A Perspective on the Prospective Use of AI in Protein Structure Prediction.

AlphaFold2 (AF2) and RoseTTaFold (RF) have revolutionized structural biology, serving as highly reliable and effective methods for predicting protein structures. This article explores their impact and limitations, focusing on their integration into experimental pipelines and their application in diverse protein classes, including membrane proteins, intrinsically disordered proteins (IDPs), and oligomers. In experimental pipelines, AF2 models help X-ray crystallography in resolving the phase problem, while complementarity with mass spectrometry and NMR data enhances structure determination and protein flexibility prediction.

. Modulates Inflammatory Markers in Human Colon and Shows a Potential Antioxidant Role in Myeloid Leukemic Cells.

(GA), a plant of the family, has long been used as a traditional cure for inflammatory and metabolic illnesses. In addition to various model studies, the current work focuses on the antioxidant and anti-inflammatory properties of GA in human colon biopsies. The phenol components in GA aqueous extract (GAAE) were identified by Liquid Chromatography-Electrospray Ionization Mass Spectrometry.

Consequences of the constitutive NOX2 activity in living cells: Cytosol acidification, apoptosis, and localized lipid peroxidation.

The phagocyte NADPH oxidase (NOX2) is a key enzyme of the innate immune system generating superoxide anions (O), precursors of reactive oxygen species. The NOX2 protein complex is composed of six subunits: two membrane proteins (gp91 and p22) forming the catalytic core, three cytosolic proteins (p67, p47 and p40) and a small GTPase Rac. The sophisticated activation mechanism of the NADPH oxidase relies on the assembly of cytosolic subunits with the membrane-bound components.

Distinct timing of neutrophil spreading and stiffening during phagocytosis.

Phagocytic cells form the first line of defense in an organism, engulfing microbial pathogens. Phagocytosis involves cell mechanical changes that are not yet well understood. Understanding these mechanical modifications promises to shed light on the immune processes that trigger pathological complications.

Rapid viscoelastic changes are a hallmark of early leukocyte activation.

To accomplish their critical task of removing infected cells and fighting pathogens, leukocytes activate by forming specialized interfaces with other cells. The physics of this key immunological process are poorly understood, but it is important to understand them because leukocytes have been shown to react to their mechanical environment. Using an innovative micropipette rheometer, we show in three different types of leukocytes that, when stimulated by microbeads mimicking target cells, leukocytes become up to 10 times stiffer and more viscous.

Membrane Dynamics and Organization of the Phagocyte NADPH Oxidase in PLB-985 Cells.

Neutrophils are the first cells recruited at the site of infections, where they phagocytose the pathogens. Inside the phagosome, pathogens are killed by proteolytic enzymes that are delivered to the phagosome following granule fusion, and by reactive oxygen species (ROS) produced by the NADPH oxidase. The NADPH oxidase complex comprises membrane proteins (NOX2 and p22), cytoplasmic subunits (p67, p47, and p40) and the small GTPase Rac.

Class I phosphoinositide 3-kinases control sustained NADPH oxidase activation in adherent neutrophils.

Phagocytes, especially neutrophils, can produce reactive oxygen species (ROS), through the activation of the NADPH oxidase (NOX2). Although this enzyme is crucial for host-pathogen defense, ROS production by neutrophils can be harmful in several pathologies such as cardiovascular diseases or chronic pulmonary diseases. The ROS production by NOX2 involves the assembly of the cytosolic subunits (p67, p47, and p40) and Rac with the membrane subunits (gp91 and p22).

Quantitative live-cell imaging and 3D modeling reveal critical functional features in the cytosolic complex of phagocyte NADPH oxidase.

Phagocyte NADPH oxidase produces superoxide anions, a precursor of reactive oxygen species (ROS) critical for host responses to microbial infections. However, uncontrolled ROS production contributes to inflammation, making NADPH oxidase a major drug target. It consists of two membranous (Nox2 and p22) and three cytosolic subunits (p40, p47, and p67) that undergo structural changes during enzyme activation.

Biosensors for spatiotemporal detection of reactive oxygen species in cells and tissues.

Redox biology has become a major issue in numerous areas of physiology. Reactive oxygen species (ROS) have a broad range of roles from signal transduction to growth control and cell death. To understand the nature of these roles, accurate measurement of the reactive compounds is required.

Phosphoinositol 3-phosphate acts as a timer for reactive oxygen species production in the phagosome.

Production of reactive oxygen species (ROS) in the phagosome by the NADPH oxidase is critical for mammalian immune defense against microbial infections and phosphoinositides are important regulators in this process. Phosphoinositol 3-phosphate (PI(3)P) regulates ROS production at the phagosome via p40 by an unknown mechanism. This study tested the hypothesis that PI(3)P controls ROS production by regulating the presence of p40 and p67 at the phagosomal membrane.

Environmental Effects on Reactive Oxygen Species Detection-Learning from the Phagosome.

Significance: Reactive oxygen species (ROS) fulfill numerous roles in biology ranging from signal transduction to the induction of cell death. To advance our understanding of these sometimes contradictory roles, quantitative, specific, and sensitive ROS measurements are required.

Recent Advances: Several organic or genetically encoded probes were successfully developed for ROS detection.

Characterization of novel store-operated calcium entry effectors.

2-Aminoethyl diphenylborinate (2-APB) is a well-known effector of the store-operated Ca(2+) entry of several cell types such as immune cells, platelets and smooth muscle cells. 2-APB has a dual effect: potentiation at 1-5μM and inhibition at >30μM. Unfortunately, it is also able to modify the activity of other Ca(2+) transporters and, thus, cannot be used as a therapeutic tool to control the leukocyte activity in diseases like inflammation.

The recruitment of p47(phox) and Rac2G12V at the phagosome is transient and phosphatidylserine dependent.

Background Information: During phagocytosis, neutrophils internalise pathogens in a phagosome and produce reactive oxygen species (ROS) by the NADPH oxidase to kill the pathogen. The cytosolic NADPH oxidase subunits p40(phox), p47(phox), p67(phox) and Rac2 translocate to the phagosomal membrane to participate in enzyme activation. The kinetics of this recruitment and the underlying signalling pathways are only partially understood.

Biochemistry of the phagosome: the challenge to study a transient organelle.

Phagocytes are specialized cells of the immune system, designed to engulf and destroy harmful microorganisms inside the newly formed phagosome. The latter is an intracellular organelle that is transformed into a toxic environment within minutes and disappears once the pathogen is destroyed. Reactive oxygen species and reactive nitrogen species are produced inside the phagosome.

Stable accumulation of p67phox at the phagosomal membrane and ROS production within the phagosome.

Production of ROS by the leukocyte NADPH oxidase is essential for the destruction of pathogenic bacteria inside phagosomes. The enzyme is a complex of cytosolic and membranous subunits that need to assemble upon activation. Biochemical data suggest that the complex is renewed continuously during activity.

Kinetic analysis of phagosomal production of reactive oxygen species.

Phagocytes produce large quantities of reactive oxygen species for pathogen killing; however, the kinetics and amplitude of ROS production on the level of individual phagosomes are poorly understood. This is mainly due to the lack of appropriate methods for quantitative ROS detection with microscopic resolution. We covalently attached the ROS-sensitive dye dichlorodihydrofluorescein (DCFH(2)) to yeast particles and investigated their fluorescence due to oxidation in vitro and in live phagocytes.

Amiloride derivatives modulate PS externalization in neutrophil-like PLB-985 cells.

During brain or cardiac ischemia/reperfusion neutrophils are recruited and activated contributing to inflammation and tissue damage. Neutrophils are removed from inflamed tissues by phosphatidylserine-dependent phagocytosis. Production of reactive oxygen species by the neutrophil NADPH-oxidase is known to affect phosphatidylserine externalization.

Potent inhibition of store-operated Ca2+ influx and superoxide production in HL60 cells and polymorphonuclear neutrophils by the pyrazole derivative BTP2.

Store-operated calcium entry (SOCE) is a key regulator in the activation of leukocytes. 3,5-Bistrifluoromethyl pyrazole (BTP) derivatives have been identified recently as inhibitors of T lymphocyte activation. The inhibitory effect of one of these compounds, N-(4-[3,5-bis(trifluoromethyl)-1H-pyrazol-1-yl]phenyl)-4-methyl-1,2,3-thiadiazole-5-carboxamide (BTP2), appears to be a result of inhibition of SOC influx.

Mouse bone marrow contains large numbers of functionally competent neutrophils.

The mouse has become an important model for immunological studies including innate immunity. Creating transgenic mice offers unique possibilities to study gene-function relationships. However, relatively little is known about the physiology of neutrophils from wild-type mice.