Light-induced global conformational change of photoactive yellow protein in solution

The light-induced global conformational change of photoactive yellow protein was directly observed by small-angle X-ray scattering (SAXS). The N-terminal 6, 15, or 23 amino acid residues were enzymatically truncated (T6, T15, or T23, respectively), and their near-UV intermediates were accumulated under continuous illumination for SAXS measurements. The Kratky plot demonstrated that illumination induced partial loss of globularity. The change in globularity was marked in T6 but very small in T15 and T23, suggesting that structural change in positions 7-15 mainly reduces the globularity. The radius of gyration (R(g)) estimated by Guinier plot was increased by 1.1 A for T6 and 0.7 A for T15 and T23 upon illumination. As T23 lacks most of the N-terminal loop, structural change in the main part composed of the PAS core, helical connector, and beta-scaffold caused an increase of R(g) by 0.7 A. The structural change of positions 7-15 caused an additional increase by 0.4 A. The decrease of R(g) upon truncation of positions 7-15 for dark state was 0.3 A, while that for the intermediate was 0.7 A, suggesting that this region moves outward on formation of the intermediate. These results indicate that a light-induced structural change of PYP takes place in the main part and N-terminal 15 amino acid residues. The former induces only dimensional increase, but the latter results in additional change in shape.     

Effect of organic anions on the photoreaction of photoactive yellow protein

In order to clarify changes in the structure and surface properties of photoactive yellow protein (PYP) upon light absorption, the spectroscopic properties and solution structure of its photo-intermediate (PYP(M)) were examined in the presence of various anions. At identical ionic strengths, citrate slowed the decay rate of PYP(M) more than acetate. Although the absorption spectrum in the dark was not affected by organic anions, citrate induced a 5-nm blue shift of the absorption maximum for PYP(M). Solution X-ray scattering experiments indicated that the radius of gyration (Rg) and apparent molecular weight in the dark were constant in all buffer systems. However, the Rg of PYP(M) in citrate buffer at high concentration was 16.2 (+/-0.2) A, while the Rg of PYP(M) in acetate buffer was 15.6 (+/-0.2) A. The apparent molecular weight increased 7% upon PYP(M) formation in citrate buffer at high concentration compared to other conditions. These results suggest that citrate molecules specifically bind to PYP(M). A cluster of basic amino acid residues with a hydrogen bond donor would be exposed upon PYP(M) formation and responsible for the specific binding of citrate.     

Can the low-resolution structures of photointermediates of bacteriorhodopsin explain their crystal structures?

To understand the molecular mechanism of light-driven proton pumps, the structures of the photointermediates of bacteriorhodopsin have been intensively investigated. Low-resolution diffraction techniques have demonstrated substantial conformational changes at the helix level in the M and N intermediates, between which there are noticeable differences. The intermediate structures at atomic resolution have also been solved by x-ray crystallography. Although the crystal structures have demonstrated local structural changes, such as hydrogen bond network rearrangements including water molecules, the large conformational changes at the helix level are not necessarily observed. Furthermore, the two reported crystal structures of an intermediate accumulated using a common method were distinct. To reconcile these apparent discrepancies, low-resolution projection maps were calculated from the crystal structures and compared to the low-resolution intermediate structures obtained using native membranes. The crystal structures can be categorized into three groups, which qualitatively correspond to the low-resolution structures of the M1-type, M2-type, and N-type determined in the native membrane. Based on these results, we conclude that at least three types of intermediate structures play a role during the photocycle.     

Escherichia coli mutator (Delta)polA is defective in base mismatch correction: the nature of in vivo DNA replication errors

We constructed a set of Escherichia coli strains containing deletions in genes encoding three SOS polymerases, and defective in MutS and DNA polymerase I (PolI) mismatch repair, and estimated the rate and specificity of spontaneous endogenous tonB(+)-->tonB- mutations. The rate and specificity of mutations in strains proficient or deficient in three SOS polymerases was compared and found that there was no contribution of SOS polymerases to the chromosomal tonB mutations. MutS-deficient strains displayed elevated spontaneous mutation rates, consisting of dominantly minus frameshifts and transitions. Minus frameshifts are dominated by warm spots at run-bases. Among 57 transitions (both G:C-->A:T and A:T-->G:C), 35 occurred at two hotspot sites. PolI-deficient strains possessed an increased rate of deletions and frameshifts, because of a deficiency in postreplicative deletion and frameshift mismatch corrections. Frameshifts in PolI-deficient strains occurred within the entire tonB gene at non-run and run sequences. MutS and PolI double deficiency indicated a synergistic increase in the rate of deletions, frameshifts and transitions. In this case, mutS-specific hotspots for frameshifts and transitions disappeared. The results suggested that, unlike the case previously known pertaining to postreplicative MutS mismatch repair for frameshifts and transitions and PolI mismatch repair for frameshifts and deletions, PolI can recognize and correct transition mismatches. Possible mechanisms for distinct MutS and PolI mismatch repair are discussed. A strain containing deficiencies in three SOS polymerases, MutS mismatch repair and PolI mismatch repair was also constructed. The spectrum of spontaneous mutations in this strain is considered to represent the spectrum of in vivo DNA polymerase III replication errors. The mutation rate of this strain was 219x10(-8), about a 100-fold increase relative to the wild-type strain. Uncorrected polymerase III replication errors were predominantly frameshifts and base substitutions followed by deletions.     

Characterization and crystallization of an IscU-type scaffold protein with bound [2Fe-2S] cluster from the hyperthermophile, aquifex aeolicus

IscU plays a key role during iron-sulphur (Fe-S) cluster biosynthesis as a scaffold for the assembly of a nascent, highly labile Fe-S cluster. Here we report the characterization of an IscU-type protein (Aa IscU) from the hyperthermophilic bacterium Aquifex aeolicus. Unlike other known homologues of IscU, expression of Aa IscU in Escherichia coli has yielded an Fe-S cluster-containing holo-protein. Biochemical and spectroscopic studies of the wild-type Aa IscU and its Asp38-to-Ala substituted (D38A) variant molecule indicate that the holo-protein forms a trimer containing substoichiometric [2Fe-2S] cluster with its stability substantially increased by a D38A substitution. The [2Fe-2S] cluster was oxygen-labile and upon loss of the cluster, the resultant apo-form dissociated into a smaller species, a mixture of monomer and dimer with the dimer form predominating. Reddish-brown crystals of holo-Aa IscU-D38A were obtained under anaerobic conditions, that gave diffractions beyond 2.0 A resolution with synchrotron radiation. The crystal belongs to the space group P2(1)2(1)2 with unit-cell parameters a = 72.6, b = 122.3, c = 62.4 A, where the asymmetric unit contains three molecules of Aa IscU. Successful crystallization of holo-Aa IscU-D38A strongly suggests that the trimer association carrying substoichiometric [2Fe-2S] cluster represents a conformationally stable oligomeric state.     

Administration of cyclooxygenase-2 inhibitor reduces joint inflammation but exacerbates osteopenia in IL-1 alpha transgenic mice due to GM-CSF overproduction

IL-1alpha transgenic (Tg) mice exhibit chronic inflammatory arthritis and subsequent osteopenia, with IL-1-induced GM-CSF playing an important role in the pathogenesis. This study analyzed the mechanisms underlying osteopenia in Tg mice, and the therapeutic effects of the cyclooxygenase-2 inhibitor celecoxib on such osteopenia. Inhibited osteoclast formation was observed in RANKL-treated bone marrow cell (BMC) cultures from Tg mice and coculture of Tg-derived BMCs and wild-type-derived primary osteoblasts (POBs). FACS analysis indicated that this inhibition was attributable to a decreased number of osteoclast precursors within Tg-derived BMCs. Moreover, in coculture of Tg-derived POBs and either Tg- or wild-type-derived BMCs, osteoclast formation was markedly inhibited because Tg-derived POBs produced abundant GM-CSF, known as a potent inhibitor of osteoclast differentiation. Histomorphometric analysis of Tg mice revealed that both bone formation and resorption were decreased, with bone formation decreased more prominently. Interestingly, administration of celecoxib resulted in further deterioration of osteopenia where bone formation was markedly suppressed, whereas bone resorption remained unchanged. These results were explained by our in vitro observation that celecoxib dose-dependently and dramatically decreased osteogenesis by Tg mouse-derived POBs in culture, whereas mRNA expressions of GM-CSF and M-CSF remained unchanged. Consequently, blockade of PGE(2) may exert positive effects on excessively enhanced bone resorption observed in inflammatory bone disease, whereas negative effects may occur mainly through reduced bone formation, when bone resorption is constitutively down-regulated as seen in Tg mice.     

Cell surface colony-stimulating factor 1 can be cleaved by TNF-alpha converting enzyme or endocytosed in a clathrin-dependent manner

CSF-1 is a hemopoietic growth factor, which plays an essential role in macrophage and osteoclast development. Alternative splice variants of CSF-1 are synthesized as soluble or membrane-anchored molecules, although membrane CSF-1 (mCSF-1) can be cleaved from the cell membrane to become soluble CSF-1. The activities involved in this proteolytic processing, also referred to as ectodomain shedding, remain poorly characterized. In the present study, we examined the properties of the mCSF-1 sheddase in cell-based assays. Shedding of mCSF-1 was up-regulated by phorbol ester treatment and was inhibited by the metalloprotease inhibitors GM6001 and tissue inhibitor of metalloproteases 3. Moreover, the stimulated shedding of mCSF-1 was abrogated in fibroblasts lacking the TNF-alpha converting enzyme (TACE, also known as a disintegrin and metalloprotease 17) and was rescued by expression of wild-type TACE in these cells, strongly suggesting that the stimulated shedding is TACE dependent. Additionally, we observed that mCSF-1 is predominantly localized to intracellular membrane compartments and is efficiently internalized in a clathrin-dependent manner. These results indicate that the local availability of mCSF-1 is actively regulated by ectodomain shedding and endocytosis. This mechanism may have important implications for the development and survival of monocyte lineage cells.     

Impaired bone fracture healing in matrix metalloproteinase-13 deficient mice

Vascular and cellular invasion into the cartilage is a critical step in the fracture healing. Matrix metalloproteinase-13 (MMP-13) is a member of the zinc-dependent endopeptidase family and plays an important role in remodeling of extracellular matrix. Therefore we investigated the possible involvement of MMP-13 in a murine model of stabilized bone fracture healing. Repair of the fracture in MMP-13 deficient (MMP-13(-/-)) mice was significantly delayed and characterized by a retarded cartilage resorption in the fracture callus. Immunohistochemistry indicated severe defects in vascular penetration and chondroclast recruitment to the fracture callus in MMP-13(-/-) mice. Consistent with the observations, the chondrocyte pellets cultured from the MMP13(-/-) mice exhibited diminished angiogenic activities when the pellets were co-cultured with endothelial cells. These results suggest that MMP-13 is crucial to the process of angiogenesis during healing of fracture, especially in the cartilage resorption process.     

Zinc, copper, and carnosine attenuate neurotoxicity of prion fragment PrP106-126

Prion diseases are progressive neurodegenerative diseases that are associated with the conversion of normal cellular prion protein (PrP(C)) to abnormal pathogenic prion protein (PrP(SC)) by conformational changes. Prion protein is a metal-binding protein that is suggested to be involved in metal homeostasis. We investigated here the effects of trace elements on the conformational changes and neurotoxicity of synthetic prion peptide (PrP106-126). PrP106-126 exhibited the formation of β-sheet structures and enhanced neurotoxicity during the aging process. The co-existence of Zn(2+) or Cu(2+) during aging inhibited β-sheet formation by PrP106-126 and attenuated its neurotoxicity on primary cultured rat hippocampal neurons. Although PrP106-126 formed amyloid-like fibrils as observed by atomic force microscopy, the height of the fibers was decreased in the presence of Zn(2+) or Cu(2+). Carnosine (β-alanyl histidine) significantly inhibited both the β-sheet formation and the neurotoxicity of PrP106-126. Our results suggested that Zn(2+) and Cu(2+) might be involved in the pathogenesis of prion diseases. It is also possible that carnosine might become a candidate for therapeutic treatments for prion diseases.     

1H, 13C, and 15N resonance assignment of photoactive yellow protein

Photoactive yellow protein (PYP) is involved in the negative phototactic response towards blue light of the bacterium Halorhodospira halophila. Here, we report nearly complete backbone and side chain ¹H, ¹³C and ¹⁵N resonance assignments at pH 5.8 and 20 °C of PYP in its electronic ground state.