Wheat gliadins contain a large amount of glutamine- and proline-rich peptides which are not hydrolyzed by human digestive peptidases and can cause autoimmune celiac disease and other forms of gluten intolerance in predisposed people. Peptidases that efficiently cleave such immunogenic peptides can be used in enzyme therapy. The stored product insect pest Tribolium castaneum efficiently hydrolyzes gliadins. The main digestive peptidase of T. castaneum is cathepsin L, which is from the papain C1 family with post-glutamine cleavage activity. We describe the isolation and characterization of T. castaneum recombinant procathepsin L (rpTcCathL1, NP_001164001), which was expressed in Pichia pastoris cells. The activation of the proenzyme was conducted by autocatalytic processing. The effects of pH and proenzyme concentration in the reaction mixture on the processing were studied. The mature enzyme retained high activity in the pH range from 5.0 to 9.0 and displayed high pH-stability from 4.0 to 8.0 at 20 °C. The enzyme was characterized according to electrophoretic mobility under native conditions, activity and stability at various pH values, a sensitivity to various inhibitors, and substrate specificity, and its hydrolytic effect on 8-, 10-, 26-, and 33-mer immunogenic gliadins peptides was demonstrated. Our results show that rTcCathL1 is an effective peptidase that can be used to develop a drug for the enzyme therapy of various types of gluten intolerance.

The calcium cation is a crucial signaling molecule involved in numerous cellular pathways. Beyond its role as a messenger or modulator in intracellular cascades, calcium’s function in excitable cells, including nerve impulse transmission, is remarkable. The central role of calcium in nervous activity has driven the rapid development of fluorescent techniques for monitoring this cation in living cells. Specifically, genetically encoded calcium indicators (GECIs) are the most in-demand molecular tools in their class. In this work, we address two issues of calcium imaging by designing indicators based on the successful GCaMP6 backbone and the fluorescent protein BrUSLEE. The first indicator variant (GCaMP6s-BrUS), with a reduced, calcium-insensitive fluorescence lifetime, has potential in monitoring calcium dynamics with a high temporal resolution in combination with advanced microscopy techniques, such as light beads microscopy, where the fluorescence lifetime limits acquisition speed. Conversely, the second variant (GCaMP6s-BrUS-145), with a flexible, calcium-sensitive fluorescence lifetime, is relevant for static measurements, particularly for determining absolute calcium concentration values using fluorescence lifetime imaging microscopy (FLIM). To identify the structural determinants of calcium sensitivity in these indicator variants, we determine their spatial structures. A comparative structural analysis allowed the optimization of the GCaMP6s-BrUS construct, resulting in an indicator variant combining calcium-sensitive behavior in the time domain and enhanced molecular brightness. Our data may serve as a starting point for further engineering efforts towards improved GECI variants with fine-tuned fluorescence lifetimes.

Roughly 1% of the global population is susceptible to celiac disease (CD)—inheritable autoimmune inflammation of the small intestine caused by intolerance to gliadin proteins present in wheat, rye, and barley grains, and called gluten in wheat. Classical treatment is a life-long gluten-free diet, which is constraining and costly. An alternative approach is based upon the development and oral reception of effective peptidases that degrade in the stomach immunogenic proline- and glutamine-rich gliadin peptides, which are the cause of the severe reaction in the intestine. In previous research, we have established that the major digestive peptidase of an insect Tribolium castaneum—cathepsin L—hydrolyzes immunogenic prolamins after Gln residues but is unstable in the extremely acidic environment (pH 2–4) of the human stomach and cannot be used as a digestive aid. In this work, using molecular dynamics simulations, we discover the probable cause of the pH instability of cathepsin L—loss of the catalytically competent rotameric state of one of the active site residues, His 275. To “fix” the correct orientation of this residue, we designed a V277A mutant variant, which extends the range of stability of the peptidase in the acidic environment while retaining most of its activity. We suggest this protein as a lead glutenase for the development of oral medical preparation that fights CD and gluten intolerance in susceptible people.

Monitoring of intracellular pH changes in situ can provide valuable information about cellular metabolism and a deeper understanding of physiological processes. Most traditional fluorescent indicators are only capable of a relative assessment of changes in the studied parameter in the cell. We associate the possibility of measuring the absolute values that characterize the analyte with the detection of the indicator signal in the time domain, where its quantitative measure is the fluorescence lifetime (tau). In this project, we test promising pH-sensitive fluorophores with labile fluorescence lifetimes—EYFP-G65T and EGFP-Y145L/S205V—both as fluorescent cores for the previously described pH indicators and as independent pH indicators. Measurement of the fluorescence attenuation kinetics of four structures (EYFP-G65T, EGFP-Y145L/S205V, SypHer3s, and SypHer3s-G65T) over a wide pH range revealed areas where tau is linearly dependent on pH. The differences in the fluorescence excitation modes of these molecules makes it possible to use them in one experimental system to assess pH changes in a wide range, 4.0–9.0. We showed that under the conditions of traditional fluorescence microscopy (in the cytoplasm of HEK293 cells), the SypHer3s-G65T indicator shows a dynamic response range approximately 3 times wider than the original SypHer3s.