Structural disorder: a tool for housekeeping proteins performing tissue-specific interactions

An interaction between a pair of proteins unique for a particular tissue is denoted as a tissue-specific interaction (TSI). Tissue-specific (TS) proteins always perform TSIs with a limited number of interacting partners. However, it has been claimed that housekeeping (HK) proteins frequently take part in TSIs. This is actually an unusual phenomenon. How a single HK protein mediates TSIs – remains an interesting yet an unsolved question. We have hypothesized that HK proteins have attained a high degree of structural flexibility to modulate TSIs efficiently. We have observed that HK proteins are selected to be intrinsically disordered compared to TS proteins. Therefore, the purposeful adaptation of structural disorder brings out special advantages for HK proteins compared to TS proteins. We have demonstrated that TSIs may play vital roles in shaping the molecular adaptation of disordered regions within HK proteins. We also have noticed that HK proteins, mediating a huge number of TSIs, have a greater portion of their interacting interfaces overlapped with the adjacent disordered segment. Moreover, these HK proteins, mediating TSIs, preferably adapt single domain (SD). We have concluded that HK proteins adapt a high degree of structural flexibility to mediate TSIs. Besides, having a SD along with structural flexibility is more economic than maintaining multiple domains with a rigid structure. This assists them in attaining various structural conformations upon binding to their partners, thereby designing an economically optimum molecular system.     

昆虫抗冻蛋白的研究进展

抗冻蛋白是一类与冰晶有亲合力,能够与冰晶结合并抑制冰晶生长的蛋白或糖蛋白。自20世纪60年代以来,研究人员已经分别从鱼类、昆虫、植物、真菌和细菌中发现多种抗冻蛋白。其中已知鱼类抗冻蛋白有5种,也是研究最详细的。但是,近几年来发现昆虫抗冻蛋白活性普遍比较高,因此受到研究人员重视,研究取得了较快的发展。主要讨论昆虫抗冻蛋白的结构特点、抗冻活性、作用机制和应用,并分析目前的研究现状提出一些待解决的问题,以期望对昆虫抗冻蛋白的研究进行比较系统化的整理。     

Comparative analysis of SET domain proteins in maize and Arabidopsis reveals multiple duplications preceding the divergence of monocots and dicots

Histone proteins play a central role in chromatin packaging, and modification of histones is associated with chromatin accessibility. SET domain [Su(var)3-9, Enhancer-of-zeste, Trithorax] proteins are one class of proteins that have been implicated in regulating gene expression through histone methylation. The relationships of 22 SET domain proteins from maize (Zea mays) and 32 SET domain proteins from Arabidopsis were evaluated by phylogenetic analysis and domain organization. Our analysis reveals five classes of SET domain proteins in plants that can be further divided into 19 orthology groups. In some cases, such as the Enhancer of zeste-like and trithorax-like proteins, plants and animals contain homologous proteins with a similar organization of domains outside of the SET domain. However, a majority of plant SET domain proteins do not have an animal homolog with similar domain organization, suggesting that plants have unique mechanisms to establish and maintain chromatin states. Although the domains present in plant and animal SET domain proteins often differ, the domains found in the plant proteins have been generally implicated in protein-protein interactions, indicating that most SET domain proteins operate in complexes. Combined analysis of the maize and Arabidopsis SET domain proteins reveals that duplication of SET domain proteins in plants is extensive and has occurred via multiple mechanisms that preceded the divergence of monocots and dicots.     

Intrinsically disordered regions have specific functions in mitochondrial and nuclear proteins

Proteins in general consist not only of globular structural domains (SDs), but also of intrinsically disordered regions (IDRs), i.e. those that do not assume unique three-dimensional structures by themselves. Although IDRs are especially prevalent in eukaryotic proteins, the functions are mostly unknown. To elucidate the functions of IDRs, we first divided eukaryotic proteins into subcellular localizations, identified IDRs by the DICHOT system that accurately divides entire proteins into SDs and IDRs, and examined charge and hydropathy characteristics. On average, mitochondrial proteins have IDRs more positively charged than SDs. Comparison of mitochondrial proteins with orthologous prokaryotic proteins showed that mitochondrial proteins tend to have segments attached at both N and C termini, high fractions of which are IDRs. Segments added to the N-terminus of mitochondrial proteins contain not only signal sequences but also mature proteins and exhibit a positive charge gradient, with the magnitude increasing toward the N-terminus. This finding is consistent with the notion that positively charged residues are added to the N-terminus of proteobacterial proteins so that the extended proteins can be chromosomally encoded and efficiently transported to mitochondria after translation. By contrast, nuclear proteins generally have positively charged SDs and negatively charged IDRs. Among nuclear proteins, DNA-binding proteins have enhanced charge tendencies. We propose that SDs in nuclear proteins tend to be positively charged because of the need to bind to negatively charged nucleotides, while IDRs tend to be negatively charged to interact with other proteins or other regions of the same proteins to avoid premature proteasomal degradation.     

Putative high mobility group (HMG) non-histone chromosomal proteins from wheat germ. Isolation, characterisation and partial sequence analysis

Four proteins have been isolated from wheat germ by methods analogous to those used to isolate HMG proteins from animal tissue. All four proteins have been shown to be chromosomal in origin. Although amino acid analyses show that three of these proteins have compositions similar to those of the mammalian HMG proteins, N-terminal sequence analyses of these proteins show an absence of sequence homology with any of the mammalian HMG proteins.     

Physical Features of Intracellular Proteins that Moonlight on the Cell Surface

Moonlighting proteins comprise a subset of multifunctional proteins that perform two or more biochemical functions that are not due to gene fusions, multiple splice variants, proteolytic fragments, or promiscuous enzyme activities. The project described herein focuses on a sub-set of moonlighting proteins that have a canonical biochemical function inside the cell and perform a second biochemical function on the cell surface in at least one species. The goal of this project is to consider the biophysical features of these moonlighting proteins to determine whether they have shared characteristics or defining features that might suggest why these particular proteins were adopted for a second function on the cell surface, or if these proteins resemble typical intracellular proteins. The latter might suggest that many other normally intracellular proteins found on the cell surface might also be moonlighting in this fashion. We have identified 30 types of proteins that have different functions inside the cell and on the cell surface. Some of these proteins are found to moonlight on the surface of multiple species, sometimes with different extracellular functions in different species, so there are a total of 98 proteins in the study set. Although a variety of intracellular proteins (enzymes, chaperones, etc.) are observed to be re-used on the cell surface, for the most part, these proteins were found to have physical characteristics typical of intracellular proteins. Many other intracellular proteins have also been found on the surface of bacterial pathogens and other organisms in proteomics experiments. It is quite possible that many of those proteins also have a moonlighting function on the cell surface. The increasing number and variety of known moonlighting proteins suggest that there may be more moonlighting proteins than previously thought, and moonlighting might be a common feature of many more proteins.     

Pentatricopeptide repeat proteins in Trypanosoma brucei function in mitochondrial ribosomes

The pentatricopeptide repeat (PPR), a degenerate 35-amino-acid motif, defines a novel eukaryotic protein family. Plants have 400 to 500 distinct PPR proteins, whereas other eukaryotes generally have fewer than 5. The few PPR proteins that have been studied have roles in organellar gene expression, probably via direct interaction with RNA. Here we show that the parasitic protozoan Trypanosoma brucei encodes 28 distinct PPR proteins, an extraordinarily high number for a nonplant organism. A comparative analysis shows that seven out of eight selected PPR proteins are mitochondrially localized and essential for oxidative phosphorylation. Six of these are required for the stabilization of mitochondrial rRNAs and, like ribosomes, are associated with the mitochondrial membranes. Furthermore, one of the PPR proteins copurifies with the large subunit rRNA. Finally, ablation of all of the PPR proteins that were tested induces degradation of the other PPR proteins, indicating that they function in concert. Our results show that a significant number of trypanosomal PPR proteins are individually essential for the maintenance and/or biogenesis of mitochondrial rRNAs.     

Calcium-dependent and -independent interactions of the S100 protein family

The S100 proteins comprise at least 25 members, forming the largest group of EF-hand signalling proteins in humans. Although the proteins are expressed in many tissues, each S100 protein has generally been shown to have a preference for expression in one particular tissue or cell type. Three-dimensional structures of several S100 family members have shown that the proteins assume a dimeric structure consisting of two EF-hand motifs per monomer. Calcium binding to these S100 proteins, with the exception of S100A10, results in an approx. 40 degrees alteration in the position of helix III, exposing a broad hydrophobic surface that enables the S100 proteins to interact with a variety of target proteins. More than 90 potential target proteins have been documented for the S100 proteins, including the cytoskeletal proteins tubulin, glial fibrillary acidic protein and F-actin, which have been identified mostly from in vitro experiments. In the last 5 years, efforts have concentrated on quantifying the protein interactions of the S100 proteins, identifying in vivo protein partners and understanding the molecular specificity for target protein interactions. Furthermore, the S100 proteins are the only EF-hand proteins that are known to form both homo- and hetero-dimers, and efforts are underway to determine the stabilities of these complexes and structural rationales for their formation and potential differences in their biological roles. This review highlights both the calcium-dependent and -independent interactions of the S100 proteins, with a focus on the structures of the complexes, differences and similarities in the strengths of the interactions, and preferences for homo- compared with hetero-dimeric S100 protein assembly.     

Poxvirus antagonism of innate immunity by Bcl-2 fold proteins

Poxviruses have evolved numerous mechanisms to evade host innate immunity. Sensory pathways that are activated by Toll-like and nucleotide receptors, as well as innate cell death pathways, are both targets of antagonism by viral proteins. Recent structural, biochemical and functional studies of poxvirus proteins have identified a family of α-helical proteins that adopt a Bcl-2 fold despite highly divergent polypeptide sequences from cellular proteins that regulate apoptosis. These newly identified proteins have assumed new roles in antagonism of NF-κB and interferon signaling pathways and interfere with the release of pro-inflammatory cytokines. Structures of isolated viral proteins and their complexes with cellular targets provide insight into the diverse ways that the Bcl-2 scaffold can be exploited for antagonism of host immunity.     

Design and discovery of metamorphic proteins

Metamorphic proteins are single amino acid sequences that reversibly interconvert between multiple, dramatically different native structures, often with distinct functions. Since the discovery of the first metamorphic proteins in the early 2000s, several additional metamorphic proteins have been identified, and it was suggested that up to 4% of proteins in the PDB may switch folds. Metamorphic proteins have been found to share common features such as marginal thermostability and inconsistencies in predicted secondary structures. Outstanding challenges in the field include the search for more metamorphic proteins and the design of new proteins that switch folds. Identification of novel metamorphic proteins in nature will improve therapeutic targeting of fold-switching proteins involved in human pathology and will enhance the design of protein-based therapies. Designed fold switching proteins have applications as biosensors, molecular switches, molecular machines, and self-assembling systems.     

RNA-binding proteins in plants: the tip of an iceberg?

RNA-binding proteins, which are involved in the synthesis, processing, transport, translation, and degradation of RNA, are emerging as important, often multifunctional, cellular regulatory proteins. Although relatively few RNA-binding proteins have been studied in plants, they are being identified with increasing frequency, both genetically and biochemically. RNA-binding proteins that regulate chloroplast mRNA stability and translation in response to light and that have been elegantly analyzed in Clamydomonas reinhardtii have counterparts with similar functions in higher plants. Several recent reports describe mutations in genes encoding RNA-binding proteins that affect plant development and hormone signaling.     

All-atom contact potential approach to protein thermostability analysis

In this paper we use all-atom potential energy to define and analyze the inter-residue contacts in mesophilic and thermophilic proteins. Fifteen families of proteins are selected and each family has two representative proteins with greatly different preferred environmental temperatures. We find that both the number and energy of the contacts defined in this way show stronger correlations with the preferred temperatures of proteins than other factors used before. We also find that the charged-polar and charged-nonpolar residue contacts not only have larger contact numbers but also have lower single contact energies. Furthermore, the most important is that most of the thermophilic proteins have more charged-polar and charged-nonpolar residue contacts than their mesophilic counterparts. This suggests that they may play an important role in the thermostability of proteins, except usual charged-charged and nonpolar-nonpolar residue contacts. Charged residues may exert their profound influence by forming contacts not only with other charged residues but also with polar or nonpolar residues, thus further increasing the strength of contact network and then the thermostability of proteins.     

Importance of hydrophobic cluster formation through long-range contacts in the folding transition state of two-state proteins

Understanding the folding pathways of proteins is a challenging task. The Phi value approach provides a detailed understanding of transition-state structures of folded proteins. In this work, we have computed the hydrophobicity associated with each residue in the folded state of 16 two-state proteins and compared the Phi values of each mutant residue. We found that most of the residues with high Phi value coincide with local maximum in surrounding hydrophobicity, or have nearby residues that show such maximum in hydrophobicity, indicating the importance of hydrophobic interactions in the transition state. We have tested our approach to different structural classes of proteins, such as alpha-helical, SH3 domains of all-beta proteins, beta-sandwich, and alpha/beta proteins, and we observed a good agreement with experimental results. Further, we have proposed a hydrophobic contact network pattern to relate the Phi values with long-range contacts, which will be helpful to understand the transition-state structures of folded proteins. The present approach could be used to identify potential hydrophobic clusters that may form through long-range contacts during the transition state.     

Identification of ubiquitinated proteins in Arabidopsis

Ubiquitin (Ub) is a small peptide that is covalently attached to proteins in a posttranslational reaction. Ubiquitination is a precise regulatory system that is present in all eukaryotic organisms and regulates the stability, the activity, the localization and the transport of proteins. Ubiquitination involves different enzymatic activities, in which the E3 ligases catalyze the last step recruiting of the target for labelling with ubiquitin. Genomic analyses have shown that the ubiquitin-proteasome system involves a large number of proteins in plants, as approximately 5% of the total protein belongs to this pathway. In contrast to the high number of E3 ligases of ubiquitin identified, very few proteins regulated by ubiquitination have been described. To solve this, we have undertaken a new proteomic approach aimed to identify proteins modified with ubiquitin. This is based on affinity purification and identification for ubiquitinated proteins using the ubiquitin binding domain (UBA) polypeptide of the P62 protein attached to agarose beads. This P62-agarose matrix is capable of specifically binding ubiquitinated proteins. These bound proteins were digested with trypsin and the peptides separated by HPLC chromatography, spotted directly onto a MALDI target and analyzed by MALDI-TOF/TOF off-line coupled LC/MALDI-MS/MS. A total of 200 putative ubiquitinated proteins were identified. From these we found that several of the putative targets were already described in plants, as well as in other organisms, as ubiquitinated proteins. In addition, we have found that some of these proteins were indeed modified with ubiquitin in vivo. Taken together, we have shown that this approach is useful for identifying ubiquitinated protein in plants.     

Yeast proteins that recognize nuclear localization sequences

A variety of peptides can mediate the localization of proteins to the nucleus. We have identified yeast proteins of 70 and 59 kD that bind to nuclear localization peptides of SV-40 T antigen, Xenopus nucleoplasmin, and the yeast proteins Ga14 and histone H2B. These proteins are assayed by the binding of peptide-albumin conjugates to proteins immobilized on nitrocellulose filters. These binding proteins fractionate with nuclei and are extractable with salt but not detergent. Radiolabeled peptide-albumin conjugates also bind to isolated nuclei; the binding is saturable and can be extracted with salt. Different nuclear localization peptides compete with each other, implying that a single class of proteins is responsible for their recognition. The 70- and 59-kD proteins have the properties expected for a receptor that would act to direct proteins to the nucleus.     

Detection and characterization of fungal-specific proteins in Saccharomyces cerevisiae

In this study, I searched for fungal-specific proteins in the genome of the budding yeast Saccharomyces cerevisiae, inferred from a comparison of amino acid sequences. I used the GTOP (Genomes to Protein structures and functions) database of the DDBJ (DNA Data Bank of Japan), which consists of 21 genomes from Archaea, 203 genomes from Bacteria, and 50 genomes from Eucarya (including 18 fungal genomes). Among 5,874 proteins of S. cerevisiae, 1,551 have homologs only in Eucarya, and 504 of the 1,551 have homologs only in fungi. To find fungal-specific proteins, homologs of the homologs have been searched repeatedly. As a result, 132 of the 504 are characterized as fungal-specific proteins. The genes encoding the 132 fungal-specific proteins are not included in the list of essential genes for viability in the S. cerevisiae genome deletion project. Among the 132 proteins, 99 are S. cerevisiae-specific, and no protein that is distributed among 10 or more of the 18 fungal species exists. In addition, most of the fungal-specific proteins are very small and functionally unknown. My results show that the fungal-specific proteins have short evolutionary histories, suggesting that S. cerevisiae produces novel proteins and that ancestral fungi also produced small proteins most of which have disappeared or have been combined with other proteins during fungal evolution.     

Structure and function of nonhistone phosphoproteins

Many nonhistone nuclear proteins have been shown to be phosphorylated and associated with a diverse range of cellular activities. The aim of this present review is to update the most recent developments in this field with respect to the traditional roles that these proteins have been postulated to play as enzymes, DNA-binding proteins, hormonal receptors, nucleosome associated proteins, and nucleolar associated proteins. In addition, evidence is presented suggesting that these proteins may also function as nuclear oncogene protein products, structural constituents of nuclear organization (e.g., lamina-nuclear matrix associated proteins), and RNA processing factors.     

Genome-wide analysis of integral membrane proteins from eubacterial,archaean, and eukaryotic organisms.

We have carried out detailed statistical analyses of integral membrane proteins of the helix-bundle class from eubacterial, archaean, and eukaryotic organisms for which genome-wide sequence data are available. Twenty to 30% of all ORFs are predicted to encode membrane proteins, with the larger genomes containing a higher fraction than the smaller ones. Although there is a general tendency that proteins with a smaller number of transmembrane segments are more prevalent than those with many, uni-cellular organisms appear to prefer proteins with 6 and 12 transmembrane segments, whereas Caenorhabditis elegans and Homo sapiens have a slight preference for proteins with seven transmembrane segments. In all organisms, there is a tendency that membrane proteins either have many transmembrane segments with short connecting loops or few transmembrane segments with large extra-membraneous domains. Membrane proteins from all organisms studied, except possibly the archaeon Methanococcus jannaschii, follow the so-called "positive-inside" rule; i.e., they tend to have a higher frequency of positively charged residues in cytoplasmic than in extra-cytoplasmic segments.