Hydrogen exchange solvent protection by an ATP analogue reveals conformational changes in ERK2 upon activation

Structural and kinetic studies have provided extensive information about the molecular mechanisms of kinase activation by phosphorylation. However, it is still unclear how changes in protein dynamics and flexibility contribute to catalytic function. Mass spectrometry was used to probe changes in hydrogen/deuterium exchange in the MAP kinase, ERK2, in the presence and absence of the ATP analogue, AMP-PNP. In both active and inactive forms of ERK2, protection from hydrogen exchange by AMP-PNP binding was observed within conserved ATP binding motifs in the N-terminal lobe, which are known to directly interact with nucleotide in various protein kinases. In contrast, higher protection from exchange by AMP-PNP was observed in active ERK2 compared to inactive ERK2, in a region corresponding to the conserved DFG motif, which is located in the C-terminal lobe and coordinates Mg2+ at the catalytic site. Thus, AMP-PNP binding simultaneously protects residues within the N and C terminus in the active form of ERK2, but not the inactive form. This demonstrates that ERK2 binds nucleotide in two modes, in which active ERK2 adopts a closed conformation following nucleotide binding in solution, while inactive ERK2 adopts an open conformation. The finding provides novel evidence that phosphorylation of ERK2 facilitates interdomain closure, allowing proper orientation between ATP and substrate to facilitate phosphoryl transfer.     

Regiospecific methylation of naringenin to ponciretin by soybean O-methyltransferase expressed in Escherichia coli

Flavonoids found in plants most likely undergo a variety of modification reactions such as hydroxylation, glycosylation, and/or methylation. Among these, O-methylation has an effect on the solubility and thus on the antimicrobial activity of the flavonoids. We analyzed the conversion of naringenin with a methyltransferase, SOMT-2, from Glycine max. SOMT-2 was expressed in Escherichia coli as a glutathion S-transferase fusion protein. E. coli harboring SOMT-2 was grown with daidzein, geninstein, apigenin, naringenin, and quercetin, respectively, and reaction products were analyzed with thin layer chromatography and HPLC. SOMT-2 could convert apigenin, daidzein, genistein, and quercetin into the corresponding 4'-O-methylated compounds such as acacetin, formononetin, biochanine A, and 4'-methylated quercetin whereas naringenin turned out to be the best substrate tested. SOMT-2 stoichiometically converted naringenin (4',5,7-trihyroxyflavanone) into a ponciretin (4'-methoxy-5,7-dihydroxyflavanone), whose structure was determined by NMR and LC/mass spectral analyses. Considering the reactions, SOMT-2 may have a regiospecific methylation activity, resulting in transforming 4'-hydroxyl group of flavonoids B-ring to 4'-methyl group.     

Epoxide formation on the aromatic B ring of flavanone by biphenyl dioxygenase of Pseudomonas pseudoalcaligenes KF707

Prokaryotic dioxygenase is known to catalyze aromatic compounds into their corresponding cis-dihydrodiols without the formation of an epoxide intermediate. Biphenyl dioxygenase from Pseudomonas pseudoalcaligenes KF707 showed novel monooxygenase activity by converting 2(R)- and 2(S)-flavanone to their corresponding epoxides (2-(7-oxabicyclo[4.1.0]hepta-2,4-dien-2-yl)-2, 3-dihydro-4H-chromen-4-one), whereby the epoxide bond was formed between C2' and C3' on the B ring of the flavanone. The enzyme also converted 6-hydroxyflavanone and 7-hydroxyflavanone, which do not contain a hydroxyl group on the B-ring, to their corresponding epoxides. In a previous report (S.-Y. Kim, J. Jung, Y. Lim, J.-H. Ahn, S.-I. Kim, and H.-G. Hur, Antonie Leeuwenhoek 84:261-268, 2003), however, we found that the same enzyme showed dioxygenase activity toward flavone, resulting in the production of flavone cis-2',3'-dihydrodiol. Extensive structural identification of the metabolites of flavanone by using high-pressure liquid chromatography, liquid chromatography/mass spectrometry, and nuclear magnetic resonance confirmed the presence of an epoxide functional group on the metabolites. Epoxide formation as the initial activation step of aromatic compounds by oxygenases has been reported to occur only by eukaryotic monooxygenases. To the best of our knowledge, biphenyl dioxygenase from P. pseudoalcaligenes KF707 is the first prokaryotic enzyme detected that can produce an epoxide derivative on the aromatic ring structure of flavanone.     

Replication fork blockage by RTS1 at an ectopic site promotes recombination in fission yeast

Homologous recombination is believed to play important roles in processing stalled/blocked replication forks in eukaryotes. In accordance with this, recombination is induced by replication fork barriers (RFBs) within the rDNA locus. However, the rDNA locus is a specialised region of the genome, and therefore the action of recombinases at its RFBs may be atypical. We show here for the first time that direct repeat recombination, dependent on Rad22 and Rhp51, is induced by replication fork blockage at a site-specific RFB (RTS1) within a 'typical' genomic locus in fission yeast. Importantly, when the RFB is positioned between the direct repeat, conservative gene conversion events predominate over deletion events. This is consistent with recombination occurring without breakage of the blocked fork. In the absence of the RecQ family DNA helicase Rqh1, deletion events increase dramatically, which correlates with the detection of one-sided DNA double-strand breaks at or near RTS1. These data indicate that Rqh1 acts to prevent blocked replication forks from collapsing and thereby inducing deletion events.     

MHC expression in a human adult stem cell line and its down-regulation by hCMV US gene transfection

Due to their unique capacity to self-renew and for multiple differentiation, stem cells are considered promising candidates for cell replacement therapy in many devastating diseases. However, studies on immune rejection, which is a major problem facing successful stem cell therapy, are rare. In this study, we examined MHC expression in the M13SV1 cell line, which has previously been shown to have stem cell properties and to be non-tumorigenic, in order to determine whether human adult stem cells might be rejected after transplantation. Our results show low expression levels of MHC class I molecules on the surface of these cells. An induction of MHC class I expression was observed when the cells were treated with IFN-gamma. Maximal induction of MHC class protein expression was observed at 48 h after treatment with concentrations above 5 ng/ml of IFN-gamma. Elevated MHC class I levels were sustained for 72 h after withdrawing IFN-gamma. Therefore, we introduced human cytomegalovirus (hCMV) US genes, which are known to be able to reduce MHC class I expression on the cell surface after infection, into M13SV1 cells. Cells transfected with the hCMV US2, US3, US6 or US11 genes exhibited a reduction (40-60%) of MHC class I expression compared with mock-transfected cells. These results suggest that human adult stem cells are capable of expressing high levels of MHC class I proteins, and thus may be rejected on transplantation unless they are modified. In addition, viral stealth mechanisms can be exploited for stem cell transplantation.     

PIKE GTPase signaling and function

PIKE (PI 3-Kinase Enhancer) is a recently identified brain specific nuclear GTPase, which binds PI 3-kinase and stimulates its lipid kinase activity. Nerve growth factor treatment leads to PIKE activation by triggering the nuclear translocation of phospholipase C-gamma1 (PLC-gamma1), which acts as a physiologic guanine nucleotide exchange factor (GEF) for PIKE through its SH3 domain. To date, three forms of PIKE have been characterized: PIKE-S, PIKE-L and PIKE-A. PIKE-S is initially identified shorter isoform. PIKE-L, a longer isoform of PIKE gene, differs from PIKE-S by C-terminal extension containing Arf-GAP (ADP ribosylation factor-GTPase Activating Protein) and two ankyrin repeats domains. In contrast to the exclusive nuclear localization of PIKE-S, PIKE-L occurs in both the nucleus and the cytoplasm. PIKE-L physiologically associates with Homer 1, an mGluR I binding adaptor protein. The Homer/PIKE-L complex couples PI 3-kinase to mGluR I and regulates a major action of group I mGluRs, prevention of neuronal apoptosis. More recently, a third PIKE isoform, PIKE-A was identified in human glioblastoma multiforme brain cancers. Unlike the brain specific PIKE-L and -S isoforms, PIKE-A distributes in various tissues. PIKE-A contains the same domains present in PIKE-L but lacks N-terminal proline-rich domain (PRD), which binds PI 3-kinase and PLC-gamma1. Instead, PIKE-A specifically binds to active Akt and upregulates its activity in a GTP-dependent manner, mediating human cancer cell invasion and preventing apoptosis. Thus, PIKE extends its roles from the nucleus to the cytoplasm, mediating cellular processes from cell invasion to programmed cell death.     

Total synthesis and antiangiogenic activity of cyclopentane analogues of fumagillol

Novel cyclopentane analogues of fumagillol were synthesized and their endothelial cell proliferation inhibitory activities were evaluated. The cyclopentane-fumagillol derivatives were synthesized from (-)-2,3-O-isopropylidene-D-erythronolactone via stereoselective glycolate Claisen rearrangement and intramolecular ester enolate alkylation as key steps.