Control of Late Development of Dictyostelium discoideum by Proteins Functioning at Early Stages

We examined two mutants of D. discoideum which are temperature-sensitive for development. At the nonpermissive temperature one mutant becomes arrested in development during the transition from the finger to the migrating slug. Temperature-shift experiment indicates that the temperature-sensitive period begins at considerably earlier tip-forming stage. The other mutant becomes arrested at the Mexican hat stage and the temperature-sensitive period coinsided with this stage. The analysis of protein synthesis by two-dimensional gels, however, showed specific changes at the nonpermissive temperature at an earlier finger-forming stage.
These results indicate the presence of a control of late development by proteins at early stages.     

Modeling the Early Stages of Phase Separation in Disordered Elastin-like Proteins

Elastin-like proteins (ELPs) are known to undergo liquid-liquid phase separation reversibly above a concentration-dependent transition temperature. Previous studies suggested that, as temperature increases, ELPs experience an increased propensity for type II β-turns. However, how the ELPs behave below the phase transition temperature itself is still elusive. Here, we investigate the importance of β-turn formation during the early stages of ELP self-association. We examined the behavior of two ELPs, a 150-repeat construct that had been investigated previously (ELP[V₅G₃A₂-150] as well as a new 40-repeat construct (ELP40) suitable for nuclear magnetic resonance measurements. Structural analysis of ELP40 reveals a disordered conformation, and chemical shifts throughout the sequence are insensitive to changes in temperature over 20°C. However, a low population of β-turn conformation cannot be ruled out based on chemical shifts alone. To examine the structural consequences of β-turns in ELPs, a series of structural ensembles of ELP[V₅G₃A₂-150] were generated, incorporating differing amounts of β-turn bias throughout the chain. To mimic the early stages of the phase change, two monomers were paired, assuming preferential interaction at β-turn regions. This approach was justified by the observation that buried hydrophobic turns are commonly observed to interact in the Protein Data Bank. After dimerization, the ensemble-averaged hydrodynamic properties were calculated for each degree of β-turn bias, and the results were compared with analytical ultracentrifugation experiments at various temperatures. We find that the temperature dependence of the sedimentation coefficient $\left(s_{20,\text{w}}^{\text{o}}\right)$ can be reproduced by increasing the β-turn content in the structural ensemble. This analysis allows us to estimate the presence of β-turns and weak associations under experimental conditions. Because disordered proteins frequently exhibit weak biases in secondary structure propensity, these experimentally-driven ensemble calculations may complement existing methods for modeling disordered proteins generally.     

水稻U-Box蛋白质在不同发育时期的表达分析

真核生物基因组中广泛存在U-Box基因,其编码蛋白大部分是泛素系统中决定底物特异性识别的E3蛋白,其构象与RING-finger极其相似．U-Box蛋白质能促进底物蛋白泛素化降解,对细胞内异常蛋白的降解及质量控制方面发挥着重要的作用．水稻基因组中有77个U-Box蛋白质,系统了解它们的表达可为功能研究提供数据．制备针对水稻U-Box蛋白质的抗体,了解水稻中U-Box蛋白质在不同发育时期的表达信息,为功能研究积累数据．选取了4个水稻U-Box蛋白质,其共同结构特点为U-Box结构在N端,C端有ARM结构．用计算机软件预测抗原决定簇,细菌体系体外表达、纯化U-Box蛋白质的片段,免疫动物制备多克隆抗体,用Western blotting检测U-Box蛋白质在水稻品种93-11苗期地上部和地下部、分蘖期根和茎、孕穗期剑叶和幼穗、开花期剑叶和穗子、成熟期剑叶和种子中的表达,并与EST数据库中公布的U-Box蛋白质EST数据进行了比较分析．体外克隆表达后,获得了纯化的蛋白质,制备的抗体特异性强,蛋白质印迹(Western blotting)检测可见一条明显的主带,其中Os06g01304和Os12g38210两个蛋白质的表观分子质量与预测分子质量相符,Os01g66130和Os08g01900两个蛋白质的表观分子质量低于预测分子质量．4个U-Box蛋白质在水稻生长发育的不同时期或部位基本上是组成型表达,且表达量接近．对NCBI上公布的来自274个文库100万条以上的EST进行分析,可以看出4个U-Box蛋白质EST的数量分布大致均匀,与Western blotting结果揭示的组成型表达平行,与ATPase、HSP81-3、EGF-1 alpha和RuBisCo等对照基因相比,U-Box基因的EST数目相对很少,说明它们属于低丰度转录的基因．选取了4个水稻U-Box蛋白质,通过抗原决定簇预测,表达片段蛋白,制备了特异性抗体,证明了这一技术路线的可行性．利用抗体对水稻不同发育时期材料进行蛋白质表达谱研究,发现这些U-Box蛋白质呈组成型表达,与EST数据揭示的结果具有平行性．所制备的抗体也为相关功能研究,如免疫共沉淀、ChIP-on-chip、Pull-down以及在抗病、抗逆反应中U-Box蛋白质的表达等,积累了 资源．     

Mobilization of Reserve Proteins During Early Stages of Seed Germination in Fagopyrum Esculentum

Hydrolysis of 13S globulin, the main storage protein in grains of common buckwheat (Fagopyrum esculentum Moench), proceeds in at least two phases during germination. The first stage, involving a limited proteolytic cleavage of the protein, is associated with increased activity of proteases having maximum activity at pH 7.6. The second stage, involving further hydrolysis of the partially cleaved protein, starts after 12 h of imbibition. During this phase, activity of proteases increased and activity maximum shifted to pH 5.6. Nevertheless, 13S globulin retains its antigenic identity till the emergence of radicle and plumule. Thus, it may not be the major source of amino acids utilized by the germinating seed during the initial stages of imbibition. This revised version was published online in July 2006 with corrections to the Cover Date.     

Order Statistics Theory of Unfolding of Multimeric Proteins

Dynamic force spectroscopy has become indispensable for the exploration of the mechanical properties of proteins. In force-ramp experiments, performed by utilizing a time-dependent pulling force, the peak forces for unfolding transitions in a multimeric protein (D)_N are used to map the free energy landscape for unfolding for a protein domain D. We show that theoretical modeling of unfolding transitions based on combining the observed first (f₁), second (f₂), …, N^th (f_N) unfolding forces for a protein tandem of fixed length N, and pooling the force data for tandems of different length, n₁ < n₂ < … < N, leads to an inaccurate estimation of the distribution of unfolding forces for the protein D, ψ_D(f). This problem can be overcome by using Order statistics theory, which, in conjunction with analytically tractable models, can be used to resolve the molecular characteristics that determine the unfolding micromechanics. We present a simple method of estimation of the parent distribution, ψ_D(f), based on analyzing the force data for a tandem (D)_n of arbitrary length n. Order statistics theory is exemplified through a detailed analysis and modeling of the unfolding forces obtained from pulling simulations of the monomer and oligomers of the all-β-sheet WW domain.     

Proteins ADP-ribosylated in Nuclei and Plasma Membrane Vesicles Isolated from Sea Urchin Embryos at Various Stages of Early Development

The ADP-ribosylations of proteins in nuclei, plasma membrane vesicles, mitochondria, microsome vesicles and the soluble fraction of sea urchin embryos isolated at various stages of development were examined by measuring the radioactivities of proteins after exposure of these subcellular fractions to [adenosine-¹⁴C]NAD or [adenylate-³²P]NAD. ADP-ribosylation of proteins was detected only in the nuclear and plasma membrane fractions. In the nuclear fraction, the rate of ADP-ribosylation of the histone fraction did not change appreciably during early development. In the TCA-insoluble protein fraction of the nuclei, the rate of ADP-ribosylation increased from fertilization to the morula stage, then decreased and again increased from the mesenchyme blastula to the late gastrula stage. After exposure of the nuclear fraction to [adenylate-³²P]NAD, a protein band with a molecular weight of 90 kDa was detected by SDS-polyacrylamide gel electrophoresis and radioautography at all stages examined. Its labeling intensity indicated that its ADP-ribosylation is higher at the morula and late gastrula stages than at other stages. In the plasma membrane fraction, proteins with molecular weights of 22 and 68 kDa were ADP-ribosylated and their rates of ADP-ribosylation hardly changed during early development.     

Proteome Analysis of Plasmodium falciparum Extracellular Secretory Antigens at Asexual Blood Stages Reveals a Cohort of Proteins with Possible Roles in Immune Modulation and Signaling

The highly co-evolved relationship of parasites and their hosts appears to include modulation of host immune signals, although the molecular mechanisms involved in the host-parasite interplay remain poorly understood. Characterization of these key genes and their cognate proteins related to the host-parasite interplay should lead to a better understanding of this intriguing biological phenomenon. The malaria agent Plasmodium falciparum is predicted to export a cohort of several hundred proteins to remodel the host erythrocyte. However, proteins actively exported by the asexual intracellular parasite beyond the host red blood cell membrane (before merozoite egress) have been poorly investigated so far. Here we used two complementary methodologies, two-dimensional gel electrophoresis/MS and LC-MS/MS, to examine the extracellular secreted antigens at asexual blood stages of P. falciparum. We identified 27 novel antigens exported by P. falciparum in the culture medium of which some showed clustering with highly polymorphic genes on chromosomes, suggesting that they may encode putative antigenic determinants of the parasite. Immunolocalization of four novel secreted proteins confirmed their export beyond the infected red blood cell membrane. Of these, preliminary functional characterization of two novel (Sel1 repeat-containing) parasite proteins, PfSEL1 and PfSEL2 revealed that they down-regulate expression of cell surface Notch signaling molecules in host cells. Also a novel protein kinase (PfEK) and a novel protein phosphatase (PfEP) were found to, respectively, phosphorylate/dephosphorylate parasite-specific proteins in the extracellular culture supernatant. Our study thus sheds new light on malaria parasite extracellular secreted antigens of which some may be essential for parasite development and could constitute promising new drug targets.Plasmodium falciparum is a wide spread protozoan parasite responsible for over a million deaths annually mainly among children in sub-Saharan Africa (1). Like other apicomplexan parasites such as Leishmania, Trypanosoma, and Toxoplasma, Plasmodia depend on a series of intricate and highly evolved adaptations that enable them to evade destruction by the host immune responses. These protozoan parasites have provided some of the best leads in elucidating the mechanisms to circumvent innate immunity and adaptive humoral and cellular immunity (2). Ingenious strategies to escape innate defenses include subversion of attack by humoral effector mechanisms such as complement lysis and lysis by other serum components (3), remodeling of phagosomal compartments in which they reside (4), modulation of host cell signaling pathways (5), and modification of the antigen-presenting and immunoregulatory functions of dendritic cells, which provide a crucial link with the adaptive immune response (6). Malaria parasites also predominantly use antigenic diversity and clonal antigenic variation to evade adaptive immunity of the host (7). Surface-associated and secreted parasite proteins are major players in host-parasite cross-talk and are advantageously used by the parasite to counter the host immune system. Proteins secreted by a wide range of parasitic pathogens into the host microenvironment result in symptomatic infections. For example, the excretory-secretory (ES)¹ products of the parasitic fluke Fasciola hepatica are key players in host-parasite interactions (8). Among the apicomplexans, proteomics analyses of rhoptry organelles of Toxoplasma gondii have revealed many novel constituents of host-parasite interactions (9).The identification and trafficking of Plasmodium proteins exported into the host erythrocyte have been subjects of recent detailed investigations. A number of studies have identified Plasmodium proteins that contain signature sequence motifs, the host cell targeting signal or the Plasmodium export element (PEXEL), that target these proteins into the infected erythrocytes (10, 11). Recent proteomics analyses have identified novel proteins in the raftlike membranes of the parasite and on the surface of infected erythrocytes (12, 13). P. falciparum translationally controlled tumor protein (PfTCTP), a homolog of the mammalian histamine-releasing factor, has been shown to be released into the culture supernatant from intact as well as ruptured infected RBCs and causes histamine release from human basophils and IL-8 secretion from eosinophils (14). However, the total spectrum of proteins actively exported by the asexual intracellular parasite beyond the host RBC membrane (before merozoite egress) has been poorly investigated so far.In the present study, we used two complementary methodologies, two-dimensional gel electrophoresis (2DE)/MS and LC-MS/MS to examine the cohort of extracellular secreted antigens (ESAs) at asexual blood stages of P. falciparum. Our findings reveal that malaria parasites secrete a number of effector molecules such as immunomodulators and signaling proteins that are potentially involved in host-parasite interactions. Prominent among these are proteins with Sel1 domain, a protein of the LCCL family, a novel protein kinase, and a novel protein phosphatase. Secreted-extracellular/iRBC surface localization of some of these proteins was validated by immunolocalization studies. We also characterized the functions of some of these proteins in the culture supernatant, thus providing an insight into the nature of some of the malaria parasite extracellular antigens.     

Molecular Dynamics of Channelrhodopsin at the Early Stages of Channel Opening

Channelrhodopsin (ChR) is a light-gated cation channel that responds to blue light. Since ChR can be readily expressed in specific neurons to precisely control their activities by light, it has become a powerful tool in neuroscience. Although the recently solved crystal structure of a chimeric ChR, C1C2, provided the structural basis for ChR, our understanding of the molecular mechanism of ChR still remains limited. Here we performed electrophysiological analyses and all-atom molecular dynamics (MD) simulations, to investigate the importance of the intracellular and central constrictions of the ion conducting pore observed in the crystal structure of C1C2. Our electrophysiological analysis revealed that two glutamate residues, Glu122 and Glu129, in the intracellular and central constrictions, respectively, should be deprotonated in the photocycle. The simulation results suggested that the deprotonation of Glu129 in the central constriction leads to ion leakage in the ground state, and implied that the protonation of Glu129 is important for preventing ion leakage in the ground state. Moreover, we modeled the 13-cis retinal bound; i.e., activated C1C2, and performed MD simulations to investigate the conformational changes in the early stage of the photocycle. Our simulations suggested that retinal photoisomerization induces the conformational change toward channel opening, including the movements of TM6, TM7 and TM2. These insights into the dynamics of the ground states and the early photocycle stages enhance our understanding of the channel function of ChR.     

A Theoretical Model for the Mechanical Unfolding of Repeat Proteins

We consider the mechanical stretching of a polypeptide chain formed by multiple interacting repeats. The folding thermodynamics and the interactions among the repeats are described by the Ising model. Unfolded repeats act as soft entropic springs, whereas folded repeats respond to a force as stiffer springs. We show that the resulting force-extension curve may exhibit a pronounced force maximum corresponding to the unfolding of the first repeat. This event is followed by the unfolding of the remaining repeats, which takes place at a lower force. As the protein extension is increased, the force-extension curve of a sufficiently long repeat protein displays a plateau, where the force remains nearly constant and the protein unfolds sequentially so that the number of unfolded repeats is proportional to the extension. Such a sequential mechanical unfolding mechanism is displayed even by the repeat proteins whose thermal denaturation is highly cooperative, provided that they are long enough. By contrast, the unfolding of short repeat progressions can be cooperative.     

Acrylamide Gel Electrophoresis of Intracellular Proteins During Early Stages of Sporulation in Bacillus subtilis

Acrylamide gel electrophoresis of unfractionated cellular extracts of Bacillus subtilis is shown to be an effective method for characterizing many of the changes in protein composition, when coupled with specific histological-type staining reactions. The results obtained here by using extracts from cells at different stages of growth and sporulation are consistent with observations from other laboratories where extensively purified and highly characterized enzymes have been studied. In several instances, the histochemical reactions can be associated with a specific enzymatic function and appear to indicate the presence of multiple molecular forms. In other instances, the data cannot be evaluated in terms of known enzyme function because the specificity of the histochemical analysis is not certain. However, the assays described permit monitoring of electrophoretic changes at the level of individual proteins within sporulating cultures. The results suggest that B. subtilis may contain two "hexokinase-like" enzymes which cease to function before sporulation is initiated. Aldolase and alanine dehydrogenase are detectable as single bands of enzyme activity during vegetative growth but as multiple molecular forms once sporulation has been initiated. Reduced nicotinamide adenine dinucleotide dehydrogenase activity is represented by an entire family of reactive species in these crude extracts, which undergo multiple changes during the early stages of sporulation. Tricarboxylic acid cycle dehydrogenase enzymes and those bands having esterase activity on alpha-naphthyl acetate show detectable changes in specific activity after cessation of exponential growth. Glucose dehydrogenase is not detectable until the sequence of changes leading to spore formation has progressed for 4 or 5 hr.     

羊布鲁氏菌的分泌蛋白质组分析

分泌蛋白是指那些分泌到细胞外的蛋白质。布鲁氏菌的分泌蛋白可能介导了病原与宿主之间的相互作用, 在布鲁氏菌的毒力方面发挥一定的作用, 但是研究方法的局限性限制了分泌蛋白的研究。本文报道了利用蛋白质组的方法来寻找羊布鲁氏菌的分泌蛋白。首先用TCA-丙酮法提取布鲁氏菌培养上清中的分泌蛋白, 双向电泳进行分离, 然后用质谱来鉴定这些蛋白, 最终鉴定到40种蛋白。通过生物信息学分析, 发现这些蛋白主要是ABC转运系统的底物结合蛋白、外膜蛋白和热休克蛋白。这些蛋白的识别不仅有助于对布鲁氏菌致病机制的理解, 而且也可为     

乌龙岭’龙眼胚胎发育时期特异性蛋白质的变化

应用IEF-SDS-PAGE技术分析龙眼胚胎分化发育过程中蛋白质组分的变化。结果表明,在各发育阶段大多数蛋白质组分的电泳图谱基本一致,但也有变化。其中花后38d存在TE1(27．1kD、p,7．3),TE2(17．5kD、pI8．2)2个特异蛋白,45d存在TE3(11．4kD、pI7．6),TE4(13．2kD、pI9．9)2个特异蛋白,52d存在TE5(22．6kD、pI7．2),TE6(18．6kD、pI8．3),TE,(23．5kD、pI3．6)3个特异蛋白。31d胚胎电泳图谱中的蛋白质点数相对较多,表明此时蛋白质旺盛合成与积累,这与蛋白含量的变化基本一致。龙眼胚胎发育过程中特异蛋白的出现或消失．对胚胎的分化发育具有重要作用。     

New Views of Old Proteins: Clarifying the Enigmatic Proteome

All human diseases involve proteins, yet our current tools to characterize and quantify them are limited. To better elucidate proteins across space, time, and molecular composition, we provide a >10 years of projection for technologies to meet the challenges that protein biology presents. With a broad perspective, we discuss grand opportunities to transition the science of proteomics into a more propulsive enterprise. Extrapolating recent trends, we describe a next generation of approaches to define, quantify, and visualize the multiple dimensions of the proteome, thereby transforming our understanding and interactions with human disease in the coming decade.