Mutations in the human TGFBI gene encoding TGFBIp have been linked to protein deposits in the cornea leading to visual impairment. The protein consists of an N-terminal Cys-rich EMI domain and four consecutive fasciclin 1 (FAS1) domains. We have compared the stabilities of wild-type (WT) human TGFBIp and six mutants known to produce phenotypically distinct deposits in the cornea. Amino acid substitutions in the first FAS1 (FAS1-1) domain (R124H, R124L, and R124C) did not alter the stability. However, substitutions within the fourth FAS1 (FAS1-4) domain (A546T, R555Q, and R555W) affected the overall stability of intact TGFBIp revealing the following stability ranking R555W>WT>R555Q>A546T. Significantly, the stability ranking of the isolated FAS1-4 domains mirrored the behavior of the intact protein. In addition, it was linked to the aggregation propensity as the least stable mutant (A546T) forms amyloid fibrils while the more stable variants generate non-amyloid amorphous deposits in vivo. Significantly, the data suggested that both an increase and a decrease in the stability of FAS1-4 may unleash a disease mechanism. In contrast, amino acid substitutions in FAS1-1 did not affect the stability of the intact TGFBIp suggesting that molecular the mechanism of disease differs depending on the FAS1 domain carrying the mutation.

In this case study, we explored quantitative ¹H NMR (qNMR), HPLC-DAD, and the Folin-Ciocalteu assay (TPC) as methods of quantifying the total phenolic content of a green macroalga, Ulva intestinalis, after optimized accelerated solvent extraction. Tentative qualitative data was also acquired after multiple steps of purification. The observed polyphenolic profile was complex with low individual concentrations. The qNMR method yielded 5.5% (DW) polyphenols in the crude extract, whereas HPLC-DAD and TPC assay yielded 1.1% (DW) and 0.4% (DW) respectively, using gallic acid as the reference in all methods. Based on the LC-MS observations of extracts and fractions, an average molar mass of 330 g/mol and an average of 4 aromatic hydrogens in each spin system was chosen for optimized qNMR calculations. Compared to the parallel numbers using gallic acid as the standard (170 g/mol, 2 aromatic H), the optimized parameters resulted in a similar qNMR result (5.3%, DW). The different results for the different methods highlight the difficulties with total polyphenolic quantification. All of the methods contain assumptions and uncertainties, and for complex samples with lower concentrations, this will be of special importance. Thus, further optimization of the extraction, identification, and quantification of polyphenols in marine algae must be researched.

Phenylketonuria (PKU) is a loss-of-function inborn error of metabolism. As many other inherited diseases the main pathologic mechanism in PKU is an enhanced tendency of the mutant phenylalanine hydroxylase (PAH) to misfold and undergo ubiquitin-dependent degradation. Recent alternative approaches with therapeutic potential for PKU aim at correcting the PAH misfolding, and in this respect pharmacological chaperones are the focus of increasing interest. These compounds, which often resemble the natural ligands and show mild competitive inhibition, can rescue the misfolded proteins by stimulating their renaturation in vivo. For PKU, a few studies have proven the stabilization of PKU-mutants in vitro, in cells, and in mice by pharmacological chaperones, which have been found either by using the tetrahydrobiopterin (BH4) cofactor as query structure for shape-focused virtual screening or by high-throughput screening of small compound libraries. Both approaches have revealed a number of compounds, most of which bind at the iron-binding site, competitively with respect to BH4. Furthermore, PAH shares a number of ligands, such as BH4, amino acid substrates and inhibitors, with the other aromatic amino acid hydroxylases: the neuronal/neuroendocrine enzymes tyrosine hydroxylase (TH) and the tryptophan hydroxylases (TPHs). Recent results indicate that the PAH-targeted pharmacological chaperones should also be tested on TH and the TPHs, and eventually be derivatized to avoid unwanted interactions with these other enzymes. After derivatization and validation in animal models, the PAH-chaperoning compounds represent novel possibilities in the treatment of PKU.