The skeleton is a dynamic organ that is constantly remodeled. Proteins secreted from bone cells, namely osteoblasts, osteocytes,and osteoclasts exert regulation on osteoblastogenesis, osteclastogenesis, and angiogenes...The skeleton is a dynamic organ that is constantly remodeled. Proteins secreted from bone cells, namely osteoblasts, osteocytes,and osteoclasts exert regulation on osteoblastogenesis, osteclastogenesis, and angiogenesis in a paracrine manner. Osteoblasts secrete a range of different molecules including RANKL/OPG, M-CSF, SEMA3A, WNT5A, and WNT16 that regulate osteoclastogenesis. Osteoblasts also produce VEGFA that stimulates osteoblastogenesis and angiogenesis. Osteocytes produce sclerostin(SOST) that inhibits osteoblast differentiation and promotes osteoclast differentiation. Osteoclasts secrete factors including BMP6, CTHRC1, EFNB2, S1P, WNT10B, SEMA4D, and CT-1 that act on osteoblasts and osteocytes, and thereby influencea A osteogenesis. Osteoclast precursors produce the angiogenic factor PDGF-BB to promote the formation of Type H vessels, which then stimulate osteoblastogenesis. Besides, the evidences over the past decades show that at least three hormones or "osteokines"from bone cells have endocrine functions. FGF23 is produced by osteoblasts and osteocytes and can regulate phosphate metabolism. Osteocalcin(OCN) secreted by osteoblasts regulates systemic glucose and energy metabolism, reproduction, and cognition. Lipocalin-2(LCN2) is secreted by osteoblasts and can influence energy metabolism by suppressing appetite in the brain.We review the recent progresses in the paracrine and endocrine functions of the secretory proteins of osteoblasts, osteocytes, and osteoclasts, revealing connections of the skeleton with other tissues and providing added insights into the pathogenesis of degenerative diseases affecting multiple organs and the drug discovery process.展开更多
Rat protein DE is an androgen-dependent cysteine-rich secretory protein (CRISP) synthesized by proximal epididymal regions. DE, also known as CRISP-1, is localized on the equatorial segment of acrosome-reacted sperm...Rat protein DE is an androgen-dependent cysteine-rich secretory protein (CRISP) synthesized by proximal epididymal regions. DE, also known as CRISP-1, is localized on the equatorial segment of acrosome-reacted spermatozoa and participates in gamete fusion through binding to egg complementary sites. Immunization of rats with DE inhibits fertility and sperm fusion ability, suggesting that DE represents a good epididymal contraceptive target. Recombinant DE fragments and synthetic peptides revealed that DE binds to the egg via a 12-amino acid region of an evolutionarily conserved motif, Signature 2 (S2). The ability of other CRISP to bind to the rat egg was correlated with their S2 amino acid sequences. Although testicular protein Tpx- 1 (CRISP-2) was capable of binding to rodent eggs, human epididymal AEG-related protein (ARP) and helothermine (from lizard saliva) were not. The S2 region presented only two substitutions in Tpx-1 and four in ARP and helothermine, compared with the DE S2, suggesting that this amino acid sequence was relevant for egg interaction. Studies with Tpx- 1 and anti-Tpx- 1 revealed the participation of this protein in gamete fusion through binding to complementary sites in the egg. In competition studies, DE reduced binding of Tpx- 1 dose-dependently, indicating that both CRISP share the egg complementary sites. That anti-DE and anti-Tpx-1 inhibit sperm-egg fusion while recognizing only the corresponding proteins, suggests functional cooperation between these homologous CRISP to ensure fertilization success. These results increase our understanding of the molecular mechanisms of gamete fusion and contribute to the development of new and safer fertility regulating methods. (Asian J Androl 2007 July; 9: 528-532)展开更多
Secretory proteins in tumor tissues are important components of the tumor microenvironment.Secretory proteins act on tumor cells or stromal cells or mediate interactions between tumor cells and stromal cells,thereby a...Secretory proteins in tumor tissues are important components of the tumor microenvironment.Secretory proteins act on tumor cells or stromal cells or mediate interactions between tumor cells and stromal cells,thereby affecting tumor progression and clinical treatment efficacy.In this paper,recent research advances in secretory proteins in malignant tumors are reviewed.展开更多
The necrotrophic fungus, Sclerotinia sclerotiorum, employs an array of cell wall-degrading enzymes(CWDEs), including cellulase, to dismantle host cell walls. However, the molecular mechanisms through which S. scleroti...The necrotrophic fungus, Sclerotinia sclerotiorum, employs an array of cell wall-degrading enzymes(CWDEs), including cellulase, to dismantle host cell walls. However, the molecular mechanisms through which S. sclerotiorum degrades cellulose remain elusive. Here, we unveil a novel secretory cellobiohydrolase, SsdchA, characterized by a signal peptide and a Glyco_hydro_7(GH7) domain. SsdchA exhibits a robust expression of during early infection stages. Interestingly, colony morphology and growth rates remain unaffected across the wild-type, SsdchA deletion strains and SsdchA overexpression strains on potato dextrose agar(PDA) medium. Nevertheless, the pathogenicity and cellobiohydrolase activity decreased in the SsdchA deletion strains, but enhanced in the SsdchA overexpression strains. Moreover,the heterologous expression of SsdchA in Arabidopsis thaliana leads to reduced cellulose content and heightened susceptibility to S. sclerotiorum. Collectively, our data underscore the pivotal role of the novel cellobiohydrolase SsdchA in the pathogenicity of S. sclerotiorum.展开更多
Vasectomy is a simple and reliable method of male contraception. A growing number of men after vasectomy request vasectomy reversal due to various reasons. The pregnancy rate is lower than the patency rate after vasov...Vasectomy is a simple and reliable method of male contraception. A growing number of men after vasectomy request vasectomy reversal due to various reasons. The pregnancy rate is lower than the patency rate after vasovasostomy and the pregnancy rate is time dependent. In this study, we evaluated the influence of reproductive tract obstruction on expression of epididymal proteins and their restoration after patency. Adult male Wistar rats were studied 30, 60 and 120 days after vasectomy, 30 days after vasovasostomy or after sham operations. Two-dimensional gel electrophoresis, mass-spectrometric technique, multidatabase search, Western blotting and real-time PCR were used to analyze the expression regulation of epididymal proteins. Total integrated intensity and total spot area of autoradiograms showed a consistent downward trend with time after obstruction, and this trend remained after patency. The intensity of the autoradiographic spots in three patency groups showed three trends: a downward trend, similar intensity and an upward trend compared with the correspondent obstruction group, respectively. Further verified experiments on human epididymis 2 (HE2), fertilization antigen-1 (FA-1), clusterin and PH20 demonstrated that compared with the correspondent obstruction group, the translation levels of HE2 and the mRNA transcription levels of HE2 showed an upward trend in patency groups, especially in the groups of obstruction for 60 days where the expression levels of HE2 were significantly upregulated after patency (P〈O.05). Reproductive tract obstruction provokes a disregulation of gene expression in the epididymis and this disregulation remained after patency. Successful reversal may recover some proteins and the recovery is time dependent, Obstruction differentially alters mRNA transcription of different proteins and the content of proteins seemed to be easier to be influenced than the gene transcription.展开更多
A number of plant pathogenic species of Phytophthora are known to produce different classes of secretory proteins during interactions with their hosts.Although several small cysteine-rich(SCR)secretory proteins,conser...A number of plant pathogenic species of Phytophthora are known to produce different classes of secretory proteins during interactions with their hosts.Although several small cysteine-rich(SCR)secretory proteins,conserved in oomycete pathogens,have been identified in Phytophthora,their specific involvement in these interactions remains unknown.In this study,an SCR effector encoded by Pn SCR82 in P.nicotianae was identified and shown to have similarities to P.cactorum phytotoxic protein,Pc F(Phytophthora cactorum Fragaria).Agroinfection with potato virus X vector,Pn SCR82,was capable of inducing plant hypersensitive cell death in Nicotiana benthamiana and Solanum lycopersicum.Real-time PCR results indicated that transiently expressed Pn SCR82 in N.benthamiana leaves activated the jasmonate,salicylic acid and ethylene signaling pathways.Transient expression of Pn SCR82 enhanced plant resisitance to P.capsici.In summary,our results demonstrated that P.nicotianae Pn SCR82 elicits defensive responses in N.benthamiana and may potentially play a significant role in future crop protection programs.展开更多
The necrotrophic fungus Rhizoctonia cerealis is the causal agent of devastating diseases of cereal crops including wheat(Triticum aestivum).We present a high-quality genome assembly of R.cerealis Rc207,a virulent stra...The necrotrophic fungus Rhizoctonia cerealis is the causal agent of devastating diseases of cereal crops including wheat(Triticum aestivum).We present a high-quality genome assembly of R.cerealis Rc207,a virulent strain causing wheat sharp eyespot.The assembly(56.36 Mb)is composed of 17.87%repeat sequences and 14,433 predicted protein-encoding genes.The Rc207 genome encodes a large and diverse set of genes involved in pathogenicity,especially rich in those encoding secreted proteins,carbohydrateactive enzymes(CAZymes),peptidases,nucleases,cytochrome P450,and secondary metabolismassociated enzymes.Most secretory protein-encoding genes,including CAZymes,peroxygenases,dehydrogenases,and cytochrome P450,were up-regulated during fungal infection of wheat.We identified 831 candidate secretory effectors and validated the functions of 10 up-regulated candidate effector proteins.Of them,nine were confirmed as necrotrophic pathogen’s effectors promoting fungal infection.Abundant potential mobile or plastic genomic regions rich in repeat sequences suggest their roles in fungal adaption and virulence-associated genomic evolution.This study provides valuable resources for further comparative and functional genomics on important fungal pathogens,and provides essential tools for development of effective disease control strategies.展开更多
Objective:To investigate the correlation between serum secretory frizzled-related protein 5(SFRP-5)expression levels and insulin resistance and glucolipid metabolism in patients with gestational diabetes mellitus(GDM)...Objective:To investigate the correlation between serum secretory frizzled-related protein 5(SFRP-5)expression levels and insulin resistance and glucolipid metabolism in patients with gestational diabetes mellitus(GDM).Methods:Baseline data were collected from 58 patients with GDM and 51 healthy controls who were admitted Affiliated Hospital of Hebei University from May 2020 to June 2022.sSTRA5 concentrations in peripheral blood of pregnant women were measured,and SFRP-5 levels in patients with different GDM types and normal controls were analyzed by logistic regression models.Results:The levels of triglyceride(TG),total cholesterol(TC),low-density lipoprotein cholesterol(LDL-C),fasting blood glucose(FBG),fasting insulin(FINS),hemoglobin A1c(HbA1c),and homeostasis model assessment-estimated insulin resistance(HOMA-IR)were higher in the observation group than in the control group,with statistically significant differences(P<0.05),while the expression levels of high-density lipoprotein cholesterol(HDL-C)and serum SFRP-5 were lower than in the control group,with statistically significant differences(P<0.05);serum SFRP-5,TG,TC,FBG,and HOMA-IR were all risk factors for GDM(P<0.05).Conclusion:Elevated serum sSTRA5 may be involved in the regulation of insulin resistance in the body and the regulation of blood glucose in the body by affecting lipid metabolism and inflammatory response.展开更多
To explore the functions of human ribonuclease 9(RNase 9),we constructed a mammalian fusion expression vector pcDNA-hRNase9,prepared recombinant human RNase 9-His fusion protein from HEK293T cells and determined its N...To explore the functions of human ribonuclease 9(RNase 9),we constructed a mammalian fusion expression vector pcDNA-hRNase9,prepared recombinant human RNase 9-His fusion protein from HEK293T cells and determined its N-terminal amino acid sequences.According to the determined mature protein,recombinant human RNase 9 was prepared in E.coli.Ribonucleolytic activity and antibacterial activity of recombinant human RNase 9 were detected,and the distribution of human RNase 9 on tissues and ejaculated spermatozoa and in vitro capacitated spermatozoa were analyzed via indirect immunofluorescence assay.The results showed that recombinant human RNase 9 did not exhibit detectable ribonucleolytic activity against yeast tRNA,but exhibited antibacterial activity,in a concentration/time dependent manner,against E.coli.Immunofluorescent analyses showed that the predicted human RNase 9 was present throughout the epididymis,but not present in other tissues examined,and human RNase 9 was also present on the entire head and neck regions of human ejaculated spermatozoa and in vitro capacitated spermatozoa.These results suggest that human RNase 9 may play roles in host defense of male reproductive tract.展开更多
Previous studies have shown that rat epididymis-specific gene HongrES1 plays important roles in sperm capacitation and fertility. In this study, we cloned the mouse homologue gene by sequence alignment and RT-PCR meth...Previous studies have shown that rat epididymis-specific gene HongrES1 plays important roles in sperm capacitation and fertility. In this study, we cloned the mouse homologue gene by sequence alignment and RT-PCR methods and designated it as mHongl. The mHongl gene is located on chromosome 12p14, spanning five exons. The cDNA sequence consists of 1257 nucleotides and encodes a 419 amino-acid protein with a predicted N-terminal signal peptide of 20 amino acids. The mHongl mRNA shows similarity with HongrES1 in the expression patterns: (i) specific expression in epididymal tissue, especially in the cauda region; and (ii) androgen-dependence but testicular fluid factor independence. Its protein product shows 71% similarity with HongrES 1 and contains a classical serpin domain as does HongrES1. A polyclonal antibody against mHongl with high specificity and sensitivity was raised. Like HongrES1, the mHongl protein shows a checker-board expression pattern in the epididymal epithelium and is secreted into the epididymal lumen. The mHongl protein shows higher glycosylation than HongrES1. Although both of them are deposited onto the sperm head surface, mHongl is localized to the equatorial segment, which is different from that of HongrES 1. The mHongl protein can be removed from the sperm membrane by high ionic strength and therefore can be classed as an extrinsic membrane protein. Collectively, we conclude that mHongl is the homologue of HongrES1 and the present work paves the way for establishing animal models to elucidate the precise functions of HongrES1 and mHongl.展开更多
A variety of glycosylphosphatidylinositol (GPI)-linked proteins are acquired on spermatozoa from epididymal luminal fluids (ELF) during sperm maturation. These proteins serve roles in immunoprotection and in key s...A variety of glycosylphosphatidylinositol (GPI)-linked proteins are acquired on spermatozoa from epididymal luminal fluids (ELF) during sperm maturation. These proteins serve roles in immunoprotection and in key steps of fertilization such as capacitation, acrosomal exocytosis and sperm-egg interactions. Their acquisition on sperm cells is mediated both by membrane vesicles (epididymosomes, EP) which were first reported to dock on the sperm surface, and by lipid carriers which facilitate the transfer of proteins associated with the membrane-free fraction of ELF. While the nonvesicular fraction is more efficient, both pathways are dependent on hydrophobic interactions between the GPI-anchor and the external lipid layer of the sperm surface. More recently proteomic and hypothesis-driven studies have shown that EP from several mammals carry transmembrane (TM) proteins, including plasma membrane Ca^2+-ATPase 4 (PMCA4). Synthesized in the testis, PMCA4 is an essential protein and the major Ca^2+ efflux pump in murine spermatozoa. Delivery of PMCA4 to spermatozoa from bovine and mouse EP during epididymal maturation and in vitro suggests that the docking of EP on the sperm surface precedes fusion, and experimental evidence supports a fusogenic mechanism for TM proteins. Fusion is facilitated by CD9, which generates fusion-competent sites on membranes. On the basis of knowledge of PMCA4's interacting partners a number of TM and membrane-associated proteins have been identified or are predicted to be present, in the epididymosomal cargo deliverable to spermatozoa. These Ca^2+-dependent proteins, undetected in proteomic studies, play essential roles in sperm motility and fertility, and their detection highlights the usefulness of the hypothesis-driven approach.展开更多
Mammalian fertilization is a complex process that involves different steps of interaction between the male and female gametes. In spite of its relevance, the molecular mechanisms underlying this process still remain t...Mammalian fertilization is a complex process that involves different steps of interaction between the male and female gametes. In spite of its relevance, the molecular mechanisms underlying this process still remain to be elucidated. The present review describes the contribution of our laboratory to the understanding of mammalian fertilization using Cysteine-Rlch Secretory Proteins (CRISP) as model molecules. Substantial evidence obtained from in vitro assays and knockout models shows that epididymal CRISP1 associates with the sperm surface with two different affinities during maturation, and participates in the regulation of signaling pathways during capacitation as well as in both sperm-zona pellucida interaction and gamete fusion. These observations can be extended to humans as judged by our findings showing that the human homolog of the rodent protein (hCRISP1) is also involved in both stages of fertilization. Evidence supports that other members of the CRISP family secreted in the testis (CRISP2), epididymis (CRISP3-4) or during ejaculation (CRISP3) are also involved in sperm-egg interaction, supporting the existence of a functional redundancy and cooperation between homolog proteins ensuring the success of fertilization. Together, our observations indicate that CRISP proteins accompany spermatozoa along their transit through both the male and female reproductive tracts. We believe these results not only contribute to a better mechanistic understanding of fertilization but also support CRISP proteins as excellent candidates for future research on infertility and contraception.展开更多
Cysteine-rich secretory protein 2 (CRISP2) is an important protein in spermatozoa that plays roles in modulating sperm flagellar motility, the acrosome reaction, and gamete fusion. Spermatozoa lacking CRISP2 exhibit...Cysteine-rich secretory protein 2 (CRISP2) is an important protein in spermatozoa that plays roles in modulating sperm flagellar motility, the acrosome reaction, and gamete fusion. Spermatozoa lacking CRISP2 exhibit low sperm motility and abnormal morphology. However, the molecular mechanisms underlying the reduction of CRISP2 in asthenoteratozoospermia (ATZ) remain unknown. In this study, low expression of CRISP2 protein rather than its mRNA was observed in the ejaculated spermatozoa from ATZ patients as compared with normozoospermic males. Subsequently, bioinformatic prediction, luciferase reporter assays, and microRNA-27a (miR-27a) transfection experiments revealed that miR-27a specifically targets CRISP2 by binding to its 3' untranslated region (3'-UTR), suppressing CRISP2 expression posttranscriptionally. Further evidence was provided by the clinical observation of high miR-27a expression in ejaculated spermatozoa from ATZ patients and a negative correlation between miR-27a expression and CRISP2 protein expression. Finally, a retrospective follow-up study supported that both high miR-27a expression and low CRISP2 protein expression were associated with low progressive sperm motility, abnormal morphology, and infertility. This study demonstrates a novel mechanism responsible for reduced CRISP2 expression in ATZ, which may offer a potential therapeutic target for treating male infertility, or for male contraception.展开更多
Background Clara cell secretory protein (CC16) is an important lung derived protective factor and may play an important role on the pathogenesis of acute lung injury (ALl) induced by endotoxemia. Growth hormone ...Background Clara cell secretory protein (CC16) is an important lung derived protective factor and may play an important role on the pathogenesis of acute lung injury (ALl) induced by endotoxemia. Growth hormone (GH) is an important anabolism hormone secreted by GH cells of the hypophysis. Previous research showed that GH would significantly exacerbate ALl induced by endotoxemia, but the mechanism is not very clear yet. Whether the effects are related to CC16 or not is undetermined. Methods One hundred and twelve male Sprague-Dawley rats were randomly divided into an ALl group and a GH group. The rats in the two groups were subdivided into seven subgroups, according to injection with lipopolysaccharides (LPS) or not, then according to different intervals of time after LPS injection; 0 hour (pre-injection of LPS, acted as control group), 0.5 hour, 1 hour, 2 hours, 4 hours, 6 hours and 24 hours for subgroups. Pulmonary alveolar septa area density (PASAD) and ploymorphonuclear cells (PMN) in the lungs were analyzed morphometrically. The levels of tumor necrosis factor (TNF) and interleukin 6 (IL-6) were determined by radioimmunoassay. To analyze the expression and activation of nuclear factor kappa B (NF-KB), the numbers of NF-KB positive cells in lungs were counted after immunofluorescence staining, and the levels of NF-KB inhibitory protein-a (IKB-a) in lung homogenates of rats were detected by Western blotting. The expression levels of CC16 mRNA in lungs of the rats with ALl were determined by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR). The levels of CC16 protein in lung homogenates were detected by Western blotting. Results Half an hour after LPS injury both the PASAD and PMN numbers in lungs of the rats with ALl began to increase significantly and peaked at 6-hour post-injury. They then began to recover and reached normal levels at 24-hour post-injury. Both the PASAD and PMN numbers in the GH group increased more significantly than those in the ALl group. The levels of TNF in lungs of the rats with ALl homogenates increased significantly 0.5-hour post-injury, peaked at 1-hour and maintained a high level until 6 hours then gradually recovered. The content of TNF in the GH group lung homogenates increased more significantly than in the ALl group post-injury. The contents of IL-6 in rat lung homogenates began to increase significantly at 1-hour post-injury, peaked at 4 hours then gradually returned to normal levels by 6 hours post-injury. The levels of IL-6 in the lung homogenates of the GH group were higher than in the ALl group at different time intervals post-injury. The number of NF-KB positive cells increased dramatically at 0.5-hour post-injury, and the fluorescence intensity was enhanced. Both peaked at 4-hour post-injury. The number of NF-KB positive cells and the enhanced intensity of fluorescence began to decrease from 6-hour post-injury, but the number of NF-KB cells at 24 hours post-injury was still higher than in the control group. The number of NF-KB cells in lungs in the GH group was significantly higher than in the LPS group at the different time intervals post-injury. The IKB-a expression in lungs of the rats with ALl homogenates decreased dramatically 0.5-hour post-injury, reaching a nadir at 4-hour post-injury and then began to recover. The levels of IKB-Q in GH group were significantly lower than those in ALl group. Both the levels of CC16 mRNA and protein in lungs of the rats with ALl began to decrease significantly 0.5-hour post-injury, reached a nadir at 6 hours, and then began to recover. Both the expression of CC16 mRNA and CC16 protein in the GH group were significantly lower than those in the ALl group at the different time intervals post-injury. Correlation analysis indicates that CC16 correlates significantly with all the indices mentioned above. Conclusions Down-regulation of CC16 expression plays a critical role in the pathogenesis of acute lung injury induced by endotoxemia. The application of GH can exacerbate the lung injury induced by endotoxemia through down-regulating the expression of CC16.展开更多
OBJECTIVE:To study the functional mechanism of Pingchuanning Decoction in treatment of airway remodeling in asthmatic rats.METHODS:Eighty healthy Wistar male rats were randomized into eight groups(n=10 rats each):Norm...OBJECTIVE:To study the functional mechanism of Pingchuanning Decoction in treatment of airway remodeling in asthmatic rats.METHODS:Eighty healthy Wistar male rats were randomized into eight groups(n=10 rats each):Normal group,Asthma model group,Dexamethasone group,Guilong Kechuanning group,Xiaoqinglong Decoction group,and Pingchuanning Decoction low-,middle-,and high-dose groups.The rats of all but the Normal group were made into asthma models through intraperitoneal injection and aerosol inhalation of ovalbumin.All treatments were administered at the first stimulation of asthma onset(third week of modeling),and the rats were killed after stimulating asthma attacks for 4 weeks.The general conditions of rats and pathomorphological changes of the lung tissues were observed.The expression of nerve growth factor(NGF) of the lung tissues was measured with immunohistochemical methods,and the content of Clara cell secretory protein(CCSP) mRNA was determined with RT-PCR.RESULTS:Compared with the Normal group,the contents of NGF and CCSP mRNA in the lung tissues of the Model group were significantly changed(P<0.01).Compared with the Model group,the indices of Pingchuanning Decoction and other treatment groups were improved to some extent(P<0.05 or P<0.01).CONCLUSIONS:Pathological changes of airway inflammation and remodeling were present in these rat asthma models.Pingchuanning Decoction had an intervention effect on these experimental models.Its functional mechanism may be related to multiple factors,including alleviation of airway inflammation,relief of bronchial smooth muscle spasm,and inhibition of airway remodeling.展开更多
Corynebacterium glutamicum represents an emerging recombinant protein expression factory due to its ideal features for protein secretion,but its applicability is harmed by the lack of an autoinduction system with tigh...Corynebacterium glutamicum represents an emerging recombinant protein expression factory due to its ideal features for protein secretion,but its applicability is harmed by the lack of an autoinduction system with tight regulation and high yield.Here,we propose a new recombinant protein manufacturing platform that leverages ethanol as both a delayed carbon source and an inducer.First,we reanalysed the native inducible promoter PICL from the acetate uptake operon and found that its limited capacity is the result of the inadequate translation initial architecture.The two strategies of bicistronic design and ribozyme-based insulator can ensure the high activity of this promoter.Next,through transcriptional engineering that alters transcription factor binding sites(TFBSs)and the first transcribed sequence,the truncated promoter PA256 with a dramatically higher transcription level was generated.When producing the superfolder green fluorescent protein(sfGFP)under 1%ethanol conditions,PA256 exhibited substantially lower protein accumulation in prophase but an approximately 2.5-fold greater final yield than the strong promoter PH36.This superior expression mode was further validated using two secreted proteins,camelid antibody fragment(VHH)and endoxylanase(XynA).Furthermore,utilizing CRISPRi technology,ethanol utilization blocking strains were created,and PA256 was shown to be impaired in the phosphotransacetylase(PTA)knockdown strains,indicating that ethanol metabolism into the tricarboxylic acid cycle is required for PA256 upregulation.Finally,this platform was applied to produce the“de novo design”protein NEO-2/15,and by introducing the N-propeptide of CspB,NEO-2/15 was effectively secreted with the accumulation 281 mg/L obtained after 24 h of shake-flask fermentation.To the best of our knowledge,this is the first report of NEO-2/15 secretory overexpression.展开更多
基金supported in part by grants from 973 Program from the Chinese Ministry of Science and Technology (MOST) (2014CB964704 and 2015CB964503)the Strategic Priority Research Program of the Chinese Academy of Sciences (Grant No. XDB19000000)the National Natural Science Foundation of China (NSFC) (31371463, 81672119, and 81725010)
文摘The skeleton is a dynamic organ that is constantly remodeled. Proteins secreted from bone cells, namely osteoblasts, osteocytes,and osteoclasts exert regulation on osteoblastogenesis, osteclastogenesis, and angiogenesis in a paracrine manner. Osteoblasts secrete a range of different molecules including RANKL/OPG, M-CSF, SEMA3A, WNT5A, and WNT16 that regulate osteoclastogenesis. Osteoblasts also produce VEGFA that stimulates osteoblastogenesis and angiogenesis. Osteocytes produce sclerostin(SOST) that inhibits osteoblast differentiation and promotes osteoclast differentiation. Osteoclasts secrete factors including BMP6, CTHRC1, EFNB2, S1P, WNT10B, SEMA4D, and CT-1 that act on osteoblasts and osteocytes, and thereby influencea A osteogenesis. Osteoclast precursors produce the angiogenic factor PDGF-BB to promote the formation of Type H vessels, which then stimulate osteoblastogenesis. Besides, the evidences over the past decades show that at least three hormones or "osteokines"from bone cells have endocrine functions. FGF23 is produced by osteoblasts and osteocytes and can regulate phosphate metabolism. Osteocalcin(OCN) secreted by osteoblasts regulates systemic glucose and energy metabolism, reproduction, and cognition. Lipocalin-2(LCN2) is secreted by osteoblasts and can influence energy metabolism by suppressing appetite in the brain.We review the recent progresses in the paracrine and endocrine functions of the secretory proteins of osteoblasts, osteocytes, and osteoclasts, revealing connections of the skeleton with other tissues and providing added insights into the pathogenesis of degenerative diseases affecting multiple organs and the drug discovery process.
文摘Rat protein DE is an androgen-dependent cysteine-rich secretory protein (CRISP) synthesized by proximal epididymal regions. DE, also known as CRISP-1, is localized on the equatorial segment of acrosome-reacted spermatozoa and participates in gamete fusion through binding to egg complementary sites. Immunization of rats with DE inhibits fertility and sperm fusion ability, suggesting that DE represents a good epididymal contraceptive target. Recombinant DE fragments and synthetic peptides revealed that DE binds to the egg via a 12-amino acid region of an evolutionarily conserved motif, Signature 2 (S2). The ability of other CRISP to bind to the rat egg was correlated with their S2 amino acid sequences. Although testicular protein Tpx- 1 (CRISP-2) was capable of binding to rodent eggs, human epididymal AEG-related protein (ARP) and helothermine (from lizard saliva) were not. The S2 region presented only two substitutions in Tpx-1 and four in ARP and helothermine, compared with the DE S2, suggesting that this amino acid sequence was relevant for egg interaction. Studies with Tpx- 1 and anti-Tpx- 1 revealed the participation of this protein in gamete fusion through binding to complementary sites in the egg. In competition studies, DE reduced binding of Tpx- 1 dose-dependently, indicating that both CRISP share the egg complementary sites. That anti-DE and anti-Tpx-1 inhibit sperm-egg fusion while recognizing only the corresponding proteins, suggests functional cooperation between these homologous CRISP to ensure fertilization success. These results increase our understanding of the molecular mechanisms of gamete fusion and contribute to the development of new and safer fertility regulating methods. (Asian J Androl 2007 July; 9: 528-532)
基金supported by National Key R&D Program of China,National Key Research Project(No.2017YFE0112100)CAMS Innovation Fund for Medical Sciences(CIFMS,No.2019-I2M-1-003)。
文摘Secretory proteins in tumor tissues are important components of the tumor microenvironment.Secretory proteins act on tumor cells or stromal cells or mediate interactions between tumor cells and stromal cells,thereby affecting tumor progression and clinical treatment efficacy.In this paper,recent research advances in secretory proteins in malignant tumors are reviewed.
基金financially supported by the National Nature Science Foundation of China (32372077)the Project of Chongqing Science and Technology Commission (CSTB2023NSCQ-MSX0355)the Fundamental Research Funds for the Central Universities (SWU120075)。
文摘The necrotrophic fungus, Sclerotinia sclerotiorum, employs an array of cell wall-degrading enzymes(CWDEs), including cellulase, to dismantle host cell walls. However, the molecular mechanisms through which S. sclerotiorum degrades cellulose remain elusive. Here, we unveil a novel secretory cellobiohydrolase, SsdchA, characterized by a signal peptide and a Glyco_hydro_7(GH7) domain. SsdchA exhibits a robust expression of during early infection stages. Interestingly, colony morphology and growth rates remain unaffected across the wild-type, SsdchA deletion strains and SsdchA overexpression strains on potato dextrose agar(PDA) medium. Nevertheless, the pathogenicity and cellobiohydrolase activity decreased in the SsdchA deletion strains, but enhanced in the SsdchA overexpression strains. Moreover,the heterologous expression of SsdchA in Arabidopsis thaliana leads to reduced cellulose content and heightened susceptibility to S. sclerotiorum. Collectively, our data underscore the pivotal role of the novel cellobiohydrolase SsdchA in the pathogenicity of S. sclerotiorum.
文摘Vasectomy is a simple and reliable method of male contraception. A growing number of men after vasectomy request vasectomy reversal due to various reasons. The pregnancy rate is lower than the patency rate after vasovasostomy and the pregnancy rate is time dependent. In this study, we evaluated the influence of reproductive tract obstruction on expression of epididymal proteins and their restoration after patency. Adult male Wistar rats were studied 30, 60 and 120 days after vasectomy, 30 days after vasovasostomy or after sham operations. Two-dimensional gel electrophoresis, mass-spectrometric technique, multidatabase search, Western blotting and real-time PCR were used to analyze the expression regulation of epididymal proteins. Total integrated intensity and total spot area of autoradiograms showed a consistent downward trend with time after obstruction, and this trend remained after patency. The intensity of the autoradiographic spots in three patency groups showed three trends: a downward trend, similar intensity and an upward trend compared with the correspondent obstruction group, respectively. Further verified experiments on human epididymis 2 (HE2), fertilization antigen-1 (FA-1), clusterin and PH20 demonstrated that compared with the correspondent obstruction group, the translation levels of HE2 and the mRNA transcription levels of HE2 showed an upward trend in patency groups, especially in the groups of obstruction for 60 days where the expression levels of HE2 were significantly upregulated after patency (P〈O.05). Reproductive tract obstruction provokes a disregulation of gene expression in the epididymis and this disregulation remained after patency. Successful reversal may recover some proteins and the recovery is time dependent, Obstruction differentially alters mRNA transcription of different proteins and the content of proteins seemed to be easier to be influenced than the gene transcription.
基金supported by the National Natural Science Foundation of China(31972218,31501590,and 31601615)the Program for Talents in Qingdao Agricultural University,China(6631114307)the Independent Innovation of Agricultural Sciences in Jiangsu Province,China(CX(18)3012)。
文摘A number of plant pathogenic species of Phytophthora are known to produce different classes of secretory proteins during interactions with their hosts.Although several small cysteine-rich(SCR)secretory proteins,conserved in oomycete pathogens,have been identified in Phytophthora,their specific involvement in these interactions remains unknown.In this study,an SCR effector encoded by Pn SCR82 in P.nicotianae was identified and shown to have similarities to P.cactorum phytotoxic protein,Pc F(Phytophthora cactorum Fragaria).Agroinfection with potato virus X vector,Pn SCR82,was capable of inducing plant hypersensitive cell death in Nicotiana benthamiana and Solanum lycopersicum.Real-time PCR results indicated that transiently expressed Pn SCR82 in N.benthamiana leaves activated the jasmonate,salicylic acid and ethylene signaling pathways.Transient expression of Pn SCR82 enhanced plant resisitance to P.capsici.In summary,our results demonstrated that P.nicotianae Pn SCR82 elicits defensive responses in N.benthamiana and may potentially play a significant role in future crop protection programs.
基金funded by the National Key Project for Research on Transgenic Biology,China(2016ZX08002001 to Zengyan Zhang)。
文摘The necrotrophic fungus Rhizoctonia cerealis is the causal agent of devastating diseases of cereal crops including wheat(Triticum aestivum).We present a high-quality genome assembly of R.cerealis Rc207,a virulent strain causing wheat sharp eyespot.The assembly(56.36 Mb)is composed of 17.87%repeat sequences and 14,433 predicted protein-encoding genes.The Rc207 genome encodes a large and diverse set of genes involved in pathogenicity,especially rich in those encoding secreted proteins,carbohydrateactive enzymes(CAZymes),peptidases,nucleases,cytochrome P450,and secondary metabolismassociated enzymes.Most secretory protein-encoding genes,including CAZymes,peroxygenases,dehydrogenases,and cytochrome P450,were up-regulated during fungal infection of wheat.We identified 831 candidate secretory effectors and validated the functions of 10 up-regulated candidate effector proteins.Of them,nine were confirmed as necrotrophic pathogen’s effectors promoting fungal infection.Abundant potential mobile or plastic genomic regions rich in repeat sequences suggest their roles in fungal adaption and virulence-associated genomic evolution.This study provides valuable resources for further comparative and functional genomics on important fungal pathogens,and provides essential tools for development of effective disease control strategies.
基金Youth Science and Technology Fund of Affiliated Hospital of Hebei University(Project number:2017Q024)Baoding City Science and Technology Plan Projects(Project number:2041ZF295)Hebei University Medical Subject Cultivation Project(Project number:2022B03)。
文摘Objective:To investigate the correlation between serum secretory frizzled-related protein 5(SFRP-5)expression levels and insulin resistance and glucolipid metabolism in patients with gestational diabetes mellitus(GDM).Methods:Baseline data were collected from 58 patients with GDM and 51 healthy controls who were admitted Affiliated Hospital of Hebei University from May 2020 to June 2022.sSTRA5 concentrations in peripheral blood of pregnant women were measured,and SFRP-5 levels in patients with different GDM types and normal controls were analyzed by logistic regression models.Results:The levels of triglyceride(TG),total cholesterol(TC),low-density lipoprotein cholesterol(LDL-C),fasting blood glucose(FBG),fasting insulin(FINS),hemoglobin A1c(HbA1c),and homeostasis model assessment-estimated insulin resistance(HOMA-IR)were higher in the observation group than in the control group,with statistically significant differences(P<0.05),while the expression levels of high-density lipoprotein cholesterol(HDL-C)and serum SFRP-5 were lower than in the control group,with statistically significant differences(P<0.05);serum SFRP-5,TG,TC,FBG,and HOMA-IR were all risk factors for GDM(P<0.05).Conclusion:Elevated serum sSTRA5 may be involved in the regulation of insulin resistance in the body and the regulation of blood glucose in the body by affecting lipid metabolism and inflammatory response.
基金The authors would like to thank Mr Shou-Xin Zhang and other members of the Research Center,Yuhuangding Hospital(Yantai,China)for technical assistance.
文摘To explore the functions of human ribonuclease 9(RNase 9),we constructed a mammalian fusion expression vector pcDNA-hRNase9,prepared recombinant human RNase 9-His fusion protein from HEK293T cells and determined its N-terminal amino acid sequences.According to the determined mature protein,recombinant human RNase 9 was prepared in E.coli.Ribonucleolytic activity and antibacterial activity of recombinant human RNase 9 were detected,and the distribution of human RNase 9 on tissues and ejaculated spermatozoa and in vitro capacitated spermatozoa were analyzed via indirect immunofluorescence assay.The results showed that recombinant human RNase 9 did not exhibit detectable ribonucleolytic activity against yeast tRNA,but exhibited antibacterial activity,in a concentration/time dependent manner,against E.coli.Immunofluorescent analyses showed that the predicted human RNase 9 was present throughout the epididymis,but not present in other tissues examined,and human RNase 9 was also present on the entire head and neck regions of human ejaculated spermatozoa and in vitro capacitated spermatozoa.These results suggest that human RNase 9 may play roles in host defense of male reproductive tract.
基金We thank Dr Qiang Liu for giving some advice and polishing the writing and thank Dr Yu-Chuan Zhou for fruitful discussions. We are also grateful to Ai- H ua Liu fur technical assistance in immunohistochemistry assay. This work was supported by grants from the National Natural Science Foundation of China (No. 30930053) and the Chinese Academy of Sciences Knowledge Innovation Program (No. KSCX2-EW-R-07).
文摘Previous studies have shown that rat epididymis-specific gene HongrES1 plays important roles in sperm capacitation and fertility. In this study, we cloned the mouse homologue gene by sequence alignment and RT-PCR methods and designated it as mHongl. The mHongl gene is located on chromosome 12p14, spanning five exons. The cDNA sequence consists of 1257 nucleotides and encodes a 419 amino-acid protein with a predicted N-terminal signal peptide of 20 amino acids. The mHongl mRNA shows similarity with HongrES1 in the expression patterns: (i) specific expression in epididymal tissue, especially in the cauda region; and (ii) androgen-dependence but testicular fluid factor independence. Its protein product shows 71% similarity with HongrES 1 and contains a classical serpin domain as does HongrES1. A polyclonal antibody against mHongl with high specificity and sensitivity was raised. Like HongrES1, the mHongl protein shows a checker-board expression pattern in the epididymal epithelium and is secreted into the epididymal lumen. The mHongl protein shows higher glycosylation than HongrES1. Although both of them are deposited onto the sperm head surface, mHongl is localized to the equatorial segment, which is different from that of HongrES 1. The mHongl protein can be removed from the sperm membrane by high ionic strength and therefore can be classed as an extrinsic membrane protein. Collectively, we conclude that mHongl is the homologue of HongrES1 and the present work paves the way for establishing animal models to elucidate the precise functions of HongrES1 and mHongl.
文摘A variety of glycosylphosphatidylinositol (GPI)-linked proteins are acquired on spermatozoa from epididymal luminal fluids (ELF) during sperm maturation. These proteins serve roles in immunoprotection and in key steps of fertilization such as capacitation, acrosomal exocytosis and sperm-egg interactions. Their acquisition on sperm cells is mediated both by membrane vesicles (epididymosomes, EP) which were first reported to dock on the sperm surface, and by lipid carriers which facilitate the transfer of proteins associated with the membrane-free fraction of ELF. While the nonvesicular fraction is more efficient, both pathways are dependent on hydrophobic interactions between the GPI-anchor and the external lipid layer of the sperm surface. More recently proteomic and hypothesis-driven studies have shown that EP from several mammals carry transmembrane (TM) proteins, including plasma membrane Ca^2+-ATPase 4 (PMCA4). Synthesized in the testis, PMCA4 is an essential protein and the major Ca^2+ efflux pump in murine spermatozoa. Delivery of PMCA4 to spermatozoa from bovine and mouse EP during epididymal maturation and in vitro suggests that the docking of EP on the sperm surface precedes fusion, and experimental evidence supports a fusogenic mechanism for TM proteins. Fusion is facilitated by CD9, which generates fusion-competent sites on membranes. On the basis of knowledge of PMCA4's interacting partners a number of TM and membrane-associated proteins have been identified or are predicted to be present, in the epididymosomal cargo deliverable to spermatozoa. These Ca^2+-dependent proteins, undetected in proteomic studies, play essential roles in sperm motility and fertility, and their detection highlights the usefulness of the hypothesis-driven approach.
文摘Mammalian fertilization is a complex process that involves different steps of interaction between the male and female gametes. In spite of its relevance, the molecular mechanisms underlying this process still remain to be elucidated. The present review describes the contribution of our laboratory to the understanding of mammalian fertilization using Cysteine-Rlch Secretory Proteins (CRISP) as model molecules. Substantial evidence obtained from in vitro assays and knockout models shows that epididymal CRISP1 associates with the sperm surface with two different affinities during maturation, and participates in the regulation of signaling pathways during capacitation as well as in both sperm-zona pellucida interaction and gamete fusion. These observations can be extended to humans as judged by our findings showing that the human homolog of the rodent protein (hCRISP1) is also involved in both stages of fertilization. Evidence supports that other members of the CRISP family secreted in the testis (CRISP2), epididymis (CRISP3-4) or during ejaculation (CRISP3) are also involved in sperm-egg interaction, supporting the existence of a functional redundancy and cooperation between homolog proteins ensuring the success of fertilization. Together, our observations indicate that CRISP proteins accompany spermatozoa along their transit through both the male and female reproductive tracts. We believe these results not only contribute to a better mechanistic understanding of fertilization but also support CRISP proteins as excellent candidates for future research on infertility and contraception.
基金The authors would like to thank Prof. Chunyan Wang, for her contributions to the samples collection, as well as thank Cheng Yang for his contributions to the figures revision. This study was supported by the Guangdong Provincial Natural Science Foundation of China (No. 2014A030313291, No. 2015A030310027 and No. 2016A030310393), the Guangdong Provincial Science and Technology Program (No. 2014A020212204), and the Science and Technology Innovation Project of Southern Medical University (QD2014N005).
文摘Cysteine-rich secretory protein 2 (CRISP2) is an important protein in spermatozoa that plays roles in modulating sperm flagellar motility, the acrosome reaction, and gamete fusion. Spermatozoa lacking CRISP2 exhibit low sperm motility and abnormal morphology. However, the molecular mechanisms underlying the reduction of CRISP2 in asthenoteratozoospermia (ATZ) remain unknown. In this study, low expression of CRISP2 protein rather than its mRNA was observed in the ejaculated spermatozoa from ATZ patients as compared with normozoospermic males. Subsequently, bioinformatic prediction, luciferase reporter assays, and microRNA-27a (miR-27a) transfection experiments revealed that miR-27a specifically targets CRISP2 by binding to its 3' untranslated region (3'-UTR), suppressing CRISP2 expression posttranscriptionally. Further evidence was provided by the clinical observation of high miR-27a expression in ejaculated spermatozoa from ATZ patients and a negative correlation between miR-27a expression and CRISP2 protein expression. Finally, a retrospective follow-up study supported that both high miR-27a expression and low CRISP2 protein expression were associated with low progressive sperm motility, abnormal morphology, and infertility. This study demonstrates a novel mechanism responsible for reduced CRISP2 expression in ATZ, which may offer a potential therapeutic target for treating male infertility, or for male contraception.
文摘Background Clara cell secretory protein (CC16) is an important lung derived protective factor and may play an important role on the pathogenesis of acute lung injury (ALl) induced by endotoxemia. Growth hormone (GH) is an important anabolism hormone secreted by GH cells of the hypophysis. Previous research showed that GH would significantly exacerbate ALl induced by endotoxemia, but the mechanism is not very clear yet. Whether the effects are related to CC16 or not is undetermined. Methods One hundred and twelve male Sprague-Dawley rats were randomly divided into an ALl group and a GH group. The rats in the two groups were subdivided into seven subgroups, according to injection with lipopolysaccharides (LPS) or not, then according to different intervals of time after LPS injection; 0 hour (pre-injection of LPS, acted as control group), 0.5 hour, 1 hour, 2 hours, 4 hours, 6 hours and 24 hours for subgroups. Pulmonary alveolar septa area density (PASAD) and ploymorphonuclear cells (PMN) in the lungs were analyzed morphometrically. The levels of tumor necrosis factor (TNF) and interleukin 6 (IL-6) were determined by radioimmunoassay. To analyze the expression and activation of nuclear factor kappa B (NF-KB), the numbers of NF-KB positive cells in lungs were counted after immunofluorescence staining, and the levels of NF-KB inhibitory protein-a (IKB-a) in lung homogenates of rats were detected by Western blotting. The expression levels of CC16 mRNA in lungs of the rats with ALl were determined by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR). The levels of CC16 protein in lung homogenates were detected by Western blotting. Results Half an hour after LPS injury both the PASAD and PMN numbers in lungs of the rats with ALl began to increase significantly and peaked at 6-hour post-injury. They then began to recover and reached normal levels at 24-hour post-injury. Both the PASAD and PMN numbers in the GH group increased more significantly than those in the ALl group. The levels of TNF in lungs of the rats with ALl homogenates increased significantly 0.5-hour post-injury, peaked at 1-hour and maintained a high level until 6 hours then gradually recovered. The content of TNF in the GH group lung homogenates increased more significantly than in the ALl group post-injury. The contents of IL-6 in rat lung homogenates began to increase significantly at 1-hour post-injury, peaked at 4 hours then gradually returned to normal levels by 6 hours post-injury. The levels of IL-6 in the lung homogenates of the GH group were higher than in the ALl group at different time intervals post-injury. The number of NF-KB positive cells increased dramatically at 0.5-hour post-injury, and the fluorescence intensity was enhanced. Both peaked at 4-hour post-injury. The number of NF-KB positive cells and the enhanced intensity of fluorescence began to decrease from 6-hour post-injury, but the number of NF-KB cells at 24 hours post-injury was still higher than in the control group. The number of NF-KB cells in lungs in the GH group was significantly higher than in the LPS group at the different time intervals post-injury. The IKB-a expression in lungs of the rats with ALl homogenates decreased dramatically 0.5-hour post-injury, reaching a nadir at 4-hour post-injury and then began to recover. The levels of IKB-Q in GH group were significantly lower than those in ALl group. Both the levels of CC16 mRNA and protein in lungs of the rats with ALl began to decrease significantly 0.5-hour post-injury, reached a nadir at 6 hours, and then began to recover. Both the expression of CC16 mRNA and CC16 protein in the GH group were significantly lower than those in the ALl group at the different time intervals post-injury. Correlation analysis indicates that CC16 correlates significantly with all the indices mentioned above. Conclusions Down-regulation of CC16 expression plays a critical role in the pathogenesis of acute lung injury induced by endotoxemia. The application of GH can exacerbate the lung injury induced by endotoxemia through down-regulating the expression of CC16.
基金Supported by the National Natural Science Fund(No. 81173187)the Natural Science Fund of Anhui Province (No.11040606M217)
文摘OBJECTIVE:To study the functional mechanism of Pingchuanning Decoction in treatment of airway remodeling in asthmatic rats.METHODS:Eighty healthy Wistar male rats were randomized into eight groups(n=10 rats each):Normal group,Asthma model group,Dexamethasone group,Guilong Kechuanning group,Xiaoqinglong Decoction group,and Pingchuanning Decoction low-,middle-,and high-dose groups.The rats of all but the Normal group were made into asthma models through intraperitoneal injection and aerosol inhalation of ovalbumin.All treatments were administered at the first stimulation of asthma onset(third week of modeling),and the rats were killed after stimulating asthma attacks for 4 weeks.The general conditions of rats and pathomorphological changes of the lung tissues were observed.The expression of nerve growth factor(NGF) of the lung tissues was measured with immunohistochemical methods,and the content of Clara cell secretory protein(CCSP) mRNA was determined with RT-PCR.RESULTS:Compared with the Normal group,the contents of NGF and CCSP mRNA in the lung tissues of the Model group were significantly changed(P<0.01).Compared with the Model group,the indices of Pingchuanning Decoction and other treatment groups were improved to some extent(P<0.05 or P<0.01).CONCLUSIONS:Pathological changes of airway inflammation and remodeling were present in these rat asthma models.Pingchuanning Decoction had an intervention effect on these experimental models.Its functional mechanism may be related to multiple factors,including alleviation of airway inflammation,relief of bronchial smooth muscle spasm,and inhibition of airway remodeling.
基金This work received funding from the National Natural Science Foundation of China(No.21878124,22078128,and 21938004)the Fundamental Research Funds for the Central Universities(No.JUSRP221032)+1 种基金the 111 Project(No.111-2-06)the national first-class discipline program of Light Industry Technology and Engineering(LITE2018-24).
文摘Corynebacterium glutamicum represents an emerging recombinant protein expression factory due to its ideal features for protein secretion,but its applicability is harmed by the lack of an autoinduction system with tight regulation and high yield.Here,we propose a new recombinant protein manufacturing platform that leverages ethanol as both a delayed carbon source and an inducer.First,we reanalysed the native inducible promoter PICL from the acetate uptake operon and found that its limited capacity is the result of the inadequate translation initial architecture.The two strategies of bicistronic design and ribozyme-based insulator can ensure the high activity of this promoter.Next,through transcriptional engineering that alters transcription factor binding sites(TFBSs)and the first transcribed sequence,the truncated promoter PA256 with a dramatically higher transcription level was generated.When producing the superfolder green fluorescent protein(sfGFP)under 1%ethanol conditions,PA256 exhibited substantially lower protein accumulation in prophase but an approximately 2.5-fold greater final yield than the strong promoter PH36.This superior expression mode was further validated using two secreted proteins,camelid antibody fragment(VHH)and endoxylanase(XynA).Furthermore,utilizing CRISPRi technology,ethanol utilization blocking strains were created,and PA256 was shown to be impaired in the phosphotransacetylase(PTA)knockdown strains,indicating that ethanol metabolism into the tricarboxylic acid cycle is required for PA256 upregulation.Finally,this platform was applied to produce the“de novo design”protein NEO-2/15,and by introducing the N-propeptide of CspB,NEO-2/15 was effectively secreted with the accumulation 281 mg/L obtained after 24 h of shake-flask fermentation.To the best of our knowledge,this is the first report of NEO-2/15 secretory overexpression.