Cofactors revisited - Predicting the impact of flavoprotein-related diseases on a genome scale.

Flavin adenine dinucleotide (FAD) and its precursor flavin mononucleotide (FMN) are redox cofactors that are required for the activity of more than hundred human enzymes. Mutations in the genes encoding these proteins cause severe phenotypes, including a lack of energy supply and accumulation of toxic intermediates. Ideally, patients should be diagnosed before they show symptoms so that treatment and/or preventive care can start immediately. This can be achieved by standardized newborn screening tests. However, many of the flavin-related diseases lack appropriate biomarker profiles. Genome-scale metabolic models can aid in biomarker research by predicting altered profiles of potential biomarkers. Unfortunately, current models, including the most recent human metabolic reconstructions Recon and HMR, typically treat enzyme-bound flavins incorrectly as free metabolites. This in turn leads to artificial degrees of freedom in pathways that are strictly coupled. Here, we present a reconstruction of human metabolism with a curated and extended flavoproteome. To illustrate the functional consequences, we show that simulations with the curated model - unlike simulations with earlier Recon versions - correctly predict the metabolic impact of multiple-acyl-CoA-dehydrogenase deficiency as well as of systemic flavin-depletion. Moreover, simulations with the new model allowed us to identify a larger number of biomarkers in flavoproteome-related diseases, without loss of accuracy. We conclude that adequate inclusion of cofactors in constraint-based modelling contributes to higher precision in computational predictions.

Enhanced cellular uptake of albumin-based lyophilisomes when functionalized with cell-penetrating peptide TAT in HeLa cells.

Lyophilisomes are a novel class of biodegradable proteinaceous nano/micrometer capsules with potential use as drug delivery carrier. Cell-penetrating peptides (CPPs) including the TAT peptide have been successfully implemented for intracellular delivery of a broad variety of cargos including various nanoparticulate pharmaceutical carriers. In the present study, lyophilisomes were modified using CPPs in order to achieve enhanced cellular uptake. Lyophilisomes were prepared by a freezing, annealing, and lyophilization method and a cystein-elongated TAT peptide was conjugated to the lyophilisomes using a heterobifunctional linker. Fluorescent-activated cell sorting (FACS) was utilized to acquire a lyophilisome population with a particle diameter smaller than 1000 nm. Cultured HeLa, OVCAR-3, Caco-2 and SKOV-3 cells were exposed to unmodified lyophilisomes and TAT-conjugated lyophilisomes and examined with FACS. HeLa cells were investigated in more detail using a trypan blue quenching assay, confocal microscopy, and transmission electron microscopy. TAT-conjugation strongly increased binding and cellular uptake of lyophilisomes in a time-dependent manner in vitro, as assessed by FACS. These results were confirmed by confocal microscopy. Transmission electron microscopy indicated rapid cellular uptake of TAT-conjugated lyophilisomes via phagocytosis and/or macropinocytosis. In conclusion, TAT-peptides conjugated to albumin-based lyophilisomes are able to enhance cellular uptake of lyophilisomes in HeLa cells.

Specific targeting of tumor cells by lyophilisomes functionalized with antibodies.

Lyophilisomes are a novel class of proteinaceous biodegradable nano/micro drug delivery capsules prepared by freezing, annealing and Iyophilization. In the present study, lyophilisomes were functionalized for active targeting by antibody conjugation in order to obtain a selective drug-carrier system. Lyophilisomes were vapor crosslinked for 2h, resulting in stable capsules, while leaving sufficient primary amines for further modification. The humanized KC4 (hKC4) antibody was conjugated to lyophilisomes to achieve specific targeting to mucin 1 (MUC1)-overexpressing tumor cells. For this, thiolated antibodies were conjugated to maleimide-activated lyophilisomes, resulting in an hKC4 specific drug targeting system toward MUC1-overexpressing human ovarian and cervical tumor cells. FACS analysis demonstrated that hKC4-conjugated lyophilisomes bound specifically to MUC1-overexpressing tumor cells (HeLa, OVCAR-3, and SKOV-3 cells), compared to MUC1-negative cells (LS174T). In addition, control non-specific IgG-conjugated lyophilisomes did not bind to MUC1-overexpressing tumor cells. When MUC1-positive and -negative cells were combined in one culture, hKC4-conjugated lyophilisomes specifically targeted MUC1-positive cells, whereas negative cells showed merely background levels. Transmission electron microscopy showed uptake of hKC4-conjugated lyophilisomes via phagocytosis or macropinocytosis. In conclusion, hKC4-conjugated albumin-based lyophilisomes represent a potential drug delivery system for targeted drug transport to MUC1-overexpressing tumor cells.