ATP2B4 is an essential gene for epidermal growth factor-induced macropinocytosis in A431 cells.

Macropinocytosis (MPC) is a large-scale endocytosis pathway that involves actin-dependent membrane ruffle formation and subsequent ruffle closure to generate macropinosomes for the uptake of fluid-phase cargos. MPC is categorized into two types: constitutive and stimuli-induced. Constitutive MPC in macrophages relies on extracellular Ca2+ sensing by a calcium-sensing receptor. However, the link between stimuli-induced MPC and Ca2+ remains unclear. Here, we find that both intracellular and extracellular Ca2+ are required for epidermal growth factor (EGF)-induced MPC in A431 human epidermoid carcinoma cells. Through investigation of mammalian homologs of coelomocyte uptake defective (CUP) genes, we identify ATP2B4, encoding for a Ca2+ pump called the plasma membrane calcium ATPase 4 (PMCA4), as a Ca2+-related regulator of EGF-induced MPC. Knockout (KO) of ATP2B4, as well as depletion of extracellular/intracellular Ca2+, inhibited ruffle closure and macropinosome formation, without affecting ruffle formation. We demonstrate the importance of PMCA4 activity itself, independent of interactions with other proteins via its C-terminus known as a PDZ domain-binding motif. Additionally, we show that ATP2B4-KO reduces EGF-stimulated Ca2+ oscillation during MPC. Our findings suggest that EGF-induced MPC requires ATP2B4-dependent Ca2+ dynamics.

A flexible linker of 8-amino acids between the membrane binding segment and the FMN domain of cytochrome P450 reductase is necessary for optimal activity.

The diflavin NADPH-cytochrome P450 reductase (CYPOR) plays a critical role in human cytochrome P450 (CYP) activity by sequentially delivering two electrons from NADPH to CYP enzymes during catalysis. Although electron transfer to forty-eight human CYP enzymes by the FMN hydroquinone of CYPOR is well-known, the role of the linker between the NH2-terminus membrane-binding domain (MBD) and FMN domain in supporting the activity of P450 enzymes remains poorly understood. Here we demonstrate that a linker with at least eight residues is required to form a functional CYPOR-CYP2B4 complex. The linker has been shortened in two amino-acid increments from Phe44 to Ile57 using site directed mutagenesis. The ability of the deletion mutants to support cytochrome P450 2B4 (CYP2B4) catalysis and reduce ferric CYP2B4 was determined using an in vitro assay and stopped-flow spectrophotometry. Steady-state enzyme kinetics showed that shortening the linker by 8-14 amino acids inhibited (63-99%) the ability of CYPOR to support CYP2B4 activity and significantly increased the Km of CYPOR for CYP2B4. In addition, the reductase mutants decreased the rate of reduction of ferric CYP2B4 (46-95%) compared to wildtype when the linker was shortened by 8-14 residues. These results indicate that a linker with a minimum length of eight residues is necessary to enable the FMN domain of reductase to interact with CYP2B4 to form a catalytically competent complex. Our study provides evidence that the length of the MBD-FMN domain linker is a major determinant of the ability of CYPOR to support CYP catalysis and drug metabolism by P450 enzymes. PREAMBLE: This manuscript is dedicated in memory of Dr. James R. Kincaid who was the doctoral advisor to Dr. Freeborn Rwere and a longtime collaborator and friend of Dr. Lucy Waskell. Dr. James R. Kincaid was a distinguished professor of chemistry specializing in resonance Raman (rR) studies of heme proteins. He inspired Dr. Rwere (a Zimbabwean native) and three other Zimbabweans (Dr. Remigio Usai, Dr. Daniel Kaluka and Ms. Munyaradzi E. Manyumwa) to use lasers to document subtle changes occurring at heme active site of globin proteins (myoglobin and hemoglobin) and cytochrome P450 enzymes. Dr. Rwere appreciate his contributions to the development of talented Black scientists from Africa.

Suppression of adaptive NK cell expansion by macrophage-mediated phagocytosis inhibited by 2B4-CD48.

Infection of mice by mouse cytomegalovirus (MCMV) triggers activation and expansion of Ly49H+ natural killer (NK) cells, which are virus specific and considered to be "adaptive" or "memory" NK cells. Here, we find that signaling lymphocytic activation molecule family receptors (SFRs), a group of hematopoietic cell-restricted receptors, are essential for the expansion of Ly49H+ NK cells after MCMV infection. This activity is largely mediated by CD48, an SFR broadly expressed on NK cells and displaying augmented expression after MCMV infection. It is also dependent on the CD48 counter-receptor, 2B4, expressed on host macrophages. The 2B4-CD48 axis promotes expansion of Ly49H+ NK cells by repressing their phagocytosis by virus-activated macrophages through inhibition of the pro-phagocytic integrin lymphocyte function-associated antigen-1 (LFA-1) on macrophages. These data identify key roles of macrophages and the 2B4-CD48 pathway in controlling the expansion of adaptive NK cells following MCMV infection. Stimulation of the 2B4-CD48 axis may be helpful in enhancing adaptive NK cell responses for therapeutic purposes.

Genetic Screening of Haploid Neural Stem Cells Reveals that Nfkbia and Atp2b4 are Key Regulators of Oxidative Stress in Neural Precursors.

Neurological diseases are expected to become the leading cause of death in the next decade. Although little is known about it, the interaction between oxidative stress and inflammation is harmful to the nervous system. To find an advanced tool for neural genetics, mouse haploid neural stem cells (haNSCs) from the somite of chimeric mouse embryos at E8.5 is established. The haNSCs present a haploid neural progenitor identity for long-term culture, promising to robustly differentiate into neural subtypes and being able to form cerebral organoids efficiently. Thereafter, haNSC mutants via a high-throughput approach and screened targets of oxidative stress is generated using the specific mutant library. Deletion of Nfkbia (the top hit among the insertion mutants) reduces damage from reactive oxygen species (ROS) in NSCs exposed to H2O2. Transcriptome analysis revealed that Atp2b4 is upregulated significantly in Nfkbia-null NSCs and is probably responsible for the observed resistance. Additionally, overexpression of Atp2b4 itself can increase the survival of NSCs in the presence of H2O2, suggesting that Atp2b4 is closely involved in this resistance. Herein, a powerful haploid system is presented to study functional genetics in neural lineages, shedding light on the screening of critical genes and drugs for neurological diseases.

Novel Tb³âº-Doped LaAl2B4O10 phosphors: Structural analysis, luminescent properties, and energy transfer mechanism.

This study explores the structural and luminescent properties of terbium (Tb³âº)-doped lanthanum aluminium borate (LaAl2B4O10, abbreviated as LAB) phosphors, a novel host lattice for Tb³âº doping. LAB:Tb³âº phosphors, with varying dopant concentrations, were synthesized using a microwave-assisted combustion synthesis approach and characterized using X-ray diffraction (XRD), Rietveld refinement, and photoluminescence spectroscopy at both room and low temperatures. The structural analysis confirmed the hexagonal crystal structure of LAB and revealed successful incorporation of Tb³âº ions without altering the fundamental lattice. Luminescence studies demonstrated that the LAB:Tb³âº phosphors show strong green emission primarily attributed to the 5D4→7F5 transition of Tb³âº. The optimal doping concentration was determined to be 5 wt% Tb³âº, which provided maximum luminescence efficiency. This concentration also allowed for a critical study of energy transfer mechanisms within the phosphor, revealing dipole-dipole interactions with a critical distance of 9.80 Å between Tb³âº ions. Additionally, the CIE chromaticity coordinates of LAB:0.05 Tb³âº were precisely determined to be (0.289, 0.4460), indicating the potential for high-quality green emission suitable for solid-state lighting and display technologies. This work not only demonstrates the potential of LAB:Tb3+ as a highly efficient green luminescent material, but also sheds light on the mechanisms responsible for energy transfer and concentration quenching.

A Theoretical Investigation of the Polyaddition of an AB2+A2+B4 Monomer Mixture.

Hyperbranched polymers (HBPs) are widely applied nowadays as functional materials for biomedicine needs, nonlinear optics, organic semiconductors, etc. One of the effective and promising ways to synthesize HBPs is a polyaddition of AB2+A2+B4 monomers that is generated in the A2+CB2, AA'+B3, A2+B'B2, and A2+C2+B3 systems or using other approaches. It is clear that all the foundational features of HBPs that are manufactured by a polyaddition reaction are defined by the component composition of the monomer mixture. For this reason, we have designed a structural kinetic model of AB2+A2+B4 monomer mixture polyaddition which makes it possible to predict the impact of the monomer mixture's composition on the molecular weight characteristics of hyperbranched polymers (number average (DPn) and weight average (DPw) degree of polymerization), as well as the degree of branching (DB) and gel point (pg). The suggested model also considers the possibility of a positive or negative substitution effect during polyaddition. The change in the macromolecule parameters of HBPs formed by polyaddition of AB2+A2+B4 monomers is described as an infinite system of kinetic equations. The solution for the equation system was found using the method of generating functions. The impact of both the component's composition and the substitution effect during the polyaddition of AB2+A2+B4 monomers on structural and molecular weight HBP characteristics was investigated. The suggested model is fairly versatile; it makes it possible to describe every possible case of polyaddition with various monomer combinations, such as A2+AB2, AB2+B4, AB2, or A2+B4. The influence of each monomer type on the main characteristics of hyperbranched polymers that are obtained by the polyaddition of AB2+A2+B4 monomers has been investigated. Based on the results obtained, an empirical formula was proposed to estimate the pg = pA during the polyaddition of an AB2+A2+B4 monomer mixture: pg = pA = (-0.53([B]0/[A]0)1/2 + 0.78)υAB2 + (1/3)1/2([B]0/[A]0)1/2, where (1/3)1/2([B]0/[A]0)1/2 is the Flory equation for the A2+B4 polyaddition, [A]0 and [B]0 are the A and B group concentration from A2 and B4, respectively, and υAB2 is the mole fraction of the AB2 monomer in the mixture. The equation obtained allows us to accurately predict the pg value, with an AB2 monomer content of up to 80%.

A novel approach in biomedical engineering: The use of polyvinyl alcohol hydrogel encapsulating human umbilical cord mesenchymal stem cell-derived exosomes for enhanced osteogenic differentiation and angiogenesis in bone regeneration.

Developing effective methods for alveolar bone defect regeneration is a significant challenge in orthopedics. Exosomes from human umbilical cord mesenchymal stem cells (HUMSC-Exos) have shown potential in bone repair but face limitations due to undefined application methods and mechanisms. To address this, HUMSC-Exos were encapsulated in polyvinyl alcohol (PVA) hydrogel (Exo@PVA) to create a novel material for alveolar bone repair. This combination enhanced the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) and human umbilical vein endothelial cells (HUVECs) more effectively than Exos alone. Additionally, Exo@PVA significantly improved alveolar bone regeneration and defect repair in rats. The microRNA-21-5p (miR-21-5p) in Exo@PVA, identified through the GEO database and analyzed via in silico methods, played a crucial role. miR-21-5p promoted BMSC osteogenic differentiation by inhibiting WWP1-mediated KLF5 ubiquitination and enhanced HUVEC angiogenesis by targeting ATP2B4. These findings underscore the potential of an Exo-based approach with PVA hydrogel scaffolds for bone defect repair, operating through the miR-21-5p/WWP1/ATP2B4 signaling axis.