钙网蛋白的免疫生物学活性研究进展

钙网蛋白(calreticulin,CRT)是内质网中的Ca2+结合蛋白,对维持细胞内的Ca2+稳态具有重要作用。CRT还可作为分子伴侣,帮助新合成的内质网蛋白正确折叠。除此之外,CRT还出现在细胞膜表面及细胞外环境中,并发挥重要的免疫调节作用。膜表面的CRT参与凋亡细胞的清除和抗肿瘤免疫应答,在T细胞活化早期也发挥重要作用。可溶性CRT出现在自身免疫病患者的血清中,且其浓度与疾病活动度相关。重组可溶性CRT腹腔注射可显著影响小鼠的Th1/Th2平衡。重组可溶性CRT分子不仅具有超强免疫刺激活性,还表现出很强的佐剂效应,提示可溶性CRT可能在自身免疫性疾病的发生与发展过程中发挥一定作用。可以预见,CRT分子的免疫生物学活性将在不久的将来成为该领域研究的一个热点。     

RIG-I Self-Oligomerization Is Either Dispensable or Very Transient for Signal Transduction

Effective host defence against viruses depends on the rapid triggering of innate immunity through the induction of a type I interferon (IFN) response. To this end, microbe-associated molecular patterns are detected by dedicated receptors. Among them, the RIG-I-like receptors RIG-I and MDA5 activate IFN gene expression upon sensing viral RNA in the cytoplasm. While MDA5 forms long filaments in vitro upon activation, RIG-I is believed to oligomerize after RNA binding in order to transduce a signal. Here, we show that in vitro binding of synthetic RNA mimicking that of Mononegavirales (Ebola, rabies and measles viruses) leader sequences to purified RIG-I does not induce RIG-I oligomerization. Furthermore, in cells devoid of endogenous functional RIG-I-like receptors, after activation of exogenous Flag-RIG-I by a 62-mer-5′ppp-dsRNA or by polyinosinic:polycytidylic acid, a dsRNA analogue, or by measles virus infection, anti-Flag immunoprecipitation and specific elution with Flag peptide indicated a monomeric form of RIG-I. Accordingly, when using the Gaussia Luciferase-Based Protein Complementation Assay (PCA), a more sensitive in cellula assay, no RIG-I oligomerization could be detected upon RNA stimulation. Altogether our data indicate that the need for self-oligomerization of RIG-I for signal transduction is either dispensable or very transient.     

HtrA Is Essential for Efficient Secretion of Recombinant Proteins by Lactococcus lactis

HtrA is a unique protease on the extracellular surface of Lactococcus lactis. It is known to take part in the proteolysis of many secreted recombinant proteins, and the mutation of htrA can lead to the complete stabilization of recombinant proteins. In this work, we have shown that htrA mutation also leads to significant reduction of the efficiency of recombinant-protein secretion. We also show that the level of HtrA can be lowered by the suppression of the acid tolerance response (ATR) in L. lactis. Instead of using an L. lactis htrA mutant, the reduction of the HtrA level in wild-type recombinant cultures of L. lactis by ATR suppression may serve as a better strategy for the production of secreted recombinant proteins.     

rhsBLyS及其突变体对小鼠血清免疫球蛋白分泌及B细胞增殖分化的影响

为了进一步研究所构建的人可溶性B淋巴细胞刺激因子 (hsBLyS)及其突变体hsBY A(Cys14 6→Ala14 6 )和hsBY V(Cys14 6→Val14 6 )在体内的生物活性 ,将 3种重组蛋白注射入BALb C小鼠体内 ,进而分析重组蛋白对小鼠血清免疫球蛋白分泌和脾脏B细胞增殖分化的影响 ,并对 3种蛋白的体内生物活性进行了比较 .结果表明 ,3种蛋白均能促进小鼠血清总IgG和IgM的分泌明显增加 ,统计学检验显示突变体rhsBY V诱导这 2种免疫球蛋白分泌的能力最强 ;3种蛋白还诱导小鼠脾脏增大、脾脏成熟B细胞的相对数量增加 .该高活性突变体hsBY V的获得提示 ,Cys14 6并非BLyS发挥正常功能所必需 ,将Cys14 6突变成Val14 6有可能提高BLyS的生物活性 .     

重组人钙网蛋白的克隆与原核表达

［摘要］目的: 克隆人钙网蛋白（calreticulin,CRT）并在E.coli中原核表达和纯化。方法：采用RT-PCR 法从人非小细胞肺腺癌A549细胞总RNA中克隆人钙网蛋白cDNA,构建CRT原核表达质粒（pET-15b/CRT）并转化E.coli 的Rossetta菌株。IPTG诱导后,表达蛋白在变性条件下经Ni-NTA 树脂亲和层析纯化,然后透析复性。分别用SDS-PAGE和Western blotting法鉴定CRT表达和纯化状态。结果：从A549细胞总RNA中成功获得人CRT cDNA克隆,重组质粒pET-15b/CRT构建正确。转化pET-15b/CRT的E.coli Rossetta诱导性表达重组人CRT蛋白,该蛋白可经Ni-NTA树脂亲和层析高度纯化。结论：成功建立了CRT原核表达和纯化的实验方法,该方法为后续的CRT蛋白功能研究奠定了基础。     

ADF/Cofilin Is Not Essential but Is Critically Important for Actin Activities during Phagocytosis in Tetrahymena thermophila

ADF/cofilin is a highly conserved actin-modulating protein. Reorganization of the actin cytoskeleton in vivo through severing and depolymerizing of F-actin by this protein is essential for various cellular events, such as endocytosis, phagocytosis, cytokinesis, and cell migration. We show that in the ciliate Tetrahymena thermophila, the ADF/cofilin homologue Adf73p associates with actin on nascent food vacuoles. Overexpression of Adf73p disrupted the proper localization of actin and inhibited the formation of food vacuoles. In vitro, recombinant Adf73p promoted the depolymerization of filaments made of T. thermophila actin (Act1p). Knockout cells lacking the ADF73 gene are viable but grow extremely slowly and have a severely decreased rate of food vacuole formation. Knockout cells have abnormal aggregates of actin in the cytoplasm. Surprisingly, unlike the case in animals and yeasts, in Tetrahymena, ADF/cofilin is not required for cytokinesis. Thus, the Tetrahymena model shows promise for future studies of the role of ADF/cofilin in vivo.     

T-cadherin Is Essential for Adiponectin-mediated Revascularization

Adipose tissue secretes protein factors that have systemic actions on cardiovascular tissues. Previous studies have shown that ablation of the adipocyte-secreted protein adiponectin leads to endothelial dysfunction, whereas its overexpression promotes wound healing. However, the receptor(s) mediating the protective effects of adiponectin on the vasculature is not known. Here we examined the role of membrane protein T-cadherin, which localizes adiponectin to the vascular endothelium, in the revascularization response to chronic ischemia. T-cadherin-deficient mice were analyzed in a model of hind limb ischemia where blood flow is surgically disrupted in one limb and recovery is monitored over 28 days by laser Doppler perfusion imaging. In this model, T-cadherin-deficient mice phenocopy adiponectin-deficient mice such that both strains display an impaired blood flow recovery compared with wild-type controls. Delivery of exogenous adiponectin rescued the impaired revascularization phenotype in adiponectin-deficient mice but not in T-cadherin-deficient mice. In cultured endothelial cells, T-cadherin deficiency by siRNA knockdown prevented the ability of adiponectin to promote cellular migration and proliferation. These data highlight a previously unrecognized role for T-cadherin in limb revascularization and show that it is essential for mediating the vascular actions of adiponectin.     

高效表达可溶性重组蛋白表达载体——pHisSUMO

目的:构建含有IL-1基因的原核表达质粒,并对其原核表达情况进行检测,验 证pHisSUMO表达载体的高效可溶性表达.方法:以质粒pMD18-T-IL-1为模板,利用PCR获得I L-1基因克隆并将其与表达载体pET、pTYB、pHisSUMO连接,重组质粒经鉴定后转化到大肠杆 菌DH5α中,并检测其蛋白表达情况.结果:仅在pHisSUMO表达系统获得了IL-1融合蛋白的 高效可溶性表达.利用Ni-NTA纯化后的融合蛋白经SUMO蛋白酶Ⅰ切割,获得了纯度较高的成熟 蛋白且不残留任何氨基酸残基.结论:实验证明pHisSUMO表达系统有助于增加外源蛋白可溶性和表达量.     

Glycan-Dependent Immunogenicity of Recombinant Soluble Trimeric Hemagglutinin

Recombinant soluble trimeric influenza A virus (IAV) hemagglutinin (sHA₃) has proven an effective vaccine antigen against IAV. Here, we investigate to what extent the glycosylation status of the sHA₃ glycoprotein affects its immunogenicity. Different glycosylation forms of subtype H5 trimeric HA protein (sH5₃) were produced by expression in insect cells and different mammalian cells in the absence and presence of inhibitors of N-glycan-modifying enzymes or by enzymatic removal of the oligosaccharides. The following sH5₃ preparations were evaluated: (i) HA proteins carrying complex glycans produced in HEK293T cells; (ii) HA proteins carrying Man₉GlcNAc₂ moieties, expressed in HEK293T cells treated with kifunensine; (iii) HA proteins containing Man₅GlcNAc₂ moieties derived from HEK293S GnTI(−) cells; (iv) insect cell-produced HA proteins carrying paucimannosidic N-glycans; and (v) HEK293S GnTI(−) cell-produced HA proteins treated with endoglycosidase H, thus carrying side chains composed of only a single N-acetylglucosamine each. The different HA glycosylation states were confirmed by comparative electrophoretic analysis and by mass spectrometric analysis of released glycans. The immunogenicity of the HA preparations was studied in chickens and mice. The results demonstrate that HA proteins carrying terminal mannose moieties induce significantly lower hemagglutination inhibition antibody titers than HA proteins carrying complex glycans or single N-acetylglucosamine side chains. However, the glycosylation state of the HA proteins did not affect the breadth of the antibody response as measured by an HA1 antigen microarray. We conclude that the glycosylation state of recombinant antigens is a factor of significant importance when developing glycoprotein-based vaccines, such as recombinant HA proteins.     

Soluble Variants of Human Recombinant Glutaminyl Cyclase

Recombinant human Glutaminyl Cyclase expressed in E. coli is produced as inclusion bodies. Lack of glycosylation is the main origin of its accumulation in insoluble aggregates. Mutation of single isolated hydrophobic amino acids into negative amino acids was not able to circumvent inclusion bodies formation. On the contrary, substitution with carboxyl-terminal residues of two or three aromatic residues belonging to extended hydrophobic patches on the protein surface provided soluble but still active forms of the protein. These mutants could be expressed in isotopically enriched forms for NMR studies and the maximal attainable concentration was sufficient for the acquisition of ¹H-¹⁵N HSQC spectra that represent the starting point for future drug development projects targeting Alzheimer’s disease.     

Transferrin Is an Essential Factor for Myelination

It has been established that oligodendrocytes, the myelin forming cells, participate in iron homeostasis through the synthesis and secretion of transferrin. Here we investigated whether a correlation exists between myelination, the commonly studied function of oligodendrocytes, and that of transferrin synthesis and secretion. We used a proteolipid protein mutant, the myelin deficient rat, whose condition is characterized by severe hypomyelination. We compared the ontogenic profile for transferrin gene expression in mutants with that of unaffected rat pups through northern blot analysis and in situ hybridization. Surprisingly, transferrin synthesis was null in mutant oligodendrocytes. Next, we demonstrated that a single apo-transferrin intraparenchymal injection administered to P5 rat pups enabled mutant oligodendrocytes to synthesize myelin basic protein and to myelinate axons, indicating that transferrin effects mutant oligodendrocyte maturation regardless of its source. Thus, transferrin availability is essential for oligodendrocyte maturation and function, and oligodencrocytes are most vulnerable to transferrin deficiency during the premyelinating stage.     

ERK Signaling Is Essential for Macrophage Development

Macrophages depend on colony stimulating factor 1 (also known as M-CSF) for their growth and differentiation, but the requirements for intracellular signals that lead to macrophage differentiation and function remain unclear. M-CSF is known to activate ERK1 and ERK2, but the importance of this signaling pathway in macrophage development is unknown. In these studies, we characterized a novel model of Erk1 ^-/- Erk2 ^flox/flox Lyz2 ^Cre/Cre mice in which the ERK2 isoform is deleted from macrophages in the background of global ERK1 deficiency. Cultures of M-CSF-stimulated bone marrow precursors from these mice yielded reduced numbers of macrophages. Whereas macrophages developing from M-CSF-stimulated bone marrow of Erk2 ^flox/flox Lyz2 ^Cre/Cre mice showed essentially complete loss of ERK2 expression, the reduced number of macrophages that develop from Erk1 ^-/- Erk2 ^flox/flox Lyz2 ^Cre/Cre bone marrow show retention of ERK2 expression, indicating selective outgrowth of a small proportion of precursors in which Cre-mediated deletion failed to occur. The bone marrow of Erk1 ^-/- Erk2 ^flox/flox Lyz2 ^Cre/Cre mice was enriched for CD11b⁺ myeloid cells, CD11b^hi Gr-1^hi neutrophils, Lin^- c-Kit⁺ Sca–1⁺ hematopoietic stem cells, and Lin^- c-Kit⁺ CD34⁺ CD16/32⁺ granulocyte-macrophage progenitors. Culture of bone marrow Lin^- cells under myeloid-stimulating conditions yielded reduced numbers of monocytes. Collectively, these data indicate that the defect in production of macrophages is not due to a reduced number of progenitors, but rather due to reduced ability of progenitors to proliferate and produce macrophages in response to M-CSF-triggered ERK signaling. Macrophages from Erk1 ^-/- Erk2 ^flox/flox Lyz2 ^Cre/Cre bone marrow showed reduced induction of M-CSF-regulated genes that depend on the ERK pathway for their expression. These data demonstrate that ERK1/ERK2 play a critical role in driving M-CSF-dependent proliferation of bone marrow progenitors for production of macrophages.     

Mitochondrial Fusion Is Essential for Steroid Biosynthesis

Although the contribution of mitochondrial dynamics (a balance in fusion/fission events and changes in mitochondria subcellular distribution) to key biological process has been reported, the contribution of changes in mitochondrial fusion to achieve efficient steroid production has never been explored. The mitochondria are central during steroid synthesis and different enzymes are localized between the mitochondria and the endoplasmic reticulum to produce the final steroid hormone, thus suggesting that mitochondrial fusion might be relevant for this process. In the present study, we showed that the hormonal stimulation triggers mitochondrial fusion into tubular-shaped structures and we demonstrated that mitochondrial fusion does not only correlate-with but also is an essential step of steroid production, being both events depend on PKA activity. We also demonstrated that the hormone-stimulated relocalization of ERK1/2 in the mitochondrion, a critical step during steroidogenesis, depends on mitochondrial fusion. Additionally, we showed that the SHP2 phosphatase, which is required for full steroidogenesis, simultaneously modulates mitochondrial fusion and ERK1/2 localization in the mitochondrion. Strikingly, we found that mitofusin 2 (Mfn2) expression, a central protein for mitochondrial fusion, is upregulated immediately after hormone stimulation. Moreover, Mfn2 knockdown is sufficient to impair steroid biosynthesis. Together, our findings unveil an essential role for mitochondrial fusion during steroidogenesis. These discoveries highlight the importance of organelles’ reorganization in specialized cells, prompting the exploration of the impact that organelle dynamics has on biological processes that include, but are not limited to, steroid synthesis.     

SipB-SipC Complex Is Essential for Translocon Formation

The delivery of effector proteins by Salmonella across the host cell membrane requires a subset of effectors secreted by the type III secretion system (TTSS) known as translocators. SipC and SipB are translocator proteins that are inserted into host membranes and presumably form a channel that translocates type III effectors into the host cell. The molecular events of how these translocators insert into the host cell membrane remain unknown. We have previously shown that the SipC C-terminal amino acid region (321–409) is required for the translocation of effectors into host cells. In this study, we demonstrate that the ability to form SipC-SipB complex is essential for their insertion into the host membrane. The SipB-interacting domain of SipC is near its C-terminal amino acid region (340–409). In the absence of SipB, SipC was not detected in the membrane fraction. Furthermore, SipC mutants that no longer interact with SipB are defective in inserting into the host cell membrane. We propose a mechanism whereby SipC binds SipB through its C-terminal region to facilitate membrane-insertion and subsequent translocon formation in the host cell membrane.     

Bone Is Not Essential for Osteoclast Activation

Background

The mechanism whereby bone activates resorptive behavior in osteoclasts, the cells that resorb bone, is unknown. It is known that α_vβ₃ ligands are important, because blockade of α_vβ₃ receptor signaling inhibits bone resorption, but this might be through inhibition of adhesion or migration rather than resorption itself. Nor is it known whether α_vβ₃ ligands are sufficient for resorption the consensus is that bone mineral is essential for the recognition of bone as the substrate appropriate for resorption.

Methodology/Principal Findings

Vitronectin- but not fibronectin-coated coverslips induced murine osteoclasts to secrete tartrate-resistant acid phosphatase, as they do on bone. Osteoclasts incubated on vitronectin, unlike fibronectin, formed podosome belts on glass coverslips, and these were modulated by resorption-regulating cytokines. Podosome belts formed on vitronectin-coated surfaces whether the substrates were rough or smooth, rigid or flexible. We developed a novel approach whereby the substrate-apposed surface of cells can be visualized in the scanning electron microscope. With this approach, supported by transmission electron microscopy, we found that osteoclasts on vitronectin-coated surfaces show ruffled borders and clear zones characteristic of resorbing osteoclasts. Ruffles were obscured by a film if cells were incubated in the cathepsin inhibitor E64, suggesting that removal of the film represents substrate-degrading behavior. Analogously, osteoclasts formed resorption-like trails on vitronectin-coated substrates. Like bone resorption, these trails were dependent upon resorbogenic cytokines and were inhibited by E64. Bone mineral induced actin rings and surface excavation only if first coated with vitronectin. Fibronectin could not substitute in any of these activities, despite enabling adhesion and cell spreading.

Conclusions/Significance

Our results show that ligands α_vβ₃ are not only necessary but sufficient for the induction of resorptive behavior in osteoclasts; and suggest that bone is recognized through its affinity for these ligands, rather than by its mechanical or topographical attributes, or through a putative ‘mineral receptor’.     

The Functional Quality of Soluble Recombinant Polypeptides Produced in Escherichia coli Is Defined by a Wide Conformational Spectrum

We have observed that a soluble recombinant green fluorescent protein produced in Escherichia coli occurs in a wide conformational spectrum. This results in differently fluorescent protein fractions in which morphologically diverse soluble aggregates abound. Therefore, the functional quality of soluble versions of aggregation-prone recombinant proteins is defined statistically rather than by the prevalence of a canonical native structure.     

Soluble Epoxide Hydrolase Dimerization Is Required for Hydrolase Activity

Soluble epoxide hydrolase (sEH) plays a key role in the metabolic conversion of the protective eicosanoid 14,15-epoxyeicosatrienoic acid to 14,15-dihydroxyeicosatrienoic acid. Accordingly, inhibition of sEH hydrolase activity has been shown to be beneficial in multiple models of cardiovascular diseases, thus identifying sEH as a valuable therapeutic target. Recently, a common human polymorphism (R287Q) was identified that reduces sEH hydrolase activity and is localized to the dimerization interface of the protein, suggesting a relationship between sEH dimerization and activity. To directly test the hypothesis that dimerization is essential for the proper function of sEH, we generated mutations within the sEH protein that would either disrupt or stabilize dimerization. We quantified the dimerization state of each mutant using a split firefly luciferase protein fragment-assisted complementation system. The hydrolase activity of each mutant was determined using a fluorescence-based substrate conversion assay. We found that mutations that disrupted dimerization also eliminated hydrolase enzymatic activity. In contrast, a mutation that stabilized dimerization restored hydrolase activity. Finally, we investigated the kinetics of sEH dimerization and found that the human R287Q polymorphism was metastable and capable of swapping dimer partners faster than the WT enzyme. These results indicate that dimerization is required for sEH hydrolase activity. Disrupting sEH dimerization may therefore serve as a novel therapeutic strategy for reducing sEH hydrolase activity.