprit behind the cellular response to multiple insults and maintaining homeostasis.Obesity is an independent disease state that has been reported as a common risk factor for multiple metabolic and microvascular diseases including nonalcoholic fatty liver disease(NAFLD),retinopathy,critical limb ischemia,and impaired angiogenesis.Sterile inflammation driven by high-fat diet,increased formation of reactive oxygen species,alteration of intracellular calcium level and associated release of inflammatory mediators,are the main common underlying forces in the pathophysiology of NAFLD,ischemic retinopathy,stroke,and aging brain.This work aims to examine the contribution of the pro-oxidative and pro-inflammatory thioredoxin interacting protein(TXNIP)to the expression and activation of NLRP3-inflammasome resulting in initiation or exacerbation of sterile inflammation in these disease states.Finally,the potential for TXNIP as a therapeutic target and whether TXNIP expression can be modulated using natural antioxidants or repurposing other drugs will be discussed.
基金This work was supported by Grants from the National Key R&D Programme of China(No.2017YFD0501103 and 2017YFD0501800)the Key Development and Research Foundation of Yunnan(No.2018BB004)the Chinese Academy of Agricultural Science and Technology Innovation Project(CAAS-XTCX2016011-01 and Y2017JC55).
文摘Receptors interaction protein 2(RIP2)is a specific adaptor molecule in the downstream of NOD2.The role of RIP2 during foot-and-mouth disease virus(FMDV)infection remains unknown.Here,our results showed that RIP2 inhibited FMDV replication and played an important role in the activation of IFN-βand NF-κB signal pathways during FMDV infection.FMDV infection triggered RIP2 transcription,while it reduced the expression of RIP2 protein.Detailed analysis showed that FMDV 2B,2C,3C^(pro),and L^(pro) proteins were responsible for inducing the reduction of RIP2 protein.3C^(pro) and L^(pro) are viral proteinases that can induce the cleavage or reduction of many host proteins and block host protein synthesis.The carboxyl terminal 105-C114 and 135-C144 regions of 2B were essential for reduction of RIP2.Our results also showed that the N terminal 1-61 region of 2C were essential for the reduction of RIP2.The 2C-induced reduction of RIP2 was dependent on inducing the reduction of poly(A)-binding protein 1(PABPC1).The interaction between RIP2 and 2C was observed in the context of viral infection,and the residues 1-61 were required for the interaction.These data clarify novel mechanisms of reduction of RIP2 mediated by FMDV.
文摘AIM:To understand the complex reaction of gastric inflammation induced by Helicobacter pylori(H pylori) in a systematic manner using a protein interaction network. METHODS:The expression of genes significantly changed on microarray during H pylori infection was scanned from the web literary database and translated into proteins.A network of protein interactions was constructed by searching the primary interactions of selected proteins.The constructed network was mathematically analyzed and its biological function was examined.In addition,the nodes on the network were checked to determine if they had any further functional importance or relation to other proteins by extending them. RESULTS:The scale-free network showing the relationship between inflammation and carcinogenesis was constructed.Mathematical analysis showed hub and bottleneck proteins,and these proteins were mostly related to immune response.The network contained pathways and proteins related to H pylori infection,such as the JAK-STAT pathway triggered by interleukins.Activation of nuclear factor (NF)-κB,TLR4,and other proteins known to function as core proteins of immune response were also found. These immune-related proteins interacted on the network with pathways and proteins related to the cell cycle,cell maintenance and proliferation,andtranscription regulators such as BRCA1,FOS,REL,and zinc finger proteins.The extension of nodes showed interactions of the immune proteins with cancer- related proteins.One extended network,the core network,a summarized form of the extended network, and cell pathway model were constructed. CONCLUSION:Immune-related proteins activated by H pylori infection interact with proto-oncogene proteins.The hub and bottleneck proteins are potential drug targets for gastric inflammation and cancer.
基金financially supported by grants from the Natural Science Foundation of Shandong Province,China(ZR2018LC005 and ZR2019BC107)the Agricultural Science and Technology Innovation Project of Shandong Academy of Agricultural Sciences,China(CXGC2022C02)。
文摘The B-box(BBX)family of proteins consists of zinc-finger transcription factors with one or two highly conserved B-box motifs at their N-termini.BBX proteins play crucial roles in various aspects of plant growth and development,including seedling photomorphogenesis,shade avoidance,flowering time,and biotic and abiotic stress responses.Previous studies have identified many different BBXs from several plant species,although the BBX family members in maize are largely unknown.Genome-wide identification and comprehensive analysis of maize BBX(ZmBBX)expression and interaction networks would therefore provide valuable information for understanding their functions.In this study,36 maize BBXs in three major clades were identified.The ZmBBXs within a given clade were found to share similar domains,motifs,and genomic structures.Gene duplication analyses revealed that the expansion of BBX proteins in maize has mainly occurred by segmental duplication.The expression levels of ZmBBXs were analyzed in various organs and tissues,and under different abiotic stress conditions.Protein–protein interaction networks of ZmBBXs were established using bioinformatic tools and verified by bimolecular fluorescence complementation(BiFC)assays.Our findings can facilitate a greater understanding of the complexity of the ZmBBX family and provide novel clues for unravelling ZmBBX protein functions.
基金Supported by NIH/NIAAA 8116Alcohol Center Grant on Liver and Pancreas P50-011999, Morphology Core
文摘Proteasome dysfunction has been repeatedly reported in alcoholic liver disease. Ethanol metabolism endproducts affect the structure of the proteasome, and, therefore, change the proteasome interaction with its regulatory complexes 19S and PA28, as well as its interacting proteins. Chronic ethanol feeding alters the ubiquitin-proteasome activity by altering the interaction between the 19S and the 20S proteasome interaction. The degradation of oxidized and damaged proteins is thus decreased and leads to accumulation of insoluble protein aggregates, such as Mallory-Denk bodies. Ethanol also affects the immunoproteasome formation. PA28a/b interactions with the 20S proteasome are decreased in the proteasome fraction isolated from the liver of rats fed ethanol chronically, thus affecting the cellular antigen presentation and defense against pathogenic agents. Recently, it has been shown that ethanol also affects the proteasome interacting proteins (PIPs). Interaction of the proteasome with Ecm29 and with deubiquitinating enzymes Rpn11, UCH37, and Usp14 has been found to decrease. However, the two UBL-ubiquitin-associated domain (UBA) PIPs p62 and valosin-containing protein are upregulated when the proteasome is inhibited. The increase of these UBL-UBA proteins, as well as the increase in Hsp70 and Hsp25 levels, compensated for the proteasome failure and helped in the unfolding/docking of misfolded proteins. Chronic alcohol feeding to rats causes a significant inhibition of the proteasome pathway and this inhibition results from a decreases of the interaction between the 20S proteasome and the regulatory complexes, PIPs, and the ubiquitin system components.
基金Supported by the National Natural Scientific Foundation, No. C03011402, No. C30070690the Research and Technique Foundation of PLA during the 9th-five year plan period, No. 98D063the Launching Foundation for Student Studying Abroad of PLA, No. 98H038and the Youth Research and Technique Foundation of PLA during the 10th-five year plan period, No. 01Q138the Research and Technique Foundation of PLA during the 10th-five year plan period, No. 01MB135
文摘AIM： To investigate the biological function of p7 protein and to look for proteins interacting with p7 protein in hepatocytes. METHODS： We constructed p7 protein bait plasmid by cloning the gene of p7 protein into pGBKTT, then transformed it into yeast AH109 （a type）. The transformed yeast was mated with yeast Y187 （α type） containing liver cDNA library plasmid, pACT2 in 2×YPDA medium. Diploid yeast was plated on synthetic dropout nutrient medium （SD/- Trp-Leu-His-Ade） containing x-α-gal for selection and screening. After extracting and sequencing of plasmids from blue colonies, we performed sequence analysis by bioinformatics. RESULTS： Fifty colonies were selected and sequenced. Among them, one colony was Homo sapiens signal sequence receptor, seven colonies were Homo sapiens H19, seven colonies were immunoglobulin superfamily containing leucine-rich repeat, three colonies were spermatid peri-nuclear RNA binding proteins, two colonies were membrane-spanning 4-domains, 24 colonies were cancer-associated antigens, four colonies were nudeoporin 214 ku and two colonies were CLL-associated antigens. CONCLUSION： The successful cloning of gene of protein interacting with p7 protein paves a way for the study of the physiological function of p7 protein and its assodated protein.
基金supported by the National Key Technology Research and Development Program of China (2018YFD1000900)the Natural Science Foundation of Shandong Province(ZR2020MC094)+1 种基金the Natural Science Foundation of Shandong Province (ZR2021QC163)Special Funds for Local Science and Technology Development Guided by the Central Committee(YDZX20203700001861)。
文摘The Ca^(2+)/CaM signal transduction pathway helps plants adapt to environmental stress. However, our knowledge on the functional proteins of C^(2+)/CaM pathway in peanut(Arachis hypogeae L.) remains limited. In the present study, a novel calmodulin 4(CaM4)-binding protein S-adenosyl-methionine synthetase 1(SAMS1) in peanut was identified using a yeast two-hybrid assay. Expression of AhSAMS1was induced by Ca^(2+), ABA, and salt stress. To elucidate the function of AhSAMS1, physiological and phenotypic analyses were performed with wild-type and transgenic materials. Overexpression of AhSAMS1increased spermidine and spermidine synthesis while decreased the contents of ethylene, thereby eliminating excessive reactive oxygen species(ROS) in transgenic lines under salt stress. AhSAMS1 reduced uptake of Na+and leakage of K+from mesophyll cells, and was less sensitive to salt stress during early seedling growth, in agreement with the induction of SOS and NHX genes Transcriptomics combined with epigenetic regulation uncovered relationships between differentially expressed genes and differentially methylated regions, which raised the salt tolerance and plants growth. Our findings support a model in which the role of AhSAMS1 in the ROS-dependent regulation of ion homeostasis was enhanced by Ca^(2+)/CaM while AhSAMS1-induced methylation was regulated by CaM, thus providing a new strategy for increasing the tolerance of plants to salt stress.
基金grants from "973" Program of China (No. 2002CB513100)National Natural Sci-ences Foundation of China (No. 30500596)
文摘To screen for novel binding proteins interacting with high-risk HPV 18 E6 oncogene, the strain AH 109 was transformed with pGBKT7-HPV 18 E6 plasmid, and subsequent transference was utilized to screen for interacting proteins with HPV 18 E6 in human Hela cDNA library. HPV 18 E6 mRNA was expressed in yeast and there was no self-activation and toxicity in AH109. Seven proteins that interacted with HPV18 E6, including transmembrane protein 87B, phosphonoformate immuno-associated protein 5, vimentin, KM-HN-1 protein, dedicator of cytokinesis 7, vaccinia related kinase 2 and a hypothetical protein, were identified. It was suggested that yeast two-hybrid system is an efficient for screening interacting proteins. The high-risk HPV 18 E6 oncogene may interact with the proteins, which may be associated with signal transduction and transcriptional control, epithelial cell invasion and migration, as well as humoral and cellular immune etc. This investigation provides functional clues for further exploration of potential oncogenesis targets for cancer biotherapy.
文摘Interactions are the essence of all biomolecules because they cannot fulfill their roles without interacting with other molecules. Hence, mapping the interactions of biomolecules can be useful for understanding their roles and functions. Furthermore, the development of molecular based systems biology requires an understanding of the biomolecular interactions. In recent years, the mapping of protein-protein interactions in different species has been reported, but few reports have focused on the large-scale mapping of protein-protein interactions in human. Here, we review the developments in protein interaction mapping and we discuss issues and strategies for the mapping of the human protein interactome.
文摘The mechanism and interaction among Rb/p16, Rb/E2F1 and HDAC1 proteins in gallbladder carcinoma were investigated. By using the immunoprecipitation method, the interactions among Rb, p16, E2F1, HDAC1 proteins in gallbladder carcinoma cell line （Mz-ChA-1） were studied. It was found that there were Rb and E2F1 proteins in the precipitates with anti-HDAC1, and there were HDAC1 and E2F1 proteins in the precipitate with anti-Rb. It was concluded that there are specific interactions among Rb, HDAC1 and E2F1 proteins in gallbladder carcinoma, indicating the existence of the direct Rb/E2F1/HDAC1 signal transduction pathway. There is no direct relationship between p16 proteins with Rb, HDAC1, and E2F1 proteins.
基金Supported by the National Natural Science Foundation of China(No.30671852)the Open Research Fund Program of the State Key Laboratory of Virology of China(Nos.2010009,2007007)the Research Fund of the Key Laboratory of Department of Education of Liaoning Province,China(No.2009S043)
文摘Avian influenza virus(AIV)nonstructural 1(NS1)gene was amplified by real-time polymerse chain reac tion(RT-PCR)and inserted into pET28a,then transformed into E.coli BL21(DE3)competent cell.With the induction of isopropyl-β-D-thiogalactoside(IPTG)and the purification of Ni-NTA column,we finally obtained purified NS1 protein.T7-phage display system was used to screen the proteins that interacted with NS1 from lung cell cDNA li brary.The selected positive clones were identified by DNA sequencing and analyzed by BLAST program in Gene Bank.Two proteins were obtained as NS1 binding proteins,Homo sapiens nucleolar and coiled-body phosphoprotein 1(NOLC1)and Homo sapiens similar to colon cancer-associated antigen.By co-immunoprecipitation and other me thods,Homo sapiens NOLC1 was found to interact with the NS1 protein,the results would provide the basis for fur ther studying biological function of NS1 protein.
文摘Regulation of the number of aetivin receptors that are present in the cell membrane plays a key role in the modulation of cellular responses to activin. In order to find the regulators, a novel protein ARIPzip, interacting with activin type II receptors, was searched and identified by using yeast two-hybrid screening. ARIPzip is a splicing variant of ARIP2. This has been discussed previously. ARIPzip can specifically interact with ActR Ⅱ A, and is widely distributed in mouse tissues. Overexpression of ARIPzip can cause the activin-induced transcriptional activities to increase in a dose-dependent manner while the overexpression of ARIV2 can decrease these activities. These data suggest that the C-terminal rezions of ARIP2 and ARIPzip are involved in the regulation of activin signaling.
基金supported by the International Science and Technology Cooperation Program of China (No. 2013DFG32910)the National Natural Science Foundation of China (Nos. 31371491 and 30971677)Zhejiang Provincial Natural Science Foundation of China (No. LZ13C120001)
文摘Zebrafish △113p53, an N-terminal truncated p53 isoform, is a p53-target gene that antagonises p53-mediated apoptotic activity. Interestingly, △113p53 does not act on p53 in a dominant-negative manner, but rather interferes with the p53 function by differentially modulating p53-target gene expression to protect cells from apoptosis. Previous studies showed that over-expressed △113p53 and p53 proteins formed a complex. However, it is not known whether endogenous p53 and △113p53 proteins also interact with each other, and if this interaction is required for △113p53 to inhibit the apoptotic activity of full-length p53. In this study, we used two available zebrafish p53 antibodies to address these questions. One, Zfp53-N, only recognises full-length p53, whereas the other, Zfp53-A7C10, detects both full-length p53 and △113p53. Using Zfp53-N for immunoprecipitation and Zfp53-A7C 10 for detection, we demonstrated that endogenous △113p53 and full-length p53 induced by a DNA-damaging drug formed a complex in vivo. Furthermore, of the six △113p53 mutants we generated with different point mutations in the oligomerisation domain, two failed to interact with p53 and lost the ability to modulate p53-target gene expression and inhibit p53-induced cell apoptosis. However, those △113p53 mutants that could interact with p53 retained the ability to antagonise the apoptotic activity of p53. Therefore, our data demonstrated that protein--protein interaction between △113p53 and p53 is essential for the anti-apoptotic function of △113p53. In addition, the two △113p53 mutants that failed to interact with p53 are also useful for the study of the mechanisms of other functions of △113p53.
基金funded by the National Natural Science Foundation of China(grant no.32260760)the Science and Technology Program of Guizhou Province(grant no.20201Z002)the Platform Construction Project of Engineering Research Center for Protected Vegetable Crops in Higher Learning Institutions of Guizhou Province(Qianjiaoji[2022]No.040).
文摘Hot pepper(Capsicum annuum var.conoides)is a significant vegetable that is widely cultivated around the world.Currently,global climate change has caused frequent severe weather events,and waterlogging stress harms the pepper industry by affecting the planting period,growth conditions,and disease susceptibility.The gene CaABI3/VP1-1 could improve pepper waterlogging tolerance.In order to explore the upstream regulatory mechanism of CaABI3/VP1-1,a high-quality standardized yeast hybrid library was successfully constructed for yeast one-,two-,and threehybrid screening using pepper‘ZHC2’as the experimental material,with a library recombinant efficiency of up to 100%.The length of inserted fragments varied from 650 to 5000 bp,the library titer was 5.18×10^(6)colony-forming units(CFU)·mL-1,and the library capacity was 1.04×10^(7)CFU of cDNA inserts.The recombinant bait plasmid was used to successfully identify 78 different proteins through the yeast one-hybrid system,including one transcription factor within the ethylene-responsive factor family and the other within the growth-regulating factor family.The interaction happened between LOC124895848 and CaABI3/VP1-1 promoter by point-to-point yeast one-hybrid experiment.The expression level of the 12 selected protein-coding genes was then evaluated by quantitative real-time polymerase chain reaction.Results indicated the protein coding genes showed different responses to waterlogging stress and that the activity of the CaABI3/VP1-1 promoter could be inhibited or activated by up-regulating or down-regulating gene expression,respectively.The identification of these proteins interacting with the promoter provides a new perspective for understanding the gene regulatory network of hot pepper operating under waterlogging stress and provides theoretical support for further analysis of the complex regulatory relationship between transcription factors and promoters.
基金This work was supported by the National Natural Science Foundation of China,Nos.31900691(to GWZ),81771331(to HSL)and 81971165(to WW)the National Basic Research Program of China(973 Program),No.2014CB542205(to WW)+5 种基金the Natural Science Foundation of Guangdong Province of China,No.2017A030313595(to HSL)the Science and Technology Program of Guangzhou,China,No.201707010370(to HSL)Project of Educational Commission of Guangdong Province of China,No.2018KQNCX013(to ZSJ)the Fundamental Research Funds for the Central Universities Project,China,No.21618304(to GWZ)Guangdong Provincial Key Research and Development Program“Precision Medicine and Stem Cell”Major Science and Technology Project,China,No.3242001(to WW)China Postdoctoral Science Foundation,No.2019M653292(to ZSJ).
文摘Cytoskeletal microtubule rearrangement and movement are crucial in the repair of spinal cord injury.Spastin plays an important role in the regulation of microtubule severing.Both spastin and collapsin response mediator proteins can regulate neurite growth and branching;however,whether spastin interacts with collapsin response mediator protein 3(CRMP3)during this process remains unclear,as is the mechanism by which CRMP3 participates in the repair of spinal cord injury.In this study,we used a proteomics approach to identify key proteins associated with spinal cord injury repair.We then employed liquid chromatography-mass spectrometry to identify proteins that were able to interact with glutathione S-transferase-spastin.Then,co-immunoprecipitation and staining approaches were used to evaluate potential interactions between spastin and CRMP3.Finally,we co-transfected primary hippocampal neurons with CRMP3 and spastin to evaluate their role in neurite outgrowth.Mass spectrometry identified the role of CRMP3 in the spinal cord injury repair process.Liquid chromatography-mass spectrometry pulldown assays identified three CRMP3 peptides that were able to interact with spastin.CRMP3 and spastin were co-expressed in the spinal cord and were able to interact with one another in vitro and in vivo.Lastly,CRMP3 overexpression was able to enhance the ability of spastin to promote neurite growth and branching.Therefore,our results confirm that spastin and CRMP3 play roles in spinal cord injury repair by regulating neurite growth and branching.These proteins may therefore be novel targets for spinal cord injury repair.The Institutional Animal Care and Use Committee of Jinan University,China approved this study(approval No.IACUS-20181008-03)on October 8,2018.
