Functional characterization of a highly specific l-arabinose transporter from Trichoderma reesei

BACKGROUND Lignocellulose biomass has been investigated as a feedstock for second generation biofuels and other value-added products. Some of the processes for biofuel production utilize cellulases and hemicellulases to convert the lignocellulosic biomass into a range of soluble sugars before fermentation with microorganisms such as yeast Saccharomyces cerevisiae. One of these sugars is L-arabinose, which cannot be utilized naturally by yeast. The first step in L-arabinose catabolism is its transport into the cells, and yeast lacks a specific transporter, which could perform this task. RESULTS We identified Trire2_104072 of Trichoderma reesei as a potential L-arabinose transporter based on its expression profile. This transporter was described already in 2007 as D-xylose transporter XLT1. Electrophysiology experiments with Xenopus laevis oocytes and heterologous expression in yeast revealed that Trire2_104072 is a high-affinity L-arabinose symporter with a Km value in the range of [Formula: see text] 0.1-0.2 mM. It can also transport D-xylose but with low affinity (Km [Formula: see text] 9 mM). In yeast, L-arabinose transport was inhibited slightly by D-xylose but not by D-glucose in an assay with fivefold excess of the inhibiting sugar. Comparison with known L-arabinose transporters revealed that the expression of Trire2_104072 enabled yeast to uptake L-arabinose at the highest rate in conditions with low extracellular L-arabinose concentration. Despite the high specificity of Trire2_104072 for L-arabinose, the growth of its T. reesei deletion mutant was only affected at low L-arabinose concentrations. CONCLUSIONS Due to its high affinity for L-arabinose and low inhibition by D-glucose or D-xylose, Trire2_104072 could serve as a good candidate for improving the existing pentose-utilizing yeast strains. The discovery of a highly specific L-arabinose transporter also adds to our knowledge of the primary metabolism of T. reesei. The phenotype of the deletion strain suggests the involvement of other transporters in L-arabinose transport in this species

A Fluorescent hPept1 Transporter Substrate for Uptake Screening

Purpose. To synthesize fluorescent analogues of hPept1 substrates, FITC-Val-OCH 3 , Lys-FITC-OH, and Lys-FITC-OCH 3 , and to characterize their hPept1 transporter-mediated uptake.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/41495/1/11095_2004_Article_475835.pd

Ovalbumin production using Trichoderma reesei culture and low-carbon energy could mitigate the environmental impacts of chicken-egg-derived ovalbumin

Ovalbumin (OVA) produced using the fungus Trichoderma reesei (Tr-OVA) could become a sustainable replacement for chicken egg white protein powder—a widely used ingredient in the food industry. Although the approach can generate OVA at pilot scale, the environmental impacts of industrial-scale production have not been explored. Here, we conducted an anticipatory life cycle assessment using data from a pilot study to compare the impacts of Tr-OVA production with an equivalent functional unit of dried chicken egg white protein produced in Finland, Germany and Poland. Tr-OVA production reduced most agriculture-associated impacts, such as global warming and land use. Increased impacts were mostly related to industrial inputs, such as electricity production, but were also associated with glucose consumption. Switching to low-carbon energy sources could further reduce environmental impact, demonstrating the potential benefits of cellular agriculture over livestock agriculture for OVA production.Peer reviewe

Amino Acid Ester Prodrugs of Floxuridine: Synthesis and Effects of Structure, Stereochemistry, and Site of Esterification on the Rate of Hydrolysis

Purpose . To synthesize amino acid ester prodrugs of floxuridine (FUdR) and to investigate the effects of structure, stereochemistry, and site of esterification of promoiety on the rates of hydrolysis of these prodrugs in Caco-2 cell homogenates.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/41502/1/11095_2004_Article_471011.pd

Comparison of Human Duodenum and Caco-2 Gene Expression Profiles for 12,000 Gene Sequences Tags and Correlation with Permeability of 26 Drugs

Purpose . To compare gene expression profiles and drug permeability differences in Caco-2 cell culture and human duodenum.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/41490/1/11095_2004_Article_450750.pd

Floxuridine Amino Acid Ester Prodrugs: Enhancing Caco-2 Permeability and Resistance to Glycosidic Bond Metabolism

The aim of this study was to synthesize amino acid ester prodrugs of 5-fluoro-2′-deoxyuridine (floxuridine) to enhance intestinal absorption and resistance to glycosidic bond metabolism.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/41510/1/11095_2005_Article_6156.pd

Designing anticancer prodrugs for targeting drug transporters and activating enzymes.

The anticancer drug, floxuridine, exhibits poor oral bioavailability partially due to extensive first pass metabolism by pyrimidine nucleoside phosphorylases to a less potent metabolite, 5-fluorouracil. The objective of this work was to design amino acid ester prodrugs of floxuridine to enhance intestinal absorption and minimize ester and glycosidic bond metabolism. In designing prodrugs to improve oral bioavailability, it was important to know what transporters or metabolizing enzymes prodrugs would encounter after reaching the absorptive surface of the intestine. Microarray expression profiling revealed that transporter expression in the Caco-2 cell model is most similar to that found in the human ileum. In terms of metabolizing enzyme expression, this model best resembles the colon. Based on peptide transporter and metabolizing enzyme expression in the intestine, floxuridine prodrugs were designed to take advantage of carrier mediated transport and resist metabolism. Twenty-seven floxuridine prodrugs were synthesized with diversity in promoiety structure, stereochemistry, and esterification site to understand the structural features involved in optimal prodrug absorption via peptide transporters, as well as how different promoieties are hydrolyzed and how they may affect parent drug metabolism. Floxuridine permeability across Caco-2 cells was enhanced up to 11-fold when the 5' position was esterified with an isoleucine or valine amino acid. The 5'-Val, 5'-Phe, 5'-Leu, and 5' -Ile prodrugs exhibited permeability values between 1--5 x 10-6 cm/s, similar to that observed with valacylovir. PEPT1 prodrug transport correlated well with permeability across Caco-2 monolayers suggesting peptide carrier enhancement. The promoiety and the esterification site most significantly influenced carboxylesterase mediated activation rates. Carboxylesterase displayed the greatest catalytic efficiency for prodrugs containing a phenylalanine residue and was less active against prodrugs containing valine or isoleucine, regardless of the esterification site. Floxuridine was rapidly cleaved by thymidine phosphorylase, whereas gemcitabine and all the ester prodrugs resisted breakdown. Therefore, the rate limiting step to prodrug metabolism is ester bond hydrolysis which subsequently releases the parent drug. This suggests that stable ester prodrugs may provide a mechanism to control undesired floxuridine metabolism. The combined strategy of increasing the intestinal uptake and reducing the metabolism of floxuridine may lead to a more effective orally administered cancer drug.Ph.D.Health and Environmental SciencesOncologyPharmaceutical sciencesUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/124853/2/3163856.pd

Mechanisms and regulation of epithelial Ca2+ absorption in health and disease

Ca2+ is essential for numerous physiological functions in our bodies. Therefore, its homeostasis is finely maintained through the coordination of intestinal absorption, renal reabsorption, and bone resorption. The Ca2+-selective epithelial channels TRPV5 and TRPV6 have been identified, and their physiological roles have been revealed: TRPV5 is important in final renal Ca2+ reabsorption, and TRPV6 has a key role in intestinal Ca2+ absorption. The TRPV5 knockout mice exhibit renal leak hypercalciuria and accordingly upregulate their intestinal TRPV6 expression to compensate for their negative Ca2+ balance. In contrast, despite their severe negative Ca2+ balance, TRPV6-null mice do not display any compensatory mechanism, thus resulting in secondary hyperparathyroidism. These results indicate that the genes for TRPV5 and TRPV6 are differentially regulated in human diseases associated with disturbed Ca2+ balance such as hypercalciuria, osteoporosis, and vitamin D-resistant rickets

Dipeptidyl peptidase IV as a potential target for selective prodrug activation and chemotherapeutic action in cancers

[Image: see text] The efficacy of chemotherapeutic drugs is often offset by severe side effects attributable to poor selectivity and toxicity to normal cells. Recently, the enzyme dipeptidyl peptidase IV (DPPIV) was considered as a potential target for the delivery of chemotherapeutic drugs. The purpose of this study was to investigate the feasibility of targeting chemotherapeutic drugs to DPPIV as a strategy to enhance their specificity. The expression profile of DPPIV was obtained for seven cancer cell lines using DNA microarray data from the DTP database, and was validated by RT-PCR. A prodrug was then synthesized by linking the cytotoxic drug melphalan to a proline-glycine dipeptide moiety, followed by hydrolysis studies in the seven cell lines with a standard substrate, as well as the glycyl-prolyl-melphalan (GP-Mel). Lastly, cell proliferation studies were carried out to demonstrate enzyme-dependent activation of the candidate prodrug. The relative RT-PCR expression levels of DPPIV in the cancer cell lines exhibited linear correlation with U95Av2 Affymetrix data (r(2) = 0.94), and with specific activity of a standard substrate, glycine-proline-p-nitroanilide (r(2) = 0.96). The significantly higher antiproliferative activity of GP-Mel in Caco-2 cells (GI(50) = 261 μM) compared to that in SK-MEL-5 cells (GI(50) = 807 μM) was consistent with the 9-fold higher specific activity of the prodrug in Caco-2 cells (5.14 pmol/min/μg protein) compared to SK-MEL-5 cells (0.68 pmol/min/μg protein) and with DPPIV expression levels in these cells. Our results demonstrate the great potential to exploit DPPIV as a prodrug activating enzyme for efficient chemotherapeutic drug targeting