Biology subjects

Kara, S.

Publications and source records attributed to Kara, S..

4 recordsLinked to original sources

bioRxiv2026

Ancestors of Arylmalonate Decarboxylase show increased Activity, Stability and Stereoselectivity

Bacterial aryl malonate decarboxylase (AMDase) generates a wide spectrum of alpha-chiral carboxylic acids in outstanding optical purity, including several non-steroidal anti-inflammatory drugs and chiral building blocks. However, extant AMDase from Bordetella bronchiseptica (BbAMDase) and related enzymes of the same family have three main limitations: (i) low operational and thermal stability, (ii) limited substrate spectrum regarding the size of the smaller substituent on the alpha-C-atom, and (iii) low stereoselectivity towards alpha-alkenyl-alpha-alkyl malonic acids. To address these limitations, we expanded the structural diversity of the AMDase family by ancestral sequence reconstruction (ASR). The phylogenetic analysis of the decarboxylase revealed conserved structural motifs and key amino acids in the hydrophobic active-site cavity, a catalytic motif crucial for activity and selectivity of the enzyme. The analysis highlighted the natural distribution of amino acid exchanges previously identified via enzyme engineering. AMDase ancestors showed higher stability, activity, and, in one case, also stereoselectivity than BbAMDase. While a 10 degree higher unfolding temperature of AMDase ancestors is a frequent ASR result, ancestor N131 exhibited an exceptional 294-fold improvement in half-life time and a 1.8-fold increased specific activity (25.1 U / mg) toward 2-methyl-2-phenyl malonate in a preparative scale application. Computational analysis attributes this improved stability to a substantial increase in surface Glu side chains, which form a protective hydration shell that enhances solubility and mitigates aggregation. Ancestor N31 formed 2-methyl-but-3-enoic acid from its corresponding malonic acid in excellent optical purity (99.7% eeR). Finally, the stereoselectivity of the ancestors was completely inverted by switching the catalytic cysteine residues (G74C/C188G).

biochemistry↗

bioRxiv2023

Role of lipid nanodomains for inhibitory FcγRIIb function

The inhibitory Fc{gamma} receptor Fc{gamma}RIIb is involved in immune regulation and is known to localize to specific regions of the plasma membrane called lipid rafts. Previous studies suggested a link between the altered lateral receptor localization within the plasma membrane and the functional impairment of the Fc{gamma}RIIb-I232T variant that is associated with systemic lupus erythematosus. Here, we conducted microsecond all-atom molecular dynamics simulations and IgG binding assays to investigate the lipid nano-environment of Fc{gamma}RIIb monomers and of the Fc{gamma}RIIb-I232T mutant within a plasma membrane model, the orientation of the Fc{gamma}RIIb ectodomain, and its accessibility to IgG ligands. In contrast to previously proposed models, our simulations indicated that Fc{gamma}RIIb does not favor a cholesterol-or a sphingolipid-enriched lipid environment. Interestingly, cholesterol was depleted for all studied Fc{gamma}RIIb variants within a 2-3 nm environment of the receptor, counteracting the usage of raft terminology for models on receptor functionality. Instead, the receptor interacts with lipids that have poly-unsaturated fatty acyl chains and with (poly-) anionic lipids within the cytosolic membrane leaflet. We also found that Fc{gamma}RIIb monomers adopt a conformation that is not suitable for binding to its IgG ligand, consistent with a lack of detectable binding of monomeric IgG in experiments on primary immune cells. However, our results propose that multivalent IgG complexes might stabilize Fc{gamma}RIIb in a binding-competent conformation. We suggest differences in receptor complex formation within the membrane as a plausible cause of the altered membrane localization or clustering and the altered suppressive function of the Fc{gamma}RIIb-I232T variant. Significance StatementOur study sheds new light on the molecular mechanisms underlying the regulation of immune signaling mediated by the inhibitory Fc{gamma} receptor (Fc{gamma}RIIb). By utilizing atomistic simulations and experimental assays, we demonstrate that Fc{gamma}RIIb interacts with specific lipids in the plasma membrane. Notably, our findings challenge the current view of membrane heterogeneity in immune cells, as Fc{gamma}RIIb is not localized in specialized membrane domains known as rafts. Rather, we propose that receptor complex formation modulates receptor localization and conformation, thereby enabling ligand binding. Our findings have important implications for understanding how immune receptors function and communicate with each other, and may provide new opportunities for developing therapeutic strategies targeting Fc{gamma}RIIb in diseases such as autoimmunity and cancer.

immunology↗

bioRxiv2023

Immune cells employ traction forces to overcome steric hindrance in 3D biopolymer networks

To reach targets outside the bloodstream, immune cells can extravasate and migrate through connective tissue. During tissue infiltration, immune cells migrate in an amoeboid fashion, characterized by weak matrix adhesions and low traction forces, that allows them to achieve high migration speeds of up to 10 {micro}m/min. How immune cells reconcile amoeboid migration with the need to overcome steric hindrance in dense matrices is currently not understood. Here we show that NK92 (natural killer) cells can switch from their default amoeboid migration mode to a contractile, mesenchymal-like migration mode when moving through fibrous human amniotic membrane (HAM) tissue. We subsequently study immune cell migration in reconstituted 3D collagen networks with known mechanical properties and pore sizes and apply time-lapse confocal reflection microscopy to obtain simultaneous measurements of migration speed, directional persistence, and cell contractility. We find that NK92 cells are highly mechanoresponsive and exert substantial acto-myosin driven, integrin-mediated contractile forces of up to 100 nN on the extracellular matrix during short contractile phases. This burst-like contractile behavior is also found in primary B, T, NK cells, neutrophils, and monocytes, and is tightly related to the fraction of cells that appear to become stuck in narrow pores of the surrounding matrix. Our results demonstrate that steric hindrance guides the rapid regulation of integrin-mediated adhesion to the ECM in a large number of immune cell subtypes.

biophysics↗

bioRxiv2021

Automated Feeding and Fermentation Phase Transitions for the Production of Unspecific Peroxygenase by Pichia pastoris

Fungal peroxygenases are promising biocatalysts for hydroxylation steps in various industry-relevant synthesis pathways. In this application note we describe a bioprocess for the production of unspecific peroxygenase (UPO) in Pichia pastoris. The process was divided in four phases, with different carbon requirements. Precise timing of culture feeding was crucial for optimal cell growth and protein expression. We demonstrate how the automation of culture feeding reduced manual work as well as the risk of process failure due to operator error.

bioengineering↗