Functional characterization of the recombinant human tumour suppressor 101F6 protein, a cytochrome b561 homologue

Abstract

Candidate human tumour suppressor gene product, 101F6 protein, is a highly hydrophobic transmembrane protein and a member of cytochrome b(561) family. Purified 101F6 protein expressed in Pichia pastoris cells showed visible absorption spectra similar but distinct from those of cytochrome b(561). Haem content analysis indicated presence of two haems B per molecule. Midpoint potentials of the purified protein were found as +109 and +26 mV for two haems, slightly lower than those for bovine chromaffin granule or plant Zea mays cytochromes b(561). Electron paramagnetic resonance (EPR) spectra in oxidized state at 5 K showed only a highly anisotropic low-spin (HALS) signal at g(z) = 3.75. However, at 15 and 20 K, another HALS-type signal appeared at g(z) = 3.65 being overlapped with that of g(z) = 3.75. The rhombic EPR signal at g(z) = 3.16 previously seen in other cytochromes b(561) was not observed, suggesting distinct haem environments. Absence of the inhibition in the electron transfer from ascorbate by a treatment of 101F6 protein with diethylpyrocarbonate showed a remarkable contrast from those of other cytochromes b(561) where the 'concerted H(+)/e(-) transfer mechanism' at the cytosolic haem centre was blocked by specific Nε-carbethoxylation of haem-coordinating imidazole, suggesting that 101F6 protein might accept electrons via a mechanism distinct from other cytochromes b(561).

Characterization of the PH1704 Protease from Pyrococcus horikoshii OT3 and the Critical Functions of Tyr120

“…1). It is reported the Cys106 of human DJ-1 which is equivalent to Cys100 in PH1704 is the most sensitive residue under the oxidative environment [33], [34]. Cys185 of Hsp31 was identified as nucleophilic site by inhibition and mutational test [5]–[7].…”

Elucidation of molecular functions of human tumor suppressor protein 101F6 by reconstitution into phospholipid bilayer nanodiscs

“…Expression and purification of 101F6 protein were conducted by employing alcohol-assimilating yeast Pichia pastoris cells and pPICZB-101F6-His8 plasmid (Recuenco et al 2013a, Recuenco et al 2013b. In brief, human 101F6 gene was incorporated into the Pichia genome and successful transformants were selected by zeocin-resistance screening.…”

Functional Assembly of Caenorhabditis elegans Cytochrome b-2 (Cecytb-2) into Phospholipid Bilayer Nanodisc with Enhanced Iron Reductase Activity

“…The linearized plasmid pPICZB-Cecytb-2-H6 with PmeI was inserted into the P. pastoris GS115 genome using EasyComp transformation protocol (Invitrogen Corp., Tokyo, Japan). Successfully transformed cells were selected on Yeast Extract Peptone Dextrose Sorbitol Medium (YPDS) agar plates containing 100-400 µg/mL Zeocin (Invitrogen Corp.) The culture for the Cecytb-2 expression was done as previously reported in [27], (Miura et al, unpublished results). The purification of the expressed Cecytb-2 was performed with Ni-Sepharose affinity column as described in [27,28] with some modification (for detailed procedures, see Supplementary file) (Miura et al, unpublished results).…”

“…Successfully transformed cells were selected on Yeast Extract Peptone Dextrose Sorbitol Medium (YPDS) agar plates containing 100-400 µg/mL Zeocin (Invitrogen Corp.) The culture for the Cecytb-2 expression was done as previously reported in [27], (Miura et al, unpublished results). The purification of the expressed Cecytb-2 was performed with Ni-Sepharose affinity column as described in [27,28] with some modification (for detailed procedures, see Supplementary file) (Miura et al, unpublished results). UV-visible absorption spectra of the purified Ceytb-2 protein were recorded in a region from 700 to 200 nm using a Shimadzu UV-2400PC spectrophotometer (Shimadzu Corp., Kyoto, Japan).…”