Biomedical subjects

Yutaka Kuroda

Publications and source records attributed to Yutaka Kuroda.

12 recordsLinked to original sources

PubMed2007

Mutational analysis of protein solubility enhancement using short peptide tags.

Protein aggregation is a common phenomenon. The preparation of highly concentrated protein samples, typically required for biophysical measurements, often involves a time consuming and tedious testing of solvent conditions for improving protein solubility. Here, in a systematic analysis, we have determined the increase in solubility upon the addition of SEP-tags (solubility enhancement peptide tags) containing, one, three, and five lysines or arginines (or six arginines) to either the N or C terminus of our low solubility model protein, bovine pancreatic trypsin inhibitor variant, BPTI-22 (a BPTI variant containing 22 alanines). As anticipated, the BPTI-22 solubility increased in direct relation to the number of charged residues contained in the SEP-tag, and without altering either the activity or the structure of the protein. The largest solubility increases were of 4.2-, 4.8-, and 6.2-folds produced by the addition, at the C terminus, of five lysine (BPTI-22-C5K), five and six arginine residues (BPTI-22-C5R and BPTI-22-C6R), respectively. The increased solubility of the tagged BPTI-22 yielded higher quality NMR spectra (hetero single quantum correlation HSQC spectra; with respect of the signal-to-noise and line shapes) in a much shorter time than for the untagged BPTI-22. Furthermore, tagged samples remained soluble for over ten days, as observed by their HSQC spectra. We believe that lysine- and arginine-based SEP-tags may provide an effective and versatile method for enhancing protein solubility.

Amino Acid Sequence↗

PubMed2006

Identification of putative domain linkers by a neural network - application to a large sequence database.

BACKGROUND: The reliable dissection of large proteins into structural domains represents an important issue for structural genomics/proteomics projects. To provide a practical approach to this issue, we tested the ability of neural network to identify domain linkers from the SWISSPROT database (101602 sequences). RESULTS: Our search detected 3009 putative domain linkers adjacent to or overlapping with domains, as defined by sequence similarity to either Protein Data Bank (PDB) or Conserved Domain Database (CDD) sequences. Among these putative linkers, 75% were "correctly" located within 20 residues of a domain terminus, and the remaining 25% were found in the middle of a domain, and probably represented failed predictions. Moreover, our neural network predicted 5124 putative domain linkers in structurally un-annotated regions without sequence similarity to PDB or CDD sequences, which suggest to the possible existence of novel structural domains. As a comparison, we performed the same analysis by identifying low-complexity regions (LCR), which are known to encode unstructured polypeptide segments, and observed that the fraction of LCRs that correlate with domain termini is similar to that of domain linkers. However, domain linkers and LCRs appeared to identify different types of domain boundary regions, as only 32% of the putative domain linkers overlapped with LCRs. CONCLUSION: Overall, our study indicates that the two methods detect independent and complementary regions, and that the combination of these methods can substantially improve the sensitivity of the domain boundary prediction. This finding should enable the identification of novel structural domains, yielding new targets for large scale protein analyses.

Algorithms↗

PubMed2006

Computer-aided NMR assay for detecting natively folded structural domains.

Structural genomics projects require strategies for rapidly recognizing protein sequences appropriate for routine structure determination. For large proteins, this strategy includes the dissection of proteins into structural domains that form stable native structures. However, protein dissection essentially remains an empirical and often a tedious process. Here, we describe a simple strategy for rapidly identifying structural domains and assessing their structures. This approach combines the computational prediction of sequence regions corresponding to putative domains with an experimental assessment of their structures and stabilities by NMR and biochemical methods. We tested this approach with nine putative domains predicted from a set of 108 Thermus thermophilus HB8 sequences using PASS, a domain prediction program we previously reported. To facilitate the experimental assessment of the domain structures, we developed a generic 6-hour His-tag-based purification protocol, which enables the sample quality evaluation of a putative structural domain in a single day. As a result, we observed that half of the predicted structural domains were indeed natively folded, as judged by their HSQC spectra. Furthermore, two of the natively folded domains were novel, without related sequences classified in the Pfam and SMART databases, which is a significant result with regard to the ability of structural genomics projects to uniformly cover the protein fold space.

Computational Biology↗

PubMed2006

Improvement of domain linker prediction by incorporating loop-length-dependent characteristics.

Protein dissection into structural domains that can fold in isolation is an important issue in both functional and structural proteomics. Here, we analyzed inter- and intradomain loop sequences (respectively named domain linker and nonlinker loops) and computed a domain linker likelihood score, which was used for developing a domain boundary prediction protocol. The analysis confirmed our previous results indicating that the amino acid composition in terms of glycine, proline, aspartic acid, asparagine, lysine, and histidine significantly differs between linker and nonlinker loops. However, a detailed examination revealed that the amino acid composition bias actually depends on the loop length. Indeed, significant frequency deviations were observed for glycine, proline, and aspartic acid in short linker and nonlinker loops, whereas deviations were observed for aspartic acid, proline, asparagine, and lysine in long linker and nonlinker loops. Finally, we incorporated this loop-length-dependent amino acid composition bias in a simple linker prediction protocol, which predicted linkers with a 40.6% specificity and a 36.1% sensitivity. These figures are 4.4 and 2.4% higher than those obtained with our former prediction protocol that does not incorporate loop-length-dependent characteristics. This result should have practical significance for experimental protein dissection, since the probability of obtaining a stably folding structural domain by randomly dissecting a protein sequence is estimated to be 12.6%.

Algorithms↗

PubMed2006

Structural and functional characterization of a mutant of Pseudocerastes persicus natriuretic peptide.

We hereby report on a mutational analysis of a novel natriuretic peptide (PNP), recently isolated by us from the Iranian snake venom. The PNP variant (mutPNP) with four substitutions (G16T, K18S, R21S, G23R) and a disulfide bonded ring shortened by 3 residues. mutPNP peptide was expressed in pET32 and purified by affinity separation on nickel resin followed by RP-HPLC chromatography. The conformation of mutPNP was characterized in solution by 1H nuclear magnetic resonance spectroscopy, where it was found that the 14-residue disulfide bonded ring, like the 17-residue ring in PNP, retains a high degree of conformational flexibility. The conformation of mutPNP bound to NPR-C receptor was predicted by homology protein structure modeling. When injected intravenously into rats, mutPNP, in contrast to PNP had no physiological effect on blood pressure or on diuresis. The loss of physiological activity is explained in terms of the modeled bound conformation and the ensemble of solution conformations obtained using the NMR constraints.

Amino Acid Sequence↗

PubMed2004

ProteoMix: an integrated and flexible system for interactively analyzing large numbers of protein sequences.

UNLABELLED: ProteoMix is a suite of JAVA programs for identifying, annotating and predicting regions of interest in large sets of amino acid sequences, according to systematic and consistent criteria. It is based on two concepts (1) the integration of results from different sequence analysis tools increases the prediction reliability; and (2) the integration protocol is critical and needs to be easily adaptable in a case-by-case manner. ProteoMix was designed to analyze simultaneously multiple protein sequences using several bioinformatics tools, merge the results of the analyses using logical functions and display them on an integrated viewer. In addition, new sequences can be added seamlessly to an analysis performed on an initial set of sequences. ProteoMix has a modular design, and bioinformatics tools are run on remote servers accessed using the Internet Simple Object Access Protocol (SOAP), ensuring the swift implementation of additional tools. ProteoMix has a user-friendly interactive graphical user interface environment and runs on PCs with Microsoft OS. AVAILABILITY: ProteoMix is freely available for academic users at http://bio.gsc.riken.jp/ProteoMix/

Database Management Systems↗

PubMed2003

Characteristics and prediction of domain linker sequences in multi-domain proteins.

To facilitate swift structural characterizations, structural genomic/proteomic projects need to divide large multidomain proteins into structural domains and to determine their structures separately. Thus, the assignment of structural domains based solely on sequence information, especially on the physico-chemical properties of the amino acid sequences, could be very helpful for such projects. In this study, we examined the characteristics of 'domain linker sequences', which are loop sequences connecting two structural domains. To this end, we prepared a set of 101 non-redundant multi-domain protein sequences with known structures, and performed an analysis of the linker sequences. The analysis revealed that the frequencies of five (Pro, Gly, Asp, Asn, Lys) amino acid residues differed significantly between the linker and non-linker loop sequences. Moreover, we observed a similar deviation for the residue pair frequencies between the two types of loop sequences. Finally, we describe an automated method, based on the above analysis, to detect loops that have high probabilities of being domain linkers in a protein sequence.

Algorithms↗

PubMed2003

Hyponatremia and/or hyperkalemia in patients treated with the standard dose of trimethoprim-sulfamethoxazole.

OBJECTIVE: High-dose trimethoprim-sulfamethoxazole (TMP-SMX) is known to cause hyperkalemia by blocking amiloride-sensitive sodium (Na) channels in distal nephrons. The purpose of this study was to establish whether the standard dose of TMP-SMX could cause electrolyte disorders. METHODS AND PATIENTS: Serum Na, potassium (K) and creatinine (Cr) levels were examined retrospectively in 53 of 77 patients prescribed TMP-SMX, before and after taking the antibiotic combination. RESULTS: Electrolyte disorders (Na < 135 mEq/l and/or K > 5.0 mEq/l) were found in 14 of the 53 patients (26.4%) during TMP-SMX treatment. The average dose was 145.7 +/- 24.9 mg/day. The dose of TMP was significantly larger in patients with electrolyte disorders (267.7 +/- 84.2 mg vs. 101.9 +/- 9.38 mg, p = 0.0024). Electrolyte disorders were also seen in 9.1% and 22.2% of patients given the low dose (TMP < 80 mg) or standard dose (TMP 80-120 mg) of TMP-SMX, respectively. Electrolyte disorders were seen in 85.7% of patients with renal dysfunction (Cr > 1.2 mg/dl), compared with 17.5% of patients with normal renal function (p = 0.0008). Logistic regression analysis showed that the dose of TMP and the presence of renal dysfunction increased the incidence of electrolyte disorders with an odds ratio of 2.35 and 80.29, respectively. CONCLUSION: Electrolyte disorders, particularly hyperkalemia and hyponatremia can be detected in patients given TMP-SMX. These disorders are more frequent in patients given high doses, but can also be detected after low-dose administration. Renal dysfunction accelerates the incidence of electrolyte disorders induced by TMP-SMX.

Anti-Infective Agents↗

PubMed2002

Solution structure of the DFF-C domain of DFF45/ICAD. A structural basis for the regulation of apoptotic DNA fragmentation.

DFF45/ICAD has dual functions in the final stage of apoptosis, by acting as both a folding chaperone and a DNase inhibitor of DFF40/CAD. Here, we present the solution structure of the C-terminal domain of DFF45, which is essential for its chaperone-like activity. The structure of this domain (DFF-C) consists of four alpha helices, which are folded in a novel helix-packing arrangement. The 3D structure reveals a large cluster of negatively charged residues on the molecular surface of DFF-C. This observation suggests that charge complementation plays an important role in the interaction of DFF-C with the positively charged catalytic domain of DFF40, and thus for the chaperone activity of DFF45. The structure of DFF-C also provides a rationale for the loss of the chaperone activity in DFF35, a short isoform of DFF45. Indeed, in DFF35, the amino acid sequence is truncated in the middle of the second alpha helix constituting the structure of DFF-C, and thus both the hydrophobic core and the cluster of negative charges are disrupted.

Amino Acid Sequence↗

PubMed2002

Marked removal of bezafibrate-induced high-density lipoprotein-cholesterol by low-density lipoprotein apheresis.

BACKGROUND: A case of marked reduction of the bezafibrate-induced increase of high-density lipoprotein (HDL)-cholesterol by low-density lipoprotein apheresis (LDL-apheresis) has not been previously reported. METHODS: A 68-year-old Japanese man with arteriosclerosis obliterans (ASO), diabetes mellitus, and hyperlipidemia underwent LDL-apheresis, followed by the concomitant bezafibrate administration. Plasma lipids of pre- and post-LDL-apheresis were measured and apolipoprotein E (apoE) localization of the pre- and post-LDL-apheresis was detected by agarose gel electrophoresis. RESULTS: Plasma concentrations of the total cholesterol, LDL-cholesterol, triglyceride, and HDL-cholesterol of pre-LDL-apheresis were 4.78 +/- 0.36, 2.74 +/- 0.24, 2.44 +/- 0.52, and 0.92 +/- 0.10 mmol/l, respectively; those of the post-LDL-apheresis were 1.94 +/- 0.31, 0.72 +/- 0.13, 0.81 +/- 0.38, and 0.86 +/- 0.11 mmol/l, respectively. LDL-apheresis reduced HDL-cholesterol by 6.4% (p=0.346). During the bezafibrate administration, plasma concentrations of the above of pre-LDL-apheresis were 5.24 +/- 0.34, 3.28 +/- 0.22, 1.26 +/- 0.25, and 1.39 +/- 0.21 mmol/l, respectively; those of the post-LDL-apheresis were 2.25 +/- 0.44, 0.80 +/- 0.12, 0.58 +/- 0.19, and 1.18 +/- 0.16 mmol/l, respectively. LDL-apheresis reduced HDL-cholesterol by 15.2% (p<0.01). Plasma apolipoprotein E detected between the prebeta- and alpha-mobility was markedly lower after the LDL-apheresis in the agarose gel electrophoresis. CONCLUSIONS: The removal of the bezafibrate induced an increase of the HDL-cholesterol by LDL-apheresis.

Aged↗

PubMed2002

Characterization and prediction of linker sequences of multi-domain proteins by a neural network.

In this paper, we describe a neural network analysis of sequences connecting two protein domains (domain linkers). The neural network was trained to distinguish between domain linker sequences and non-linker sequences, using a SCOP-defined domain library. The analysis indicated that a significant difference existed between domain linkers and non-linker regions, including intra-domain loop regions. Moreover, the resulting Hinton diagram showed a position-dependent amino acid preference of the domain linker sequences, and implied their non-random nature. We then applied the neural network to predict domain linkers in multi-domain protein sequences. As the result of a Jack-knife test, 58% of the predicted regions matched actual linker regions (specificity), and 36% of the SCOP-derived domain linkers were predicted (sensitivity). This prediction efficiency is superior to simpler methods derived from secondary structure prediction that assume that long loop regions are putative domain linkers. Altogether, these results suggest that domain linkers possess local characteristics different from those of loop regions.

Amino Acid Sequence↗

PubMed2001

Abdominal Aortic Aneurysm Surgery in Patients with Cardiac and Renal Complications: Retrograde Anastomosis Using a Ringed Y-graft.

When performing high-risk abdominal aortic aneurysm (AAA), aortic cross-clamp time was reduced to a mean of 9 minutes by performing retrograde anastomosis using a ringed Y-graft (RYG). Retrograde anastomosis with RYG was performed in nine patients, (eight men and one woman) with a mean age of 74 years (range: 65-82 years). Three patients had angina pectoris and chronic renal failure, two had angina pectoris, one had thoracoabdominal aortic aneurysm and chronic renal failure, one had renal failure, one had aortic regurgitation, and one had aortic stenosis. First, the right common, external, and internal iliac arteries were clamped, then, the right limb of the graft was anastomosed to the common iliac artery or external iliac artery. Next, the aorta and left common iliac artery were clamped, and a longitudinal incision was made in the aneurysm. The proximal end of the RYG was inserted into the aorta and blood flow was resumed. Finally, the left limb of the graft was anastomosed to the left common iliac artery or external iliac artery. The mean aortic cross-clamp time was 9 minutes (range: 8-18 minutes). There were no cardiac complications during surgery. The mean operating time was 3:34 hours (range: 3:05-4:35 hours), and the blood loss averaged 1156 ml (range: 200-2000 ml). None of the patients developed postoperative complications and all of them have remained well after discharge. Retrograde anastomosis using RYG is one type of surgery that could be used in cases of high-risk patients with AAA. </hea

Journal Article↗