UCP3 expressed in yeast is primarily localized in extramitochondrial particles

Previously it was concluded (1) that, differently from UCP1, on expression in Saccharomyces cerevisiae, UCP3, and UCP3 short (UCP3s) are in a deranged state, allowing for unregulated uncoupling. Here we show that the bulk of UCP3 and UCP3s is in extramitochondrial aggregates whether expressed with high or medium expression vectors. The evidence is based on the insolubility of most UCP3 and UCP3s in nonionic detergents such as Triton X100, in contrast to UCP1. Using very high expression vector, macroscopic evidence for extramitochondrial UCP3 containing particles is a viscous white sediment surrounding the mitochondrial fraction which contains UCP3 as inclusion body type aggregate. Together with the previous data it is concluded that uncoupling due to small amounts of incorporated, deranged, and nucleotide insensitive UCP3 prevents incorporation of the bulk of UCP3 into mitochondria. This finding also provides a simple and stringent assay for the state of heterologously expressed in mitochondrial membrane proteins.     

Critical role of the residue size at position 87 in H2O2- dependent substrate hydroxylation activity and H2O2 inactivation of cytochrome P450BM-3

Replacement of phenylalanine 87 with alanine or glycine (mutant F87A or F87G) greatly increased the H2O2-supported substrate hydroxylation activity of cytochrome P450BM-3, whose original H2O2-supported activity is hardly detectable. On the other hand, replacement of phenylalanine 87 with valine (mutant F87V) did not. In the oxidation of p-nitrophenoxydodecanoic acid (12-pNCA), the turnover numbers of the mutant F87A in the presence of NADPH and O2, or H2O2 were 493 and 162 nmol/min/nmol, respectively. The H2O2-supported F87A hydroxylation activity was further confirmed with free fatty acids as substrates. Moreover, the stability of F87A in H2O2 solutions also largely increased. The order of the stability of the wild type (WT), F87A, and their substrate (12-pNCA)-binding complexes in H2O2 solutions listed from high to low was F87A, WT, F87A substrate-binding complex, and WT substrate-binding complex. We propose that the free space size in the vicinity of the heme iron significantly influences P450BM-3 H2O2-supported activity and H2O2 inactivation.     

Juxtamembrane region of the amino terminus of the corticotropin releasing factor receptor type 1 is important for ligand interaction

The functional properties of the amino terminus (NT) of the corticotropin releasing factor (CRF) receptor type 1 (R1) were studied by use of murine (m) CRFR1 and rat (r) parathyroid hormone (PTH)/parathyroid hormone-related peptide receptor (PTH1R) chimeras. The chimeric receptor CXP, in which the NT of mCRFR1 was annealed to the TMs of PTH1R, and the reciprocal hybrid, PXC, bound radiolabeled analogues of sauvagine and PTH(3--34), respectively. Neither hybrid bound radiolabeled CRF or PTH(1--34). CRF and PTH(1--34) weakly stimulated intracellular cAMP accumulation in COS-7 cells transfected with PXC and CXP, respectively. Thus the NT is required for ligand binding and the TMs are required for agonist-stimulated cAMP accumulation. Replacing individual intercysteine segments of PXC with their mCRFR1 counterparts did not rescue CRF or sauvagine radioligand binding or stimulation of cAMP accumulation. Replacement of residues 1--31 of mCRFR1 with their PTH1R counterparts resulted in a chimeric receptor, PEC, which had normal CRFR1 functional properties. In addition, a series of chimeras (F1PEC--F6PEC) were generated by replacement of the NT intercysteine residues of PEC with their PTH1R counterparts. Only F1PEC, F2PEC, and F3PEC showed detectable CRF and sauvagine radioligand binding. All of the PEC chimeras except F5PEC increased cAMP accumulation. These data indicate that the Cys(68)(-)Glu(109) domain is important for binding and that the Cys(87)(-)Cys(102) region plays an important role in CRFR1 activation.     

Apolipoprotein A-II/A-I ratio is a key determinant in vivo of HDL concentration and formation of pre-beta HDL containing apolipoprotein A-II

Overexpression of human apolipoprotein A-II (apo A-II) in mice induced postprandial hypertriglyceridemia and marked reduction in plasma HDL concentration and particle size [Boisfer et al. (1999) J. Biol. Chem. 274, 11564-11572]. We presently compared lipoprotein metabolism in three transgenic lines displaying plasma concentrations of human apo A-II ranging from normal to 4 times higher, under ad libitum feeding and after an overnight fast. Fasting dramatically decreased VLDL and lowered circulating human apo A-II in transgenic mice; conversely, plasma HDL levels increased in all genotypes. The apo A-I content of HDL was inversely related to the expression of human apo A-II, probably reflecting displacement of apo A-I by an excess of apo A-II. Thus, the molar ratios of apo A-II/A-I in HDL were significantly higher in fed as compared with fasted animals of the same transgenic line, while endogenous LCAT activity concomitantly decreased. The number and size of HDL particles decreased in direct proportion to the level of human apo A-II expression. Apo A-II was abundantly present in all HDL particles, in contrast to apo A-I mainly present in large ones. Two novel findings were the presence of pre-beta migrating HDL transporting only human apo A-II in the higher-expressing mice and the increase of plasma HDL concentrations by fasting in control and transgenic mice. These findings highlight the reciprocal modifications of VLDL and HDL induced by the feeding-fasting transition and the key role of the molar ratio of apo A-II/A-I as a determinant of HDL particle metabolism and pre-beta HDL formation.     

Characterization of a naturally occurring trans-splicing intein from Synechocystis sp. PCC6803

A naturally occurring trans-splicing intein from the dnaE gene of Synechocystis sp. PCC6803 (Ssp DnaE intein) was used to characterize the intein-catalyzed splicing reaction. Trans-splicing/cleavage reactions were initiated by combining the N-terminal splicing domain of the Ssp DnaE intein containing five native N-extein residues and maltose binding protein as the N-extein with the C-terminal Ssp DnaE intein splicing domain (E(C)) with or without thioredoxin fused in-frame to its carboxy terminus. Observed rate constants (k(obs)) for dithiothreitol-induced N-terminal cleavage, C-terminal cleavage, and trans-splicing were (1.0 +/- 0.5) x 10(-3), (1.9 +/- 0.9) x 10(-4), and (6.6 +/- 1.3) x 10(-5) s(-1), respectively. Preincubation of the intein fragments showed no change in k(obs), indicating association of the two splicing domains is rapid relative to the subsequent steps. Interestingly, when E(C) concentrations were substoichiometric with respect to the N-terminal splicing domain, the levels of N-terminal cleavage were equivalent to the amount of E(C), even over a 24 h period. Activation energies for N-terminal cleavage and trans-splicing were determined by Arrhenius plots to be 12.5 and 8.9 kcal/mol, respectively. Trans-splicing occurred maximally at pH 7.0, while a slight increase in the extent of N-terminal cleavage was observed at higher pH values. This work describes an in-depth kinetic analysis of the splicing and cleavage activity of an intein, and provides insight for the use of the split intein as an affinity domain.     

Trapping conformational intermediate states in the reaction center protein from photosynthetic bacteria

In protein, conformational changes are often crucial for function but not easy to observe. Two functionally relevant conformational intermediate states of photosynthetic reaction center protein (RCs) are trapped and characterized at low temperature. RCs frozen in the dark do not allow electron transfer from the reduced primary quinone, Q(A)(-), to the secondary quinone, Q(B). In contrast, RCs frozen under illumination in the product (P(+)Q(A)Q(B)(-)) state, with the oxidized electron donor, P(+), and reduced Q(B)(-), return to the ground state at cryogenic temperature in a conformation that allows a high yield of Q(B) reduction. Thus, RCs frozen under illumination are found to be trapped above the ground state in a conformation that allows product formation. When the temperature is raised above 120 K, the protein relaxes to an inactive conformation which is different from the RCs frozen in the dark. The activation energy for this change is 87 +/- 8 meV, and the active and inactive states differ in energy by only 16 +/- 3 meV. Thus, there are several conformational substates along the reaction coordinate with different transition temperatures. The ground state spectra of the RCs in active and inactive conformations report differences in the intraprotein electrostatic field, demonstrating that the dipole or charge distribution has changed. In addition, the electrochromic shift associated with the Q(A)(-) to Q(B) electron transfer at low temperature was characterized. The electron-transfer rate from Q(B)(-) to P(+) was measured at cryogenic temperature and is similar to the rate at room temperature, as expected for an exothermic, electron tunneling reaction in RCs.     

Involvement of the MAP kinase pathways in induction of GADD45 following UV radiation

Indian hedgehog (Ihh) is highly expressed in prehypertrophic chondrocytes in vivo and has been proposed to regulate the proliferation and maturation of chondrocytes and bone collar formation in the growth plate. In high-density cultures of rabbit growth-plate chondrocytes, Ihh mRNA was also expressed at the highest level in the prehypertrophic stage. To explore endogenous factors that regulate Ihh expression in chondrocytes, we examined the effects of various growth factors on Ihh mRNA expression in this system. Retinoic acid (RA) and bone morphogenetic protein-2 enhanced Ihh mRNA expression, whereas PTH/PTH-related peptide (PTHrP) markedly suppressed Ihh expression. RA at more than 10(-8) M induced the expression of Ihh and Patched 1 (Ptc1) within 3 h, before it increased the type X collagen mRNA level at 6-24 h. Cycloheximide blocked the up-regulation of Ihh by RA, indicating the requirement of de novo protein synthesis for this stimulation. These findings suggest that RA is involved in the up-regulation of Ihh during endochondral bone formation. In contrast to RA, PTH (1-84) at 10(-7) M abolished the mRNA expression of Ihh and Ptc1 within 2-4 h, before it suppressed the expression of type X collagen at 12-24 h. The inhibition of Ihh expression by PTH (1-84) did not require de novo protein synthesis. PTH (1-34), PTHrP (1-34), and (Bu)(2)cAMP also suppressed Ihh expression. On the other hand, Ihh has been reported to induce PTHrP synthesis in the perichondrium. Consequently, the direct inhibitory action of PTH/PTHrP on Ihh appears to be a negative feedback mechanism that prevents excess PTHrP accumulation in cartilage.     

Uncoupling the senescent phenotype from telomere shortening in hydrogen peroxide-treated fibroblasts

Normal human cells have a limited replicative potential and inevitably reach replicative senescence in culture. Replicatively senescent cells show multiple molecular changes, some of which are related to the irreversible growth arrest in culture, whereas others resemble the changes occurring during the process of aging in vivo. Telomeres shorten as a result of cell replication and are thought to serve as a replicometer for senescence. Recent studies show that young cells can be induced to develop features of senescence prematurely by damaging agents, chromatin remodeling, and overexpression of ras or the E2F1 gene. Accelerated telomere shortening is thought to be a mechanism of premature senescence in some models. In this work, we test whether the acquisition of a senescent phenotype after mild-dose hydrogen peroxide (H(2)O(2)) exposure requires telomere shortening. Treating young HDFs with 150 microM H(2)O(2) once or 75 microM H(2)O(2) twice in 2 weeks causes long-term growth arrest, an enlarged morphology, activation of senescence-associated beta-galactosidase, and elevated expression of collagenase and clusterin mRNAs. No significant telomere shortening was observed with H(2)O(2) at doses ranging from 50 to 200 microM. Weekly treatment with 75 microM H(2)O(2) also failed to induce significant telomere shortening. Failure of telomere shortening correlated with an inability to elevate p16 protein or mRNA in H(2)O(2)-treated cells. In contrast, p21 mRNA was elevated over 40-fold and remained at this level for at least 2 weeks after a pulse treatment of H(2)O(2). The role of cell cycle checkpoints centered on p21 in premature senescence induced by H(2)O(2) is discussed here.