Extracellular vesicles (EVs) are membrane particles secreted from cells into all body fluids. Several EV populations exist differing in size and cellular origin. Using differential centrifugation EVs pelleting at 14,000 g (“microvesicles” (MV)) and 100,000 g (“exosomes”) are distinguishable by protein markers. Neutral sphingomyelinase (nSMase) inhibition has been shown to inhibit exosome release from cells and has since been used to study their functional implications. How nSMases (also known as SMPD2 and SMPD3) affect the basal secretion of MVs is unclear. Here we investigated how SMPD2/3 impact both EV populations. SMPD2/3 inhibition by GW4869 or RNAi decreases secretion of exosomes, but also increases secretion of MVs from the plasma membrane. Both populations differ significantly in metabolite composition and Wnt proteins are specifically loaded onto MVs under these conditions. Taken together, our data reveal a novel regulatory function of SMPD2/3 in vesicle budding from the plasma membrane and clearly suggest that – despite the different vesicle biogenesis – the routes of vesicular export are adaptable.

1 Mycophenolic acid (MPA) is primarily metabolized to a phenolic glucuronide (MPAG) as well as to two further minor metabolites: an acyl glucuronide (AcMPAG) and a phenolic glucoside (MPAG1s). This study presents investigations of the formation of these metabolites by human liver (HLM), kidney (HKM), and intestinal (HIM) microsomes, as well as by recombinant UDPglucuronosyltransferases. 2 HLM (n=5), HKM (n=6), HIM (n=5) and recombinant UGTs were incubated in the presence of either UDP-glucuronic acid or UDP-glucose and various concentrations of MPA. Metabolite formation was followed by h.p.l.c. 3 All microsomes investigated formed both MPAG and AcMPAG. Whereas the e ciency of MPAG formation was greater with HKM compared to HLM, AcMPAG formation was greater with HLM than HKM. HIM showed the lowest glucuronidation e ciency and the greatest interindividual variation. The capacity for MPAGls formation was highest in HKM, while no glucoside was detected with HIM. 4 HKM produced a second metabolite when incubated with MPA and UDP-glucose, which was labile to alkaline treatment. Mass spectrometry of this metabolite in the negative ion mode revealed a molecular ion of m/z 481 compatible with an acyl glucoside conjugate of MPA. 5 All recombinant UGTs investigated were able to glucuronidate MPA with K M values ranging from 115.3 to 275.7 mM l 71 and V max values between 29 and 106 pM min 71 mg protein 71 . 6 Even though the liver is the most important site of MPA glucuronidation, extrahepatic tissues particularly the kidney may play a signi®cant role in the overall biotransformation of MPA in man. Only kidney microsomes formed a putative acyl glucoside of MPA.

Background: Tacrolimus is metabolized predominantly to 13-O-demethyltacrolimus in the liver and intestine by cytochrome P450 3A (CYP3A). Patients with high concentrations of CYP3A5, a CYP3A isoenzyme polymorphically produced in these organs, require higher doses of tacrolimus, but the exact mechanism of this association is unknown. Methods: cDNA-expressed CYP3A enzymes and a bank of human liver microsomes with known CYP3A4 and CYP3A5 content were used to investigate the contribution of CYP3A5 to the metabolism of tacrolimus to 13-O-demethyltacrolimus as quantified by liquid chromatography-tandem mass spectrometry. Results: Demethylation of tacrolimus to 13-O-demethyltacrolimus was the predominant clearance reaction. Calculated K m and V max values for CYP3A4, CYP3A5, and CYP3A7 cDNA-expressed microsomes were 1.5 mol/L and 0.72 pmol ⅐ (pmol P450) ؊1 ⅐ min ؊1 , 1.4 mol/L and 1.1 pmol ⅐ (pmol P450) ؊1 ⅐ min ؊1 , and 6 mol/L and 0.084 pmol ⅐ (pmol P450) ؊1 ⅐ min ؊1 , respectively. Recombinant CYP3A5 metabolized tacrolimus with a catalytic efficiency (V max /K m ) that was 64% higher than that of CYP3A4. The contribution of CYP3A5 to 13-O-demethylation of tacrolimus in human liver microsomes varied from 1.5% to 40% (median, 18.8%). There was an inverse association between the contribution of CYP3A5 to 13-O-demethylation and the amount of 3A4 protein (r ‫؍‬ 0.90; P <0.0001). Mean 13-O-demethylation clearances in CYP3A5 high and low expressers, estimated by the parallel-tube liver model,

The secreted proteolytic activity of Aspergillus fumigatus is of potential importance as a virulence factor and in the industrial hydrolysis of protein sources. The A. fumigatus genome contains sequences that could encode a five-member gene family that produces proteases in the sedolisin family (MEROPS S53). Four putative secreted sedolisins with a predicted 17-to 20-amino-acid signal sequence were identified and termed SedA to SedD. SedA produced heterologously in Pichia pastoris was an acidic endoprotease. Heterologously produced SedB, SedC, and SedD were tripeptidyl-peptidases (TPP) with a common specificity for tripeptide-p-nitroanilide substrates at acidic pHs. Purified SedB hydrolyzed the peptide Ala-Pro-Gly-Asp-Arg-Ile-Tyr-Val-HisPro-Phe to Arg-Pro-Gly, Asp-Arg-Ile, and Tyr-Val-His-Pro-Phe, thereby confirming TPP activity of the enzyme. SedB, SedC, and SedD were detected by Western blotting in culture supernatants of A. fumigatus grown in a medium containing hemoglobin as the sole nitrogen source. A degradation product of SedA also was observed. A search for genes encoding sedolisin homologues in other fungal genomes indicates that sedolisin gene families are widespread among filamentous ascomycetes.

This study demonstrates an interaction between mycophenolate mofetil and rifampin, which is a result of induction of MPA glucuronidation and possibly also rifampin-associated alterations in MRP2-mediated transport of MPAG and AcMPAG. This interaction should be taken into account when rifampin or other drugs influencing pregnane X receptor activity are coadministered with mycophenolate mofetil.