Takashi Okuno

Conserved Pore Residues in the AAA Protease FtsH Are Important for Proteolysis and its Coupling to ATP Hydrolysis

Like other AAA proteins, Escherichia coli FtsH, a membrane-bound AAA protease, contains highly conserved aromatic and glycine residues (Phe228 and Gly230) that are predicted to lie in the central pore region of the hexamer. The functions of Phe228 and Gly230 were probed by site-directed mutagenesis. The results of both in vivo and in vitro assays indicate that these conserved pore residues are important for FtsH function and that bulkier, uncharged/apolar residues are essential at position 228. None of the point mutants, F228A, F228E, F228K, or G230A, was able to degrade σ32, a physiological substrate. The F228A mutant was able to degrade casein, an unfolded substrate, although the other three mutants were not. Mutation of these two pore residues also affected the ATPase activity of FtsH. The F228K and G230A mutations markedly reduced ATPase activity, whereas the F228A mutation caused a more modest decrease in this activity. The F228E mutant was actually more active ATPase. The substrates, σ32 and casein, stimulated the ATPase activity of wild type FtsH. The ATPase activity of the mutants was no longer stimulated by casein, whereas that of the three Phe228 mutants, but not the G230A mutant, remained σ32-stimulatable. These results suggest that Phe228 and Gly230 in the predicted pore region of the FtsH hexamer have important roles in proteolysis and its coupling to ATP hydrolysis.

Oxidation of cyclohexane with hydrogen peroxide catalysed by copper(II) complexes containing N,N-bis(2-pyridylmethyl)-β-alanineamide ligands

Abstract In the presence of hydrogen peroxide, a mononuclear copper(II) complex, [Cu(bdpg)Cl]Cl [where (bdpg) represents N,N-bis(2-pyridylmethyl)-β-alanine-amide] exhibits a much higher activity for the oxygenation of cyclohexane than other analogs. Crystal structure determinations and spectroscopic data indicate that the amide group in the (bdpg) ligand, which can interact with a hydroperoxide-ion η1-bonded to copper(II) plays an important role in activation of hydrogen peroxide, leading to the facile oxygenation of cyclohexane.

Allelic characterization of the leaf-variegated mutation var2 identifies the conserved amino acid residues of FtsH that are important for ATP hydrolysis and proteolysis.

Arabidopsis var1 and var2 mutants exhibit leaf variegation. VAR1 and VAR2 encode similar FtsH metalloproteases (FtsH5 and FtsH2, respectively). We have previously found many variegated mutants to be allelic to var2. Each mutant was shown to express a different degree of variegation, and the formation of white sectors was enhanced in severely variegated alleles when these alleles were grown at low temperature. VAR1/FtsH5 and VAR2/FtsH2 levels were mutually affected even in the weak alleles, confirming our previous observation that the two proteins form a hetero complex. In this study, the sites of the mutations in these var2 alleles were determined. We isolated eight point mutations. Five alleles resulted in an amino acid substitution. Three of the five amino acid substitutions occurred in Walker A and B motifs of the ATP-binding site, and one occurred in the central pore motif. These mutations were considered to profoundly suppress the ATPase and protease activities. In contrast, one mutation was found in a region that contained no obvious signature motifs, but a neighboring sequence, Gly–Ala–Asp, was highly conserved among the members of the AAA protein family. Site-directed mutagenesis of the corresponding residue in E. coli FtsH indeed showed that this residue is necessary for proper ATP hydrolysis and proteolysis. Based on these results, we propose that the conserved Gly–Ala–Asp motif plays an important role in FtsH activity. Thus, characterization of the var2 alleles could help to identify the physiologically important domain of FtsH.

An AAA protease FtsH can initiate proteolysis from internal sites of a model substrate, apo-flavodoxin.

Escherichia coli FtsH, which belongs to the AAA (ATPases associated with diverse cellular activities) family, is an ATP‐dependent and membrane‐bound protease. FtsH degrades misassembled membrane proteins and a subset of cytoplasmic regulatory proteins. It has been proposed that ATP‐dependent proteases unfold substrate proteins and initiate a processive proteolysis from either terminus of the substrate polypeptide. We have found that FtsH degrades E. coli apo‐flavodoxin (apo‐Fld) but not holo‐Fld containing non‐covalently bound flavin mononucleotide (FMN). A mutant Fld carrying a substitution of Tyr94 to Asp (FldYD) with a lower affinity for FMN was efficiently degraded by FtsH. To elucidate the directionality of FldYD degradation by FtsH, we constructed several FldYD fusion proteins with glutathione S‐transferase (GST), green fluorescent protein (GFP), or both GST and GFP. It was found that FtsH was able to initiate degradation of the FldYD moiety even when it was sandwiched by GST and GFP. Evidence indicated that FtsH can initiate proteolysis of GST‐FldYD‐GFP from the FldYD moiety by translocating an internal loop to the protease chamber in an ATP‐dependent manner and that, at least, the proteolysis in the C to N direction proceeds processively.

DNA degradation by the copper(II) complex with tripodal-ligands containing peptide group

Abstract Copper-based transition metal complexes which perform single-strand scission of DNA in a site-specific manner have been designed. The complexes Cu(dpg)+ and Cu(dpgt)Cl+ (H(dpg) and (dpgt) represent bis(2-pyridylmethyl) derivatives of glycylglycine and glycylglycylglycine, respectively, were prepared in this study and its crystal structures were determined. We have found that Cu(dpgt)Cl+ and Cu(dpg)+ complexes convert supercoiled plasmid DNA into a mixture of nicked and linear forms in the presence of hydrogen peroxide, whereas related Cu(II) complexes, which lack a peptide group, produce complete degradation of DNA under the same experimental conditions.

FtsH Protease-Mediated Regulation of Various Cellular Functions

FtsH, a member of the AAA (ATPases associated with a variety of cellular activities) family of proteins, is an ATP-dependent protease of ∼71 kDa anchored to the inner membrane. It plays crucial roles in a variety of cellular processes. It is responsible for the degradation of both membrane and cytoplasmic substrate proteins. Substrate proteins are unfolded and translocated through the central pore of the ATPase domain into the proteolytic chamber, where the polypeptide chains are processively degraded into short peptides. FtsH is not only involved in the proteolytic elimination of unnecessary proteins, but also in the proteolytic regulation of a number of cellular functions. Its role in proteolytic regulation is achieved by one of two approaches, either the cellular levels of a regulatory protein are controlled by processive degradation of the entire protein, or the activity of a particular substrate protein is modified by processing. In the latter case, protein processing requires the presence of a stable domain within the substrate. Since FtsH does not have a robust unfolding activity, this stable domain is sufficient to abort processive degradation of the protein - resulting in release of a stable protein fragment.

FtsH cleavage of non-native conformations of proteins

FtsH is a peculiar prokaryotic protease with low unfoldase activity. Different reports have proposed that FtsH substrates could be either tagged proteins or proteins of low stability. We show here that FtsH degradation of 31 point mutants of Anabaena apoflavodoxin is inversely proportional to their conformational stabilities, and that the same applies to other substrate proteins. In contrast, highly stable proteins such as GST and holoflavodoxin are not degraded at all. Attempts to identify sequence tags signaling for degradation in apoflavodoxin fragments have been unsuccessful. Apoflavodoxin adopts three conformations: native, partly unfolded and fully unfolded. It is revealing that degradation of the 31 variants is proportional to the molar fraction of fully unfolded molecules and inversely proportional to the fraction of stable apoflavodoxin molecules. This indicates that FtsH, rather than unfolding the protein, acts on the fraction that is already unfolded.

Comparison of intracellular localization of Nubp1 and Nubp2 using GFP fusion proteins

Nubp1 (also known as Nbp35) and Nubp2 (also known as Cfd1) proteins are known to be responsible for regulating centrosome duplication in mouse and ribosome biogenesis in yeast. Nubp proteins contribute to diverse physiological functions. It is thought that Nubp1 and Nubp2 proteins interact with each other and regulate their functions. However, little is known about the intracellular localization of Nubp proteins. In this study, we compared the intracellular localization of human Nubp1 and Nubp2 by fusing these proteins with green fluorescent protein (GFP) in HeLa cells. The nuclear transfer of Nubp1–GFP, where GFP was fused to the C-terminus, was not observed. However, GFP–Nubp1, where GFP was fused to the N-terminus, did accumulate in the nucleus. In addition, GFP-modification at the N-terminal of Nubp2 induced nuclear transformation. Our data suggest that the C-terminal region of Nubp1 is important for nuclear transfer and the N-terminal of Nubp2 contributes to the morphology of the nucleus.

Electron delocalization in binuclear manganese(III/IV) complexes with di-μ-oxo bridges

Abstract Solvent-dependent (solvent; acetonitrile, N , N -dimethylformamide, N , N -dimethylacetamide, dimethylsulfoxide and methanol) solution ESR spectra of binuclear manganese(III/IV) compounds with di-μ-oxo bridges have been studied. The binuclear manganese(III/IV) compounds used in this study [ligand; tris(2-pyridylmethyl)amine, 1,4,8,11 - tetraazacyclotetradecane, N , N ′-dimethyl- N , N ′-bis(2-pyridylmethyl)ethylene - diamine and N , N -bis(2-pyridylmethyl)glycine] generally exhibit solvent-dependent solution ESR spectra at room temperature; for example, the complex with tris(2-pyridylmethyl)amine shows a 16-line ESR spectrum in acetonitrile at room temperature, but a 6-line ESR spectrum is observed in the N , N -dimethylformamide solution. Studies on the temperature dependency of the ESR spectra (range 77–295 K) clearly showed that the 6-line ESR signal observed can be attributed to a spin-trapped dimeric species with a di-μ-oxo bridge, rather than a monomeric one, and that the two species, which show 16-line and 6-line ESR signals, are in an equilibrium in the temperature range studied.

Isolation of two geometrical isomers of bis(µ-acetato)(µ-oxo)diiron(III) complexes with tridentate ligands and their activities for functionalization of cyclohexane in the presence of hydrogen peroxide

Two geometrical isomers of bis(µ-acetato)(µ-oxo)diiron(III) complexes with tridentate ligands were isolated and characterized by X-ray crystallography and their activities for cyclohexane functionalization in the presence of hydrogen peroxide examined.