Peripheral-Type Benzodiazepine Receptor Function in Cholesterol Transport. Identification of a Putative Cholesterol Recognition/Interaction Amino Acid Sequence and Consensus Pattern

Abstract

In steroid-synthesizing cells, like the MA-10 mouse tumor Leydig cells, the peripheral-type benzodiazepine receptor (PBR) is an outer mitochondrial membrane protein involved in the regulation of cholesterol transport from the outer to the inner mitochondrial membrane, the rate-determining step in steroid biosynthesis. Expression of PBR in Escherichia coli DE3 cells, which have no PBR, no cholesterol, and do not make steroids, induced the ability to take up cholesterol in a time-dependent, temperature-sensitive, and energy-independent manner. These cells took up no other steroids tested. Addition of the high affinity PBR ligand PK 11195 to cholesterol-loaded membranes, obtained from cells transfected with PBR, resulted in the release of the uptaken cholesterol. Expression in DE3 cells of mutant PBRs demonstrated that deletions in the cytoplasmic carboxy-terminus dramatically reduced the cholesterol uptake function of PBR, although it retained full capacity to bind PK 11195. Site-directed mutagenesis in the carboxy-terminal region of PBR demonstrated that bacteria expressing the mutant PBR proteins PBR(Y153S) and PBR(R156L) do not accumulate cholesterol, suggesting that amino acids Y153 and R156 are involved in the interaction of the receptor with cholesterol. Considering these results, we postulate the existence of a common cholesterol recognition/interaction amino acid consensus pattern (-L/V-(X)(1-5)-Y-(X)(1-5)-R/K-). Indeed, we found this amino acid consensus pattern in all proteins shown to interact with cholesterol. In conclusion, these data suggest that the expression of PBR confers the ability to take up and release, upon ligand activation, cholesterol. Considering the widespread occurrence of this protein and its tissue and cell specific subcellular localization, these results suggest a more general role of PBR in intracellular cholesterol transport and compartmentalization.

Aggregatibacter actinomycetemcomitans Leukotoxin Utilizes a Cholesterol Recognition/Amino Acid Consensus Site for Membrane Association

“…We have shown that LtxA binds specifically to cholesterol-containing membranes in a unique mechanism with a strong cholesterol-dependent dissociation rate but a cholesterol-independent association rate (Fig. 2) ), were identified as potential cholesterol binding sites, based upon the CRAC motif, (L/V)X 1-5 YX 1-5 (R/K) (60,61). Although both sites are juxtaposed to the LtxA hydrophobic domain, the requirements for a CRAC motif are quite flexible, and therefore, not all predicted CRAC sites bind cholesterol or possess an in vivo function.…”

Camel Milk: An Important Natural Adjuvant

“…The potential of camel milk lactoferrin for its ability to inhibit the proliferation of the colon cancer cell line has been reported [42]. The concentration of lysozyme, lactoferrin and immunoglobulins in camel milk is higher than in bovine or buffalo milk [30,68]. The antimicrobial activity of these molecules is, however, lost in camel milk after heat treatment at 100°C for 30 min [25].…”

“…The hypocholesterolemia mechanism of camel milk is still unclear, but different hypotheses have been proposed, including: interaction between bioactive peptides derived from camel milk proteins and cholesterol, which result in cholesterol reduction [68], and the presence of orotic acid in camel milk which is thought to be responsible for lowering cholesterol level in human subjects [17] and in rats [96].…”

Identification of Novel Cholesterol-binding Regions in Kir2 Channels

“…With the critical residues that affect cholesterol sensitivity being primarily non-polar aliphatic, the cholesterol-binding regions that we have identified differ from both the CRAC and the CCM cholesterol binding motifs, which include an aromatic residue and a positively charged residue in addition to a non-polar aliphatic residue. Specifically, the CRAC motif is -(L/V)X 1-5 YX 1-5 (R/K)-, where X 1-5 represents 1-5 residues of any amino acid (28,29). The CCM cholesterol binding motif includes sites on adjacent helices: (W/Y)(I/V/L)(K/R) on one helix and (F/Y/R) on the second helix (32).…”

“…Recent studies have shown that a well known cholesterol binding motif, the cholesterol recognition amino acid consensus (CRAC) motif (28,29), is found in TRPV1 channels (21) and in BK channels (30), representing putative cholesterol binding sites. CRAC motifs are also found in several types of AChR, but it has been suggested that it is not the CRAC itself but rather its inverted sequence, CARC, that may be responsible for cholesterol interactions with AChR (31).…”