Purification of a Recombinant Human PEX1/PEX6 AAA+ ATPase Complex from HEK293TT Cells.

The heteromeric complex of the two AAA+ ATPases PEX1 and PEX6 is involved in the export of the monoubiquitinated import receptor PEX5 from the peroxisomal membrane. Mutations in this complex make up for over 60% of the patients with Peroxisomal Biogenesis Disorders. To have better options for the treatment of the milder mutations we purified the human PEX1/PEX6 complex after overexpression of plasmids encoding tagged proteins from HEK293TT cells.

Chemically monoubiquitinated PEX5 binds to the components of the peroxisomal docking and export machinery.

Peroxisomal matrix proteins contain either a peroxisomal targeting sequence 1 (PTS1) or a PTS2 that are recognized by the import receptors PEX5 and PEX7, respectively. PEX5 transports the PTS1 proteins and the PEX7/PTS2 complex to the docking translocation module (DTM) at the peroxisomal membrane. After cargo release PEX5 is monoubiquitinated and extracted from the peroxisomal membrane by the receptor export machinery (REM) comprising PEX26 and the AAA ATPases PEX1 and PEX6.

Towards the molecular mechanism of the integration of peroxisomal membrane proteins.

The correct topogenesis of peroxisomal membrane proteins is a crucial step for the formation of functioning peroxisomes. Although this process has been widely studied, the exact mechanism with which it occurs has not yet been fully characterized. Nevertheless, it is generally accepted that peroxisomes employ three proteins - Pex3, Pex19 and Pex16 in mammals - for the insertion of peroxisomal membrane proteins into the peroxisomal membrane.

Progesterone rapidly down-regulates the biosynthesis of 5-lipoxygenase products in human primary monocytes.

5-Lipoxygenase (5-LO), the key enzyme in the biosynthesis of pro-inflammatory leukotrienes (LTs) from arachidonic acid, is regulated by androgens in human neutrophils and monocytes accounting for sex differences in LT formation. Here we show that progesterone suppresses the synthesis of 5-LO metabolites in human primary monocytes. 5-LO product formation in monocytes stimulated with Ca(2+)-ionophore A23187 or with lipopolysaccharide/formyl peptide was suppressed by progesterone at concentrations of 10-100 nM in cells from females and at 1 μM in cells from males.