热休克转录因子1的抗炎症作用

热休克转录因子1(heat shock factor 1, HSF1)是调节细胞保护性应激蛋白--热休克蛋白表达的主要转录因子,可被热应激、氧化应激等多种理化因素激活.近年研究表明,HSF1具有抗炎症作用:HSF1可抑制TNFα、IL-1β、M-CSF等致炎因子表达,促进IL-10等抗炎因子表达,并降低NF-κB、AP-1等致炎转录因子的活性.HSF1上调热休克蛋白和抑制炎症的双重活性,提示其很可能是联系应激反应和炎症反应的重要因子.     

单核细胞趋化蛋白诱导蛋白-1的研究进展

单核细胞趋化蛋白诱导蛋白-1（MCPIP1）是最近发现的一类具有免疫调节作用的CCCH型锌指家族分子。MCPIP1可被LPS、IL-1β或MCP-1等多种炎性因子刺激表达,可通过下调炎症因子（如IL-6、IL-12p40等）表达,从而负向调控炎症过程。MCPIP1的作用机制主要是作为RNA酶调节某些炎性因子mRNA或pre-miRNA的降解。此外,MCPIP1也可作为去泛素化酶靶向TNF受体相关蛋白（TRAFs）成员,从而负向调控JNK和NF-κB信号活化。将从MCPIP1分子的发现、基因和蛋白质结构、生物学功能、表达调控以及临床应用前景等几个方面进行阐述。     

ASK1激活的分子机制与相关疾病

凋亡信号调节激酶1(Apoptosis signal-regulating kinase 1,ASK1)是细胞丝裂原活化蛋白激酶激酶激酶(mitogen-activated protein kinase kinase kinase,MAP3Ks)家族成员之一,在调节细胞凋亡过程中起到非常重要的作用.在正常细胞中,ASK1的活化受到严格的控制,如苏氨酸/丝氨酸磷酸化和去磷酸化、蛋白-蛋白相互作用等.多种应激和促炎因子能激活ASK1,因此在多种生理和病理过程中都有活化的ASK1的参与.     

MICA/B分子研究进展

MICA/B基因具有高度多态性,所编码的蛋白是NKG2D的主要配体,MICA/B蛋白结合NKG2D后介导NK和T细胞等的活化及细胞杀伤效应.应激条件下,MICA/B蛋白高表达于细胞表面,并以游离的sMICA/B蛋白分子形式进入细胞外液.越来越多的研究证实MICA/B分子与肿瘤、感染、移植排斥、自身免疫病等多种炎性疾病相关,因此对其表达调控的研究是当前的热点.主要从MICA/B分子与疾病相关性、表达调控及其临床应用等方面近年来的研究进展进行综述.     

脂氧素A4受体在炎症中的作用

脂氧素A4受体是一种经典的G蛋白偶联受体;其表达在微观上受到转录和翻译水平的调节,在宏观上受到促炎与抗炎介质的调节以及药物、年龄等其它因素的影响。该受体主要表达于白细胞,结合多种激动性配体,调节炎症反应,在炎症中具有重要作用。本综述着重介绍脂氧素A4受体在炎症中所起的作用,并对该受体的称谓进行了简单的整理。     

肝素的抗炎作用与抗细胞粘附调节

肝素类药物具有抗凝以外的包括抗炎在内的多种生物学活性。炎症反应是多种因子、细胞参与的复杂的病生理过程,其物质基础是粘附分子介导的白细胞粘附及其粘附级联反应。近来研究证实肝素抗炎机制主要与抗细胞粘附调节有关,肝素通过竞争抑制L—、P—选择素与其配基sLe^X的结合,阻止白细胞粘附活化及调抑炎症级联反应,进而起到抗炎作用。对肝素抗炎机制的深入研究,将有助于进一步阐明抗粘附／抗炎的作用机制。     

热休克蛋白70在心血管疾病中的保护作用

热休克蛋白(heat shock protein70,HSP70)是HSP家族中重要成员,在生物细胞中含量最高,可诱导性最强,具有保护细胞免受刺激损伤,促进受损细胞修复及抗炎、抗凋亡、耐受缺血/缺氧损伤等多种生物学功能。许多研究发现在心肌组织中HSP70表达升高可减轻心肌细胞损伤程度,利于损伤心肌细胞的恢复,在预防和延缓心血管疾病中起到重要作用。因此,热休克蛋白70诱导剂在心血管疾病的防治中具有潜在的临床价值。本文主要对HSP70在心血管疾病中的保护作用进行综述。     

综述:炎症小体信号通路的负调控

炎症小体(inflammasomes)活化后产生的IL-1β和IL-18等促炎因子对天然免疫和适应性免疫具有重要作用.炎症小体持续活化可引起促炎因子过度表达,导致慢性炎症和自身免疫疾病的发生.正常生理状态下,机体存在多种炎症小体负调机制,以维持免疫反应平衡.病理状态下,感染机体的病原微生物通过多种途径抑制炎症小体信号通路的活化及促炎因子的产生,以利于免疫逃逸.本文综述了机体和病原微生物对炎症小体信号通路的负调控机理.阐明炎症小体信号通路负调控机制将为感染性疾病及其他炎症小体相关炎症性疾病的治疗提供策略.     

干旱胁迫下不同抗旱水平陆地棉的叶片蛋白质组学比较研究

为了探讨陆地棉品种抗旱机理,以陆地棉抗旱品种‘中H177’和不抗旱品种‘中S9612’为材料,运用双向电泳结合质谱技术,分析干旱胁迫下不同陆地棉三叶期叶片蛋白质组分差异变化。结果表明:干旱胁迫下,不同陆地棉叶片蛋白表达差异较大;‘中H177’出现30个差异表达蛋白质点,‘中S9612’出现47个差异表达蛋白质点,只在‘中H177’表达差异的蛋白点11个,只在‘中S9612’表达差异的蛋白点28个,差异表达一致蛋白点8个,表达不一致蛋白点11个。质谱共鉴定出43个差异表达蛋白;功能分类分析表明,干旱胁迫蛋白参与光合作用、物质与能量代谢、抗逆相关蛋白、物质运输和活性氧清除;Rubisco活化酶和能量代谢相关蛋白ATP合成酶类表达差异最大。研究结果可以初步为陆地棉抗旱机理的探讨提供一定的理论基础。     

脂联素调节糖脂代谢相关信号通路的研究进展

脂联素是一种主要由脂肪组织分泌的脂肪细胞因子,具有调节糖脂代谢、增强胰岛素敏感性、抗炎和抗动脉粥样硬化等多种作用。在脂联素介导的信号通路中,脂联素首先与脂联素受体(AdipoR)位于膜外的羧基端结合,再通过AdipoR膜内的氨基端与信号接头蛋白结合,进而激活下游的多条信号通路,其中腺苷酸活化蛋白激酶(AMPK)是脂联素信号通路中的关键分子,活化的AMPK可以使其下游的乙酰辅酶A羧化酶(ACC)、p38丝裂原活化蛋白激酶(p38 MAPK)、磷脂酰肌醇3激酶(PI3K)等多种胞质信号分子磷酸化,介导细胞能量代谢。本文重点综述了脂联素通过AMPK调节糖脂代谢的信号通路的研究进展。     

The role of thrombomodulin lectin-like domain in inflammation

Thrombomodulin (TM) is a cell surface glycoprotein which is widely expressed in a variety of cell types. It is a cofactor for thrombin binding that mediates protein C activation and inhibits thrombin activity. In addition to its anticoagulant activity, recent evidence has revealed that TM, especially its lectin-like domain, has potent anti-inflammatory function through a variety of molecular mechanisms. The lectin-like domain of TM plays an important role in suppressing inflammation independent of the TM anticoagulant activity. This article makes an extensive review of the role of TM in inflammation. The molecular targets of TM lectin-like domain have also been elucidated. Recombinant TM protein, especially the TM lectin-like domain may play a promising role in the management of sepsis, glomerulonephritis and arthritis. These data demonstrated the potential therapeutic role of TM in the treatment of inflammatory diseases.     

Effects of a novel peptide derived from human thrombomodulin on endotoxin-induced uveitis in vitro and in vivo

Thrombomodulin (TM) is a single-transmembrane glycoprotein receptor for thrombin, which is best known as a cofactor for thrombin-mediated activation of anticoagulant protein C. C-type lectin-like domain (CTLD) of TM has distinct coagulation/fibrinolysis-independent anti-inflammatory properties. Here we found anti-inflammatory effects of a novel peptide (GC31) from CTLD of TM in endotoxin-induced uveitis, which was characterized by a reduction of leukocyte counts, protein concentration, tumor necrosis factor (TNF)-α and monocyte chemoattractant protein (MCP)-1 levels in aqueous humor. Through in vitro experiments, we further found that GC31 suppressed TNF-α and interleukin (IL)-6 expressions in lipopolysaccharide (LPS)-stimulated macrophage-like RAW264.7 cells and interrupted LPS-induced nuclear factor-κB (NF-κB) activation. These data indicate a beneficial role of peptide GC31 in preventing intraocular inflammatory response, especially uveitis.     

The fourth epidermal growth factor-like domain of thrombomodulin interacts with the basic exosite of protein C

Thrombomodulin (TM) functions as a cofactor to enhance the rate of protein C activation by thrombin approximately 1000-fold. The molecular mechanism by which TM improves the catalytic efficiency of thrombin toward protein C is not known. Molecular modeling of the protein C activation based on the crystal structure of thrombin in complex with the epidermal growth factor-like domains 4, 5, and 6 of TM (TM456) predicts that the binding of TM56 to exosite 1 of thrombin positions TM4 so that a negatively charged region on this domain juxtaposes a positively charged region of protein C. It has been hypothesized that electrostatic interactions between these oppositely charged residues of TM4 and protein C facilitate a proper docking of the substrate into the catalytic pocket of thrombin. To test this hypothesis, we have constructed several mutants of TM456 and protein C in which charges of the putative interacting residues on both TM4 (Asp/Glu) and protein C (Lys/Arg) have been reversed. Results of TM-dependent protein C activation studies by such a compensatory mutagenesis approach support the molecular model that TM4 interacts with the basic exosite of protein C.     

High affinity agonistic metal ion binding sites within the melanocortin 4 receptor illustrate conformational change of transmembrane region 3

We created a molecular model of the human melanocortin 4 receptor (MC4R) and introduced a series of His residues into the receptor protein to form metal ion binding sites. We were able to insert micromolar affinity binding sites for zinc between transmembrane region (TM) 2 and TM3 where the metal ion alone was able to activate this peptide binding G-protein-coupled receptor. The exact conformation of the metal ion interactions allowed us to predict the orientation of the helices, and remodeling of the receptor protein indicated that Glu100 and Ile104 in TM2 and Asp122 and Ile125 in TM3 are directed toward a putative area of activation of the receptor. The molecular model suggests that a rotation of TM3 may be important for activation of the MC4R. Previous models of G-protein-coupled receptors have suggested that unlocking of a stabilizing interaction between the DRY motif, in the cytosolic part of TM3, and TM6 is important for the activation process. We suggest that this unlocking process may be facilitated through creation of a new interaction between TM3 and TM2 in the MC4R.     

Inactivation of thrombomodulin by ionizing radiation in a cell-free system: possible implications for radiation responses in vascular endothelium

Normal tissue radiation injury is associated with loss of vascular thromboresistance, notably because of deficient levels of endothelial thrombomodulin (TM). TM is located on the luminal surface of most endothelial cells and has critical anticoagulant and anti-inflammatory functions. Chemical oxidation of a specific methionine residue (Met388) at the thrombin-binding site in TM reduces its main functional activity, i.e., the ability to activate protein C. We examined whether exposure to ionizing radiation affects TM in a similar manner. Full-length recombinant human TM, a construct of epidermal growth factor-like domains 4-6, which are involved in protein C activation, and a synthetic peptide containing the methionine of interest were exposed to gamma radiation in a cell-free system, i.e., a system not confounded by TM turnover or ectodomain shedding. The influence of radiation on functional activity was assessed with the protein C activation assay; formation of a TM-thrombin complex was assessed with surface plasmon resonance (Biacore), and oxidation of Met388 was assessed by HPLC and confirmed by mass spectroscopy. Exposure to radiation caused a dose-dependent reduction in protein C activation, impaired TM-thrombin complex formation, and oxidation of Met388. These results demonstrate that ionizing radiation adversely affects the TM molecule. Our findings may have relevance to normal tissue toxicity in clinical radiation therapy as well as to the development of radiation syndromes in the non-therapeutic radiation exposure setting.     

Activation of the luteinizing hormone receptor following substitution of Ser-277 with selective hydrophobic residues in the ectodomain hinge region

Glycoprotein hormone receptors are G protein-coupled receptors with ligand-binding ectodomains consisting of leucine-rich repeats. The ectodomain is connected by a conserved cysteine-rich hinge region to the seven transmembrane (TM) region. Gain-of-function mutants of luteinizing hormone (LH) and thyroid-stimulating hormone receptors found in patients allowed identification of residues important for receptor activation. Based on constitutively active mutations at Ser-281 in the hinge region of the thyroid-stimulating hormone receptor, we mutated the conserved serine in the LH (S277I) and follicle-stimulating hormone receptors (S273I) and observed increased basal cAMP production and ligand affinity by mutant receptors. For the LH receptor, conversion of Ser-277 to all natural amino acids led to varying degrees of receptor activation. Hydropathy index analysis indicated that substitution of neutral serine with selective nonpolar hydrophobic residues (Leu>Val>Met>Ile) confers constitutive receptor activation whereas serine deletion or substitution with charged Arg, Lys, or Asp led to defective receptor expression. Furthermore, mutation of the angular proline near Ser-273 to flexible Gly also led to receptor activation. The findings suggest the ectodomain of glycoprotein hormone receptors constrain the TM region. Point mutations in the hinge region of these proteins, or ligand binding to these receptors, could cause conformational changes in the TM region that result in G(s) activation.     

Uropathogenic E. coli induce different immune response in testicular and peritoneal macrophages: implications for testicular immune privilege

Infertility affects one in seven couples and ascending bacterial infections of the male genitourinary tract by Escherichia coli are an important cause of male factor infertility. Thus understanding mechanisms by which immunocompetent cells such as testicular macrophages (TM) respond to infection and how bacterial pathogens manipulate defense pathways is of importance. Whole genome expression profiling of TM and peritoneal macrophages (PM) infected with uropathogenic E. coli (UPEC) revealed major differences in regulated genes. However, a multitude of genes implicated in calcium signaling pathways was a common feature which indicated a role of calcium-dependent nuclear factor of activated T cells (NFAT) signaling. UPEC-dependent NFAT activation was confirmed in both cultured TM and in TM in an in vivo UPEC infectious rat orchitis model. Elevated expression of NFATC2-regulated anti-inflammatory cytokines was found in TM (IL-4, IL-13) and PM (IL-3, IL-4, IL-13). NFATC2 is activated by rapid influx of calcium, an activity delineated to the pore forming toxin alpha-hemolysin by bacterial mutant analysis. Alpha-hemolysin suppressed IL-6 and TNF-α cytokine release from PM and caused differential activation of MAP kinase and AP-1 signaling pathways in TM and PM leading to reciprocal expression of key pro-inflammatory cytokines in PM (IL-1α, IL-1β, IL-6 downregulated) and TM (IL-1β, IL-6 upregulated). In addition, unlike PM, LPS-treated TM were refractory to NFκB activation shown by the absence of degradation of IκBα and lack of pro-inflammatory cytokine secretion (IL-6, TNF-α). Taken together, these results suggest a mechanism to the conundrum by which TM initiate immune responses to bacteria, while maintaining testicular immune privilege with its ability to tolerate neo-autoantigens expressed on developing spermatogenic cells.     

Vascular Immunotargeting to Endothelial Determinant ICAM-1 Enables Optimal Partnering of Recombinant scFv-Thrombomodulin Fusion with Endogenous Cofactor

The use of targeted therapeutics to replenish pathologically deficient proteins on the luminal endothelial membrane has the potential to revolutionize emergency and cardiovascular medicine. Untargeted recombinant proteins, like activated protein C (APC) and thrombomodulin (TM), have demonstrated beneficial effects in acute vascular disorders, but have failed to have a major impact on clinical care. We recently reported that TM fused with an scFv antibody fragment to platelet endothelial cell adhesion molecule-1 (PECAM-1) exerts therapeutic effects superior to untargeted TM. PECAM-1 is localized to cell-cell junctions, however, whereas the endothelial protein C receptor (EPCR), the key co-factor of TM/APC, is exposed in the apical membrane. Here we tested whether anchoring TM to the intercellular adhesion molecule (ICAM-1) favors scFv/TM collaboration with EPCR. Indeed: i) endothelial targeting scFv/TM to ICAM-1 provides ∼15-fold greater activation of protein C than its PECAM-targeted counterpart; ii) blocking EPCR reduces protein C activation by scFv/TM anchored to endothelial ICAM-1, but not PECAM-1; and iii) anti-ICAM scFv/TM fusion provides more profound anti-inflammatory effects than anti-PECAM scFv/TM in a mouse model of acute lung injury. These findings, obtained using new translational constructs, emphasize the importance of targeting protein therapeutics to the proper surface determinant, in order to optimize their microenvironment and beneficial effects.     

The nematode leucine-rich repeat-containing, G protein-coupled receptor (LGR) protein homologous to vertebrate gonadotropin and thyrotropin receptors is constitutively active in mammalian cells

The receptors for LH, FSH, and TSH belong to the large G protein-coupled, seven-transmembrane protein family and are unique in having a large N-terminal extracellular (ecto-) domain containing leucine-rich repeats important for interactions with the large glycoprotein hormone ligands. Recent studies indicated the evolution of an expanding family of homologous leucine-rich repeat-containing, G protein-coupled receptors (LGRs), including the three known glycoprotein hormone receptors; mammalian LGR4 and LGR5; and LGRs in sea anemone, fly, and snail. We isolated nematode LGR cDNA and characterized its gene from the Caenorhabditis elegans genome. This receptor cDNA encodes 929 amino acids consisting of a signal peptide for membrane insertion, an ectodomain with nine leucine-rich repeats, a seven-TM region, and a long C-terminal tail. The nematode LGR has five potential N-linked glycosylation sites in its ectodomain and multiple consensus phosphorylation sites for protein kinase A and C in the cytoplasmic loop and C tail. The nematode receptor gene has 13 exons; its TM region and C tail, unlike mammalian glycoprotein hormone receptors, are encoded by multiple exons. Sequence alignments showed that the TM region of the nematode receptor has 30% identity and 50% similarity to the same region in mammalian glycoprotein hormone receptors. Although human 293T cells expressing the nematode LGR protein do not respond to human glycoprotein hormones, these cells exhibited major increases in basal cAMP production in the absence of ligand stimulation, reaching levels comparable to those in cells expressing a constitutively activated mutant human LH receptor found in patients with familial male-limited precocious puberty. Analysis of cAMP production mediated by chimeric receptors further indicated that the ectodomain and TM region of the nematode LGR and human LH receptor are interchangeable and the TM region of the nematode LGR is responsible for constitutive receptor activation. Thus, the identification and characterization of the nematode receptor provides the basis for understanding the evolutionary relationship of diverse LGRs and for future analysis of mechanisms underlying the activation of glycoprotein hormone receptors and related LGRs.