Yuri Mukai

Large-scale analysis of human alternative protein isoforms: pattern classification and correlation with subcellular localization signals

We investigated human alternative protein isoforms of >2600 genes based on full-length cDNA clones and SwissProt. We classified the isoforms and examined their co-occurrence for each gene. Further, we investigated potential relationships between these changes and differential subcellular localization. The two most abundant patterns were the one with different C-terminal regions and the one with an internal insertion, which together account for 43% of the total. Although changes of the N-terminal region are less common than those of the C-terminal region, extension of the C-terminal region is much less common than that of the N-terminal region, probably because of the difficulty of removing stop codons in one isoform. We also found that there are some frequently used combinations of co-occurrence in alternative isoforms. We interpret this as evidence that there is some structural relationship which produces a repertoire of isoformal patterns. Finally, many terminal changes are predicted to cause differential subcellular localization, especially in targeting either peroxisomes or mitochondria. Our study sheds new light on the enrichment of the human proteome through alternative splicing and related events. Our database of alternative protein isoforms is available through the internet.

PROTEIN SUBCELLULAR LOCALIZATION PREDICTION

ORGANIZATION. Section 1. We first provide the motivation for prediction of protein subcellular localization sites, as well as discuss changes being brought about by progress in proteomics. Section 2. After that, we describe the biology of protein subcellular location. In particular, we explain the principle of protein sorting signals. Section 3. Then we present several experimental techniques for determining protein subcellular localization sites. The techniques surveyed include traditional methods such as immunofluorescence microscopy, as well as large-scale methods such as green fluorescent protein. Section 4. Next we mention some of the general issues involved in predicting protein subcellular localization, such as what are the sites? how many sites per protein? how good are the predictions? and so on. We also discuss the distinction between features that reflect causal influences on localization versus features that merely reflect correlation with localization.

Sensitive Detection of Protein―Lipid Interaction Change on Bacteriorhodopsin Using Dodecyl β-D-Maltoside

A light-driven proton pump bacteriorhodopsin (bR) forms a two-dimensional hexagonal lattice with about 10 archaeal lipids per monomer bR on purple membrane (PM) of Halobacterium salinarum. In this study, we found that the weakening of the bR-lipid interaction on PM by addition of alcohol can be detected as the significant increase of protein solubility in a nonionic detergent, dodecyl β-D-maltoside (DDM). The protein solubility in DDM was also increased by bR-lipid interaction change accompanied by structural change of the apoprotein after retinal removal and was about 7 times higher in the case of completely bleached membrane than that of intact PM. Interestingly, the cyclic and milliseconds order of structural change of bR under light irradiation also led to increasing the protein solubility and had a characteristic light intensity dependence with a phase transition. These results indicate that there is a photointermediate in which bR-lipid interaction has been changed by its dynamic structural change. Because partial delipidation of PM by CHAPS gave minor influence for the change of the protein solubility compared to intact PM in both dark and light conditions, it is suggested that specific interactions of bR with some lipids which remain on PM even after delipidation treatment have a key role for the change of solubility in DDM induced by alcohol binding, ligand release, and photon absorption on bR.

Discrimination of Mammalian GPI-Anchored Proteins by Hydropathy and Amino Acid Propensities

The glycosylphosphatidylinositol (GPI) attachment is a most important post-translational modification of proteins that plays essential roles in promoting the biochemical activities of eukaryotic cells. Described here is an analysis of the amino acid properties of mammalian GPI-anchored proteins (GPI-APs) and the development of an innovative method of detecting them. GPI-APs are characterized by two high-hydropathy regions: the signal peptide, located inside the Endoplasmic Reticulum (ER), and the GPI attachment signal, a sequence adjacent to the GPI-anchoring site (the ω-site). Especially in sequence analysis of known GPI-APs, there were some distinct aspects of the amino acid propensities around the ω-sites. Therefore, a method of detecting GPI-APs was developed based on hydropathy profiles and a position-specific scoring matrix (PSSM) calculated by position-specific amino acid propensities. First, sequences of GPI-APs and negative controls, determined by screening based on hydropathy and residue volume profiles, were aligned based on residue volume profiles in the C-terminal region, and the position-specific amino acid propensities of each group were calculated according to their alignment positions. Then, a PSSM was devised using the amino acid propensities of GPI-APs and negative controls, and discrimination scores were estimated for each dataset. Based on these scores at a threshold was fixed for each dataset. GPI-APs were detected with 81.1% sensitivity and a 0.818 success rate in an optimized calculation region determined by adjusting the window size of this region using a 5-fold dataset. The results indicate that a PSSM around the ω-site can effectively discriminate GPI-APs.

Discrimination of Golgi Type II Membrane Proteins Based on Their Hydropathy Profiles and the Amino Acid Propensities of Their Transmembrane Regions

Membrane proteins in the Golgi apparatus play important roles in biological functions, predominantly as catalysts related to post-translational modification of protein oligosaccharides. We succeeded in extracting the characteristics of Golgi type II membrane proteins computationally by comparison with those of Golgi no retention proteins, which are mainly localized in the plasma membrane. Golgi type II membrane proteins were detected by combining hydropathy alignment and a position-specific score matrix of the amino acid propensities around the transmembrane region. We achieved 96.2% sensitivity, 93.5% specificity, and a 0.949 success rate in a self-consistency test. In a 5-fold cross-validation test, 88.0% sensitivity, 85.5% specificity, and a 0.867 success rate were achieved.

Partially induced transition from horizontal to vertical orientation of helical peptides at the air–water interface and the structure of their monolayers transferred on the solid substrates

To apply the Langmuir-Blodgett (LB) technique as a platform for investigating the fundamental properties of amphiphilic peptides (APs), we have investigated the structure of LB films using the APs. To vertically orient the helical APs like transmembrane proteins in the membrane, the primary structure of the APs was designed to have two domains: a hydrophilic domain (three amino acids) and a hydrophobic domain (ca. 20 amino acids). However, we are still far from having full control of their orientation. This study reports the contribution of the subphase temperature to the change in the orientation of helical APs. When the surface pressure-area isotherm of AP was observed at the subphase temperature at 41.5°C, the isotherm exhibited a plateau, implying that a phase transition of the monolayer at the air-water interface occurred. Circular dichroism (CD) spectra of the monolayers transferred on the solid substrates revealed that the orientation of the helices changed at the pressure, where the plateau of the isotherm was observed. This change was not observed at 21.5°C, i.e., the horizontal alignment of helixes was maintained. Atomic force microscopy (AFM) was used to systematically investigate the surface structure of the monolayers transferred at different surface pressures. A structural model of the monolayer that did not contradict with the results obtained by the three different techniques (the isotherm, CD spectroscopy, and AFM) was derived, and it was concluded that the horizontally oriented helices partially changed their orientation to vertical upon compression in the plateau region of the isotherm.

Characteristics of Halorhodopsin–Bacterioruberin Complex from Natronomonas pharaonis Membrane in the Solubilized System

Halorhodopsin is a retinal protein with a seven-transmembrane helix and acts as an inward light-driven Cl(-) pump. In this study, structural state of the solubilized halorhodopsin (NpHR) from the biomembrane of mutant strain KM-1 of Natronomonas pharaonis in nonionic detergent was investigated. A gel filtration chromatography monitored absorbances at 280 and 504 nm corresponding to the protein and a lipid soluble pigment of bacterioruberin (BR), respectively, has clearly detected an oligomer formation of the NpHRs and a complex formation between the NpHR and BR in the solubilized system. A molar ratio of NpHR:BR in the solubilized complex was close to 1:1. Further SDS-PAGE analysis of the solubilized NpHR cross-linked by 1% glutaraldehyde has revealed that the NpHR forms homotrimer in detergent system. Although this trimeric structure was stable in the presence of NaCl, it was dissociated to the monomer by the heat treatment at 45 °C in the desalted condition. The same tendency has been reported in the case of trimeric NpHR expressed heterologously on the E. coli membrane, leading to a conclusion that the change of strength of the trimeric association dependent on the ion binding is a universal feature of the NpHR. Interestingly, the trimer dissociation on the NpHR was accompanied by the complete dissociation of the BR molecule from the protein, indicated that the cavity formed by the NpHR protomers in the trimeric conformation is important for tight binding of the BR. Because the binding affinity for Cl(-) and the resistance to hydroxylamine under light illumination showed only minor differences between the NpHR in the solubilized state and that on the biomembrane, the influences of solubilization to the tertiary structure and function of the protein are thought to be minor. This NpHR-BR complex in the solubilized system has a potential to be a good model system to investigate the intermolecular interaction between the membrane protein and lipid.

Identification of mammalian GPI-anchored proteins based on amino acid propensities in their GPI attachment signal sequences

Attachment of glycosylphosphatidylinositol (GPI) is one of the most important posttranslational modifications, playing an important role in vital eukaryote activities. GPI-anchored proteins (GPI-APs) are characterized by a pro-peptide of hydrophobic residues and small amino acid residues near the GPI-anchoring site. Here, we describe a new method for identifying GPI-APs based on hydropathy profiles and a position-specific scoring matrix (PSSM). First, the sequences of mammalian GPI-APs from the UniProt Knowledgebase/Swiss-Prot protein sequence database release 54.0 were scanned for their average hydropathy with several different window sizes. Hydrophobic regions were observed not only in the signal-peptide but also in the pro-peptide at the C-terminus. Non-GPI-anchored proteins (non-GPI-APs) with similar hydropathy profiles to those of the GPI-APs were used as the negative control. The sequences were aligned according to the residue sizes in the C-terminal region, and the position-specific amino acid propensities were analyzed according to their alignment positions in both the GPI-APs and the negative controls. The PSSM was devised using each amino acid propensity and a matching score was estimated for each dataset. The accuracy achieved in discriminating between GPI-APs and the negative controls was evaluated, and the GPI-APs were detected with 96.5% sensitivity and 97.3% specificity on a self-consistency test and with 85.0% sensitivity and 92.7% specificity on a 4-fold cross-validation test.

Signal-anchor sequences are an essential factor for the Golgi-plasma membrane localization of type II membrane proteins

ABSTRACT Despite studies of the mechanism underlying the intracellular localization of membrane proteins, the specific mechanisms by which each membrane protein localizes to the endoplasmic reticulum, Golgi apparatus, and plasma membrane in the secretory pathway are unclear. In this study, a discriminant analysis of endoplasmic reticulum, Golgi apparatus and plasma membrane-localized type II membrane proteins was performed using a position-specific scoring matrix derived from the amino acid propensity of the sequences around signal-anchors. The possibility that the sequence around the signal-anchor is a factor for identifying each localization group was evaluated. The discrimination accuracy between the Golgi apparatus and plasma membrane-localized type II membrane proteins was as high as 90%, indicating that, in addition to other factors, the sequence around signal-anchor is an essential component of the selection mechanism for the Golgi and plasma membrane localization. These results may improve the use of membrane proteins for drug delivery and therapeutic applications. GRAPHICAL ABSTRACT The sequence around the signal-anchor is an essential component of the selection mechanism for Golgi and plasma membrane protein localization.

A discrimination method for Golgi type II membrane proteins based on the hydropathy alignment and the position-specific score matrix around their transmembrane regions

Golgi membrane proteins contribute protein glycosylation, which is one of post-translational modification. It plays an important role in the cellular processes such as cell-cell adhesion, signal transfer, and subcellular localization. In this regard, the development of a computational method to discrimination of Golgi membrane proteins from the mammal genomes is desired. In this study, we succeeded in the feature extraction of the characteristics of Golgi type II membrane proteins (GLs) by comparison with post-Golgi type II membrane proteins (PGs) except the Golgi retention type mainly localized in the plasma membrane. The nonredundant datasets of GLs (344 sequences) and PGs (356 sequences) were obtained from Swiss-Prot release 57.0. GLs were detected by combining hydropathy alignment and position-specific score matrix (PSSM) around the transmembrane region. Each sequences were aligned by superpositioning the highest average hydrophobicity position. The PSSM was estimated based on position-specific amino acid propensities of the alignment position in the region of −14 to +18. Our method can discriminated GLs from PGs with 96.2% sensitivity and 93.5% specificity in a self-consistency test. Furthermore, 89.8% sensitivity and 87.0% specificity were achieved in 5-fold cross-validation test.