Potential energy function for continuous state models of globular proteins.

One of the approaches to protein structure prediction is to obtain energy functions which can recognize the native conformation of a given sequence among a zoo of conformations. The discriminations can be done by assigning the lowest energy to the native conformation, with the guarantee that the native is in the zoo. Well-adjusted functions, then, can be used in the search for other (near-) natives. Here the aim is the discrimination at relatively high resolution (RMSD difference between the native and the closest nonnative is around 1 A) by pairwise energy potentials. The potential is trained using the experimentally determined native conformation of only one protein, instead of the usual large survey over many proteins. The novel feature is that the native structure is compared to a vastly wider and more challenging array of nonnative structures found not only by the usual threading procedure, but by wide-ranging local minimization of the potential. Because of this extremely demanding search, the native is very close to the apparent global minimum of the potential function. The global minimum property holds up for one other protein having 60% sequence identity, but its performance on completely dissimilar proteins is of course much weaker.     

Characterization of the unfolding process of lipocalin-type prostaglandin D synthase

We found that low concentrations of guanidine hydrochloride (GdnHCl, <0.75 M) or urea (<1.5 M) enhanced the enzyme activity of lipocalin-type prostaglandin (PG) D synthase (L-PGDS) maximally 2.5- and 1.6-fold at 0.5 M GdnHCl and 1 M urea, respectively. The catalytic constants in the absence of denaturant and in the presence of 0.5 M GdnHCl or 1 m urea were 22, 57, and 30 min(-1), respectively, and the K(m) values for the substrate, PGH(2), were 2.8, 8.3, and 2.3 microm, respectively, suggesting that the increase in the catalytic constant was mainly responsible for the activation of L-PGDS. The intensity of the circular dichroism (CD) spectrum at 218 nm, reflecting the beta-sheet content, was also increased by either denaturant in a concentration-dependent manner, with the maximum at 0.5 M GdnHCl or 1 M urea. By plotting the enzyme activities against the ellipticities at 218 nm of the CD spectra of L-PGDS in the presence or absence of GdnHCl or urea, we found two states in the reversible folding process of L-PGDS: one is an activity-enhanced state and the other, an inactive state. The NMR analysis of L-PGDS revealed that the hydrogen-bond network was reorganized to be increased in the activity-enhanced state formed in the presence of 0.5 M GdnHCl or 1 m urea and to be decreased but still remain in the inactive intermediate observed in the presence of 2 M GdnHCl or 4 M urea. Furthermore, binding of the nonsubstrate ligands, bilirubin or 13-cis-retinal, to L-PGDS changed from a multistate mode in the native form of L-PGDS to a simple two-state mode in the activity-enhanced form, as monitored by CD spectra of the bound ligands. Therefore, L-PGDS is a unique protein whose enzyme activity and ligand-binding property are biphasically altered during the unfolding process by denaturants.     

Serum paraoxonase activity decreases in rheumatoid arthritis

OBJECTIVE: To estimate the alterations of paraoxonase 1 (PON1) and high-density lipoprotein (HDL) in rheumatoid arthritis (RA). DESIGN AND METHODS: We investigated the serum enzyme activity and concentration of PON1 and their relationship with serum lipids, high-density lipoprotein (HDL) parameters, and acute phase reactants of serum amyloid A (SAA) and C-reactive protein (CRP) in patients with RA. RESULTS: Serum paraoxonase (PON) activity was significantly decreased in RA patients (n = 64, 131 +/- 53 micro mol/min/L) compared with healthy subjects (n = 155, 164 +/- 59) despite the absence of any difference in serum lipid levels between the two groups. This decrease of serum PON activity in RA patients was found in every genotype (Q/Q, Q/R, R/R) of PON1 at 192 Q/R. There was a different distribution in PON1 Q/R genotypes between RA patients and healthy subjects, and RA patients exhibited less (44%) positive PON1-Q than did the healthy subjects (66%). In a further investigation of age- and gender-matched subgroups of RA (n = 25) and healthy subjects (n = 25), not only serum PON activity, but also lecithin-cholesterol acyltransferase (LCAT) was found to be significantly decreased in RA patients (125 +/- 61 micro mol/min/L, 63.2 +/- 17.2 nmol/ml/hr/37 degrees C) than in healthy subjects (169 +/- 67, 74.7 +/- 19.5), respectively. PON1 and LCAT as well as HDL constituent apolipoprotein (apo) AI and apo AII, were altered significantly in RA patients. CONCLUSIONS: Acute-phase HDL, which is remodeled structurally and functionally in RA, might be less anti-atherogenic due to the impairment of original HDL function. These alterations of HDL in RA patients may explain in part the reported increase in cardiovascular mortality in patients with RA.     

Spinally delivered N-, P/Q- and L-type Ca2+-channel blockers potentiate morphine analgesia in mice

We studied the antinociceptive effects induced at the spinal level by N-, P/Q- and L-type voltage-dependent Ca2+-channel (VDCC) blockers given alone or in combination with morphine, the test responses being the algesic ones induced by acute thermal and mechanical stimuli. When given alone, intrathecal omega-agatoxin IVA (P/Q-type blocker) produced a potent dose-dependent inhibition in the tail-flick and tail-pressure over the dose range 0.33-33 pmol/mouse. Omega-conotoxin GVIA (N-type blocker) also produced dose-dependent inhibitions, but its antinociception against thermal stimuli was weaker than against mechanical stimuli. Calciseptine (L-type blocker) slightly reduced both nociceptive responses, but only at 33 pmol. At their subthreshold doses, intrathecal omega-agatoxin IVA, omega-conotoxin GVIA and calciseptine each significantly enhanced morphine analgesia in the tail-flick and tail-pressure tests, the rank order of potencies being N-> or =P/Q->L-type. These results indicate that combining a low-dose VDCC blocker, especially the N- or P/Q-type, with morphine may be a very useful way of minimizing the dose of morphine and may reduce side effects.     

Essential 110Cys in active site of membrane-associated prostaglandin E synthase-2

The amino acid sequence of membrane-associated prostaglandin (PG) E synthase-2 (mPGE synthase-2), which has a broad specificity in its thiol requirement for a catalytic activity, has the consensus region from 104Leu to 120Leu found in glutaredoxin and of thioredoxin. The sequence of Cys-x-x-Cys in the consensus region is the active site for thioredoxin and mPGE synthase-2 also has this amino acid sequence (110Cys-x-x-113Cys). The mutation from 110Cys to Ser or the double mutation from 110Cys and 113Cys to Ser caused loss of PGE synthase activity, whereas the single mutation from 113Cys to Ser did not affect the enzyme activity. These results indicate that 110Cys, but not 113Cys, is the essential amino acid in the active site of mPGE synthase-2. 110Cys is an important amino acid in PGE synthase activity and plays the critical role as Cys at the same position in redoxin. Moreover, we found that the reduced form of lipoic acid (dihydrolipoic acid) serves as one of the natural activators of mPGE synthase-2 in the cells.     

Characteristic domain motion in the ribosome recycling factor revealed by 15N NMR relaxation experiments and molecular dynamics simulations

The backbone dynamics of ribosome recycling factor (RRF) from Escherichia coli in water were characterized by (15)N NMR relaxation analysis and molecular dynamics (MD) simulation. RRF is composed of two domains connected by a joint region that consists of two peptide chains, such that the overall structure seems to mimic that of tRNA. MD trajectories indicated that the relative orientation of domains varies on the nanosecond time scale. We analyzed the observed (15)N T(1), T(2), and NOE using an extended model-free spectral density function in which the domain motions with a nanosecond time scale were considered. At 30 degrees C, the order parameters of slow motion () were determined to be approximately 0.9 for domain I and 0.7 for domain II, respectively. These values indicate that domain I is nearly fixed on the molecular diffusion frame, and domain II is wobbling in a cone for which the semi-angle is about 30 degrees.     

Rat tripeptidyl peptidase I: molecular cloning, functional expression, tissue localization and enzymatic characterization

We purified tripeptidyl peptidase I (TPP I) to homogeneity from a rat kidney lysosomal fraction and determined its physicochemical properties, including its molecular weight, substrate specificity and partial amino acid sequence. The molecular weight of the enzyme was calculated to be 280,000 and 290,000 by non-denaturing PAGE and gel filtration, respectively, and to be 43 000 and 46 000 on SDS-PAGE in the absence and presence of beta-ME, respectively. These findings suggest that the enzyme is composed of six identical subunits. The Km, Vmax, kcat and kcat/Km values of TPP I at optimal pH (pH 4.0) were 680 microM, 3.7 micromol x mg(-1) x min(-1), 33.1 s(-1) and 4.87 x 10(4) s(-1) x M(-1) for Ala-Ala-Phe-MCA, respectively. TPP I was significantly inhibited by PCMBS and HgCl2, and moderately by DFP. These findings also suggest that TPP I is an exotype serine peptidase that is regulated by SH reagent. TPP I released the tripeptide Arg-Val-Tyr from angiotensin III more rapidly than from Ala-Ala-Phe-MCA, and also released Gly-Asn-Leu from neuromedin B with the same velocity as from Ala-Ala-Phe-MCA. Angiotensin III and neuromedin B have recently been found to be good natural substrates for lysosomal TPP I. Furthermore, we determined the rat liver cDNA structure and deduced the amino acid sequence. The cDNA, designated as lambdaRTI-1, is composed of 2485 bp and encodes 563 amino acids in the coding region. By Northern blot analysis, the order for TPP I mRNA expression was kidney > or = liver > heart > brain > lung > spleen > skeletal muscle and testis. In parallel experiments, the TPP I antigen was detected in various rat tissues by immunohistochemical staining.     

Enhancement of oxidative damage to cultured cells and Caenorhabditis elegans by mitochondrial electron transport inhibitors

The mechanisms that lead to mitochondrial damage under oxidative stress conditions were examined in primary and cultured cells as well as in the nematode Caenorhabditis elegans (C. elegans) treated simultaneously with electron transport inhibitors and oxygen gas. Oxygen loading enhanced the damage of PC 12 cells by thenoyltrifluoroacetone (TTFA, a complex II inhibitor), but did not by rotenone (a complex I inhibitor), antimycin (a complex III inhibitor), and sodium azide (a complex IV inhibitor). In primary hepatocytes, the enhancement was observed with the addition of sodium azide and rotenone, but not by TTFA or antimycin. In the nematode, only rotenone and TTFA enhanced the sensitivity under hyperoxia. These results demonstrate that highly specific inhibitors of electron transport can induce oxygen hypersensitivity in cell levels such as PC 12 cells and primary hepatocytes, and animal level of C. elegans. In addition the cell damage is different dependent on cell type and organism.