Expression and Function of ABC Proteins in Fish Intestine

In fish, the intestine is fundamental for digestion, nutrient absorption, and other functions like osmoregulation, acid-base balance, and excretion of some metabolic products. These functions require a large exchange surface area, which, in turn, favors the absorption of natural and anthropogenic foreign substances (xenobiotics) either dissolved in water or contained in the food. According to their chemical nature, nutrients, ions, and water may cross the intestine epithelium cells’ apical and basolateral membranes by passive diffusion or through a wide array of transport proteins and also through endocytosis and exocytosis. In the same way, xenobiotics can cross this barrier by passive diffusion or taking advantage of proteins that transport physiological substrates. The entry of toxic substances is counterbalanced by an active efflux transport mediated by diverse membrane proteins, including the ATP binding cassette (ABC) proteins. Recent advances in structure, molecular properties, and functional studies have shed light on the importance of these proteins in cellular and organismal homeostasis. There is abundant literature on mammalian ABC proteins, while the studies on ABC functions in fish have mainly focused on the liver and, to a minor degree, on the kidney and other organs. Despite their critical importance in normal physiology and as a barrier to prevent xenobiotics incorporation, fish intestine’s ABC transporters have received much less attention. All the ABC subfamilies are present in the fish intestine, although their functionality is still scarcely studied. For example, there are few studies of ABC-mediated transport made with polarized intestinal preparations. Thus, only a few works discriminate apical from basolateral transport activity. We briefly describe the main functions of each ABC subfamily reported for mammals and other fish organs to help understand their roles in the fish intestine. Our study considers immunohistochemical, histological, biochemical, molecular, physiological, and toxicological aspects of fish intestinal ABC proteins. We focus on the most extensively studied fish ABC proteins (subfamilies ABCB, ABCC, and ABCG), considering their apical or basolateral location and distribution along the intestine. We also discuss the implication of fish intestinal ABC proteins in the transport of physiological substrates and aquatic pollutants, such as pesticides, cyanotoxins, metals, hydrocarbons, and pharmaceutical products.

The protease SplB of Staphylococcus aureus targets host complement components and inhibits complement-mediated bacterial opsonophagocytosis

Staphylococcus aureus is an opportunistic pathogen that can cause life-threatening infections, particularly in immunocompromised individuals. The high-level virulence of S. aureus largely relies on its diverse and variable collection of virulence factors and immune-evasion proteins, including the six serine protease-like proteins SplA-SplF. Spl proteins are expressed by most clinical isolates of S. aureus , but little is known about the molecular mechanisms by which these proteins modify the host’s immune response for the benefit of the bacteria. Here, we identify SplB as a protease that inactivates central human complement proteins, i.e., C3, C4, and the activation fragments C3b and C4b, by preferentially cleaving their α-chains. SplB maintained its proteolytic activity in human serum, degrading C3 and C4. SplB further cleaved the components of the terminal complement pathway, C5, C6, C7, C8, and C9. By contrast, the important soluble human complement regulators, Factor H and C4BP, as well as C1q, were left intact. Thereby SplB reduced C3b-mediated opsonophagocytosis by human neutrophils as well as C5b-9 deposition on the bacterial surface. In conclusion, we identified the first physiological substrates of the S. aureus extracellular protease SplB. This enzyme inhibits all three complement pathways and blocks opsonophagocytosis. Thus, SplB can be considered as a novel staphylococcal complement-evasion protein. Importance Success of bacterial pathogens in immunocompetent humans depends on control and inactivation of host immunity. S aureus , like many other pathogens, efficiently blocks host complement attack early in infection. Aiming to understand the role of the S. aureus -encoded orphan proteases SplA-SplD, we asked whether these proteins play a role in immune escape. We found that SplB inhibits all three-complement activation pathways as well as the lytic terminal complement pathway. This blocks opsonophagocytosis of the bacteria by neutrophils. We also clarified the molecular mechanisms: SplB cleaves the human complement proteins C3, C4, C5, C6, C7, C8 C9 as well as Factor B, but not the complement inhibitors Factor H and C4BP. Thus we identified the first physiological substrates of the extracellular protease SplB of S. aureus and characterize SplB as a novel staphylococcal complement-evasion protein.

Impact of Type II LRRK2 inhibitors on signalling and mitophagy

Much effort has been devoted to the development of selective inhibitors of the LRRK2 as a potential treatment for LRRK2 driven Parkinson's disease. In this study we first compare the properties of Type I (GSK3357679A and MLi-2) and Type II (GZD-824, Rebastinib and Ponatinib) kinase inhibitors that bind to the closed or open conformations of the LRRK2 kinase domain, respectively. We show that Type I and Type II inhibitors suppress phosphorylation of Rab10 and Rab12, key physiological substrates of LRRK2 and also promote mitophagy, a process suppressed by LRRK2. Type II inhibitors also display higher potency towards wild type LRRK2 compared to pathogenic mutants. Unexpectedly, we find that Type II inhibitors, in contrast to Type I compounds, fail to induce dephosphorylation of a set of well-studied LRRK2 biomarker phosphorylation sites at the N-terminal region of LRRK2, including Ser935. These findings emphasize that the biomarker phosphorylation sites on LRRK2 are likely reporting on the open vs closed conformation of LRRK2 kinase and that only inhibitors which stabilize the closed conformation induce dephosphorylation of these biomarker sites. Finally, we demonstrate that the LRRK2[A2016T] mutant which is resistant to MLi-2 Type 1 inhibitor, also induces resistance to GZD-824 and Rebastinib suggesting this mutation could be exploited to distinguish off target effects of Type II inhibitors. Our observations provide a framework of knowledge to aide with the development of more selective Type II LRRK2 inhibitors.

Impact of Type II LRRK2 inhibitors on signalling and mitophagy

Much effort has been devoted to the development of selective inhibitors of the LRRK2 as a potential treatment for LRRK2 driven Parkinson's disease. In this study we first compare the properties of Type I (GSK3357679A and MLi-2) and Type II (Ponatinib and GZD-824) kinase inhibitors that bind to the closed or open conformations of the LRRK2 kinase domain, respectively. We show that Type I and Type II inhibitors suppress phosphorylation of Rab10 and Rab12, key physiological substrates of LRRK2 and also promote mitophagy, a process suppressed by LRRK2. Unexpectedly, we strikingly find that Type II inhibitors, in contrast to Type I compounds, fail to induce dephosphorylation of a set of well-studied LRRK2 biomarker phosphorylation sites at the N-terminal region of LRRK2, including Ser935. Type II inhibitors also display higher potency towards wild type LRRK2 compared to pathogenic mutants. These findings emphasize that the biomarker phosphorylation sites on LRRK2 are likely reporting on the open-closed conformation of LRRK2 kinase and that only inhibitors which stabilize the closed conformation induce dephosphorylation of these biomarker sites. Our observations provide a framework of knowledge to aide with the development of more selective Type II LRRK2 inhibitors.

Does alcohol affect emotional face processing via interoceptive pathways?

Background: Our brain uses interoceptive signals from the body to shape how we perceive emotions in others; however, whether interoceptive signals can be manipulated to alter emotional perceptions is unknown. Alcohol has acute effects both on emotional processing and on the physiological substrates supporting interoception. In this registered report, we examine whether alcohol administration triggers physiological changes that alter interoceptive signals and manipulate emotional face processing. Such knowledge will broaden understanding of the mechanisms by which alcohol affects emotional face processing.Methods: Participants (n=36) will be administered an alcohol or placebo beverage. Cardiovascular physiology will be recorded before and after administration. Participants will complete two behavioral tasks in which they view emotional faces presented in synchrony with different phases of the cardiac cycle (i.e., systole, diastole). This manipulation creates an index of how interoceptive signals amplify emotional face processing. Hypotheses: We hypothesize that, compared to placebo, alcohol administration will disrupt the cardiac amplification of emotional face processing. We further explore whether this disruption depends on the nature and magnitude of changes in cardiovascular physiology after alcohol administration.

Biochemical Characterization of Two Polygalacturonases Purified from the Digestive Juice of the Snail Limicolaria flammea

Polygalacturonases constitute the major part of pectinase preparations for many bioprocess purposes. Investigation on the digestive juice of snail Limicolaria flammea led to purification of two polygalacturonases named PG1 and PG2. Properties of these enzymes were examined to explore their potential in biotechnology applications. A three steps procedure including size exclusion, anion and cation exchange and hydrophobic interaction chromatography were used for purification. The enzymes PG1 and PG2 had native molecular weights of approximately 46 and 86 kDa, respectively and functioned both as monomeric structures. The purified polygalacturonases PG1 and PG2 showed optimum hydrolysis activities at 50°C in sodium acetate buffer pH 5.6. The common inhibitor of the two purified polygalacturonases activity were Mn2+, Ca2+, Zn2+, EDTA, SDS and L-cystein. NH3+ stimulate the polygalacturonase PG1 while Ba2+ was an activator for polygalacturonase PG2. Substrate specificity indicated that these enzymes hydrolyse a broad range of pectin from different sources. The highest activity of PG1 was observed with apple pectin and lemon pectin while PG2 showed its highest activity with orange pectin. The catalytic efficiency of PG1 was highest for lemon pectin (0.125 µmol/min/mL) and orange pectin (0.124 µmol/min/mL). PG2 displayed highest catalytic efficiency (0.325 µmol/min/mL) towards orange pectin. These results suggest that orange and lemon pectin would be the potential physiological substrates of the two purified enzymes.

Mapping specificity, cleavage entropy, allosteric changes and substrates of blood proteases in a high-throughput screen

Proteases are among the largest protein families and critical regulators of biochemical processes like apoptosis and blood coagulation. Knowledge of proteases has been expanded by the development of proteomic approaches, however, technology for multiplexed screening of proteases within native environments is currently lacking behind. Here we introduce a simple method to profile protease activity based on isolation of protease products from native lysates using a 96FASP filter, their analysis in a mass spectrometer and a custom data analysis pipeline. The method is significantly faster, cheaper, technically less demanding, easy to multiplex and produces accurate protease fingerprints. Using the blood cascade proteases as a case study, we obtain protease substrate profiles that can be used to map specificity, cleavage entropy and allosteric effects and to design protease probes. The data further show that protease substrate predictions enable the selection of potential physiological substrates for targeted validation in biochemical assays.