Targeting neuronal lysosomal dysfunction caused by ß-glucocerebrosidase deficiency with an enzyme-based brain shuttle construct.

Mutations in glucocerebrosidase cause the lysosomal storage disorder Gaucher's disease and are the most common risk factor for Parkinson's disease. Therapies to restore the enzyme's function in the brain hold great promise for treating the neurological implications. Thus, we developed blood-brain barrier penetrant therapeutic molecules by fusing transferrin receptor-binding moieties to ß-glucocerebrosidase (referred to as GCase-BS). We demonstrate that these fusion proteins show significantly increased uptake and lysosomal efficiency compared to the enzyme alone. In a cellular disease model, GCase-BS rapidly rescues the lysosomal proteome and lipid accumulations beyond known substrates. In a mouse disease model, intravenous injection of GCase-BS leads to a sustained reduction of glucosylsphingosine and can lower neurofilament-light chain plasma levels. Collectively, these findings demonstrate the potential of GCase-BS for treating GBA1-associated lysosomal dysfunction, provide insight into candidate biomarkers, and may ultimately open a promising treatment paradigm for lysosomal storage diseases extending beyond the central nervous system.

Cell wall glycosylation in Staphylococcus aureus: targeting the tar glycosyltransferases.

Peptidoglycan (PG) is the major structural polymer of the bacterial cell wall. The PG layer of gram-positive bacterial pathogens such as Staphylococcus aureus (S. aureus) is permeated with anionic glycopolymers known as wall teichoic acids (WTAs) and lipoteichoic acids (LTAs). In S. aureus, the WTA backbone typically consists of repeating ribitol-5-phosphate units, which are modified by enzymes that introduce glycosylation as well as amino acids at different locations. These modifications are key determinants of phage adhesion, bacterial biofilm formation and virulence of S. aureus. In this review, we examine differences in WTA structures in gram-positive bacteria, focusing in particular on three enzymes, TarM, TarS, and TarP that glycosylate the WTA of S. aureus at different locations. Infections with S. aureus pose an increasing threat to human health, particularly through the emergence of multidrug-resistant strains. Recently obtained structural information on TarM, TarS and TarP has helped to better understand the strategies used by S. aureus to establish resistance and to evade host defense mechanisms. Moreover, structures of complexes with poly-RboP and its analogs can serve as a platform for the development of new inhibitors that could form a basis for the development of antibiotic agents.

A fusion protein consisting of the exopeptidases PepN and PepX-production, characterization, and application.

Nowadays, general and specific aminopeptidases are of great interest, especially for protein hydrolysis in the food industry. As shown previously, it is confirmed that the general aminopeptidase N (PepN; EC 3.4.11.2) and the proline-specific peptidase PepX (EC 3.4.14.11) from Lactobacillus helveticus ATCC 12046 show a synergistic effect during protein hydrolysis which results in high degrees of hydrolysis and reduced bitterness. To combine both activities, the enzymes were linked and a fusion protein called PepN-L1-PepX (FUS-PepN-PepX) was created. After production and purification, the fusion protein was characterized. Some of its biochemical characteristics were altered in favor for an application compared to the single enzymes. As an example, the optimum temperature for the PepN activity increased from 30 °C for the single enzyme to 35 °C for FUS-PepN. In addition, the temperature stability of PepX was higher for FUS-PepX than for the single enzyme (50 % compared to 40 % residual activity at 50 °C after 14 days, respectively). In addition, the disulfide bridge-reducing reagent ß-mercaptoethanol did not longer inactivate the FUS-PepN activity. Furthermore, the K M values decreased for both enzyme activities in the fusion protein. Finally, it was found that the synergistic hydrolysis performance in a casein hydrolysis was not reduced for the fusion protein. The increase of the relative degree of hydrolysis of a prehydrolyzed casein solution was the same as it was for the single enzymes. As a benefit, the resulting hydrolysate showed a strong antioxidative capacity (ABTS-IC50 value: 5.81 µg mL(-1)).