Physiological and pathophysiological aspects of gastrointestinal peptide hormones.

The author made a review about the origin, the biochemistry the physiological and pathological roles of gastrointestinal peptide hormones. They originate from the APUD cell system, chemically from the ancient growth hormone, or placental lactogen. The theoretical prosecgastrin's first sequencies form the "secretin family", the tail sequencies form the "gastrin family". The author describes many details of their effects on the different gastrointestinal organs, they behave mainly antagonistic way to each other. Finally a discussions is given about their role in the development of peptic ulcer, in the WDHA syndrome and in malabsorption.     

A mutation in the receiver domain of the Agrobacterium tumefaciens transcriptional regulator VirG increases its affinity for operator DNA

Early genetic analysis of alternate recombination pathways in Escherichia coli identified the RecE recombination pathway and the required exonuclease VIII encoded by the recE gene. Observations that not ail recombination events promoted by the RecE pathway require recA suggest the existence of an additional homologous pairing protein besides RecA in E. coli. Genetic and biochemical analysis of the recE gene region indicates there are two partially overlapping genes, recE and recT, encoding at least two proteins: exoVIII and the RecT protein. Biochemical analysis has shown that the RecT protein, in combination with exoVIII, promotes homologous pairing and strand exchange in reactions containing linear duplex DNA and homologous, circular, single-stranded DNA as substrates. This reaction occurs in the absence of any high-energy cofactor. These two proteins, RecT and exoVIII, appear to be members of a second class of homologous pairing proteins that are required in genetic recombination and differ from the class of homologous pairing proteins that includes RecA. Members of this second class of proteins appear to include both bacteriophage-encoded proteins and proteins from eukaryotes and their viruses.     

The complete amino acid sequences of ribosomal proteins L17, L27, and S9 from Bacillus stearothermophilus

The complete primary structures of proteins L17, L27 and S9 extracted from the Bacillus stearothermophilus ribosomes with 1 M NaCl and purified to homogeneity by column chromatography have been determined. The amino acid sequences of these proteins are compared to those of the homologous ribosomal proteins from Escherichia coli. The number of identical amino acid residues between the homologous proteins lies between 33-55%.     

Amino acid sequence homology applied to the prediction of protein secondary structures, and joint prediction with existing methods

The assumption that homologous segments in different proteins may share a similar conformation is applied to the prediction of secondary structures in proteins. Sequences homologous to a target protein are searched, without allowing any gap, and compared against a number of reference proteins of known three-dimensional structure, and then a conformational state (alpha, beta or coil) for each residue of the protein is predicted by looking at the secondary structure of corresponding homologous segments. This prediction is done in a statistical rather than 'deterministic' way, by assigning the most probable conformation state among homologous data to each residue site of a target protein. A test application for 22 sample proteins yields 60% correctness on the average, a better value in comparison with two other existing methods. Joint prediction combining three methods into one is shown to increase the reliability up to 70%, when only the regions identically predicted with the three methods are taken into account. Application of the present method to 10 proteins of unknown structure is demonstrated.     

Homologous recombination-mediated double-strand break repair

Exchange of DNA strands between homologous DNA molecules via recombination ensures accurate genome duplication and preservation of genome integrity. Biochemical studies have provided insights into the molecular mechanisms by which homologous recombination proteins perform these essential tasks. More recent cell biological experiments are addressing the behavior of homologous recombination proteins in cells. The challenge ahead is to uncover the relationship between the individual biochemical activities of homologous recombination proteins and their coordinated action in the context of the living cell.     

Regulation of DNA strand exchange in homologous recombination

Homologous recombination, the exchange of DNA strands between homologous DNA molecules, is involved in repair of many structural diverse DNA lesions. This versatility stems from multiple ways in which homologous DNA strands can be rearranged. At the core of homologous recombination are recombinase proteins such as RecA and RAD51 that mediate homology recognition and DNA strand exchange through formation of a dynamic nucleoprotein filament. Four stages in the life cycle of nucleoprotein filaments are filament nucleation, filament growth, homologous DNA pairing and strand exchange, and filament dissociation. Progression through this cycle requires a sequence of recombinase-DNA and recombinase protein-protein interactions coupled to ATP binding and hydrolysis. The function of recombinases is controlled by accessory proteins that allow coordination of strand exchange with other steps of homologous recombination and that tailor to the needs of specific aberrant DNA structures undergoing recombination. Accessory proteins are also able to reverse filament formation thereby guarding against inappropriate DNA rearrangements. The dynamic instability of the recombinase-DNA interactions allows both positive and negative action of accessory proteins thereby ensuring that genome maintenance by homologous recombination is not only flexible and versatile, but also accurate.     

A new method for identification of protein (sub)families in a set of proteins based on hydropathy distribution in proteins

Structural similarity among proteins is reflected in the distribution of hydropathicity along the amino acids in the protein sequence. Similarities in the hydropathy distributions are obvious for homologous proteins within a protein family. They also were observed for proteins with related structures, even when sequence similarities were undetectable. Here we present a novel method that employs the hydropathy distribution in proteins for identification of (sub)families in a set of (homologous) proteins. We represent proteins as points in a generalized hydropathy space, represented by vectors of specifically defined features. The features are derived from hydropathy of the individual amino acids. Projection of this space onto principal axes reveals groups of proteins with related hydropathy distributions. The groups identified correspond well to families of structurally and functionally related proteins. We found that this method accurately identifies protein families in a set of proteins, or subfamilies in a set of homologous proteins. Our results show that protein families can be identified by the analysis of hydropathy distribution, without the need for sequence alignment.     

Characterization of Lovastatin biosynthetic cluster proteins in Aspergillus terreus strain ATCC 20542

Aspergillus terreus is a filamentous ascomycota, which is prominent for its production of lovastatin, an antihypercholesterolemic drug. The commercial importance of lovastatin with annual sales of billions of dollars made us to focus on lovastatin biosynthetic cluster proteins. The analysis of these lovastatin biosynthetic cluster proteins with different perspectives such as physicochemical property, structure based analysis and functional studies were done to find out the role and function of every protein involved in the lovastatin biosynthesis pathway. Several computational tools are used to predict the physicochemical properties, secondary structural features, topology, patterns, domains and cellular location. There are 8 unidentified proteins in lovastatin biosynthetic cluster, in which 6 proteins have homologous partners, and annotation transfer is done based on the closely related homologous genes, and their structures are also modeled. The two other proteins that do not have homologous partners are predicted as PQ loop repeat protein that may be involved in glycosylation machinery and as thiolase-acyl activity by the integrated functional analysis approach.     

Chemical shift homology in proteins

The degree of chemical shift similarity for homologous proteins has been determined from a chemical shift database of over 50 proteins representing a variety of families and folds, and spanning a wide range of sequence homologies. After sequence alignment, the similarity of the secondary chemical shifts of C protons was examined as a function of amino acid sequence identity for 37 pairs of structurally homologous proteins. A correlation between sequence identity and secondary chemical shift rmsd was observed. Important insights are provided by examining the sequence identity of homologous proteins versus percentage of secondary chemical shifts that fall within 0.1 and 0.3 ppm thresholds. These results begin to establish practical guidelines for the extent of chemical shift similarity to expect among structurally homologous proteins.     

Detecting distant homologies of mosaic proteins. Analysis of the sequences of thrombomodulin, thrombospondin complement components C9, C8 alpha and C8 beta, vitronectin and plasma cell membrane glycoprotein PC-1

Recognition of homologies may give hints about the structure and function of proteins; therefore, we are developing strategies to aid sequence comparisons. Detecting homology of mosaic proteins is especially difficult since the modules constituting these proteins are usually distantly related and their homology is not readily recognized by conventional computer programs. In the present work we show that the rules of the evolution of mosaic proteins can guide the identification of modules of mosaic proteins and can delineate the group of sequences in which the presence of homologous sequences may be expected. By this approach we can concentrate the search for homology to a limited group of sequences; thus ensuring a more intense and more fruitful search. The power of this approach is illustrated by the fact that it could detect homologies not identified by earlier methods of sequence comparison. In this paper we show that thrombomodulin contains a domain homologous with animal lectins, that complement components C9, C8 alpha and C8 beta have modules homologous with one of the repeat units of thrombospondin and that the somatomedin B module of vitronectin is homologous with the internal repeats of plasma cell membrane glycoprotein PC-1.     

基于结构比较的蛋白质同源模建系统及其评估Ⅱ侧链的安装

至今,有关蛋白质侧链的同源模建,除了在本体模板上安装侧链和少数限制条件下在同源模板上安装侧链的报道外,系统的研究和实施似乎还未见报道。本软件系统ＰＭＯＤＥＬＩＮＧ采用同源移植和“死端排除“相结合的侧链安装策略,对与模板蛋白相应践基具有相似大小和形状的目标残基采用直接移植的方法。其余铡链则用广义“死端排除定则”安装。经众多蛋白的测试,达到了较好的模建品质。     

基于结构比较的蛋白质同源模建系统及其评估Ⅱ侧链的安装

至今,有关蛋白质侧链的同源模建,除了在本体模板上安装侧链和少数限制条件下在同源模板上安装侧链的报道外,系统的研究和实施似乎还未见报道。本软件系统ＰＭＯＤＥＬＩＮＧ采用同源移植和“死端排除“相结合的侧链安装策略,对与模板蛋白相应践基具有相似大小和形状的目标残基采用直接移植的方法。其余铡链则用广义“死端排除定则”安装。经众多蛋白的测试,达到了较好的模建品质。     

Regulation of double-strand break-induced mammalian homologous recombination by UBL1, a RAD51-interacting protein

Mammalian RAD51 protein plays essential roles in DNA homologous recombination, DNA repair and cell proliferation. RAD51 activities are regulated by its associated proteins. It was previously reported that a ubiquitin-like protein, UBL1, associates with RAD51 in the yeast two-hybrid system. One function of UBL1 is to covalently conjugate with target proteins and thus modify their function. In the present study we found that non-conjugated UBL1 forms a complex with RAD51 and RAD52 proteins in human cells. Overexpression of UBL1 down-regulates DNA double-strand break-induced homologous recombination in CHO cells and reduces cellular resistance to ionizing radiation in HT1080 cells. With or without overexpressed UBL1, most homologous recombination products arise by gene conversion. However, overexpression of UBL1 reduces the fraction of bidirectional gene conversion tracts. Overexpression of a mutant UBL1 that is incapable of being conjugated retains the ability to inhibit homologous recombination. These results suggest a regulatory role for UBL1 in homologous recombination.     

Common stressor proteins in bacteria

Research data on common stressor proteins of bacteria obtained during recent 10 years are updated and analyzed. Bacteria of one and the same species were shown to give similar response to the action of different stressors; the main stressor proteins of different bacteria appeared to be homologous; bacteria have cross protection from different stressors. In addition, some common stressor proteins of bacteria were found to be homologous with human antigens that is of great importance for immunobiotechnology.     

Primary structures of five ribosomal proteins from the archaebacterium Halobacterium marismortui and their structural relationships to eubacterial and eukaryotic ribosomal proteins

The complete amino acid sequences of ribosomal proteins L9, L20, L21/22, L24 and L32 from the archaebacterium Halobacterium marismortui were determined. The comparison of the sequences of these proteins with those from other organisms revealed that proteins L21/22 and L24 are homologous to ribosomal protein Yrp29 from yeast and L19 from rat, respectively, and that H. marismortui L20 is homologous to L30 from eubacteria. H. marismortui ribosomal protein L9 showed sequence homology to both L29 from yeast and L15 from eubacteria. No homologous protein was found for H. marismortui L32. These results are discussed with respect to the phylogenetic relationship between eubacteria, archaebacteria and eukaryotes.     

Construction of a series of ompF-ompC chimeric genes by in vivo homologous recombination in Escherichia coli and characterization of the translational products.

OmpF and OmpC are major outer membrane proteins. Although they are homologous proteins, they function differently in several respects. As an approach to elucidate the submolecular structures that determine the difference, a method was developed to construct a series of ompF-ompC chimeric genes by in vivo homologous recombination between these two genes, which are adjacent on a plasmid. The genomic structures of these chimeric genes were determined by restriction endonuclease analysis and nucleotide sequence determination. In almost all cases, recombination took place between the corresponding homologous regions of the ompF and ompC genes. Many of the chimeric genes produced proteins that migrated to various positions between the OmpF and OmpC proteins on polyacrylamide gel. On the basis of the results, a domain contributing to the mobility difference the OmpF and OmpC proteins was identified. Some chimeric genes did not accumulate outer membrane proteins, despite the fact that the fusion of the ompF and ompC genes was in frame. Bacterial cells possessing the chimeric proteins were also tested as to their sensitivity to phages which require either OmpF or OmpC as a receptor component. The chimeric proteins were either of the OmpF or OmpC type with respect to receptor activity. Based on the observations, the roles of submolecular domains in the structure, function, and biogenesis of the OmpF and OmpC proteins are discussed.     

Diverse proteins homologous to inositol monophosphatase.

Bovine inositol monophosphatase (IMP) and several homologous proteins were found to share two sequence motifs with bovine inositol polyphosphate 1-phosphatase (IPP). These motifs may correspond to binding sites within IMP and IPP for inositol phosphates or for lithium, since both substances are bound by these proteins. This suggests that the proteins homologous to IMP, which have diverse biological roles but whose function is not clear, may act by enhancing the synthesis or degradation of phosphorylated compounds.     

Effect of ligand orientation on the redox potential of homologous proteins

Using experimental g-values of homologous cytochromes isolated from the horse heart and bacterial cyt-c-551 from Pseudomonas aeruginosa, the electron energy difference of redox-orbital of Fe(III) ion of heme was calculated during reduction. Data are in a good accordance with experimental redox-potential values for these proteins. The model gives opportunity to predict redox-potential values for other homologous proteins.     

Efficient production of secreted proteins by Aspergillus : progress, limitations and prospects

Filamentous fungi are widely used for the production of homologous and heterologous proteins but, compared to homologous proteins, the levels of production of heterologous proteins are usually low. During the last 5 years, the levels of production of heterologous proteins have been drastically improved by fusing the corresponding gene to the 3' end of a homologous gene, encoding a well-secreted protein such as glucoamylase. Nevertheless, little research has been carried out to determine the limitations that hamper heterologous protein production. Recently we have carried out a detailed analysis of the levels of production of several proteins and glucoamylase fusion proteins in defined recombinant Aspergillus awamori strains. In this review we will focus on the use of filamentous fungi for the production of heterologous, especially non-fungal, proteins. In particular, the effect of gene-fusion strategies will be reviewed. Furthermore, the remaining limitations in heterologous protein production and suggestions for improvement strategies for overproduction of these protein will be discussed. Received: 5 July 1996 / Accepted: 6 September 1996