NMR structure of CXCR3 binding chemokine CXCL11 (ITAC)

CXCL11 (ITAC) is one of three chemokines known to bind the receptor CXCR3, the two others being CXCL9 (Mig) and CXCL10 (IP-10). CXCL11 differs from the other CXCR3 ligands in both the strength and the particularities of its receptor interactions: It has a higher affinity, is a stronger agonist, and behaves differently when critical N-terminal residues are deleted. The structure of CXCL11 was determined using solution NMR to allow comparison with that of CXCL10 and help elucidate the source of the differences. CXCL11 takes on the canonical chemokine fold but exhibits greater conformational flexibility than has been observed for related chemokines under the same sample conditions. Unlike related chemokines such as IP-10 and IL-8, ITAC does not appear to form dimers at millimolar concentrations. The origin for this behavior can be found in the solution structure, which indicates a beta-bulge in beta-strand 1 that distorts the dimerization interface used by other CXC chemokines.     

Andrographolide inhibits prostate cancer by targeting cell cycle regulators,CXCR3 and CXCR7 chemokine receptors

Despite state of the art cancer diagnostics and therapies offered in clinic, prostate cancer (PCa) remains the second leading cause of cancer-related deaths. Hence, more robust therapeutic/preventive regimes are required to combat this lethal disease. In the current study, we have tested the efficacy of Andrographolide (AG), a bioactive diterpenoid isolated from Andrographis paniculata, against PCa. This natural agent selectively affects PCa cell viability in a dose and time-dependent manner, without affecting primary prostate epithelial cells. Furthermore, AG showed differential effect on cell cycle phases in LNCaP, C4-2b and PC3 cells compared to retinoblastoma protein (RB^?/?) and CDKN2A lacking DU-145 cells. G2/M transition was blocked in LNCaP, C4-2b and PC3 after AG treatment whereas DU-145 cells failed to transit G1/S phase. This difference was primarily due to differential activation of cell cycle regulators in these cell lines. Levels of cyclin A2 after AG treatment increased in all PCa cells line. Cyclin B1 levels increased in LNCaP and PC3, decreased in C4-2b and showed no difference in DU-145 cells after AG treatment. AG decreased cyclin E2 levels only in PC3 and DU-145 cells. It also altered Rb, H3, Wee1 and CDC2 phosphorylation in PCa cells. Intriguingly, AG reduced cell viability and the ability of PCa cells to migrate via modulating CXCL11 and CXCR3 and CXCR7 expression. The significant impact of AG on cellular and molecular processes involved in PCa progression suggests its potential use as a therapeutic and/or preventive agent for PCa.     

大肠杆菌二氢叶酸还原酶基因folA的克隆、表达纯化及分子间交联

利用PCR技术从大肠杆菌DH5α中获取二氢叶酸还原酶(DHFR)基因folA。用限制性内切酶BamHI与PstI将该片段插入到克隆载体pUC18上,DNA测序鉴定目的基因。而后再将该基因亚克隆到表达载体pTrcHisC上,IPTG诱导表达重组蛋白。在非变性条件下,用TALON金属亲和层析树脂纯化含组氨酸标记的重组DHFR。纯化产物在热诱导条件下行SDSPAGE分析,除23000大小的单体外,还出现了交联的二聚体和多聚体;而当反应体系中含有还原剂β-巯基乙醇时,二聚体和多聚体都被减弱。推断蛋白质在热诱导条件下二级结构发生改变而产生交联,并且有二硫键的参与。     

CXCL7-induced macrophage infiltration in lung tumor is independent of CXCR2 expression: CXCL7-induced macrophage chemotaxis in LLC tumors

Chemokines play diverse roles in modulating the immune response during tumor development. Levels of CXC chemokine ligand 7 (CXCL7) protein vary during tumorigenesis, and the evidence suggests that this chemokine serves as a novel biomarker of early-stage lung cancer. We investigated the effect of CXCL7 gene expression on the infiltration of myeloid cells into the tumor microenvironment in Lewis lung carcinoma (LLC). Tumors established from LLC cells overexpressing CXCL7 (CXCL7-LLC tumors) increased the infiltration of CD206⁺ M2 macrophages at the early stages of tumorigenesis. This infiltration was independent of CXCR2 expression on either tumor cells or macrophages. CXCL7-LLC tumors developed faster than control-LLC tumors (IRES-LLC tumor) did. The extent of CD4⁺ T cell, CD8⁺ T cell, and natural killer T cell infiltration was similar between the two tumor groups. Our findings suggest that CXCL7 attracts macrophages especially at the tumor site and may accelerate lung tumor development in the early stages.     

Monomeric structure of the cardioprotective chemokine SDF‐1/CXCL12

The chemokine stromal cell‐derived factor‐1 (SDF‐1/CXCL12) directs leukocyte migration, stem cell homing, and cancer metastasis through activation of CXCR4, which is also a coreceptor for T‐tropic HIV‐1. Recently, SDF‐1 was shown to play a protective role after myocardial infarction, and the protein is a candidate for development of new anti‐ischemic compounds. SDF‐1 is monomeric at nanomolar concentrations but binding partners promote self‐association at higher concentrations to form a typical CXC chemokine homodimer. Two NMR structures have been reported for the SDF‐1 monomer, but only one matches the conformation observed in a series of dimeric crystal structures. In the other model, the C‐terminal helix is tilted at an angle incompatible with SDF‐1 dimerization. Using a rat heart explant model for ischemia/reperfusion injury, we found that dimeric SDF‐1 exerts no cardioprotective effect, suggesting that the active species is monomeric. To resolve the discrepancy between existing models, we solved the NMR structure of the SDF‐1 monomer in different solution conditions. Irrespective of pH and buffer composition, the C‐terminal helix remains tilted at an angle with no evidence for the perpendicular arrangement. Furthermore, we find that phospholipid bicelles promote dimerization that necessarily shifts the helix to the perpendicular orientation, yielding dipolar couplings that are incompatible with the NOE distance constraints. We conclude that interactions with the alignment medium biased the previous structure, masking flexibility in the helix position that may be essential for the distinct functional properties of the SDF‐1 monomer.     

趋化因子CXCL12 及其受体CXCR4 在胶质瘤中的研究进展

CXCL12是趋化因子家族成员之一,是能够特异性结合其受体CXCR4发挥趋化性作用的细胞因子。最初,CXCL12及CXCR4被发现于炎症细胞,参与机体炎症、免疫等病理反应。接下来的几年中发现,它在机体发育、成熟过程中也有重要作用。如今,大量研究表明它与肿瘤的生长、侵袭及转移密切相关。据报道,在乳腺癌、肺癌、卵巢癌等二十余种肿瘤组织中发现CXCL12及CXCR4的表达,其中也包括中枢系统肿瘤-胶质瘤。CXCL12/CXCR4参与胶质瘤生长过程的多个步骤,包括肿瘤增殖、侵袭、转移等。有实验指出,转移灶的CXCR4表达水平较原发灶高,CXCR4有可能成为抑制胶质瘤生长、转移的重要靶目标。     

CXCL12/CXCR4/CXCR7轴在子宫内膜异位症中的研究进展

子宫内膜异位症(endometriosis, EMT)是常见的妇科疾病,发病率高,且有年轻化的趋势。因其治疗困难且复发率高,严重影响了女性的生活质量和生育能力。研究发现趋化因子CXCL12与其受体CXCR4和CXCR7在恶性肿瘤中起重要作用。虽然EMT为良性疾病,但有恶性肿瘤的生物学特征,近来发现CXCL12/CXCR4/CXCR7轴可以影响子宫内膜异位症的定植、侵袭和转移。本文就当前国内外研究CXCL12/CXCR4/CXCR7轴在EMT发生发展过程中的作用进行了综述,旨在为EMT的治疗找到新靶点。     

Rat chemokine CXCL11: Structure, tissue distribution, function and expression in cardiac transplantation models

CXCL11 is thought to play a critical role in allograft rejection. To clarify the role of CXCL11 in the rat transplantation model, we cloned CXCL11 cDNA from rat liver tissue and used it to study CXCL11 structure, function and expression. The rat CXCL11 gene encodes a protein of 100 amino acids and spans approximately a 2.8 kb DNA segment containing 4 exons in the protein coding region. Tissue distribution of rat CXCL11 was analyzed by quantitative RT-PCR and showed that rat CXCL11 mRNA is expressed in various tissues and, in particular, at high levels in the spleen and lymph nodes. COS-1 cells were transfected with a plasmid vector encoding rat CXCL11 and used to study CXCL11 effects on cell migration and internalization of CXCR3, the CXCL11 receptor. The recombinant CXCL11 showed chemotactic properties and induced CXCR3 internalization in CD4⁺ T cells. Expression of CXCL11 mRNA also was measured in rat acute (ACI to LEW) and chronic (LEW to F344) heart transplant rejection models. CXCL11 mRNA expression in allografts increased in both models, compared with controls, and was primarily observed in infiltrating macrophages and donor endothelial cells. These results indicate that, like the other CXCR3 chemokines, rat CXCL11 seems to have a role in the homing of CD4⁺ T cells in both acute and chronic rejection models of heart allotransplantation.     

G-protein-coupled receptor independent, immunomodulatory properties of chemokine CXCL9

Certain chemokines possess anti-angiogenic and antibacterial activity, in addition to their ability to recruit leukocytes. Herein, we demonstrate that CXCL9/MIG induces the expression, by a monocytic cell line and peripheral blood mononuclear cells, of a variety of chemokines including CXCL8/IL-8, CCL3/MIP-1α, CCL4/MIP-1β, CCL2/MCP-1 in a pertussis toxin insensitive manner. Similarly, another cationic chemokine CCL20/MIP-3α, but not the non-cationic chemokines CCL2 or CCL3, stimulated monocytic cells to produce substantial amounts of CXCL8 and CCL3. Microarray experiments demonstrated that CXCL9, but not CCL2, induced the expression of hundreds of genes, many of which have known or proposed immunomodulatory functions. Induction of CXCL8 required the p38 and ERK1/2 mitogen-activated protein kinases but not NFκB, JAK-STAT or JNK signaling pathways. These results collectively demonstrate that CXCL9 has immunomodulatory functions that are not mediated through a G-protein coupled receptor and may possess additional roles in host defenses against infection.     

Structural and dynamic effects of alpha-helix deletion in Sso7d: implications for protein thermal stability

Sso7d is a 62-residue protein from the hyperthemophilic archaeon Sulfolobus solfataricus with a denaturation temperature close to 100 degrees C around neutral pH. An engineered form of Sso7d truncated at leucine 54 (L54Delta) is significantly less stable, with a denaturation temperature of 53 degrees C. Molecular dynamics (MD) studies of Sso7d and its truncated form at two different temperatures have been performed. The results of the MD simulations at 300 K indicate that: (1) the flexibility of Sso7d chain at 300 K agrees with that detected from X-ray and NMR structural studies; (2) L54Delta remains stable in the native folded conformation and possesses an overall dynamic behavior similar to that of the parent protein. MD simulations performed at 500 K, 10 ns long, indicate that, while Sso7d is in-silico resistant to high temperature, the truncated variant partially unfolds, revealing the early phases of the thermal unfolding pathway of the protein. Analysis of the trajectories of L54Delta suggests that the unzipping of the N-terminal and C-terminal beta-strands should be the first event of the unfolding pathway, and points out the regions more resistant to thermal unfolding. These findings allow one to understand the role played by specific interactions connecting the two ends of the chain for the high thermal stability of Sso7d, and support recent hypotheses on its folding mechanism emerged from site-directed mutagenesis studies.     

The relevance of the chemokine receptor ACKR3/CXCR7 on CXCL12-mediated effects in cancers with a focus on virus-related cancers

Recent studies have highlighted the importance of understanding the molecular determinants of CXCL12-mediated effects in cancers. Once previously thought to interact exclusively with CXCR4, CXCL12 also binds with high affinity to CXCR7 (recently renamed ACKR3), which belongs to an atypical chemokine receptor family whose members fail to activate G_αi proteins but interact with β-arrestins. In addition to its capacity to control CXCL12 bioavailability, ACKR3 can either enhance or dampen CXCR4-mediated signaling and activity. In light of the most recent findings, we have examined the role of ACKR3 in cancer, including a subset of virus-related cancers.     

趋化因子CXCL12及其受体CXCR4在胶质瘤中的研究进展

CXCL12是趋化因子家族成员之一,是能够特异性结合其受体CXCR4发挥趋化性作用的细胞因子。最初,CXCL12及CXCR4被发现于炎症细胞,参与机体炎症、免疫等病理反应。接下来的几年中发现,它在机体发育、成熟过程中也有重要作用。如今,大量研究表明它与肿瘤的生长、侵袭及转移密切相关。据报道,在乳腺癌、肺癌、卵巢癌等二十余种肿瘤组织中发现CXCL12及CXCR4的表达,其中也包括中枢系统肿瘤-胶质瘤。CXCL12/CXCR4参与胶质瘤生长过程的多个步骤,包括肿瘤增殖、侵袭、转移等。有实验指出,转移灶的CXCR4表达水平较原发灶高,CXCR4有可能成为抑制胶质瘤生长、转移的重要靶目标。     

Overexpression of CXCL12 chemokine up-regulates connexin and integrin expression in mesenchymal stem cells through PI3K/Akt pathway

Abstract

Mesenchymal stem cells offer several potential advantages over other types of stem cells for cardiac repair. Nevertheless, poor survival of donor cells is one of the major concerns that hampers a better prognosis. Integrins, which involved in cell/extracellular matrix (ECM) interaction and connexins (Cxs), with a dual role as an anti-apoptotic and gap-junctional protein, can effectively resolve this issue. CXCL12, a member of the chemokine CXC subfamily, may play a role in stem cell survival and proliferation. CXCL12 activates several signaling pathways in stem cells, particularly the survival kinase, PI3K/Akt, which is also an important mediator of integrins and Cxs. Based on these characteristics of CXCL12, we investigated the potential of CXCL12 overexpression to induce integrin and connexin expression via PI3K/Akt pathway. Mesenchymal stem cells were transfected with adenovirus for increasing CXCL12 secretion. Membranous integrin and Cx expression as well as Akt expression levels were evaluated using Western blot analysis. Transfection resulted in increased CXCL12 in situ. Increased CXCL12 elevated membrane Cx43, Cx45, and integrin αVβ3 expression, as well as Cx phosphorylaton, which was activated by PI3K/Akt pathway. This mechanism may serve to improve mesenchymal stem cell viability in host tissue.     

大肠杆菌L-酒石酸脱氢酶b亚基体外分子交联

通过PCR技术扩增大肠杆菌L-酒石酸脱氢酶b亚基(L-tartrate dehydratase beta subunit, TtdB)野生型与Cys/Ser突变型目的基因, 构建带6×His标签的诱导型表达载体pTrcHisC-TtdB。重组蛋白以包含体形式存在, 应用TALON固定化金属亲和树脂(Immobilized metal affinity chromatography, IMAC)以变性的方法纯化, 通过分步透析逐步去除变性剂的方法复性, 复性率可达70%。将复性后的两种蛋白通过热诱导去折叠和氧化重折叠方法进行体外蛋白质分子交联实验。SDS-PAGE分析表明: 野生型TtdB在其变性的临界温度反应时, 出现交联二聚体和多聚体; 在氧化重折叠后SDS-PAGE前加入100 mmol/L DTT时, 交联强度明显减弱。这种DTT打不开的交联即为异肽键交联; 若在其氧化重折叠反应液中加入DTT则没有任何交联。突变型TtdB在与野生型TtdB相同的热诱导去折叠条件下, 完全没有二聚体和多聚体的形成。     

Spatiotemporal expression and functional implication of CXCL14 in the developing mice cerebellum

Cerebellar granule neurons migrate from the external granule cell layer (EGL) to the internal granule cell layer (IGL) during postnatal morphogenesis. This migration process through 4 different layers is a complex mechanism which is highly regulated by many secreted proteins. Although chemokines are well-known peptides that trigger cell migration, but with the exception of CXCL12, which is responsible for prenatal EGL formation, their functions have not been thoroughly studied in granule cell migration. In the present study, we examined cerebellar CXCL14 expression in neonatal and adult mice. CXCL14 mRNA was expressed at high levels in adult mouse cerebellum, but the protein was not detected. Nevertheless, Western blotting analysis revealed transient expression of CXCL14 in the cerebellum in early postnatal days (P1, P8), prior to the completion of granule cell migration. Looking at the distribution of CXCL14 by immunohistochemistry revealed a strong immune reactivity at the level of the Purkinje cell layer and molecular layer which was absent in the adult cerebellum. In functional assays, CXCL14 stimulated transwell migration of cultured granule cells and enhanced the spreading rate of neurons from EGL microexplants. Taken together, these results revealed the transient expression of CXCL14 by Purkinje cells in the developing cerebellum and demonstrate the ability of the chemokine to stimulate granule cell migration, suggesting that it must be involved in the postnatal maturation of the cerebellum.     

Clinical significance and biological functions of chemokine CXCL3 in head and neck squamous cell carcinoma

CXCL3 plays extensive roles in tumorigenesis in various types of human cancers through its roles in tumor cell differentiation, invasion, and migration. However, the mechanisms of CXCL3 in head and neck squamous cell carcinoma (HNSCC) remain unclear. In our study, multiple databases were used to explore the expression level, prognostic value, and related mechanisms of CXCL3 in human HNSCC through bioinformatic methods. We also performed further experiments in vivo and in vitro to evaluate the expression of CXCL3 in a human head and neck tissue microarray and the underlying effect mechanisms of CXCL3 on the tumor biology of HNSCC tumor cells. The result showed that the expression level of CXCL3 in patients with HNSCC was significantly higher as compared with that in normal tissues (P<0.05). Kaplan–Meier survival analysis demonstrated that patients with high CXCL3 expression had a lower overall survival rate (P=0.038). CXCL3 was further identified as an independent prognostic factor for HNSCC patients by Cox regression analysis, and GSEA exhibited that several signaling pathways including Apoptosis, Toll-like receptor, Nod-like receptor, Jak-STAT, and MAPK signaling pathways may be involved in the tumorigenesis of HNSCC. CAL27 cells overexpressing or HNSCC cells treated with exogenous CXCL3 exhibited enhanced cell malignant behaviors, whereas down-regulating CXCL3 expression resulted in decreased malignant behaviors in HSC4 cells. In addition, CXCL3 may affect the expression of several genes, including ERK1/2, Bcl-2, Bax, STAT3, and NF-κB. In summary, our bioinformatics and experiment findings effectively suggest the information of CXCL3 expression, roles, and the potential regulatory network in HNSCC.     

Chemokine CXCL4 interactions with extracellular matrix proteoglycans mediate widespread immune cell recruitment independent of chemokine receptors

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