Interactions of Plasmodium berghei-Infected Erythrocytes with Complement1

Incubation of radioactively labeled parasitized (Plasmodium berghei) erythrocytes (PE) with adherent peritoneal exudate cells in the presence of 10% (v/v) fresh mouse serum (NMS) resulted in the uptake of a proportion of radioactive material (PE). Inactivation of the added serum by heat or zymosan treatment resulted in diminished uptake of radioactivity. These results suggest that PE activated complement. Incubation of fresh NMS with PE reduced the hemolytic complement level of the serum as shown by its subsequent decreased ability to lyse antibody-coated rabbit red blood cells. No such effect was found when uninfected erythrocytes from either infected or uninfected blood were preincubated with fresh NMS. Thus, PE or PE-derived material activated complement. Addition of EGTA during incubation of fresh NMS with PE did not inhibit the decrease in complement level. This indicated that complement was activated by the alternative pathway. Complement levels decreased even when fresh NMS and PE were incubated in the presence of EDTA (which inhibits both classical and alternative pathway activation), suggesting that a complement activating factor (or a complement inhibitor) was released from the PE. However, lysis of PE after incubation with either fresh rabbit or guinea pig serum did not occur unless anti-mouse erythrocyte antibody was added. The production of a complement-activating factor by PE might explain part of the decreasing complement levels during infection and might enable the parasite to escape from a complement-mediated defense mechanism of the host.     

Structural Alteration of the Membrane of Erythrocytes Infected with Plasmodium falciparum1

Human erythrocytes infected with five strains of Plasmodium falciparum and Aotus erythrocytes infected with three strains of P. falciparum were studied by thin-section and freeze-fracture electron microscopy. All strains of P. falciparum we studied induced electron-dense conical knobs, measuring 30–40 nm in height and 90–100 nm in diameter on erythrocyte membranes. Freeze-fracture demonstrated that the knobs were distributed over the membrane of both human and Aotus erythrocytes. A distinct difference was seen between the intramembrane particle (IMP) distribution over the knobs of human and Aotus erythrocyte membranes. There was no change in IMP distribution in infected human erythrocyte membranes, but infected Aotus erythrocytes showed an aggregation of IMP over the P face of the knobs with a clear zone at the base. This difference in IMP distribution was related only to the host species and not to parasite strains. Biochemical analysis demonstrated that a higher proportion of band 3 was bound to the cytoskeleton of uninfected Aotus erythrocytes than uninfected human erythrocytes after Triton X-100 extraction. This may account for the different effects of P. falciparum infection on IMP distribution in the two different cell types.     

夏氏疟原虫入侵时小鼠红细胞膜唾液酸糖蛋白的改变

夏氏疟原虫入侵时小鼠红细胞膜唾液酸糖蛋白的改变刘俊凡,卢义钦（湖南医科大学生化教研室,长沙４１Ｏ０７８〕ＲｏｎａｌｄＬ．ＮｏｇｅｌＯｌｇａＯ．Ｂｌｕｍｅｎｆｅｌｄ（爱因斯坦医学院内科血液学研究室和生化学系,美国纽约１０４６１）关键词夏氏疟原虫;红细胞...     

Characterization of Plasmodium vivax Early Transcribed Membrane Protein 11.2 and Exported Protein 1

In Plasmodium, the membrane of intracellular parasites is initially formed during invasion as an invagination of the red blood cell surface, which forms a barrier between the parasite and infected red blood cells in asexual blood stage parasites. The membrane proteins of intracellular parasites of Plasmodium species have been identified such as early-transcribed membrane proteins (ETRAMPs) and exported proteins (EXPs). However, there is little or no information regarding the intracellular parasite membrane in Plasmodium vivax. In the present study, recombinant PvETRAMP11.2 (PVX_003565) and PvEXP1 (PVX_091700) were expressed and evaluated antigenicity tests using sera from P. vivax-infected patients. A large proportion of infected individuals presented with IgG antibody responses against PvETRAMP11.2 (76.8%) and PvEXP1 (69.6%). Both of the recombinant proteins elicited high antibody titers capable of recognizing parasites of vivax malaria patients. PvETRAMP11.2 partially co-localized with PvEXP1 on the intracellular membranes of immature schizont. Moreover, they were also detected at the apical organelles of newly formed merozoites of mature schizont. We first proposed that these proteins might be synthesized in the preceding schizont stage, localized on the parasite membranes and apical organelles of infected erythrocytes, and induced high IgG antibody responses in patients.     

Phosphorylation of Mouse Melanopsin by Protein Kinase A

The visual pigment melanopsin is expressed in intrinsically photosensitive retinal ganglion cells (ipRGCs) in the mammalian retina, where it is involved in non-image forming light responses including circadian photoentrainment, pupil constriction, suppression of pineal melatonin synthesis, and direct photic regulation of sleep. It has recently been shown that the melanopsin-based light response in ipRGCs is attenuated by the neurotransmitter dopamine. Here, we use a heterologous expression system to demonstrate that mouse melanopsin can be phosphorylated by protein kinase A, and that phosphorylation can inhibit melanopsin signaling in HEK cells. Site-directed mutagenesis experiments revealed that this inhibitory effect is primarily mediated by phosphorylation of sites T186 and S287 located in the second and third intracellular loops of melanopsin, respectively. Furthermore, we show that this phosphorylation can occur in vivo using an in situ proximity-dependent ligation assay (PLA). Based on these data, we suggest that the attenuation of the melanopsin-based light response by dopamine is mediated by direct PKA phosphorylation of melanopsin, rather than phosphorylation of a downstream component of the signaling cascade.     

Staining of Plasmodium yoelii-Infected Mouse Erythrocytes with the Fluorescent Dye Rhodamine 1231

The cationic permeant fluorescent dye rhodamine 123 (R123) was used to stain Plasmodium yoelii-infected mouse erythrocytes. Fluorescence microscopic observations demonstrated that the parasite, but not the matrix of the infected erythrocyte, accumulated the dye. Differences in fluorescence intensity could not be found at the various developmental stages of the parasite; however, quantitation of the cell-associated dye revealed an increase in R123 uptake with parasite development. The retention of the parasite-associated dye, as measured by fluorescence microscopy and spectrophotometry after extraction of R123 with butanol, was markedly reduced by treatment of the infected erythrocytes with a proton ionophore, carbonyl cyanide m-chlorophenylhydrazone (CCCP), and an inhibitor of proton ATPase, dicyclohexylcarbodiimide (DCCD). These results indicate that the accumulation and retention of R123 in P. yoelii reflect the parasite membrane potential and suggest that the parasite plasma membrane has a membrane potential-generating proton pump.     

溴氰菊酯对鼠脑蛋白质磷酸化作用的影响

以小鼠大脑碎片与[γ-~(32)P]ATP一起保温,观察到溴氰菊酯对蛋白1—3磷酸化的刺激作用和对4、5磷酸化的抑制作用,表明溴氰菊酯对大脑蛋白质磷酸化产生了影响。从鼠脑分离了C、D、S三个组分,分别进行的蛋白质磷酸化试验结果表明,C、D组分可能是重要的磷酸化部位。 蛋白1、2、3的磷酸化明显地受到溴氰菊酯的刺激,这三个蛋白质可能是“蛋白Ⅲb”的几种形式。溴氰菊酯对“蛋白Ⅲb”磷酸化的刺激,可能会影响神经末梢的神经激素释放,从而影响到与其相关的某些神经功能。     

The Protein Phosphorylation Landscape of Mouse Spermatids during Spermiogenesis

The characteristic tadpole shape of sperm is formed from round spermatids via spermiogenesis, a process which results in dramatic morphological changes in the final stage of spermatogenesis in the testis. Protein phosphorylation, as one of the most important post‐translational modifications, can regulate spermiogenesis; however, the phosphorylation events taking place during this process have not been systematically analyzed. In order to better understand the role of phosphorylation in spermiogenesis, large‐scale phosphoproteome profiling is performed using IMAC and TiO₂ enrichment. In total, 13 835 phosphorylation sites, in 4196 phosphoproteins, are identified in purified mouse spermatids undergoing spermiogenesis in two biological replicates. Overall, 735 testis‐specific proteins are identified to be phosphorylated, and are expressed at high levels during spermiogenesis. Gene ontology analysis shows enrichment of the identified phosphoproteins in terms of histone modification, cilium organization, centrosome and the adherens junction. Further characterization of the kinase‐substrate phosphorylation network demonstrates enrichment of phosphorylation substrates related to the regulation of spermiogenesis. This global protein phosphorylation landscape of spermiogenesis shows wide phosphoregulation across a diverse range of processes during spermiogenesis and can help to further characterize the process of sperm generation. All MS data are available via ProteomeXchange with the identifier PXD011890.     

Malaria Parasite-Infected Erythrocytes Secrete PfCK1, the Plasmodium Homologue of the Pleiotropic Protein Kinase Casein Kinase 1

Casein kinase 1 (CK1) is a pleiotropic protein kinase implicated in several fundamental processes of eukaryotic cell biology. Plasmodium falciparum encodes a single CK1 isoform, PfCK1, that is expressed at all stages of the parasite’s life cycle. We have previously shown that the pfck1 gene cannot be disrupted, but that the locus can be modified if no loss-of-function is incurred, suggesting an important role for this kinase in intra-erythrocytic asexual proliferation. Here, we report on the use of parasite lines expressing GFP- or His-tagged PfCK1 from the endogenous locus to investigate (i) the dynamics of PfCK1 localisation during the asexual cycle in red blood cells, and (ii) potential interactors of PfCK1, so as to gain insight into the involvement of the enzyme in specific cellular processes. Immunofluorescence analysis reveals a dynamic localisation of PfCK1, with evidence for a pool of the enzyme being directed to the membrane of the host erythrocyte in the early stages of infection, followed by a predominantly intra-parasite localisation in trophozoites and schizonts and association with micronemes in merozoites. Furthermore, we present strong evidence that a pool of enzymatically active PfCK1 is secreted into the culture supernatant, demonstrating that PfCK1 is an ectokinase. Our interactome experiments and ensuing kinase assays using recombinant PfCK1 to phosphorylate putative interactors in vitro suggest an involvement of PfCK1 in many cellular processes such as mRNA splicing, protein trafficking, ribosomal, and host cell invasion.     

小鼠肝质膜蛋白质的生物信息学研究

随着高通量蛋白质组研究技术的发展,使用生物信息学方法对鉴定出的蛋白质进行批量的物理化学性质和功能属性的研究显得越来越重要．对2-DE分离的小鼠肝质膜中鉴定的209个蛋白质运用生物信息学方法进行了一系列的功能属性分析,包括统计分析ProtParam软件计算出的209个蛋白质的理论相对分子质量、等电点以及疏水值的分布情况,使用TMHMM预测蛋白质的跨膜区数目,运用系统发生谱方法预测蛋白质相互作用网络,根据其相互作用网络预测部分未知蛋白质的功能．     

Protein Kinase A-dependent Phosphorylation of Rap1 Regulates Its Membrane Localization and Cell Migration

The small G protein Rap1 can mediate “inside-out signaling” by recruiting effectors to the plasma membrane that signal to pathways involved in cell adhesion and cell migration. This action relies on the membrane association of Rap1, which is dictated by post-translational prenylation as well as by a stretch of basic residues within its carboxyl terminus. One feature of this stretch of acidic residues is that it lies adjacent to a functional phosphorylation site for the cAMP-dependent protein kinase PKA. This phosphorylation has two effects on Rap1 action. One, it decreases the level of Rap1 activity as measured by GTP loading and the coupling of Rap1 to RapL, a Rap1 effector that couples Rap1 GTP loading to integrin activation. Two, it destabilizes the membrane localization of Rap1, promoting its translocation into the cytoplasm. These two actions, decreased GTP loading and decreased membrane localization, are related, as the translocation of Rap1-GTP into the cytoplasm is associated with its increased GTP hydrolysis and inactivation. The consequences of this phosphorylation in Rap1-dependent cell adhesion and cell migration were also examined. Active Rap1 mutants that lack this phosphorylation site had a minimal effect on cell adhesion but strongly reduced cell migration, when compared with an active Rap1 mutant that retained the phosphorylation site. This suggests that optimal cell migration is associated with cycles of Rap1 activation, membrane egress, and inactivation, and requires the regulated phosphorylation of Rap1 by PKA.     

Increased Phosphorylation of a Membrane Protein Consequent to Amygdaloid Kindling

The phosphorylation of both particulate and soluble proteins in the amygdala was examined in electrically kindled rats. In animals receiving electrical stimulation in the left amygdala for 5-6 days that displayed electrical after-discharges but no motor seizures, no changes were observed in the phosphorylation of either particulate or soluble proteins. In animals stimulated for 20-21 days where major motor seizures were produced, the phosphorylation of a protein having a molecular weight of 45,000 ( 45K ) was markedly increased. The phosphorylation of this protein was increased in both the right (unstimulated) and left (stimulated) amygdala. Major motor seizures induced by electroconvulsive shocks, however, did not alter phosphorylation of this protein. Phosphorylation of the 45K protein was stimulated by calcium and calmodulin. The 45K protein is a major phosphoprotein of amygdala, representing 3.2% of the total particulate phosphoproteins in control animals and 7.4% in the kindled animals. In the presence of calcium-calmodulin, 16.2% of net protein phosphorylation was accounted for by the 45K protein.     

The Merozoite Surface Protein 1 Complex Is a Platform for Binding to Human Erythrocytes by Plasmodium falciparum

Plasmodium falciparum is the causative agent of the most severe form of malaria in humans. The merozoite, an extracellular stage of the parasite lifecycle, invades erythrocytes in which they develop. The most abundant protein on the surface of merozoites is merozoite surface protein 1 (MSP1), which consists of four processed fragments. Studies indicate that MSP1 interacts with other peripheral merozoite surface proteins to form a large complex. Successful invasion of merozoites into host erythrocytes is dependent on this protein complex; however, the identity of all components and its function remain largely unknown. We have shown that the peripheral merozoite surface proteins MSPDBL1 and MSPDBL2 are part of the large MSP1 complex. Using surface plasmon resonance, we determined the binding affinities of MSPDBL1 and MSPDBL2 to MSP1 to be in the range of 2–4 × 10⁻⁷ m. Both proteins bound to three of the four proteolytically cleaved fragments of MSP1 (p42, p38, and p83). In addition, MSPDBL1 and MSPDBL2, but not MSP1, bound directly to human erythrocytes. This demonstrates that the MSP1 complex acts as a platform for display of MSPDBL1 and MSPDBL2 on the merozoite surface for binding to receptors on the erythrocyte and invasion.     

蛋白磷酸酯酶2A调节微管相关蛋白1b的磷酸化

微管相关蛋白MAP1b的生物学活性受其磷酸化修饰的调节,后者则受相应的蛋白激酶和蛋白磷酸酯酶(PP)调控.为研究蛋白磷酸酯酶在脑内对MAP1b磷酸化的调控作用,采用有代谢活性的大鼠脑片作为模型,分别应用冈田酸(okadaic acid)和cyclosporin A选择性地抑制PP2A 和PP2B活性,来研究其对脑内蛋白磷酸酯酶MAP1b磷酸化的调控.采用特异性的MAP1bⅠ型磷酸化依赖性抗体522和免疫印迹技术检测MAP1bⅠ型磷酸化.结果表明,当PP2A被okadaic acid选择性抑制后,MAP1bⅠ型磷酸化明显增加.而PP2B被选择性地抑制后,MAP1b磷酸化的变化不大.免疫组化染色显示,MAP1b广泛分布于鼠大脑神经元和突起中,与对照组相比,在PP2A抑制的脑片中抗体522的免疫活性在神经元中明显升高.上述结果表明,PP2A是脑中调控MAP1bⅠ型磷酸化的主要蛋白磷酸酯酶.     

检控蛋白Rad17在小鼠睾丸精子发生过程中的表达及其磷酸化变化

Rad17是细胞周期检控点信号转导过程中的一个关键检控蛋白,在DNA损伤检控和DNA复制检控中具有重要功能。但Rad17在细胞减数分裂中的检控作用还不是很清楚。因细胞减数分裂在睾丸组织中非常活跃,应用Western印迹检测Rad17在不同发育时期的小鼠睾丸组织中的表达及其磷酸化水平,并应用免疫组化的方法检测小鼠睾丸组织不同时期生殖细胞内Rad17的表达变化。结果显示Rad17在小鼠睾丸组织内高表达,而在肝、肾等组织中表达水平较低;Rad17在不同周龄的小鼠睾丸组织中均高水平表达,但在4周龄以后的小鼠睾丸组织中其磷酸化水平明显升高;免疫组化结果显示Rad17在精原细胞、精母细胞的细胞核中高表达,但在成熟精子细胞中消失。这些结果提示Rad17在小鼠睾丸生殖细胞减数分裂过程中也起重要检控作用。     

An Exported Heat Shock Protein 40 Associates with Pathogenesis-Related Knobs in Plasmodium falciparum Infected Erythrocytes

Cell surface structures termed knobs are one of the most important pathogenesis related protein complexes deployed by the malaria parasite Plasmodium falciparum at the surface of the infected erythrocyte. Despite their relevance to the disease, their structure, mechanisms of traffic and their process of assembly remain poorly understood. In this study, we have explored the possible role of a parasite-encoded Hsp40 class of chaperone, namely PFB0090c/PF3D7_0201800 (KAHsp40) in protein trafficking in the infected erythrocyte. We found the gene coding for PF3D7_0201800 to be located in a chromosomal cluster together with knob components KAHRP and PfEMP3. Like the knob components, KAHsp40 too showed the presence of PEXEL motif required for transport to the erythrocyte compartment. Indeed, sub-cellular fractionation and immunofluorescence analysis (IFA) showed KAHsp40 to be exported in the erythrocyte cytoplasm in a stage dependent manner localizing as punctuate spots in the erythrocyte periphery, distinctly from Maurer's cleft, in structures which could be the reminiscent of knobs. Double IFA analysis revealed co-localization of PF3D7_0201800 with the markers of knobs (KAHRP, PfEMP1 and PfEMP3) and components of the PEXEL translocon (Hsp101, PTEX150). KAHsp40 was also found to be in a complex with KAHRP, PfEMP3 and Hsp101 as confirmed by co-immunoprecipitation assay. Our results suggest potential involvement of a parasite encoded Hsp40 in chaperoning knob assembly in the erythrocyte compartment.     

Some Qualitative Aspects of Spleen Cell Transfer Studies on Plasmodium berghei-Infected Rats*

SYNOPSIS. Adoptive immunity to Plasmodium berghei was transferred by intraperitoneal injections into rats, never before exposed to this parasite, of either 2 × 10⁷ or 2 × 10⁸, but not of 2 × 10⁶, spleen cells from syngenic rats which had recovered from a primary P. berghei infection. When the spleen cells from the latter animals were kept at 47 C for 45 min they remained alive, but no longer were able to transfer protection. The capacity to transfer adoptive immunity was not found in spleen cells from adult rats capable of age immunity. On the other hand, this capacity was found in spleen cells from rats that had suffered a very transient parasitemia (> 1% peak parasitemia).     

Alterations in Phosphorylation of Hepatocyte Ribosomal Protein S6 Control Plasmodium Liver Stage Infection

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Rapid Changes in Plasma Membrane Protein Phosphorylation during Initiation of Cell Wall Digestion

Plasma membrane vesicles from wild carrot cells grown in suspension culture were isolated by aqueous two-phase partitioning, and ATP-dependent phosphorylation was measured with [γ-³²P]ATP in the presence and absence of calcium. Treatment of the carrot cells with the cell wall digestion enzymes, driselase, in a sorbitol osmoticum for 1.5 min altered the protein phosphorylation pattern compared to that of cells treated with sorbitol alone. Driselase treatment resulted in decreased phosphorylation of a band of M_r 80,000 which showed almost complete calcium dependence in the osmoticum treated cells; decreased phosphorylation of a band of M_r 15,000 which showed little calcium activation, and appearance of a new band of calcium-dependent phosphorylation at M_r 22,000. These effects appeared not to be due to nonspecific protease activity and neither in vivo nor in vitro exposure to driselase caused a significant loss of Coomassie blue-staining bands on the gels of the isolated plasma membranes. However, protein phosphorylation was decreased. Adding driselase to the in vitro reaction mixture caused a general decrease in the membrane protein phosphorylation either in the presence or absence of calcium which did not mimic the in vivo response. Cells labeled in vivo with inorganic ³²P also showed a response to the Driselase treatment. An enzymically active driselase preparation was required for the observed responses.