Identification of TFG- and Autophagy-Regulated Proteins and Glycerophospholipids in B Cells.

Autophagy supervises the proteostasis and survival of B lymphocytic cells. Trk-fused gene (TFG) promotes autophagosome-lysosome flux in murine CH12 B cells, as well as their survival. Hence, quantitative proteomics of CH12tfgKO and WT B cells in combination with lysosomal inhibition should identify proteins that are prone to lysosomal degradation and contribute to autophagy and B cell survival. Lysosome inhibition via NH4Cl unexpectedly reduced a number of proteins but increased a large cluster of translational, ribosomal, and mitochondrial proteins, independent of TFG. Hence, we propose a role for lysosomes in ribophagy in B cells. TFG-regulated proteins include CD74, BCL10, or the immunoglobulin JCHAIN. Gene ontology (GO) analysis reveals that proteins regulated by TFG alone, or in concert with lysosomes, localize to mitochondria and membrane-bound organelles. Likewise, TFG regulates the abundance of metabolic enzymes, such as ALDOC and the fatty acid-activating enzyme ACOT9. To test consequently for a function of TFG in lipid metabolism, we performed shotgun lipidomics of glycerophospholipids. Total phosphatidylglycerol is more abundant in CH12tfgKO B cells. Several glycerophospholipid species with similar acyl side chains, such as 36:2 phosphatidylethanolamine and 36:2 phosphatidylinositol, show a dysequilibrium. We suggest a role for TFG in lipid homeostasis, mitochondrial functions, translation, and metabolism in B cells.

Mitochondrial respiration in B lymphocytes is essential for humoral immunity by controlling the flux of the TCA cycle.

To elucidate the function of oxidative phosphorylation (OxPhos) during B cell differentiation, we employ CD23Cre-driven expression of the dominant-negative K320E mutant of the mitochondrial helicase Twinkle (DNT). DNT-expression depletes mitochondrial DNA during B cell maturation, reduces the abundance of respiratory chain protein subunits encoded by mitochondrial DNA, and, consequently, respiratory chain super-complexes in activated B cells. Whereas B cell development in DNT mice is normal, B cell proliferation, germinal centers, class switch to IgG, plasma cell maturation, and T cell-dependent as well as T cell-independent humoral immunity are diminished. DNT expression dampens OxPhos but increases glycolysis in lipopolysaccharide and B cell receptor-activated cells. Lipopolysaccharide-activated DNT-B cells exhibit altered metabolites of glycolysis, the pentose phosphate pathway, and the tricarboxylic acid cycle and a lower amount of phosphatidic acid. Consequently, mTORC1 activity and BLIMP1 induction are curtailed, whereas HIF1α is stabilized. Hence, mitochondrial DNA controls the metabolism of activated B cells via OxPhos to foster humoral immunity.

Fast and effective chromatographic separation of polymersomes from proteins by multimodal chromatography.

Artificial vesicles made of block copolymers, so-called polymersomes, represent a versatile chassis for the creation of functionalized nanocompartments with a wide range of biotechnological applications. The specific application depends on the biomolecules - usually proteins - that are positioned in the interior, in the membrane or on the surface of the vesicles. However, not all added proteins are integrated into the vesicles during the usual manufacturing processes of polymersomes. Excess proteins must therefore be removed. The separation techniques currently used for this, however, are associated with decisive disadvantages, such as damaged vesicles, long process times, or small sample volumes that can be processed. To overcome these drawbacks, we investigated the applicability of Capto™ Core 700 resin for polymersome purification. Polymersomes were not damaged or otherwise affected by passage through the column verified by hollow fiber flow field flow fractionation technique. Using three proteins with divergent physico-chemical properties as examples, it was demonstrated that different types of unentrapped proteins were efficiently removed from polymersome dispersions. The dynamic binding capacities in the presence of polymersomes varied between 9.5 and 16.5 mg per mL resin for the proteins applied. The technique can be used for small and large sample volumes alike. In addition, it can be used without special laboratory equipment. This adds a new and easy-to-use purification method for polymer vesicles to the repertoire that will also facilitate the large-scale production of functionalized polymersomes.

Three Steps, Two Enzymes, One Pot, but a Multitude of Nanocompartments: Combined Cycles of Kinetic Resolutions and Re-racemization with Incompatible Biocatalysts.

Deracemizations are clearly preferable to kinetic resolutions in the production of chiral molecules from racemates, as they allow up to 100% chemical and optical yield. Here we present a new process route for multienzymatic deracemizations that is relevant for reaction systems with incompatible reaction conditions of the biocatalysts. This often applies to combinations of lipases used for stereoselective acylation and solvent-sensitive racemases. By encapsulating a model racemase in polymeric vesicles, it was protected from inactivation by the organic solvent up to phase proportions of 99%. As high yields in the lipase reaction required either water proportions well below 1% or racemase-denaturating acyl donor concentrations, a one-pot reaction was implemented through the sequential use of lipase and racemase-containing nanocompartments. This strategy allowed us to perform two kinetic resolutions with intermittent re-racemization in one pot yielding 72% (0.72 mM after 120 h) of an enantiopure product.

Polymersomes as Nanoreactors Enabling the Application of Solvent-Sensitive Enzymes in Different Biphasic Reaction Setups.

There is an increasing interest in biocatalysis to perform chemical reactions in biphasic systems, consisting of an aqueous phase and a water-immiscible organic solvent or ionic liquid. In most cases, the hydrophobic phase is used as reservoir for poorly water-soluble substrates or for in situ product removal. However, many enzymes are solvent-sensitive and cannot be used in such systems. In this study, the solvent-sensitive enzyme mandelate racemase is exemplarily protected from the organic phase by its entrapment in (crosslinked) polymersomes. The covalent crosslinking of the individual chains of the block copolymer poly(2-methyloxazoline)15 -poly(dimethylsiloxane)68 -poly(2-methyloxazoline)15 via terminal methacrylates leads to enhanced membrane stability. This effect is especially pronounced for long-time incubation in the presence of organic solvents and ionic liquids. By using a gentle polymerization initiator at its minimal necessary concentration, the prior encapsulated enzymes remain intact during crosslinking. Although the insertion of natural channel proteins into the membrane improves the mass transport into the vesicles, it is non-essential. Mandelate racemase in (crosslinked) polymersomes remains active in different highly dispersed biphasic systems for more than 24 h. The free enzyme, on the other hand, gets completely inactivated within 1 h, thus illustrating the potential of polymersomes as nanoreactors in biphasic reaction setups.