Über die Bindungsart der amidierten Reste derβ-Aminodicarbonsäuren in Eiweißstoffen

Summary Gliadin and chymotrypsinogen, respectively, were treated with alkaline sodium hypochlorite solution equivalent to the carbonamide groups present, and then hydrolysed by acid. The proteins treated with dilute alkaline solution have been assayed in control experiments for their content of N-terminal amino acids. The degradation products identified by paper chromatography seem to indicate that in chymotrypsinogen the amido groups of the amino dicarboxylic acids represent asparaginyl (Ia) and glutaminyl (Ib) residues. In gliadin, besides these residues, the presence of isoglutaminyl residues (IVb) also seems to be probable.     

Proteolysis in protein import and export: signal peptide processing in eu- and prokaryotes.

Numerous proteins in pro- and eukaryotes must cross cellular membranes in order to reach their site of function. Many of these proteins carry signal sequences that are removed by specific signal peptidases during, or shortly after, membrane transport. Signal peptidases have been identified in the rough endoplasmic reticulum, the matrix and inner membrane of mitochondria, the stroma and thylakoid membrane of chloroplasts, the bacterial plasma membrane and the thylakoid membrane of cyanobacteria. The composition of these peptidases varies between one and several subunits. No site-specific inhibitors are known for the majority of these enzymes. Accordingly, signal peptidases recognize structural motifs rather than linear amino acid sequences. Such motifs have become evident by employing extensive site-directed mutagenesis to investigate the anatomy of signal sequences. Analysis of the reaction specificities and the primary sequences of several signal peptidases suggests that the enzymes of the endoplasmic reticulum, the inner mitochondrial membrane and the thylakoid membrane of chloroplasts all have evolved from bacterial progenitors.     

Proteolysis in protein import and export: Signal peptide processing in eu-and prokaryotes

Numerous proteins in pro-and eukaryotes must cross cellular membranes in order to reach their site of function. Many of these proteins carry signal sequences that are removed by specific signal peptidases during, or shortly after, membrane transport. Signal peptidases have been identified in the rough endoplasmic reticulum, the matrix and inner membrane of mitochondria, the stroma and thylakoid membrane of chloroplasts, the bacterial plasma membrane and the thylakoid membrane of cyanobacteria. The composition of these peptidases varies between one and several subunits. No site-specific inhibitors are known for the majority of these enzymes. Accordingly, signal peptidases recognize structural motifs rather than linear amino acid sequences. Such motifs have become evident by employing extensive site-directed mutagenesis to investigate the anatomy of signal sequences. Analysis of the reaction specificities and the primary sequences of several signal peptidases suggests that the enzymes of the endoplasmic reticulum, the inner mitochondrial membrane and the thylakoid membrane of chloroplasts all have evolved from bacterial progenitors.     

The role of synapsins in neuronal development

The synapsins, the first identified synaptic vesicle-specific proteins, are phosphorylated on multiple sites by a number of protein kinases and are involved in neurite outgrowth and synapse formation as well as in synaptic transmission. In mammals, the synapsin family consists of at least 10 isoforms encoded by 3 distinct genes and composed by a mosaic of conserved and variable domains. The synapsins are highly conserved evolutionarily, and orthologues have been found in invertebrates and lower vertebrates. Within nerve terminals, synapsins are implicated in multiple interactions with presynaptic proteins and the actin cytoskeleton. Via these interactions, synapsins control several mechanisms important for neuronal homeostasis. In this review, we describe the main functional features of the synapsins, in relation to the complex role played by these phosphoproteins in neuronal development.     

Sterol carrier protein-2: structure reveals function

The multiple actions of sterol carrier protein-2 (SCP-2) in intracellular lipid circulation and metabolism originate from its gene and protein structure. The SCP-x/pro-SCP-2 gene is a fusion gene with separate initiation sites coding for 15-kDa pro-SCP-2 (no enzyme activity) and 58-kDa SCP-x (a 3-ketoacyl CoA thiolase). Both proteins share identical cDNA and amino acid sequences for 13-kDa SCP-2 at their C-termini. Cellular 13-kDa SCP-2 derives from complete, posttranslational cleavage of the 15-kDa pro-SCP-2 and from partial posttranslational cleavage of 58-kDa SCP-x. Putative physiological functions of SCP-2 have been proposed on the basis of enhancement of intermembrane lipid transfer (e.g., cholesterol, phospholipid) and activation of enzymes involved in fatty acyl CoA transacylation (cholesterol esters, phosphatidic acid) in vitro, in transfected cells, and in genetically manipulated animals. At least four important SCP-2 structural domains have been identified and related to specific functions. First, the 46-kDa N-terminal presequence present in 58-kDa SCP-x is a 3-ketoacyl-CoA thiolase specific for branched-chain acyl CoAs. Second, the N-terminal 20 amino acid presequence in 15-kDa pro-SCP-2 dramatically modulates the secondary and tertiary structure of SCP-2 as well as potentiating its intracellular targeting coded by the C-terminal peroxisomal targeting sequence. Third, the N-terminal 32 amino acids form an amphipathic a-helical region, one face of which represents a membrane-binding domain. Positively charged amino acid residues in one face of the amphipathic helices allow SCP-2 to bind to membrane surfaces containing anionic phospholipids. Fourth, the hydrophobic faces of the N-terminal amphipathic a helices along with beta strands 4, 5, and helix D form a ligand-binding cavity able to accommodate multiple types of lipids (e. g., fatty acids, fatty acyl CoAs, cholesterol, phospholipids, isoprenoids). Two-dimensional 1H-15N heteronuclear single quantum coherence spectra of both apo-SCP-2 and of the 1:1 oleate-SCP-2 complex, obtained at pH 6.7, demonstrated the homogenous formation of holo-SCP-2. While comparison of the apo- and holoprotein amide fingerprints revealed about 60% of the resonances remaining essentially unchanged, 12 assigned amide residues underwent significant chemical-shift changes upon oleic acid binding. These residues were localized in three regions: the juncture of helices A and B, the mid-section of the beta sheet, and the interface formed by the region of beta strands 4, 5, and helix D. Circular dichroism also showed that these chemical-shift changes, upon oleic acid binding, did not alter the secondary structure of SCP-2. The nuclear magnetic resonance chemical shift difference data, along with mapping of the nearby hydrophobic residues, showed the oleic acid-binding site to be comprised of a pocket created by the face of the beta sheet, helices A and B on one end, and residues associated with beta strands 4, 5, and helix D at the other end of the binding cavity. Furthermore, the hydrophobic nature of the previously ill-defined C-terminus suggested that these 20 amino acids may form a 'hydrophobic cap' which closes around the oleic acid upon binding. Thus, understanding the structural domains of the SCP-x/pro-SCP-2 gene and its respective posttranslationally processed proteins has provided new insights into their functions in intracellular targeting and metabolism of lipids.     

Yeast prions and human prion-like proteins: sequence features and prediction methods

Prions are self-propagating infectious protein isoforms. A growing number of prions have been identified in yeast, each resulting from the conversion of soluble proteins into an insoluble amyloid form. These yeast prions have served as a powerful model system for studying the causes and consequences of prion aggregation. Remarkably, a number of human proteins containing prion-like domains, defined as domains with compositional similarity to yeast prion domains, have recently been linked to various human degenerative diseases, including amyotrophic lateral sclerosis. This suggests that the lessons learned from yeast prions may help in understanding these human diseases. In this review, we examine what has been learned about the amino acid sequence basis for prion aggregation in yeast, and how this information has been used to develop methods to predict aggregation propensity. We then discuss how this information is being applied to understand human disease, and the challenges involved in applying yeast prediction methods to higher organisms.     

Expansion of amino acid homo-sequences in proteins: Insights into the role of amino acid homo-polymers and of the protein context in aggregation

Expansion of amino acid homo-sequences, such as polyglutamines or polyalanines, in proteins has been directly implicated in various degenerative diseases through a mechanism of protein misfolding and aggregation. However, it is still unclear how the nature of the expansion and the protein context influence the tendency of a protein to aggregate. Here, we have addressed these questions using spinocerebellar ataxia type-3 (ATX3) protein, the best characterised of the polyglutamine proteins, chosen as a model system. Using a transfected mammalian cell line, we demonstrate that ATX3 aggregation is noticeably reduced by deletion or replacement of regions other than the polyglutamine tract. The nature of the amino acid homo-sequences also has a strong influence on aggregation. From our studies, we draw general conclusions on the effect of the protein architecture and of the amino acid homo-sequence on pathology. Received 3 March 2006; received after revision 19 April 2006; accepted 22 May 2006     

Isolation of an antifreeze peptide from the Antarctic sponge <Emphasis Type="Italic">Homaxinella balfourensis</Emphasis>

Polar plants and animals survive in subzero waters (-2 degrees C) and many of these marine organisms produce antifreeze proteins (AFPs) to better adapt themselves to these conditions. AFPs prevent the growth of ice crystals which disrupt cellular membranes and destroy cells by inhibiting crystallization of water within the organism. The hydrophilic extract of an Antarctic sponge Homaxinella balfourensis exhibited a non-colligative freezing point depression effect on the crystal morphology of water. The extract was purified by repeated reverse phase high-pressure liquid chromatography, then assayed and shown to contain several AFPs. The major peptide was isolated, analyzed using matrix-assisted laser desorption ionization mass spectrometry and the partial structure of the peptide identified through amino acid sequencing. AFPs have potential applications in agriculture, medicine and the food industry.     

Free amino acids in the adult citrus brown mite,Eutetranychus orientalis (Klein)

Summary Amino acids contained in extracts of adultEutetranychus orientalis were separated and determined quantitatively by 2-dimensional paper chromatography. 14 amino acids were identified. Asparagine, ornithine, histidine, lysine, aspartic acid, serine and glycine were the major components of the free amino acid pool, comprising 83.94% of the total content.     

Purification and partial characterization of two lectins fromMomordica charantia

Summary 2 different lectins have been purified from the seeds ofMomordica charantia by gel-filtration and ion-exchange chromatography. These 2 lectins appear to be composed of 2 subunits of 26,000 daltons. Protein fraction I, but not II, showed agglutinating activity toward human type-O red blood cells. The amino acid compositions and amino-terminal sequences of these two homologous proteins are quite different.Acknowledgment. The skillful assistance of Ms M. Diane Forde is greatly acknowledged. Part of this work was supported by NIH grants CA18621 and AI09810 when the author was at Mount Sinai School of Medicine.     

Small,novel proteins from the mistletoe <Emphasis Type="Italic">Phoradendron tomentosum</Emphasis> exhibit highly selective cytotoxicity to human breast cancer cells

Four novel proteins (phoratoxins C–F) have been isolated from the North American mistletoe Phoradendron tomentosum. The amino acid sequences of these phoratoxins were determined unambiguously using a combination of Edman degradation and trypsin enzymatic digestion, and by electrospray ionization tandem mass spectrometry sequencing. Phoratoxins C, E and F consist of 46 amino acid residues; and phoratoxin D of 41. All proteins had six cysteines, similar to the earlier described phoratoxins A and B, which are thionins. The cytotoxicity of each protein was evaluated in a human cell line panel that represented several cytotoxic drug-resistance mechanisms. For the half-maximal inhibitory concentrations (IC₅₀ values) of the different cell lines in the panel, correlation with those of standard drugs was low. The most potent cytotoxic phoratoxin C was further tested on primary cultures of human tumor cells from patients. The solid tumor samples from breast cancer cells were 18 times more sensitive to phoratoxin C than the tested hematological tumor samples. Received 30 September 2002; received after revision 28 October 2002; accepted 7 November 2002 RID="*" ID="*"Corresponding author.     

Compatible solutes of halophilic eubacteria: molecular principles,water-solute interaction,stress protection

Compatible solutes are best described as organic osmolytes responsible for osmotic balance and at the same time compatible with the cells' metabolism. A comprehensive survey (using HPLC and NMR methods) on halophilic/halotolerant eubacteria has revealed the full diversity of compatible solutes employed in nature. Molecular principles derived from the spectrum of compounds found in the bacterial world may be summarized as follows. Compatible solutes are polar, highly soluble molecules and uncharged at physiological pH. With the exception of proline (a proteinogenic amino acid) they are characterized as amino acid derivatives of the following types: betaines, ectoines, N-acetylated diamino acids and N-derivatized carboxamides of glutamine. Using nearinfrared spectroscopy we have also been able to demonstrate that compatible solutes are strong water-structure formers and as such probably excluded from the hydration shell of proteins. This preferential exclusion probably explains their function as effective stabilizers of the hydration shell of native proteins (protection against heating freezing and drying). Hence these typical products of halophilic eubacteria have a considerable potential as stabilizing/protecting agents on both molecular and whole-cell level. Thorough understanding of common structural principles and fundamental water-solute interactions will ultimately enable us to design novel highly efficient stress protectants and stabilizers of biomolecules.     

Functions of fatty acid binding proteins

Summary Cytosolic fatty acid binding proteins (FABP) belong to a gene family of which eight members have been conclusively identified. These 14–15 kDa proteins are abundantly expressed in a highly tissue-specific manner. Although the functions of the cytosolic FABP are not clearly established, they appear to enhance the transfer of long-chain fatty acids between artificial and native lipid membranes, and also to have a stimulatory effect on a number of enzymes of fatty acid metabolism in vitro. These findings, as well as the tissue expression, ligand binding properties, ontogeny and regulation of these proteins provide a considerable body of indirect evidence supporting a broad role for the FABP in the intracellular transport and metabolism of long-chain fatty acids. The available data also support the existence of structure- and tissue-specific specialization of function among different members of the FABP gene family. Moreover, FABP may also have a possible role in the modulation of cell growth and proliferation, possibly by virtue of their affinity for ligands such as prostaglandins, leukotrienes and fatty acids, which are known to influence cell growth activity. FABP structurally unrelated to the cytosolic gene family have also been identified in the plasma membranes of several tissues (FABP_pm). These proteins have not been fully characterized to date, but strong evidence suggests that they function in the transport of long-chain fatty acids across the plasma membrane.     

Influence of nitrogen components of wine on the growth of lactic acid bacteria]

Three different fractions of nitrogen compounds have been extracted from wine: proteins, peptides, amino acids. They have been tested to support the growth of malolactic bacteria in acid medium: only the peptidic fraction supports this growth.     

Biogenesis of prokaryotic pores.

The prokaryotic pore-forming proteins are synthesized in the cytoplasm, and are assembled in their functional form in the outer membrane. They begin to traverse the cytoplasmic membrane via the SecY/SecA export pathway, which is shared also by periplasmic proteins. The sorting signals that direct these proteins to the outer membrane could be present in the three-dimensional conformations of the proteins, but some results suggest that they may be present in short, contiguous sequences. Outer membrane proteins share a rather hydrophilic amino acid composition, and appear to be rich in beta-sheets (with the exception of lipoproteins). This observation as well as the demonstration of periplasmic export intermediates favor the secretion pathway through the periplasm, as opposed to export through fusion sites between the inner and the outer membrane, but such intermediates have not yet been observed with the wild type proteins under physiological conditions.     

Functions of fatty acid binding proteins

Cytosolic fatty acid binding proteins (FABP) belong to a gene family of which eight members have been conclusively identified. These 14-15 kDa proteins are abundantly expressed in a highly tissue-specific manner. Although the functions of the cytosolic FABP are not clearly established, they appear to enhance the transfer of long-chain fatty acids between artificial and native lipid membranes, and also to have a stimulatory effect on a number of enzymes of fatty acid metabolism in vitro. These findings, as well as the tissue expression, ligand binding properties, ontogeny and regulation of these proteins provide a considerable body of indirect evidence supporting a broad role for the FABP in the intracellular transport and metabolism of long-chain fatty acids. The available data also support the existence of structure- and tissue-specific specialization of function among different members of the FABP gene family. Moreover, FABP may also have a possible role in the modulation of cell growth and proliferation, possibly by virtue of their affinity for ligands such as prostaglandins, leukotrienes and fatty acids, which are known to influence cell growth activity. FABP structurally unrelated to the cytosolic gene family have also been identified in the plasma membranes of several tissues (FABPpm). These proteins have not been fully characterized to date, but strong evidence suggest that they function in the transport of long-chain fatty acids across the plasma membrane.     

基于一种动态特征选择融合算法的蛋白质结构类预测

本文根据氨基酸理化性质，基于氨基酸组成成分与自相关函数相结合特征提取法从非同源蛋白质序列中提取七个特征集，采用局部正确性的动态特征选择算法进行多特征组合来预测蛋白质结构类，并与各个特征集进行了比较。结果表明，DFS_LA算法的预测总精度较各个特征集均有不同程度的提高。Jackknife检验下，DFS_LA算法的预测总精度为82．80％，比COMP特征集提高8．91％；独立测试检验下，DFS_LA算法的预测总精度为86．67％，比COMP特征集提高11．67％。这说明DFS_A算法可有效提高结构类预测精度，多特征组合能在一定程度上更多地反映蛋白质的空间结构信息。     

Regulatory mechanisms involved in modulating RGS function

Regulator of G-Protein Signaling (RGS) refers to a conserved 120–125 amino acid motif that was first identified by its ability to negatively regulate G-Protein-Coupled Receptor (GPCR) signalling. Mechanistically, RGSs were found to regulate GPCR responses by binding to and stimulating the GTPase activity of the receptor-activated GTP-bound G α subunits. There are now over 25 mammalian RGSs containing proteins that are reported to carry out a variety of functions, many of which are unrelated to GPCR signalling. RGS proteins range in size from small proteins that contain little more than an RGS box to very large proteins that contain a variety of domains. The selectivity of function of the RGS proteins is attributable to the divergence of the RGS sequences as well as the presence of a variety of functional motifs, which allow them to interact with other proteins. Here we focus on the RGSs that are involved in modulating GPCR signalling by reviewing the diversity of the mechanisms involved in regulating these RGSs. Received 9 February 2006; received after revision 4 May 2006; accepted 22 May 2006     

Cytoplasmic14C-labeled arginine basic proteins of Ehrlich ascites tumour cells

Summary With the use of¹⁴C-arginine it is shown that among soluble cytoplasmic proteins of Ehrlich ascites tumour cells there appear basic proteins rich in arginine with the content of amino acid higher than 14%. The amount of these proteins is about 10%. The role of arginine-rich basic proteins in the cytoplasma is briefly discussed.Supported by Polish National Cancer Programe PRC Project No. 1318/13. The authors are grateful to Mrs Anna Zbroch and Elzbieta Lipczyska for skillful technical assistance, during the course of this work.