DING proteins; novel members of a prokaryotic phosphate-binding protein superfamily which extends into the eukaryotic kingdom

PstS proteins are the cell-bound phosphate-binding elements of the ubiquitous bacterial ABC phosphate uptake mechanisms. Primary and tertiary structures, characteristic of pstS proteins, are conserved in proteins, which are expressed in secretory operons and induced by phosphate deprivation, in Pseudomonas species. There are two subsets of these proteins; AP proteins, which are alkaline phosphatases, and DING proteins, named for their N-terminal sequence, which are phosphate-binding proteins. Both form elements of a proposed phosphate-scavenging system in pseudomonads. DING proteins have also been isolated from many eukaryotic sources, and are associated with both normal and pathological functions in mammals. Their phosphate-binding function suggests a role in biomineralization, but the ability to bind other ligands may be related to signal transduction in eukaryotes. Though it has been claimed that all such proteins may originate from pseudomonads, many eukaryotic DING proteins have unique features which are incompatible with a bacterial origin.     

Primary structures of ribosomal proteins from the archaebacterium Halobacterium marismortui and the eubacterium Bacillus stearothermophilus.

Approximately 40 ribosomal proteins from each Halobacterium marismortui and Bacillus stearothermophilus have been sequenced either by direct protein sequence analysis or by DNA sequence analysis of the appropriate genes. The comparison of the amino acid sequences from the archaebacterium H marismortui with the available ribosomal proteins from the eubacterial and eukaryotic kingdoms revealed four different groups of proteins: 24 proteins are related to both eubacterial as well as eukaryotic proteins. Eleven proteins are exclusively related to eukaryotic counterparts. For three proteins only eubacterial relatives-and for another three proteins no counterpart-could be found. The similarities of the halobacterial ribosomal proteins are in general somewhat higher to their eukaryotic than to their eubacterial counterparts. The comparison of B stearothermophilus proteins with their E coli homologues showed that the proteins evolved at different rates. Some proteins are highly conserved with 64-76% identity, others are poorly conserved with only 25-34% identical amino acid residues.     

An electrophoretic study of proteins of the ovary,fat body,and haemolymph in the migratory grasshopper, Melanoplus sanguinipes

Electrophoretic separation of saline extracts from the ovary revealed 14 proteins. Twelve proteins were detected in the fat body, of which seven had electrophoretic mobilities identical to those in the ovary. Similarly, eight of 16 proteins in the haemolymph of vitellogenic females ahad electrophoretically identical counterparts in the ovary. As these proteins accumulate in the haemolymph of ovariectomized females, the findings suggest that most yolk proteins are synthesized in the fat body. Although most female haemolymph proteins are present in males, two of the predominant yolk protiens are absent and represent female-specific proteins.Although certain proteins accumulate in the haemolymph of allatectomized females, the major ovarian proteins are absent or present in low concentrations. However, 48 hr after allatectomized females are treated with a juvenile hormone analogue, the haemolymph protein pattern resembles that of a normal female. This suggests that the corpora allata stimulate the synthesis of female-specific and other vitellogenic proteins. The median neurosecretory cells (mNSC) are also necessary for synthesis of female-specific proteins. Furthermore, proteins which are present in allatectomized females are absent in mNSC-cauterized insects suggesting that the mNSC stimulate general protein synthesis.     

Signal-anchored proteins follow a unique insertion pathway into the outer membrane of mitochondria

Signal-anchored proteins are a class of mitochondrial outer membrane proteins that expose a hydrophilic domain to the cytosol and are anchored to the membrane by a single transmembrane domain in the N-terminal region. Like the vast majority of mitochondrial proteins, signal-anchored proteins are synthesized on cytosolic ribosomes and are subsequently imported into the organelle. We have studied the mechanisms by which precursors of these proteins are recognized by the mitochondria and are inserted into the outer membrane. The import of signal-anchored proteins was found to be independent of the known import receptors, Tom20 and Tom70, but to require the major Tom component, Tom40. In contrast to precursors destined to internal compartments of mitochondria and those of outer membrane beta-barrel proteins, precursors of signal-anchored proteins appear not to be inserted via the general import pore. Taken together, we propose a novel pathway for insertion of these proteins into the outer membrane of mitochondria.     

A survey of putative secreted and transmembrane proteins encoded in the C. elegans genome

ABSTRACT: BACKGROUND: Almost half of the Caenorhabditis elegans genome encodes proteins with either a signal peptide or a transmembrane domain. Therefore a substantial fraction of the proteins are localized to membranes, reside in the secretory pathway or are secreted. While these proteins are of interest to a variety of different researchers ranging from developmental biologists to immunologists, most of secreted proteins have not been functionally characterized so far. RESULTS: We grouped proteins containing a signal peptide or a transmembrane domain using various criteria including evolutionary origin, common domain organization and functional categories. We found that putative secreted proteins are enriched for small proteins and nematode-specific proteins. Many secreted proteins are predominantly expressed in specific life stages or in one of the two sexes suggesting stage- or sex-specific functions. More than a third of the putative secreted proteins are upregulated upon exposure to pathogens, indicating that a substantial fraction may have a role in immune response. Slightly more than half of the transmembrane proteins can be grouped into broad functional categories based on sequence similarity to proteins with known function. By far the largest groups are channels and transporters, various classes of enzymes and putative receptors with signaling function. CONCLUSION: Our analysis provides an overview of all putative secreted and transmembrane proteins in C. elegans. This can serve as a basis for selecting groups of proteins for large-scale functional analysis using reverse genetic approaches.     

ON SOME GENERAL PROPERTIES OF PROTEINS

1. The processes of denaturation and coagulation of hemoglobin are like those of other proteins. 2. When hemoglobin is denatured it is probably depolymerized into hemochromogen. 3. When other proteins are denatured they, too, are probably depolymerized. Conversely, native proteins can be regarded as aggregates of denatured proteins. 4. The globins and histones are to be regarded as denatured proteins rather than as a distinct group of proteins. 5. The factors affecting the equilibrium between native and denatured proteins have been considered. 6. A non-polar group is uncovered when a protein is denatured. 7. It has been shown that judged by the two most sensitive tests for the specificity of proteins, it is only when proteins are in the native form that they are highly specific.     

Synthesis, modification and structural binding of heat-shock proteins in tomato cell cultures

Synthesis of about 30 acidic and 18 basic heat-shock proteins (hsps) is induced in suspension cultures of tomato (Lycopersicon peruvianum) if subjected to supraoptimal temperature conditions (35-40 degrees C). A characteristic aspect of the plant heat-shock response is the formation of cytoplasmic granular aggregates, heat-shock granules, containing distinct heat-shock proteins as major structural components and, in addition, several hitherto undetected minor acidic and basic heat-shock proteins. Structural binding of heat-shock proteins, i.e. assembly of heat-shock granules, is dependent on the persistance of supraoptimal temperature conditions. Despite the ongoing synthesis also at 25 degrees C, e.g. in pulse heat-shocked cultures, these proteins are accumulated exclusively in soluble form. Individual heat-shock proteins are characterized by their kinetics of synthesis and are classified by their compartmentation behaviour into class A proteins (exclusively found in soluble form, e.g. hsps 95 and 80), class B proteins (5-10% bound to heat-shock granules, e.g. hsps 70, 68), class C proteins (30-80% bound to heat-shock granules, e.g. hsps 21, 17, 15) and class D proteins, which are minor heat-shock proteins only detected in structure-bound form. Major representatives are modified proteins, i.e. hsps 95, 80, 70 and 68 are phosphorylated and hsps 80, 74, 70 and 17 are methylated proteins (numbers 70, 80 etc. refer to 10(-3) Mr). Under heat-shock conditions synthesis of the proteins detected in control cells (25 degrees C proteins) exhibits two patterns. There are proteins with continued and proteins with discontinued synthesis. Synthesis of most of the latter proteins is resumed very rapidly after shift-down to 25 degrees C, even in the presence of actinomycin D. We conclude that reversible segregation of distinct mRNA species from the translation apparatus contributes to the heat-shock-specific pattern of protein synthesis in plants also.     

粟酒裂殖酵母全基因组中含信号肽蛋白质的研究

对粟酒裂殖酵母全基因组3条染色体上的4,997个蛋白序列进行了全局性的分析,利用signalP3.0软件分析这些蛋白的N-末端信号肽序列, 预测有N-末端分泌信号肽序列的蛋白196个;利用TMpred 软件分析跨膜结构, 预测跨膜蛋白117个; 使用PrositeScan程序分析膜脂蛋白的脂结合位点, 预测有膜脂结合蛋白13个, 进而预测分泌性蛋白序列66个。使用Target P分析66个分泌蛋白的蛋白序列, 研究这些蛋白在细胞中的定位。这些分泌蛋白的功能涉及粟酒裂殖酵母的营养、生殖、细胞间以及细胞与环境间的交流等许多方面, 对细胞的生存和繁殖有重要意义, 在系统生物学的研究中有重要参考价值。粟酒裂殖酵母分泌组的研究也将为粟酒裂殖酵母作为药物筛选模型以及开发为外源蛋白表达的宿主提供基础。     

Homologous sequences in proteins with different functions

Data on the primary structure of approximately 250 proteins and regulatory peptides are presented. It was shown that functionally different proteins contain homologous sequencies. Growth hormone molecule contains homologous sequencies to several proteins. On the whole, polypeptide hormones more frequently, as compared to other proteins, contained sequencies, which are homologous to fragments of proteins with various functions. Evolutionary aspects of multiple relations between proteins are discussed.     

2-DE技术中疏水性和碱性蛋白质的研究进展

双向凝胶电泳(2-DE)具有高分辨率、高通量等特点,已被广泛地用于蛋白质组的分离．但是它在分离疏水性蛋白质和碱性蛋白质时却遇到了极大的挑战．然而,疏水性与碱性蛋白质在全蛋白质中占相当大的比例,且具有很重要的生物学意义．因而,近年来,越来越多的研究者将目标瞄准这些蛋白质,并且取得了一些令人鼓舞的进展：用亚细胞预分离技术,顺序提取法等方法来富集疏水性蛋白质,用一些新的有效的增溶剂如硫脲,ASB一14等来改善疏水性蛋白质的溶解,应用这些技术2一DE可分辨出总平均疏水值达O．80的蛋白质;在碱性蛋白质分离方面,通过等电聚焦预处理,使用窄pH梯度胶条等大大地改善了碱性蛋白质在2-DE中的分离,能分辨出等电点达11．7的蛋白质．现对2-DE技术中疏水性和碱性蛋白质分离的研究进展进行综述．     

Small,basic antifungal proteins secreted from filamentous ascomycetes: a comparative study regarding expression,structure, function and potential application

Peptides and proteins with antimicrobial activity are produced throughout all kingdoms in nature, from prokaryotes to lower and higher eukaryotes, including fungi, plants, invertebrates and vertebrates. These proteins contribute to an important constitutive or induced defense mechanism of the producer against microorganisms. According to their variety in structure and function, these proteins are classified arbitrarily into groups that are based on their mechanism of action, their structure and their similarity to other known proteins. The present review focuses on a new group of antimicrobial proteins, namely small, basic and cysteine-rich antifungal proteins, which are secreted from filamentous fungi of the group Ascomycetes. These proteins are encoded by orthologous genes and exhibit both similarities and differences concerning their species-specificity, primary structure, protein activity and target sites. The properties of these proteins, their possible mode of action and their potential application for human benefits are discussed in comparison with other already well known antimicrobial proteins.     

Recombination of protein fragments: a promising approach toward engineering proteins with novel nanomechanical properties

Combining single molecule atomic force microscopy (AFM) and protein engineering techniques, here we demonstrate that we can use recombination-based techniques to engineer novel elastomeric proteins by recombining protein fragments from structurally homologous parent proteins. Using I27 and I32 domains from the muscle protein titin as parent template proteins, we systematically shuffled the secondary structural elements of the two parent proteins and engineered 13 hybrid daughter proteins. Although I27 and I32 are highly homologous, and homology modeling predicted that the hybrid daughter proteins fold into structures that are similar to that of parent protein, we found that only eight of the 13 daughter proteins showed beta-sheet dominated structures that are similar to parent proteins, and the other five recombined proteins showed signatures of the formation of significant alpha-helical or random coil-like structure. Single molecule AFM revealed that six recombined daughter proteins are mechanically stable and exhibit mechanical properties that are different from the parent proteins. In contrast, another four of the hybrid proteins were found to be mechanically labile and unfold at forces that are lower than the approximately 20 pN, as we could not detect any unfolding force peaks. The last three hybrid proteins showed interesting duality in their mechanical unfolding behaviors. These results demonstrate the great potential of using recombination-based approaches to engineer novel elastomeric protein domains of diverse mechanical properties. Moreover, our results also revealed the challenges and complexity of developing a recombination-based approach into a laboratory-based directed evolution approach to engineer novel elastomeric proteins.     

Partial purification and characterization of GDP dissociation stimulator (GDS) for the rho proteins from bovine brain cytosol

A novel type of regulatory proteins for the rho proteins (rhoA p21 and rhoB p20), ras p21-like small GTP-binding proteins (G proteins), are partially purified from bovine brain cytosol. These regulatory proteins, named rho GDP dissociation stimulator (GDS) 1 and -2, stimulate the dissociation of GDP from rhoA p21 and rhoB p20. rho GDS1 and -2 are inactive for other ras p21/ras p21-like small G proteins including c-Ha-ras p21, smg p21B, and smg p25A. Since we have previously shown that the rate limiting step for the GDP/GTP exchange reaction of the rho proteins is the dissociation of GDP from these proteins, the present results suggest that rho GDS1 and -2 stimulate the GDP/GTP exchange reaction of the rho proteins. rho GDS1 and -2 are distinct from the GAP- and GDI-types of regulatory proteins for the rho proteins previously purified from bovine brain cytosol. rho GAP stimulates the GTPase activity of the rho proteins and rho GDI inhibits the GDP/GTP exchange reaction of the rho proteins. The present results together with these earlier observations indicate that the rho proteins are regulated by at least three different types of regulatory proteins, GDS, GDI, and GAP.     

Transport of cytoplasmically synthesized proteins into the mitochondria in a cell free system from Neurospora crassa.

Synthesis and transport of mitochondrial proteins were followed in a cell-free homogenate of Neurospora crassa in which mitochondrial translation was inhibited. Proteins synthesized on cytoplasmic ribosomes are transferred into the mitochondrial fraction. The relative amounts of proteins which are transferred in vitro are comparable to those transferred in whole cells. Cycloheximide and puromycin inhibit the synthesis of mitochondrial proteins but not their transfer into mitochondria. The transfer of immunoprecipitable mitochondrial proteins was demonstrated for matrix proteins, carboxyatractyloside-binding protein and cytochrome c. Import of proteins into mitochondria exhibits a degree of specificity. The transport mechanism differentiates between newly synthesized proteins and preexistent mitochondrial proteins, at least in the case of matrix proteins. In the cell-free homogenate membrane-bound ribosomes are more active in the synthesis of mitochondrial proteins than are free ribosomes. The finished translation products appear to be released from the membrane-bound ribosomes into the cytosol rather than into the membrane vesicles. The results suggest that the transport of cytoplasmically synthesized mitochondrial proteins is essentially independent of cytoplasmic translation; that cytoplasmically synthesized mitochondrial proteins exist in an extramitochondrial pool prior to import; that the site of this pool is the cytosol for at least some of the mitochondrial proteins; and that the precursors in the extramitochondrial pool differ in structure or conformation from the functional proteins in the mitochondria.     

大肠埃希氏杆菌UTI89分泌蛋白组的预测分析

目的：从大肠埃希氏杆菌UTI89基因组中筛选出全部潜在的分泌蛋白并进行初步研究。方法：使用SignalP3.0、TatP1.0、 SecretomeP2.0等蛋白分析软件对5211个ORF进行预测;对筛选出的信号肽及分泌蛋白的基本特征进行统计学分析;使用Blast 2 Sequences进行同源性分析。结果：共筛选出432个sec途径分泌蛋白,19个Tat途径分泌蛋白,386个非经典分泌蛋白;信号肽、分泌蛋白平均长度分别为25.5aa、282.8aa;信号肽中出现频率最高的3种氨基酸依次为L、A、S;仅有两个信号肽的氨基酸序列完全相同,相应的分泌蛋白高度同源。结论：大肠埃希氏杆菌UTI89基因组中有837个ORF可能编码分泌蛋白;分泌蛋白集中在500aa以下;组成信号肽的氨基酸相对保守,多数为疏水氨基酸;信号肽变异性较大,含相同信号肽的蛋白可能由同源基因编码。     

Ribosomal proteins in halobacteria

The amino acid sequences of 16 ribosomal proteins from archaebacterium Halobacterium marismortui have been determined by a direct protein chemical method. In addition, amino acid sequences of three proteins, S11, S18, and L25, have been established by DNA sequencing of their genes as well as by protein sequencing. Comparison of their sequences with those of ribosomal proteins from other organisms revealed that proteins S14, S16, S19, and L25 are related to both eukaryotic and eubacterial ribosomal proteins, being more homologous to eukaryotic than eubacterial counterparts, and proteins S12, S15, and L16 are related to only eukaryotic ribosomal proteins. Furthermore, some proteins are found to be similar to only eubacterial proteins, whereas other proteins show no homology to any other known ribosomal proteins. Comparisons of amino acid compositions between halophilic and nonhalophilic ribosomal proteins revealed that halophilic proteins gain aspartic and glutamic acid residues and significantly lose lysine and arginine residues. In addition, halophilic proteins seem to lose isoleucine as compared with Escherichia coli ribosomal proteins.     

The cuticle proteins of Drosophila melanogaster: stage specificity

Five stage-specific cuticles are produced during the development of Drosophila. Urea-soluble proteins were extracted from each developmental stage and compared by gel electrophoresis. Proteins from first and second instar cuticle are identical except for minor differences in two proteins. Each subsequent stage, third instar, pupa, and adult, has a unique set of cuticle proteins. Qualitative changes within stages are seen in proteins from third instar and adult cuticle. Third instar cuticle proteins can be divided into “early” [proteins 2a, 3, 4, 5, 7, and 8] and “late” [proteins 2 and 1] groups. Adult cuticle proteins change in relative amounts during pharate adult development and change mobility at eclosion. The lower abdominal pupal cuticle lacks a protein found in the pupal cuticle covering the head and thorax. Cuticle proteins from each stage are immunologically related. Nonetheless, electrophoretic variants of three larval proteins do not affect any major changes in the electrophoretic mobility of proteins from other stages. We propose that each stage (except first and second instar) has proteins encoded by discrete genes.     

Ankyrin repeat-containing proteins in Arabidopsis: characterization of a novel and abundant group of genes coding ankyrin-transmembrane proteins

Ankyrin repeats are present in a great variety of proteins of eukaryotes, prokaryotes and some viruses and they function as protein-protein interaction domains. We have search for all the ankyrin repeats present in Arabidopsis proteins and determined their consensus sequence. We identified a total of 509 ankyrin repeats present in 105 proteins. Ankyrin repeat containing proteins can be classified in 16 groups of structurally similar proteins. The most abundant group contains proteins with ankyrin repeats and transmembrane domains (AtANKTM). Sequence similarity analysis indicates that these proteins are divided in six families. Some of the AtAnkTm genes are organized in tandem arrays and others are present in duplicated parts of the Arabidopsis genome. The expression of several AtAnkTm genes was analyzed resulting in a wide variety of expression patterns even within the same family. The likely functions of these proteins are discussed in comparison with the known functions of proteins with similar organization in other species.