Participation of epididymal cysteine-rich secretory proteins in sperm-egg fusion and their potential use for male fertility regulation

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）

Structure and function of epididymal protein cysteine-rich secretory protein-1

Cysteine-rich secretory protein-1 （CRISP-1） is a glycoprotein secreted by the epididymal epithelium. It is a member of a large family of proteins characterized by two conserved domains and a set of 16 conserved cysteine residues. In mammals, CRISP-1 inhibits sperm-egg fusion and can suppress sperm capacitation. The molecular mechanism of action of the mammalian CRISP proteins remains unknown, but certain non-mammalian CRISP proteins can block ion channels. In the rat, CRISP-1 comprises two forms referred to as Proteins D and E. Recent work in our laboratory demonstrates that the D form of CRISP-1 associates transiently with the sperm surface, whereas the E form binds tightly. When the spermatozoa are washed, the E form of CRISP-1 persists on the sperm surface after all D form has dissociated. Cross-linking studies demonstrate different protein-protein interaction patterns for D and E, although no binding partners for either protein have yet been identified. Mass spectrometric analyses revealed a potential post-translational modification on the E form that is not present on the D form. This is the only discernable difference between Proteins D and E, and presumably is responsible for the difference in behavior of these two forms of rat CRISP- 1. These studies demonstrate that the more abundant D form interacts with spermatozoa transiently, possibly with a specific receptor on the sperm surface, consistent with a capacitation-suppressing function during sperm transit and storage in the epididymis, and also confirm a tightly bound population of the E form that could act in the female reproductive tract. （Asian J Androl 2007 July; 9： 508-514）

Inhibitory Effect of Dexamethasone on Expression of Cysteine-rich 61 Protein in Airway Epithelial Cells of Allergic Mouse Models

Summary： In order to study whether cysteine-rich 61 protein （cyr61） is involved in the pathogenesis of asthma and its relation to airway inflammation, the effect of dexamethasone （Dxm） on the expression of cyr61 in the lung tissues of asthmatic mice was investigated. Forty BALB/c mice were divided into asthma group （n=15）, control group （n=10） and Dxm group （n=15）. The asthma group was sensitized and challenged by ovalbumin （OVA）. The mice in Dxm group were intraperitoneally administered with Dxm after OVA challenge. The expression of cyr61 in the lung tissues was detected by using immuno- histochemistry, and that of eotaxin protein in the bronchoalveolar lavage fluid （BALF） by using en- zyme-linked immunosorbent assay （ELISA）. The number of inflammatory cells in BALF was also ana- lyzed. The results showed that the cyr61 expression was highest in asthma group （P〈0.05）, followed by Dxm group （P〈0.05） and control group. The cyr61 had a positive correlation with the total nucleated cells （r=0.867, P〈0.05）, especially eosinophils （r=0.856, P〈0.05）, and eotaxin level （r=0.983, P〈0.05） in the BALF. Our findings suggested that cyr61 is expressed in airway epithelial cells and has a positive correlation with eotaxin and number of airway infiltrating eosinophils.

PnSCR82,a small cysteine-rich secretory protein of Phytophthora nicotianae,can enhance defense responses in plants

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.

A Novel Trypsin Inhibitor-Like Cysteine-Rich Peptide from the Frog Lepidobatrachus laevis Containing ProteinaseInhibiting Activity

Various bio-active substances in amphibian skins play important roles in survival of the amphibians.Many protease inhibitor peptides have been identified from amphibian skins,which are supposed to negatively modulate the activity of proteases to avoid premature degradation or release of skin peptides,or to inhibit extracellular proteases produced by invading bacteria.However,there is no information on the proteinase inhibitors from the frog Lepidobatrachus laevis which is unique in South America.In this work,a cDNA encoding a novel trypsin inhibitor-like(TIL)cysteine-rich peptide was identified from the skin cDNA library of L.laevis.The 240-bp coding region encodes an 80-amino acid residue precursor protein containing 10 half-cysteines.By sequence comparison and signal peptide prediction,the precursor was predicted to release a 55-amino acid mature peptide with amino acid sequence,IRCPKDKIYKFCGSPCPPSCKDLTPNCIAVCKKGCFCRDGTVDNNHGKCVKKENC.The mature peptide was named LL-TIL.LL-TIL shares significant domain similarity with the peptides from the TIL supper family.Antimicrobial and trypsin-inhibitory abilities of recombinant LL-TIL were tested.Recombinant LL-TIL showed no antimicrobial activity,while it had trypsin-inhibiting activity with aKi of 16.5178 lM.These results suggested there was TIL peptide with proteinase-inhibiting activity in the skin of frog L.laevis.To the best of our knowledge,this is the first report of TIL peptide from frog skin.

Cloning and expression of the Chinese wheat mosaic virus RNA2 coat protein read-through and 19 ku cysteine-rich domains and localization of these proteins

The 5’-terminal (RTn) and 3’-terminal (RTc) halves of the coat protein readthrough domain and the 19 ku cysteine-rich protein of Chinese wheat mosaic virus (CWMV) were amplified by RT-PCR, cloned and expressed in E. coll. Antisera and monoclonal antibodies against these proteins were prepared by immunising these purified proteins to mice. Detection of RTn, RTc and 19 ku proteins in CWMV infected wheat sap and leaf tissue indicated that the RTn and RTc proteins were distributed on the surface of virus particles whereas the 19 ku protein was in the cytoplasm of the infected wheat cells.

MicroRNA-27a-mediated repression of cysteine-rich secretory protein 2 translation in asthenoteratozoospermic patients

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.

Therapeutic effects of the extract of Sancao Formula, a Chinese herbal compound, on imiquimod-induced psoriasis via cysteine-rich protein 61

Objective: Psoriasis is a common chronic inflammatory skin disease that is prone to recurrence, and the proinflammatory factor, cysteine-rich protein 61(Cyr61), is important in its pathophysiology. Long-term clinical practice has shown that Sancao Formula(SC), a Chinese herbal compound, is effective in the treatment of psoriasis, but the precise mechanism remains unknown. In this study, we investigate the mechanism by which SC extract alleviates imiquimod(IMQ)-induced psoriasis.Methods: The expression of Cyr61 in psoriatic lesions and normal healthy skin was detected using immunohistochemical analysis to investigate the biological role of Cyr61 in models of psoriatic inflammation. A psoriatic mouse model was established by topical application of IMQ, and the effect of topical application of SC extract was evaluated using the psoriasis area and severity index(PASI) score,hematoxylin-eosin staining, and histopathological features of the skin. Next, a HaCaT cell inflammation model was established using interferon-γ(IFN-γ), and the effect of SC extract on the mRNA and protein levels of Cyr61 and intercellular cell adhesion molecule-1(ICAM-1) was confirmed using Western blot and quantitative real-time polymerase chain reaction analyses.Results: Immunohistochemical staining showed that the expression of Cyr61 in psoriatic lesions was higher than that in normal skin samples(78.26% vs 41.18%, P < 0.05), and the number of Cyr61-positive cells in psoriatic lesions was also significantly higher than in normal skin(18.66 ± 2.51 vs4.33 ± 1.52, P < 0.05). Treatment in mice with IMQ-induced psoriasis showed that SC extract could significantly improve the inflammatory phenotype, PASI score(10.875 ± 0.744 vs 3.875 ± 0.582, P < 0.05),and pathological features compared with those in IMQ model group;SC treatment was also associated with decreased levels of Cyr61 and ICAM-1. In the IFN-γ-induced inflammatory cell model, the mRNA and protein levels of Cyr61 and ICAM-1 were upregulated, while the SC extract downregulated the levels of Cyr61 and ICAM-1.Conclusion: The results provide a theoretical basis for the involvement of Cyr61 in the pathogenesis of psoriasis, and suggest that SC should be used to target Cyr61 for the prevention of psoriasis recurrence.

An activated form of NB-ARC protein RLS1 functions with cysteine-rich receptor-like protein RMC to trigger cell death in rice

A key event that follows pathogen recognition by a resistance(R)protein containing an NB-ARC(nucleotide-binding adaptor shared by Apaf-1,R proteins,and Ced-4)domain is hypersensitive response(HR)-type cell death accompanied by accumulation of reactive oxygen species and nitric oxide.However,the integral mechanisms that underlie this process remain relatively opaque.Here,we show that a gain-offunction mutation in the NB-ARC protein RLS1(Rapid Leaf Senescence 1)triggers high-light-dependent HR-like cell death in rice.The RLS1-mediated defense response is largely independent of salicylic acid accumulation,NPR1(Nonexpressor of Pathogenesis-Related Gene 1)activity,and RAR1(Required for Mla12 Resistance 1)function.A screen for suppressors of RLS1 activation identified RMC(Root Meander Curling)as essential for the RLS1-activated defense response.RMC encodes a cysteine-rich receptor-like secreted protein(CRRSP)and functions as an RLS1-binding partner.Intriguingly,their co-expression resulted in a change in the pattern of subcellular localization and was sufficient to trigger cell death accompanied by a decrease in the activity of the antioxidant enzyme APX1.Collectively,our findings reveal an NBARC-CRRSP signaling module that modulates oxidative state,the cell death process,and associated immunity responses in rice.

CCN1在氧诱导小鼠视网膜新生血管形成中的表达及意义

目的:研究富含半胱氨酸蛋白61(CCN1/Cyr61)在氧诱导小鼠视网膜新生血管(retinal neovascularization,RNV)中的表达及意义,探讨特异性抑制CCN1对RNV形成的抑制作用。方法:取C57BL/6J小鼠200只,随机分为对照组、高氧组、高氧对照组和CCN1治疗组,每组各50只。高氧对照组和CCN1治疗组分别玻璃体腔内注射空载体质粒和CCN1siRNA表达质粒。ADP酶视网膜铺片观察视网膜血管形态,HE染色计数突破视网膜内界膜的新生血管内皮细胞核数,免疫组织化学、Western blot和RT-PCR法检测CCN1蛋白及mRNA的表达情况。结果:高氧组和高氧对照组视网膜可见大片无灌注区和大量突破内界膜的新生血管内皮细胞核(25.25±1.26个;23.12±1.16个),CCN1治疗组较高氧组和高氧对照组的无灌注区及新生血管内皮细胞核数(8.47±1.15个)明显减少。高氧组和高氧对照组较对照组相比,CCN1蛋白及mRNA表达显著增高,CCN1治疗组较高氧组和高氧对照组显著减弱,均有统计学意义(均为P<0.05)。结论:CCN1的异常表达可能与RNV形成密切相关,特异性抑制CCN1能有效抑制RNV的形成,为预防和治疗ROP提供新的思路及对策。

SIRT1 and stem cells: In the forefront with cardiovascular disease, neurodegeneration and cancer

Cardiovascular disease, nervous system disorders, and cancer in association with other diseases such as diabetes mellitus result in greater than sixty percent of the global annual deaths. These noncommunicable diseases also affect at least one-third of the population in low and middle-income countries and lead to hypertension, elevated cholesterol, malignancy, and neurodegenerative disorders such as Alzheimer's disease and stroke. With the climbing lifespan of the world's population, increased prevalence of these disorders is expected requiring the development of new therapeutic strategies against these disabling disease entities. Targeting stem cellproliferation for cardiac disease, vascular disorders, cancer, and neurodegenerative disorders is receiving great enthusiasm, especially those that focus upon SIRT1, a mammalian homologue of the yeast silent information regulator-2. Modulation of the cellular activity of SIRT1 can involve oversight by nicotinamide/nicotinic acid mononucleotide adenylyltransferase, mammalian forkhead transcription factors, mechanistic of rapamycin pathways, and cysteine-rich protein 61, connective tissue growth factor, and nephroblastoma over-expressed gene family members that can impact cytoprotective outcomes. Ultimately, the ability of SIRT1 to control the programmed cell death pathways of apoptosis and autophagy can determine not only cardiac, vascular, and neuronal stem cell development and longevity, but also the onset of tumorigenesis and the resistance against chemotherapy. SIRT1 therefore has a critical role and holds exciting prospects for new therapeutic strategies that can offer reparative processes for cardiac, vascular, and nervous system degenerative disorders as well as targeted control of aberrant cell growth during cancer.

Cyr61/CTGF/Nov family proteins in gastric carcinogenesis

Gastric cancer(GC)is the second leading cause of cancer-related death.The poor survival rate may reflect the relatively aggressive tumor biology of GC.Recently,the importance of the tumor microenvironment in carcinogenesis has emerged.In the tumor microenvironment,tumor cells and the surrounding stromal cells aberrantly secrete matricellular proteins capable of modulating carcinogenesis and regulating metastasis.The Cyr61/CTGF/Nov(CCN)proteins are a family of matricellular proteins with variable roles in many physiological and pathological processes.The evidence suggests that CCN family proteins contribute to GC carcinogenic processes.Here,we briefly review recent research on the effects of CCN family proteins in GC carcinogenesis and the development of new targeted agents in this field.

Stretch-induced Expression of CYR61 Increases the Secretion of IL-8 in A549 Cells via the NF-κβ/Iκβ Pathway

Mechanical ventilation （MV） with large tidal volumes can increase lung alveolar permeability and initiate inflammatory responses, resulting in ventilator-induced lung injury （VILI）. The mechanisms of the injurious effects of MV and the genetic susceptibility remain unclear. VILI-related genes such as cysteine-rich angiogenic inducer 61 （Cyr61） have been demonstrated to play a detrimental role in the aggressive ventilation strategies. In the present study, we investigated the involvement of Cyr61 in the VILI and the underlying mechanism. A549 cells were exposed to cyclic stretch of varying durations and then the mRNA and protein levels of Cyr61 were measured by real-time PCR and Western blotting, respectively. Additionally, after exposure ofA549 cells to cyclic stretch for 5 min to 1 h, the expression levels of nuclear factor kappaB （NF-κβ） and IL-8 were detected by ELISA and Western blotting. Thereafter, Cyr61 expression was depressed in A549 cells with the siRNA pGenesill. 1-Cyr61-3 before the cyclic stretch, and IL-8 secretion and the activation of NF- κB pathways were probed by ELISA and Western blotting, respectively. Moreover, a NF- κB inhibitor （PDTC） and an activator （TNF） were used before mechanical stretch. Realtime PCR and ELISA were performed to detect the mRNA and protein of IL-8, respectively. The results showed that the mechanical cyclic stretch led to increased Cyr61 expression at mRNA and protein levels in A549 cells. Additionally, cyclic stretch also mobilized NF- κB from the cytoplasm to the nucleus and increased IL-8 secretion in A549 cells. The inhibition of Cyr61 blocked the NF-κB activation and IL-8 secretion in response to cyclic stretch. Inhibition of NF-κB attenuated the mRNA and protein expression of IL-8 in A549 cells transfected with Cyr61 siRNA. It was suggested that Cyr61/NF-κB signaling pathway mediates the upregulation of IL-8 in response to cyclic stretch in A594 cells. These findings support the hypothesis that Cyr61 plays a critical role in acute lung inflammation triggered by mechanical strain.

Cyr61 Alleviates Cholangitis by Inhibiting Cytotoxic Effects of CD8^(+) T Cells on Biliary Epithelial Cells

Objective:Primary biliary cholangitis(PBC)is a chronic progressive cholestatic liver disease.In recent years,researchers have found that cysteine-rich angiogenic inducer 61(Cyr61,also known as CCN1)has a potential role in reducing portal inflammation in patients with PBC.This study aimed to explore the relationship between Cyr61 and PBC to provide new ideas and an experimental basis for the clinical treatment of PBC.Methods:After induction of the overexpression of Cyr61 in a mouse model of PBC using recombinant adenovirus,hematoxylin and eosin staining and pathological scores were used to indicate intrahepatic inflammation and bile duct damage.Real-time PCR was used to detect changes in inflammation-related cytokines in the liver.To further study the mechanism,we assessed whether Cyr61 protects bile duct epithelial cells from cytotoxic effects.Results:Serum and hepatic Cyr61 levels were increased in the murine model of PBC.Overexpression of Cyr61 alleviated hepatic inflammation and bile duct injury in vivo.Cyr61 inhibited the cytotoxic effects of CD8^(+)T cells by acting on biliary epithelial cells(BECs)in vitro.Conclusion:Our results provide novel insight into the pathogenesis of PBC and suggest that Cyr61 plays a dominant role in the cytotoxic effects on BECs in PBC.Consequently,therapeutic strategies targeting Cyr61 could be a potent therapy for PBC.

Association of Single Nucleotide Polymorphisms in Exon 3 of Porcine LMCD1 Gene with Meat Quality and Carcass Traits

LIM domain proteins are found to be important regulators in cell growth, cell fate determination, cell differentiation, and remodelling of the cell cytoskeleton by their interaction with some structural proteins, kinases, transcriptional regulators, etc. The presence of LIM domains in LMCD1 gene implies it may be involved in skeletal muscle protein-protein interactions. This study was to investigate polymorphisms of LIM and cysteine-rich domain 1 （LMCD1） gene and its effect on meat quality and carcass traits in pig. The polymorphism （G294A） in exon 3 region of porcine LMCD1 gene, which is synonymous mutation, was genotyped in the population of 178 F, pigs of a Large White × Meishan resource family. Statistical results indicated the distribution of allele G （with a A → G mutation） and A （without mutation）. Analysis of variance showed that the polymorphism of LMCD1 gene was associated with variation in several carcass traits of interest for pig breeding. Some carcass traits and meat quality traits are close to significance by association. An analysis of more animals is necessary to analyze the polymorphisms in exon 3 of porcine LMCD1 gene if it was selected as a marker for the pig carcass traits.

Broad-spectrum ginsentides are principal bioactives in unraveling the cure-all effects of ginseng

Stress and illness connection is complex and involves multiple physiological systems.Panax ginsengs,reputed for their broad-spectrum“cure-all”effect,are widely prescribed to treat stress and related illnesses.However,the identity of ginseng’s“cure-all”medicinal compounds that relieve stress remains unresolved.Here,we identify ginsentides as the principal bioactives that coordinate multiple systems to restore homeostasis in response to stress.Ginsentides are disulfide-rich,cell-penetrating and proteolytic-stable microproteins.Using affinity-enrichment mass spectrometry target identification together with in vitro,ex vivo and in vivo validations,we show that highly purified or synthetic ginsentides promote vasorelaxation by producing nitric oxide through endothelial cells via intracellular PI3K/Akt signaling pathway,alleviate a1-adrenergic receptor overactivity by reversing phenylephrine-induced constriction of aorta,decrease monocyte adhesion to endothelial cells via CD166/ESAM/CD40 and inhibit P2Y12 receptors to reduce platelet aggregation.Orally administered ginsentides were effective in animal models to reduce ADP-induced platelet aggregation,to prevent collagen and adrenalineinduced pulmonary thrombosis as well as anti-stress behavior of tail suspension and forced swimming tests in mice.Together,these results strongly suggest that ginsentides are the principal panacea compounds of ginsengs because of their ability to target multiple extra-and intra-cellular proteins to reverse stress-induced damages.

Male-Female Crosstalk during Pollen Germination, Tube Growth and Guidance, and Double Fertilization

Sperm cells of flowering plants are non-motile and thus require transportation to the egg apparatus via the pollen tube to execute double fertilization. During its journey, the pollen tube interacts with various sporophytic cell types that support its growth and guide it towards the surface of the ovule. The final steps of tube guidance and sperm delivery are controlled by the cells of the female gametophyte. During fertilization, cell-cell communication events take place to achieve and maximize reproductive success. Additional layers of crosstalk exist, including self-recognition and specialized processes to prevent self-fertilization and consequent inbreeding. In this review, we focus on intercellular communication between the pollen grain/pollen tube including the sperm cells with the various sporophytic maternal tissues and the cells of the female gametophyte. Polymorphic-secreted peptides and small proteins, especially those belonging to various subclasses of small cysteine-rich proteins （CRPs）, reactive oxygen species （ROS）/NO signaling, and the second messenger Ca2＋, play center stage in most of these processes.