Crystal structure of a hypothetical protein, TTHA0829 from Thermus thermophilus HB8, composed of cystathionine-ß-synthase (CBS) and aspartate-kinase chorismate-mutase tyrA (ACT) domains.

TTHA0829 from Thermus thermophilus HB8 has a molecular mass of 22,754 Da and is composed of 210 amino acid residues. The expression of TTHA0829 is remarkably elevated in the latter half of logarithmic growth phase. TTHA0829 can form either a tetrameric or dimeric structure, and main-chain folding provides an N-terminal cystathionine-ß-synthase (CBS) domain and a C-terminal aspartate-kinase chorismate-mutase tyrA (ACT) domain. Both CBS and ACT are regulatory domains to which a small ligand molecule can bind. The CBS domain is found in proteins from organisms belonging to all kingdoms and is observed frequently as two or four tandem copies. This domain is considered as a small intracellular module with a regulatory function and is typically found adjacent to the active (or functional) site of several enzymes and integral membrane proteins. The ACT domain comprises four ß-strands and two α-helices in a ßαßßαß motif typical of intracellular small molecule binding domains that help control metabolism, solute transport and signal transduction. We discuss the possible role of TTHA0829 based on its structure and expression pattern. The results imply that TTHA0829 acts as a cell-stress sensor or a metabolite acceptor.

Genuine functions of P-glycoprotein (ABCB1).

P-glycoprotein (P-gp, ABCB1, MDR1) was recognized as a drug-exporting protein from cancer cells three decade ago. Apart from the multidrug transporter side effects of P-gp, normal physiological functions of P-gp have been reported. P-gp could be responsible for translocating platelet-activating factor (PAF) across the plasma membrane and PAF inhibited drug transport mediated by P-gp in cancer cells. P-gp regulated the translocation of sphingomyelin (SM) and GlcCer, and short chain C(6)-NBD-GlcCer was found in the apical medium of P-gp cells exclusively and not in the basolateral membrane. SM plays an important role in the esterification of cholesterol. High expression of P-gp prevents stem-cell differentiation, leading to the proliferation and amplification of this cell repertoire, and functional P-gp plays a fundamental role in regulating programmed cell death, apoptosis. The transporter function of P-gp is therefore necessary to protect cells from death. P-gp can translocate both C(6)-NBD-PC and C(6)-NBD-PE across the apical membrane. This PC translocation was also confirmed with [(3)H]choline radioactivity. Progesterone is not transported by P-gp, but blocks P-gp-mediated efflux of other drugs and P-gp can mediate the transport of a variety of steroids. Cells transfected with human P-gp esterified more cholesterol. P-gp might also be involved in the transport of cytokines, particularly IL-1beta, IL-2, IL-4 and IFNgamma, out of activated normal lymphocytes into the surrounding medium. P-gp expression is also associated with a volume-activated chloride channel, thus P-gp is bifunctional with both transport and channel regulators. We also present information about P-gp polymorphism and new structural concepts, "gate" and "twist", of the P-gp structure.

Study of oxidized lipids as endogenous substrates of P-gp (ABCB1).

We studied endogenous substrates for P-glycoprotein (P-gp) in an oxidative reaction mixture of ceramides, phospholipids, sphingolipids, or GM1-gangliosides (GM1-G). Extracts from the reaction mixture of galactocerebrosides (GalCer), sphingomyelin (SM) , lactocerebrosides (LactoCer), and asolectine (AS) with 0.3% hydrogen peroxide exhibited significant ATPase activity of P-gp of 7.6, 7.8, 5.3, and 4.7 nmol/min/mg protein, respectively, at a concentration of 10 microg equivalent/ml, but not GalCer, SM, LactoCer, and AS themselves. Meanwhile, both GM1-G and its oxidized product showed ATPase activity of 3.7 nmol/min/mg protein at a concentration of 0.75 microM. Phosphatidylcholine, phosphatidylethanolamine, phophatidylserine, triglyceride, and cholesterol did not show P-gp activity. When reactive oxygen species, such as hydrogen peroxide, exceed the ability of antioxidant defense systems to remove it from living cells, SM, GalCer, LactoCer, and AS could react with it; therefore, it is possible for these oxidized lipids to play as substrates for P-gp in living cells. This finding should be a milestone to search a new physiological P-gp function.