Searching for protein-protein interaction sites and docking by the methods of molecular dynamics, grid scoring, and the pairwise interaction potential of amino acid residues

In CAPRI Rounds 1 and 2, we assumed that because there are many ionic charges that weaken electrostatic interaction forces in living cells, the hydrophobic interaction force might be important entropically. As a result of Rounds 1 and 2, the predictions for binding sites and geometric centers were acceptable, but those of the binding axes were poor, because only the largest benzene cluster was used for generating the initial docking structures. These were generated by fitting of benzene clusters formed on the surface of receptor and ligand. In CAPRI Rounds 3-5, the grid-scoring sum on the protein-protein interaction surface and the pairwise potential of the amino acid residues, which were indicated as coming easily into the protein-protein interaction regions, were used as the calculation methods, along with the smaller benzene clusters that participated in benzene cluster fitting. Good predicted models were obtained for Targets 11 and 12. When the modeled receptor proteins were superimposed on the experimental structures, the smallest ligand root-mean-square deviation (RMSD) values corresponding to the RMSD between the model and experimental structures were 6.2 A and 7.3 A, respectively.     

Biosynthetic origin of the carbon skeleton and nitrogen atom of pamamycin-607, a nitrogen-containing polyketide

The biosynthesis of pamamycin-607 (PM-607), a sixteen-membered macrodiolide compound, was studied with 13C- and 15N-labeled precursor units in Streptomyces alboniger. Feeding experiments with 13C-labeled acetate or propionate indicate that the carbon skeleton of PM-607 was derived from six acetate, four propionate and three succinate units. MS analyses of 15N-labeled PM-607 suggest that the nitrogen atom in PM-607 was derived from the alpha-amino group of an amino acid.     

Albumin clusters: structurally defined protein tetramer and oxygen carrier including thirty-two iron(II) porphyrins

Recombinant human serum albumin (rHSA) clusters have been synthesized and physicochemically characterized. Cross-linking between the Lys groups of the core albumin and a unique Cys-34 of the shell albumins with an N-succinimidyl-6-[3'-(2-pyridyldithio)propionamido]hexanoate produced the structurally defined rHSA trimer and tetramer. MALDI-TOF-MS showed a single peak with the triple and quadruple masses of rHSA. Their molar ellipticities and the isoelectric points (pI = 4.8) are all identical to those of the monomer, suggesting that the essential structures of the albumin units were intact. TEM observations demonstrated a uniform morphology of the rHSA tetramer with a diameter of 20-30 nm. The circulation half-life (tau1/2) of the 125I-labeled rHSA tetramer in rat (5.5 h) was significantly longer than that of the monomer (2.3 h) due to the low ratio of the distribution phase (alpha-phase). A total of 24 and 32 molecules of the synthetic iron(II) porphyrins (FePs) are incorporated into the hydrophobic cavities of the rHSA trimer and tetramer, respectively, producing huge artificial hemoproteins. These albumin-heme clusters can reversibly bind and release O2 under physiological conditions (37 degrees C, pH 7.3) and showed similar O2-binding properties (O2-binding affinity, association and dissociation rate constants) to those of the corresponding monomer. A large volume of O2 can be chemically dissolved into the albumin-heme cluster solutions relative to the monomeric rHSA-FeP when the molar concentration of the albumin scaffold is identical.     

Rice proteome analysis: a step toward functional analysis of the rice genome

The technique of proteome analysis using 2-DE has the power to monitor global changes that occur in the protein complement of tissues and subcellular compartments. In this review, we describe construction of the rice proteome database, the cataloging of rice proteins, and the functional characterization of some of the proteins identified. Initially, proteins extracted from various tissues and organelles were separated by 2-DE and an image analyzer was used to construct a display or reference map of the proteins. The rice proteome database currently contains 23 reference maps based on 2-DE of proteins from different rice tissues and subcellular compartments. These reference maps comprise 13 129 rice proteins, and the amino acid sequences of 5092 of these proteins are entered in the database. Major proteins involved in growth or stress responses have been identified by using a proteomics approach and some of these proteins have unique functions. Furthermore, initial work has also begun on analyzing the phosphoproteome and protein-protein interactions in rice. The information obtained from the rice proteome database will aid in the molecular cloning of rice genes and in predicting the function of unknown proteins.     

Quantification of lipid alkyl radicals trapped with nitroxyl radical via HPLC with postcolumn thermal decomposition

Lipid alkyl radicals generated from polyunsaturated fatty acids via chemical or enzymatic H-abstraction have been a pathologically important target to quantify. In the present study, we established a novel method for the quantification of lipid alkyl radicals via nitroxyl radical spin-trapping. These labile lipid alkyl radicals were converted into nitroxyl radical-lipid alkyl radical adducts using 3-carbamoyl-2,2,5,5-tetramethyl-3-pyrroline-N-oxyl (CmdeltaP) (a partition coefficient between octanol and water is approximately 3) as a spin-trapping agent. The resulting CmdeltaP-lipid alkyl radical adducts were determined by HPLC with postcolumn online thermal decomposition, in which the adducts were degraded into nitroxyl radicals by heating at 100 degrees C for 2 min. The resulting nitroxyl radicals were selectively and sensitively detected by electrochemical detection. With the present method, we, for the first time, determined the lipid alkyl radicals generated from linoleic acid, linolenic acid, and arachidonic acid via soybean lipoxygenase-1 or the radical initiator 2,2'-azobis(2,4-dimethyl-valeronitrile).     

Density-dependent induction and suppression of soldier differentiation in an aphid social system

We investigated the mechanism underlying the control of soldier production in colonies of a social aphid, Tuberaphis styraci, which has a sterile soldier caste in the second instar. High aphid density was shown to induce soldier production in T. styraci. Analysis of natural colonies revealed that the soldier proportion tended to increase with aphid density but reached a plateau. Artificial diet experiments identified a similar plateau of soldier proportion under high-density conditions. In order to gain insights into the controlling mechanism of soldier production, the effect of soldiers on reproducing adult aphids was examined using the artificial diet system. It was experimentally demonstrated that soldier production was suppressed by coexisting soldiers, whereas coexisting non-soldiers facilitated soldier production. These results suggested that caste ratio in the colony of T. styraci is controlled by positive and negative feedbacks consisting of density-dependent induction and suppression of soldier differentiation.     

Definition of sequence requirements for latency-associated nuclear antigen 1 binding to Kaposi's sarcoma-associated herpesvirus DNA

In latent infection, Kaposi's sarcoma-associated herpesvirus (KSHV) latency-associated nuclear antigen 1 (LANA1)-specific binding to KSHV terminal repeat DNA mediates multicopy episome persistence. We now use electrophoretic mobility shift assays to investigate LANA1 binding to its 20-bp cognate sequence. Mutations at positions 6, 7, and 8 ((6)CCC(8)) severely reduced LANA1 binding, whereas mutations at other positions only modestly reduced binding. Since (6)CCC(8) is in the 5' half of an inverted repeat sequence, these results are consistent with an asymmetric role for the inverted repeat in LANA1 binding.     

Poly(ethylene glycol)-conjugated human serum albumin including iron porphyrins: surface modification improves the O2-transporting ability

Artificial O2-carrying hemoprotein composed of human serum albumin including tetrakis(o-amidophenyl)porphinatoiron(II) (Fe4P or Fe3P) [HSA-FeXP] has been modified by maleimide- or succinimide-terminated poly(ethylene glycol) (PEG), and the formed PEG bioconjugates have been physicochemically characterized. 2-Iminothiolane (IMT) reacted with the amino groups of Lys to create active thiol groups, which bind to alpha-maleimide-omega-methoxy PEG [Mw: 2-kDa (PEG(M2)), 5-kDa (PEG(M5))]. On the other hand, alpha-succinimidyl-omega-methoxy PEG [Mw: 2-kDa (PEG(S2)), 5-kDa (PEG(S5))] directly binds to Lys residues. MALDI-TOF MS of the PEG-conjugated HSA-FeXP showed distinct molecular ion peaks, which provide an accurate number of the PEG chains. In the case of PEG(MY)(HSA-FeXP), the spectroscopic assay of the thiol groups also provided the mean of the binding numbers of the polymers, and the degree of the modification was controlled by the ratio of [IMT]/[HSA]. The viscosity and colloid osmotic pressures of the 2-kDa PEG conjugates (phosphate-buffered saline solution, [HSA] = 5 g dL(-1)) were almost the same as that of the nonmodified one, whereas the 5-kDa PEG binding increased the rheological parameters. The presence of flexible polymers on the HSA surface retarded the association reaction of O2 to FeXP and stabilized the oxygenated complex. Furthermore, PEG(MY)(HSA-FeXP) exhibited a long circulation lifetime of FeXP in rats (13-16 h). On the basis of these results, it can be concluded that the surface modification of HSA-FeXP by PEG has improved its comprehensive O2-transporting ability. In particular the PEG(MY)(HSA-FeXP) solution could be a promising material for entirely synthetic O2-carrying plasma expander as a red cell substitute.     

Autophagy and neurodegeneration

The proteasome and lysosome are sophisticated apparatuses capable of shredding unnecessary proteins in eukaryotic cells. The proteasome and its partner ubiquitin (which functions as a destination signal for proteolysis) play crucial roles in selective breakdown of not only short-lived regulatory proteins but also abnormal proteins that need to be rapidly eliminated from the cells. It is generally accepted that deficits of the proteasome-ubiquitin system are associated with various neurodegenerative diseases, since ubiquitin-positive inclusions frequently appear in neurons of patients and mice models of neurodegenerative diseases. However, investigators working in the field of neuronal diseases have focused their attention in recent years on autophagy (Greek for "the eating of oneself") following the recent discovery that ablation of autophagy leads to accumulation of ubiquitin-positive inclusions, which are the pathological hallmark of neurodegenerative diseases. Here we discuss the consequences of autophagy deficiency in neurons.