人巨细胞病毒潜伏感染机制的研究进展

人巨细胞病毒(HCMV)的潜伏感染在人群中极为普遍。在儿科学领域,潜伏感染的巨细胞病毒激活后,可能导致死胎、流产、胎儿畸形、生长发育迟缓等一系列严重后果。在病毒潜伏感染过程中,机体会通过免疫反应或诱导宿主细胞凋亡等方式清除病毒。然而,在病毒与宿主共同进化的漫长过程中,病毒会调控自身基因的表达、宿主细胞微环境及免疫杀伤作用,从而达到与长期宿主共存的目的。目前研究揭示,HCMV的潜伏感染可能与病毒立即早期启动子沉默、病毒干扰宿主细胞凋亡、病毒免疫逃逸及非编码RNA调控机制有关。本文将从以上四个方面对HCMV潜伏感染相关机制的研究进展进行总结。     

人巨细胞病毒潜伏感染机制的研究进展

人巨细胞病毒可通过原发感染或者潜伏感染再激活而广泛传播.基于对人巨细胞病毒AD169株和Towne株基因测序的完成,以及人巨细胞病毒的基因功能研究及其相关动物模型如小鼠模型、猪模型、恒河猴模型等的研究,人巨细胞病毒感染的潜伏机制研究取得了一定进展.本文从人巨细胞病毒的感染机制、免疫应答和免疫逃避三方面对人巨细胞病毒潜伏感染机制的研究现状进行简要综述.     

人巨细胞病毒感染对树突细胞功能的影响

人巨细胞病毒(HCMV)对人多为潜伏感染,但在免疫受损人群中,该病毒感染后会引起严重的后果.人巨细胞病毒感染人体后,可从多条途径干扰宿主细胞的生长、分化,诱导细胞恶变;人巨细胞病毒的IE基因具有抑制凋亡的功能.树突细胞是机体内功能最强的抗原呈递细胞,在免疫应答中发挥重要作用.HCMV感染后,能下调树突细胞表面的主要组织相容性复合物Ⅰ、Ⅱ类分子,CD80,CD86和CD40的表达,也使黏附分子如ICAM-3、ICAM-2等的表达发生改变.由此,人巨细胞病毒逃避了细胞免疫应答,引起持续感染或潜伏感染.     

逃避CD8~+T细胞反应是巨细胞病毒重复感染的关键

虽然宿主有针对巨细胞病毒(CMV)的特异性体液和细胞免疫,但它能重复感染已感染的宿主,具体机制还不清楚。该研究证明,CMV需要逃避CD8+T细胞的免疫反应才能重复感染已感染过CMV的恒河猴,这种逃逸是通过病毒编码的主要组织相容性复合物(MHC)I类抗原呈递的抑制剂(特别是与人同源的CMV US2、3、6和11)实现的。相反,干扰MHC-I对初次感染恒河猴及在瞬时去除CD8+T细胞已感染     

人巨细胞病毒博弈宿主免疫进展

人巨细胞病毒(HCMV)感染在人群中极其普遍,病毒一旦侵入机体,将长期存在于体内,且具有潜伏-活化的生物学特性。在病毒与宿主共同进化的漫长过程中,病毒靶向性的产生了多种免疫逃避机制,通过编码病毒自身免疫调节分子,参与调控机体主要组织相容性复合体、细胞免疫、体液免疫、细胞因子及趋化因子等方面的功能,以躲避宿主的免疫杀伤作用。HCMV的免疫调节基因被认为在病毒的致病机制中扮演重要角色。本文将对近年来有关HCMV的免疫调控机制研究作一综述,从病毒编码的免疫调节分子功能的角度并结合本实验室的相关研究成果,探讨病毒与宿主免疫的相互作用过程,从病毒干预宿主免疫关键分子作用的角度映射机体对抗病毒的免疫机理。     

检测人巨细胞病毒的免疫PCR技术的建立

人巨细胞病毒(human cytomegalovirus,HCMV)是一种广泛传播的病毒,初次感染后,病毒潜伏存在于体内,但可被激活出现复发感染。诊断HCMV近期活动性感染,主要依靠实验室检测。传统的病毒分离、血清中抗CMV-IgM及CMV抗原等的检测,敏感性差,易漏诊;核酸杂交、PCR等方法虽然敏感性高,但既可检出复制的病毒DNA,也可检出潜伏、整合     

人巨细胞病毒潜伏-再激活感染的研究进展

人巨细胞病毒(HCMV)感染大多呈亚临床或潜伏状态.当宿主免疫功能减弱时,潜伏的病毒可被激活,出现明显的临床症状,甚至是致死性的.本文概述了HCMV引起潜伏感染的机制、潜伏的组织细胞,以及再激活的诱因,并对今后的研究方向进行了展望.     

核酸杂交技术在人巨细胞病毒研究中的应用

人巨细胞病毒Human Cy tomegalo-virus(HCMV)感染非常普遍,常引起不显性感染或潜伏感染;但当器官移植者、早产儿、早期孕妇、免疫抑制和免疫缺陷患者受到CMV感染后可出现明显的临床症状和严重后果。目前探求一种敏感、     

巨细胞病毒检测与鉴定研究进展

巨细胞病毒(CMV)多引起潜伏感染,而且其所致疾病的临床表现多为非特异性的,因此,建立早期快速、灵敏特异、可靠的检测和鉴定方法是至关重要的。本文从细胞培养、病毒抗原血症的检测、特异性抗体的检测和病毒核酸的检测四个方面就近年来CMV的检测和鉴定方法研究进展进行了简要的概述。     

CAR-T免疫细胞疗法在抗病毒感染中的研究进展

目前,世界上大多数病毒感染没有针对性的特效药,尤其是具有潜伏感染能力的病毒,如乙型肝炎病毒(hepatitis B virus, HBV)、巨细胞病毒(cytomegalovirus, CMV)、Epstein-Barr病毒(epstein-barr virus,EBV)、人类免疫缺陷病毒(human immunodeficiency virus, HIV)等,严重威胁人类健康。这些病毒感染宿主细胞后,与宿主蛋白互作,导致宿主细胞在其表面表达一些特异性的感染或潜伏标志蛋白,可以成为CAR-T细胞疗法(chimeric antigen receptor T-cell immunotherapy, CAR-T)的靶点,为难治性病毒性疾病的治愈提供了新的方向。     

The M33 Chemokine Receptor Homolog of Murine Cytomegalovirus Exhibits a Differential Tissue-Specific Role during In Vivo Replication and Latency

M33, encoded by murine cytomegalovirus (MCMV), is a member of the UL33 homolog G-protein-coupled receptor (GPCR) family and is conserved across all the betaherpesviruses. Infection of mice with recombinant viruses lacking M33 or containing specific signaling domain mutations in M33 results in significantly diminished MCMV infection of the salivary glands. To determine the role of M33 in viral dissemination and/or infection in other tissues, viral infection with wild-type K181 virus and an M33 mutant virus, ΔM33B_T2, was characterized using two different routes of inoculation. Following both intraperitoneal (i.p.) and intranasal (i.n.) inoculation, M33 was attenuated for infection of the spleen and pancreas as early as 7 days after infection. Following i.p. inoculation, ΔM33B_T2 exhibited a severe defect in latency as measured by a diminished capacity to reactivate from spleens and lungs in reactivation assays (P < 0.001). Subsequent PCR analysis revealed markedly reduced ΔM33B_T2 viral DNA levels in the latently infected spleens, lungs, and bone marrow. Following i.n. inoculation, latent ΔM33B_T2 viral DNA was significantly reduced in the spleen and, in agreement with results from i.p. inoculation, did not reactivate from the spleen (P < 0.001). Furthermore, in vivo complementation of ΔM33B_T2 virus replication and/or dissemination to the salivary glands and pancreas was achieved by coinfection with wild-type virus. Overall, our data suggest a critical tissue-specific role for M33 during infection in the salivary glands, spleen, and pancreas but not the lungs. Our data suggest that M33 contributes to the efficient establishment or maintenance of long-term latent MCMV infection.Since the discovery of the G protein-coupled receptors (GPCRs) encoded by the betaherpesviruses, there has been intense speculation on the biological role these viral proteins play during infection (15, 16, 22). Human cytomegalovirus (HCMV), a betaherpesvirus, is a ubiquitous pathogen that asymptomatically infects humans and establishes a long-term persistent infection. HCMV is life-threatening, however, to immunocompromised individuals, such as neonates, AIDS patients, and transplant recipients. HCMV, similar to a number of herpesviruses, encodes viral genes that are predicted to impact virus-host interactions that may promote efficient long-term infection of the host. The CMVs encode genes for proteins that potentially enhance viral dissemination and replication and promote immune evasion by mimicry of host functions that influence the conditions of primary infection, the virus-specific immune response, and even long-term host control of persistent or latent infection (reviewed in references 1, 44, and 68).HCMV encodes four GPCRs (UL33, UL78, US28, and US27) which share homology to host chemokine receptors (16). This suggests that these virally encoded chemokine receptors may function similarly to their cellular receptor counterparts. Chemokines are chemoattractant cytokines that bind and activate chemokine receptors that are on the surfaces of cells. Host chemokine receptors then mediate the activation of cellular signaling pathways and cell migration to sites of inflammation by transmitting signals through G proteins (56, 70). In humans, approximately 50 chemokines and 20 chemokine receptors have been identified, many of which have close homologs in mice and other species (39). Chemokines are divided into two classes, lymphoid chemokines, which are constitutively expressed and involved in lymphoid tissue organization, and inflammatory chemokines, which are induced following infection and part of the inflammatory response (21, 39, 51). Growing evidence indicates that chemokines play a critical role in the host response to infection and inflammation during both the innate and adaptive immune responses (26), thus suggesting that the betaherpesviruses have “hijacked” the chemokine receptors from the host genome to subvert or alter these responses during infection. Besides chemokine receptors, HCMV also encodes a CXC chemokine (UL146) that induces the migration of neutrophils (48); a second CXC chemokine homolog (UL147) whose function is not yet known; a viral CC chemokine (UL131) that is critical for infection of macrophages, endothelial cells, and epithelial cells (25, 57, 73); and a RANTES decoy protein (72). A CC chemokine (vMCK or m131/129) is also encoded by murine CMV (MCMV), and a homolog in rat CMV ([RCMV] r131) that promotes monocyte-associated viremia (20, 37, 59, 60). The MCMV m131/129 chemokine was shown to recruit myelomonocytic progenitors from the bone marrow, perhaps to facilitate cell-type-specific viremia (46). Clearly, the CMVs have invested a great deal of effort into manipulating or subverting the host chemokine system, thus making it reasonable to speculate that these viral members of the chemokine system play an important role during CMV pathogenesis.Of the HCMV-encoded GPCRs, US28 has been well characterized in vitro and functions as a bona fide chemokine receptor, whereas much less is known about the receptor activity of US27, UL33, and UL78. US28 binds and sequesters CC chemokines, induces smooth-muscle cell migration, and constitutively activates signaling pathways (5, 7-9, 42, 52, 64, 67, 71). US28 and US27 are found only in primate CMVs, whereas both UL33 and UL78 are highly conserved across all betaherpesvirus genomes, suggesting an important evolutionary function for UL33 and UL78 during CMV infection. Two other betaherpesviruses, human herpesviruses 6 and 7 (HHV6 and HHV7), encode homologs to the UL33 and UL78 receptors, U12 and U51, respectively. The U12 receptors of HHV6 and HHV7 (34, 45, 66) and the HHV6-encoded U51 receptor (22) exhibit chemokine binding activity. UL33, along with its rodent CMV homologs, M33 (MCMV) and R33 (RCMV), constitutively activates signaling pathways (13, 23, 71). M33 induces smooth-muscle cell migration (39), similar to US28-mediated smooth-muscle cell migration (64). Thus, members of the UL33 family potentially function during viral infection by modulating or influencing the composition of leukocytes at sites of infection, the migration of infected cells or infiltrating leukocytes, or modulation of intracellular signaling pathways.Due to the species specificity of CMV, the in vivo role of the HCMV-encoded GPCRs cannot be addressed. However, the importance of UL33 and UL78 for viral dissemination and virulence in vivo has been indicated by disruption of the viral homologs in MCMV and RCMV (6, 19, 36, 47). Disruption of the UL33, M33, and R33 genes demonstrated that they are dispensable for replication in vitro, indicating that the UL33 family members are not required for replication or cell entry in at least some cell types (6, 19, 40). Infection of mice with M33-deficient MCMV or infection of rats with R33-deficient RCMV results in highly attenuated viruses and diminished infection of the salivary glands. The RCMV R33 protein also appears to play a role in virulence since rats infected with an R33 deletion virus had a lower mortality rate (6). More recently, constitutive M33-mediated activation of signaling pathways was shown to be essential for MCMV infection of salivary glands (14). Significantly, the UL33 protein partially rescued the defect in salivary gland infection attributed to disruption of M33, indicating the evolutionary conservation of function between the HCMV (UL33) and MCMV (M33) chemokine receptor homologs.In this paper, the role of M33 is further investigated using two routes of infection to assess viral dissemination and viral replication kinetics at different tissue sites, the numbers of infected cells following infection, and the possibility that M33 plays a role during latent infection. In addition to the critical role that M33 plays in salivary gland infection, this study reveals that M33 is important for MCMV infection of the spleen and the pancreas but not the lungs. Significantly, our studies provide preliminary evidence that disruption of M33 leads to reduced latent viral load in the spleen, lungs, and bone marrow, perhaps due to defects in the establishment and/or maintenance of latent infection. Lastly, we demonstrate that the tissue defects observed during acute infection with an M33 mutant virus (ΔM33B_T2) can be complemented in vivo when mice are coinfected with ΔM33B_T2 virus and wild-type MCMV. Taken together, our findings indicate that M33 plays a critical tissue-specific role during acute MCMV infection and, importantly, contributes to the efficient establishment or maintenance of latent MCMV infection.     

趋化因子及其受体基因家族的系统进化分析

通过分析现有的趋化因子和趋化因子受体的氨基酸序列,用距离法和最简约法构建了聚类图,探讨了趋化因子和趋化因子受体基因家族的系统演化特征。可见基因家族成员的分化早于脊椎动物的分化。不同物种的同一种基因的聚类关系能较好地反映物种经因子受体的进化速度不同,其中ＣＸＣＲ４的进化速率最低。趋化因子和趋化因子受体可能都起源于少数几个原始的基因,病毒编码与寄主相似的趋化因子或受体是进化过程中分子模拟的结果。     

Chemokine receptors

Although chemokines were originally defined as host defense proteins it is now clear that their repertoire of functions extend well beyond this role. For example chemokines such as MGSA have growth regulatory properties while members of the CXC chemokine family can be mediators or inhibitors of angiogenesis and may be important targets for oncology. Recent work shows that the chemokine receptor CXCR4 and its cognate ligand SDF play important roles in the development of the immune, circulatory and central nervous systems. In addition, chemokine receptors play an important role in the pathogenesis of the AIDS virus, HIV-1. Taken together these findings expand the biological importance of chemokines from that of simple immune modulators to a much broader biological role than was at first appreciated and these and other properties of the chemokine receptor family are discussed in detail in this review.     

A Cytohesin Homolog in Dictyostelium Amoebae

Background

Dictyostelium, an amoeboid motile cell, harbors several paralogous Sec7 genes that encode members of three distinct subfamilies of the Sec7 superfamily of Guanine nucleotide exchange factors. Among them are proteins of the GBF/BIG family present in all eukaryotes. The third subfamily represented with three members in D. discoideum is the cytohesin family that has been thought to be metazoan specific. Cytohesins are characterized by a Sec7 PH tandem domain and have roles in cell adhesion and migration.

Principal Findings

Dictyostelium SecG exhibits highest homologies to the cytohesins. It harbors at its amino terminus several ankyrin repeats that are followed by the Sec7 PH tandem domain. Mutants lacking SecG show reduced cell-substratum adhesion whereas cell-cell adhesion that is important for development is not affected. Accordingly, multicellular development proceeds normally in the mutant. During chemotaxis secG⁻ cells elongate and migrate in a directed fashion towards cAMP, however speed is moderately reduced.

Significance

The data indicate that SecG is a relevant factor for cell-substrate adhesion and reveal the basic function of a cytohesin in a lower eukaryote.     

Surprises from an Unusual CLC Homolog

The chloride channel (CLC) family is distinctive in that some members are Cl⁻ ion channels and others are Cl⁻/H⁺ antiporters. The molecular mechanism that couples H⁺ and Cl⁻ transport in the antiporters remains unknown. Our characterization of a novel bacterial homolog from Citrobacter koseri, CLC-ck2, has yielded surprising discoveries about the requirements for both Cl⁻ and H⁺ transport in CLC proteins. First, even though CLC-ck2 lacks conserved amino acids near the Cl⁻-binding sites that are part of the CLC selectivity signature sequence, this protein catalyzes Cl⁻ transport, albeit slowly. Ion selectivity in CLC-ck2 is similar to that in CLC-ec1, except that SO₄²⁻ strongly competes with Cl⁻ uptake through CLC-ck2 but has no effect on CLC-ec1. Second, and even more surprisingly, CLC-ck2 is a Cl⁻/H⁺ antiporter, even though it contains an isoleucine at the Glu_in position that was previously thought to be a critical part of the H⁺ pathway. CLC-ck2 is the first known antiporter that contains a nonpolar residue at this position. Introduction of a glutamate at the Glu_in site in CLC-ck2 does not increase H⁺ flux. Like other CLC antiporters, mutation of the external glutamate gate (Glu_ex) in CLC-ck2 prevents H⁺ flux. Hence, Glu_ex, but not Glu_in, is critical for H⁺ permeation in CLC proteins.The chloride channel (CLC) family includes both Cl⁻ ion channels and Cl⁻/H⁺ antiporters (1). The ion channels allow Cl⁻ to diffuse passively down an electrochemical gradient, and antiporters couple the movement of chloride and protons in opposite directions across cellular membranes. So far, the only known CLC structures are those of antiporters (2–4). On the basis of sequence similarity and functional studies, it is thought that the basic structures of the ion channels and antiporters are similar, and that slight structural differences account for these diverse functions. Understanding how the CLC family has evolved to allow proteins of similar structure to carry out two distinct mechanisms remains a critical goal.In the Escherichia coli antiporter CLC-ec1, two glutamates, Glu_ex (E148) and Glu_in (E203), are absolutely required for H⁺ transport (5,6). Glu_ex is conserved in both CLC ion channels and antiporters. Glu_in is conserved only in antiporters and is instead a hydrophobic valine in all of the known ion channels. Hence, it was proposed that both Glu_in and Glu_ex are necessary to transfer protons through CLC antiporters (6). Studies of the CLC-4 and CLC-5 antiporters supported the notion that Glu_in and Glu_ex play critical roles in H⁺ transport (7,8). Surprisingly, however, recent experiments revealed that although the red algae homolog CmCLC contains a threonine at the Glu_in position, it is still Cl⁻/H⁺ antiporter (3). It is unknown whether this threonine has a shifted pKa that allows it to transfer protons or whether the H⁺ transport in CmCLC does not require a protonatable residue at this position. Further blurring the role of Glu_in, the CLC-0 ion channel, which contains a valine at the Glu_in position, requires slow transmembrane H⁺ transport for channel gating (9).To probe the molecular requirements for Cl⁻ and H⁺ transport in CLC proteins, we characterized a novel homolog from Citrobacter koseri called CLC-ck2. CLC-ck2 is 21% identical and 37% similar in amino acid sequence to CLC-ec1. CLC-ck2 contains an isoleucine at the Glu_in position, and hence we originally hypothesized that this protein would act as an ion channel. Additionally, CLC-ck2 lacks several amino acids that coordinate the central and internal Cl⁻-binding sites in CLC-ec1, most notably the GSGIP motif (Fig. S1 in the Supporting Material). With genomic information now revealing >1000 putative CLC homologs, we find that CLC-ck2 is not unique—several other uncharacterized homologs also lack these regions. To our knowledge, ours is the first study to characterize the function of a homolog missing these regions.Using Cl⁻ flux assays, we first sought to determine whether CLC-ck2 could catalyze Cl⁻ transport (10). With CLC-ck2-containing vesicles, slow but significant Cl⁻ efflux was observed upon addition of valinomycin (Vln; Fig. 1 A, blue trace). In control vesicles lacking CLC-ck2, no significant Cl⁻ flux was observed (Fig. 1 A, black). The CLC-ec1 inhibitor 4,4′-octanamidostilbene-2,2′-disulfonate (OADS) (11) completely inhibited Cl⁻ flux (Fig. 1 A, green). The Cl⁻ unitary turnover rate for wild-type CLC-ck2 was 31 ± 5 s⁻¹ (mean ± SE, n = 5). This rate is ∼2 orders of magnitude less than the Cl⁻ flux through the CLC-ec1 antiporter, and is much slower transport than expected for an ion channel. However, it is a similar to the rate catalyzed by the cyanobacterium antiporter CLC-sy1 (4).Open in a separate windowFigure 1(A) Representative Cl⁻ flux assays. Cl⁻ efflux was initiated by addition of Vln. Triton X-100 was added to disrupt liposomes and release all intracellular Cl⁻. The insert shows an expanded view of the efflux immediately after addition of Vln. (B) Representative H⁺ flux assays demonstrate Cl⁻-driven H⁺ influx. H⁺ flux was initiated by the addition of Vln. The H⁺ gradient was collapsed at the end by the addition of FCCP.To test whether CLC-ck2 is a Cl⁻ ion channel or Cl⁻/H⁺ antiporter, we performed H⁺ flux assays as previously described (10). If vesicles contain a Cl⁻/H⁺ antiporter, the efflux of Cl⁻ upon addition of Vln will drive the movement of protons into the vesicles against their concentration gradient. If vesicles contain a Cl⁻ ion channel, however, no movement of protons will be observed upon addition of Vln. We found that CLC-ck2 showed significant Cl⁻-driven H⁺ uptake. Fig. 1 B illustrates uphill movement of protons in the presence of a Cl⁻ gradient. H⁺ influx, like Cl⁻ efflux, was inhibited by the presence of OADS. These assays are not quantitative enough to determine Cl⁻/H⁺ stoichiometry. However, they qualitatively demonstrate that CLC-ck2 acts as a Cl⁻/H⁺ antiporter even though it lacks Glu_in.If Glu_in is important for maximizing H⁺ flux, we would expect that introducing a glutamate at the Glu_in position would increase the H⁺ flux observed through CLC-ck2. However, we found that the I175E mutation did not significantly alter H⁺ or Cl⁻ flux (Fig. 2). Hence, Glu_in does not enhance H⁺ transport through CLC-ck2.Open in a separate windowFigure 2Unitary turnover rates, calculated from initial velocities after addition of Vln in (A) Cl⁻ and (B) H⁺ flux assays. Reconstitutions contained 5–38 μg protein/mg lipid. Bars represent the mean ± SE for three to 17 assays.The external glutamate gate Glu_ex is conserved and required for H⁺ transport in all known CLC antiporters (5,7). To determine whether Glu_ex is also essential for H⁺ transport in CLC-ck2, we made the E122Q mutation. This mutant can still transport chloride but fails to move protons (Fig. 2). This mutant protein was not very stable in micelles, precipitating over the course of hours, and thus the unitary turnover rates shown in Fig. 2 represent lower limits. Nevertheless, this result is consistent with observations in other CLC antiporters and suggests that Glu_ex is important for H⁺ transport in all CLC antiporters.Because CLC-ck2 lacks amino acids that coordinate the Cl⁻ ions in the structure of CLC-ec1, we wondered whether the ion selectivity might differ. Indeed, the plant atCLC-a homolog has a single change in this region that makes it selective for NO₃⁻ over Cl⁻ (12). To determine the ion selectivity of CLC-ck2, we used radioactive uptake assays (11). In these assays, the amount of ³⁶Cl⁻ exchanged into CLC-ck2-containing vesicles loaded with cold Cl⁻ is measured as a function of time. Various anions were added to the extravesicular solution to test which ions were transported in preference to the ³⁶Cl⁻. A decrease in radioactive uptake indicates that the anion is permeant and/or blocks CLC-ck2. Fig. 3 A plots the amount of ³⁶Cl⁻ uptake with each of the various ions added; the ion selectivity (or block) was SO₄²⁻ ≫ Cl⁻ > NO₃⁻ > SCN⁻ >Br⁻ > F⁻ > P_i⁻ ≈ I⁻ ≫ isethionate⁻. This selectivity is similar to that of CLC-ec1 (13), with one noticeable exception: SO₄²⁻. SO₄²⁻ had no effect on CLC-ec1, but strongly competed with Cl⁻ uptake through CLC-ck2 (Fig. 3 B). Hence, the selectivity filter of CLC-ck2 is similar enough to other CLCs to transport Cl⁻, NO₃⁻, and Br⁻ as expected. However, further investigation is required to determine the structural differences that must underlie the distinct disparity in SO₄²⁻ permeability and/or block.Open in a separate windowFigure 3Ion selectivity of CLC-ck2. (A) Liposomes reconstituted with CLC-ck2 were screened for selectivity against various test ions in the presence of 1 mM ³⁶Cl⁻ at pH 4.5. All test ions were present at 10 mM, except for isethionate, which was present at 20 mM to confirm that it is inert. After 10 min, the radioactivity counts were measured to determine total ³⁶Cl⁻ uptake (for isethionate, uptake was stopped after 20 min). Counts were normalized with respect to liposome uptake in the absence of an external test ion. Bars represent the mean ± SE for three assays. (B) Comparison of effects of external sulfate on CLC-ec1 and CLC-ck2 on radioactive update assays, normalized as in part A.This study reveals that Glu_in is not essential for Cl⁻- coupled H⁺ transport in CLC-ck2, in direct contrast to the previous conclusion that the protonatable side chain of the glutamate is directly involved in the H⁺ transport pathway (14). Thus, our result brings into question the location of the H⁺ permeation pathway. The protons must be transferred via other protonatable residues or water molecules. The residue adjacent to Glu_in (E202 in CLC-ec1) is conserved in CLC-ck2. Unfortunately, mutation of this glutamate (E174F) in CLC-ck2 resulted in unstable protein that could not be characterized in functional studies. Using the structure of CLC-ec1 as a guide, we see no other obvious protonatable residues in CLC-ck2 available to transfer protons from the intracellular side to Glu_ex. One possibility is that H⁺ transport may require a water wire. The idea of a water wire is not new. In CLC-ec1, there is an ∼15 Å gap between Glu_in and Glu_ex, and it has never been clear exactly how protons cross this gap. Recent molecular-dynamics studies have supported the idea that the Glu_in in CLC-ec1 may help to position water molecules for a water wire to transfer protons to the extracellular glutamate (15). If indeed the role of Glu_in is simply to position water molecules properly to transfer protons, subtle changes in other parts of the structure could allow this water wire to exist in the absence of Glu_in. This could also explain how the eukaryotic CmCLC homolog, which has a threonine at the Glu_in position, is able to act as a coupled transporter as well. We have not yet been able to determine the structure of CLC-ck2 to understand how the lack of conserved amino acids near the Cl⁻-binding sites affects the structure. This study will inspire future work to investigate the molecular mechanism of CLC-ck2 and CLC-ck2 homologs in greater detail.     

趋化因子及其受体对血管内皮细胞的作用

趋化因子是机体内一类重要的生物活性物质,参与多种生理病理活动的调控。趋化因子可通过对血管内皮细胞的趋化作用,引起血管内皮细胞增殖、迁移、毛细血管形成而促进血管生成;部分趋化因子可通过凋亡和抑制多种促血管生成因子的活性而发挥抑制血管生成的作用。现将趋化因子及其受体对血管内皮细胞的作用进行综述。