Chemogenetic approaches to dissect the role of H2O2 in redox-dependent pathways using genetically encoded biosensors

Chemogenetic tools are recombinant enzymes that can be targeted to specific organelles and tissues. The provision or removal of the enzyme substrate permits control of its biochemical activities. Yeast-derived enzyme D-amino acid oxidase (DAAO) represents the first of its kind for a substrate-based chemogenetic approach to modulate H2O2 concentrations within cells. Combining these powerful enzymes with multiparametric imaging methods exploiting genetically encoded biosensors has opened new lines of investigations in life sciences. In recent years, the chemogenetic DAAO approach has proven beneficial to establish a new role for (patho)physiological oxidative stress on redox-dependent signaling and metabolic pathways in cultured cells and animal model systems. This mini-review covers established or emerging methods and assesses newer approaches exploiting chemogenetic tools combined with genetically encoded biosensors.

A Novel Gelatinase from Marine Flocculibacter collagenilyticus SM1988: Characterization and Potential Application in Collagen Oligopeptide-Rich Hydrolysate Preparation

Although the S8 family in the MEROPS database contains many peptidases, only a few S8 peptidases have been applied in the preparation of bioactive oligopeptides. Bovine bone collagen is a good source for preparing collagen oligopeptides, but has been so far rarely applied in collagen peptide preparation. Here, we characterized a novel S8 gelatinase, Aa2_1884, from marine bacterium Flocculibacter collagenilyticus SM1988T, and evaluated its potential application in the preparation of collagen oligopeptides from bovine bone collagen. Aa2_1884 is a multimodular S8 peptidase with a distinct domain architecture from other reported peptidases. The recombinant Aa2_1884 over-expressed in Escherichia coli showed high activity toward gelatin and denatured collagens, but no activity toward natural collagens, indicating that Aa2_1884 is a gelatinase. To evaluate the potential of Aa2_1884 in the preparation of collagen oligopeptides from bovine bone collagen, three enzymatic hydrolysis parameters, hydrolysis temperature, hydrolysis time and enzyme-substrate ratio (E/S), were optimized by single factor experiments, and the optimal hydrolysis conditions were determined to be reaction at 60 ℃ for 3 h with an E/S of 400 U/g. Under these conditions, the hydrolysis efficiency of bovine bone collagen by Aa2_1884 reached 95.3%. The resultant hydrolysate contained 97.8% peptides, in which peptides with a molecular weight lower than 1000 Da and 500 Da accounted for 55.1% and 39.5%, respectively, indicating that the hydrolysate was rich in oligopeptides. These results indicate that Aa2_1884 likely has a promising potential application in the preparation of collagen oligopeptide-rich hydrolysate from bovine bone collagen, which may provide a feasible way for the high-value utilization of bovine bone collagen.

Molecular Weight Analysis of Blue Shark (Prionace glauca) Collagen Hydrolysates by GPC-LS. Effect of High Molecular Weight Hydrolysates on Fibroblast Cultures: mRNA Collagen Type I Expression and Synthesis

High molecular weight (Mw) collagen hydrolysates have been demonstrated to produce a higher synthesis of collagen type I mRNA. Mw determination is a key factor maximizing the effect of collagen hydrolysates on collagen type I synthesis by fibroblasts. This work aimed to achieve a high average Mw in Blue Shark Collagen Hydrolysate, studying different hydrolysis parameters by GPC-LS analysis and testing its effect on mRNA Type I collagen expression. Analysis revealed differences in blue shark collagen hydrolysates Mw depending on hydrolysis conditions. Papain leads to obtaining a significantly higher Mw hydrolysate than Alcalase at different times of hydrolysis and at different enzyme/substrate ratios. Besides, the time of the hydrolysis factor is more determinant than the enzyme/substrate ratio factor for obtaining a higher or lower hydrolysate Mw when using Papain as the enzyme. Contrary, Alcalase hydrolysates resulted in similar Mw with no significant differences between different conditions of hydrolysis assayed. Blue shark collagen hydrolysate showing the highest Mw showed neither cytotoxic nor proliferation effect on fibroblast cell culture. Besides, it exhibited an increasing effect on both mRNA expression and pro-collagen I production.

N-glycosylation modulates enzymatic activity of Trypanosoma congolense trans-sialidase

Trypanosomes cause the devastating disease trypanosomiasis, in which the action of trans-sialidase (TS) enzymes harbored on their surface is a key virulence factor. TS are highly N-glycosylated, but the biological functions of the glycans remain elusive. In this study, we investigated the influence of N-glycans on the enzymatic activity and structure stability of TconTS1, a recombinant TS from the African parasite Trypanosoma congolense. MALDI-TOF MS revealed that eight asparagine sites were glycosylated with high-mannose type N-glycans. Deglycosylation of TconTS1 led to a 5-fold decrease in substrate affinity but to the same conversion rate relative to the untreated enzyme. After deglycosylation, no changes in secondary structure elements were observed in circular dichroism experiments. Molecular dynamics simulations revealed interactions between the highly flexible N-glycans and some conserved amino acids belonging to the catalytic site. These interactions led to conformational changes, possibly enhancing substrate accessibility and promoting enzyme/substrate complex stability. The here-observed modulation of catalytic activity via the N-glycan shield may be a structure-function relationship intrinsic of several members of the TS family.

eIF5A-Independent Role of DHPS in p21CIP1 and Cell Fate Regulation

Deoxyhypusine synthase (DHPS) catalyzes the first step of hypusination of the elongation translation factor 5A (eIF5A), and these two proteins have an exclusive enzyme–substrate relationship. Here we demonstrate that DHPS has a role independent of eIF5A hypusination in A375 and SK-MEL-28 human melanoma cells, in which the extracellular signal regulated kinase 1/2 (ERK1/2) pathway is deregulated. We found that RNA interference of DHPS induces G0/G1 cell cycle arrest in association with increased p21CIP1 expression in these cells whereas eIF5A knockdown induces cell death without increasing p21CIP1 expression. Interestingly, p21CIP1 knockdown switched DHPS knockdown-induced growth arrest to cell death in these cells, suggesting a specific relation between DHPS and p21CIP1 in determining cell fate. Surprisingly, ectopic expression of DHPS-K329R mutant that cannot hypusinate eIF5A abrogated DHPS knockdown-induced p21CIP1 expression in these cells, suggesting a non-canonical role of DHPS underlying the contrasting effects of DHPS and eIF5A knockdowns. We also show that DHPS knockdown induces p21CIP1 expression in these cells by increasing CDKN1A transcription through TP53 and SP1 in an ERK1/2-dependent manner. These data suggest that DHPS has a role independent of its ability to hypusinate eIF5A in cells, which appears to be important for regulating p21CIP1 expression and cell fate.

Influence of Enzyme Concentration and Hydrolysis Time on Soluble Protein Content of Protein Hydrolysate Prepared from Apple Snail (Pila ampullacea)

The objective of this study was to evaluate soluble protein content of protein hydrolysates obtained by enzymatic hydrolysis of apple snail using a trypsin enzyme. Apple snail were collected from traditional market at Pabean-Sidoarjo. Trypsin enzyme was used in enzymatic hydrolysis. The two variables, enzyme/substrate (E/S) ( 0.01, 0.05, 0.1) ratio and hydrolysis time (3 h, 6 h, 9 h, 12 h, 15 h, 18 h) and was used to produce the apple snail hydrolysate. The result showed that soluble protein content was about 2.3%-4.52%. The increase E/S ratio and hydrolysis time, the higher soluble protein content values was. The highest total soluble protein was achieved E/S 0.1 ratio at 12 h, 4.52%. But, after 12 h hydrolysis time, soluble protein was decreased. Optimum treatment to hydrolyzing apple snail using trypsin enzyme was E3H4 treated (E/S 0.1 ratio and 3 h)

Lysosomal Storage Disorders

Lysosomes are membrane-bound organelles that degrade various macromolecules. Lysosomal storage diseases are a clinically, enzymatically, and genetically heterogeneous group of disorders resulting from intracellular accumulation of substrates. Mechanisms of lysosomal storage disorders include 1) primary deficiency of specific hydrolases; 2) defects in activator proteins required for enzyme-substrate interactions in posttranslational modification of enzymes or in transport of the substrate from lysosomes; and 3) abnormalities of fusion between autophagic vacuoles and lysosomes. Substrate accumulation is slowly progressive, leading to considerable morbidity and mortality.

Exponential Amplification by Redox Cross-Catalysis and Unmasking of Doubly Protected Molecular Probes

The strength of autocatalytic reactions lies in their ability to provide a powerful means of molecular amplification, which can be very useful for improving the analytical performances of a multitude of analytical and bioanalytical methods. However, one of the major difficulties in designing an efficient autocatalytic amplification system is the requirement for reactants that are both highly reactive and chemically stable in order to avoid limitations imposed by undesirable background amplifications. In the present work, we devised a reaction network based on a redox cross-catalysis principle, in which two catalytic loops activate each other. The first loop, catalyzed by H2O2, involves the oxi-dative deprotection of a naphthylboronate ester probe into a redox-active naphthohydroquinone, which in turn catalyzes the production of H2O2 by redox cycling in the presence of a reducing enzyme/substrate couple. We present here a set of new molecular probes with improved reactivity and stability, resulting in particularly steep sigmoidal kinetic traces and enhanced discrimination between specific and nonspecific responses. This translates into the sensitive de-tection of H2O2 down to a few nM in less than 10 minutes or a redox cycling compound such as the 2-amino-3-chloro-1,4-naphthoquinone H2O2 down to 50 pM in less than 30 minutes. The critical reason leading to these remarkably good performances is the extended stability stemming from the double masking of the naphthohydroquinone core by two boronate groups, a counterintuitive strategy if we consider the need for two equivalents of H2O2 for full deprotection. An in-depth study of the mechanism and dynamics of this complex reaction network is conducted in order to better understand, predict and optimize its functioning. From this investigation, the time response as well as detection limit are found highly dependent on pH, nature of buffer, and concentration of the reducing enzyme.

Deep learning allows genome-scale prediction of Michaelis constants from structural features

The Michaelis constant KM describes the affinity of an enzyme for a specific substrate and is a central parameter in studies of enzyme kinetics and cellular physiology. As measurements of KM are often difficult and time-consuming, experimental estimates exist for only a minority of enzyme–substrate combinations even in model organisms. Here, we build and train an organism-independent model that successfully predicts KM values for natural enzyme–substrate combinations using machine and deep learning methods. Predictions are based on a task-specific molecular fingerprint of the substrate, generated using a graph neural network, and on a deep numerical representation of the enzyme’s amino acid sequence. We provide genome-scale KM predictions for 47 model organisms, which can be used to approximately relate metabolite concentrations to cellular physiology and to aid in the parameterization of kinetic models of cellular metabolism.