bims-noxint 2022-05-08 papers

Issue of 2022‒05‒08
six papers selected by
Laia Caja Puigsubira
Uppsala University

Biochim Biophys Acta Mol Cell Res. 2022 Apr 27. pii: S0167-4889(22)00067-2. [Epub ahead of print] 119276

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

Hana Valenta, Sophie Dupré, Mouna Abdesselem, Tania Bizouarn, Laura Baciou, Oliver Nüsse, Ariane Deniset-Besseau, Marie Erard.

The phagocyte NADPH oxidase (NOX2) is a key enzyme of the innate immune system generating superoxide anions (O2•-), precursors of reactive oxygen species. The NOX2 protein complex is composed of six subunits: two membrane proteins (gp91phox and p22phox) forming the catalytic core, three cytosolic proteins (p67phox, p47phox and p40phox) 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. A chimeric protein, called 'Trimera', composed of the essential domains of the cytosolic proteins p47phox (aa 1-286), p67phox (aa 1-212) and full-length Rac1Q61L, enables a constitutive and robust NOX2 activity in cells without the need of any stimulus. We employed Trimera as a single activating protein of the phagocyte NADPH oxidase in living cells and examined the consequences on the cell physiology of this continuous and long-term NOX activity. We showed that the sustained high level of NOX activity causes acidification of the intracellular pH, triggers apoptosis and leads to local peroxidation of lipids in the membrane. These local damages to the membrane correlate with the strong tendency of the Trimera to clusterize in the plasma membrane observed by FRET-FLIM microscopy.

Keywords: FRET-FLIM; Intracellular pH; Lipid peroxidation; NADPH oxidase; Oxidative stress; ROS

DOI: https://doi.org/10.1016/j.bbamcr.2022.119276

Sci Total Environ. 2022 Apr 30. pii: S0048-9697(22)02654-7. [Epub ahead of print] 155558

Suppression of NADPH oxidase 4 inhibits PM2.5-induced cardiac fibrosis through ROS-P38 MAPK pathway.

Meiqiong Wu, Qisong Xing, Huiling Duan, Guohua Qin, Nan Sang.

Fine particulate matter (PM2.5) has been consistently linked to cardiovascular diseases, and cardiac fibrosis plays a crucial role in the occurrence and development of heart diseases. It is reported that NOX4-dependent redox signaling are responsible for TGFβ-mediated profibrotic responses. The current study was designed to explore the possible mechanisms of cardiac fibrosis by PM2.5 both in vitro and in vivo. Female C57BL/6 mice received PM2.5 (3 mg/kg b.w.) exposure with/without NOX4 inhibitor (apocynin, 25 mg/kg b.w.) or ROS scavenger (NALC, 50 mg/kg b.w.), every other day, for 4 weeks. H9C2 cells were incubated with PM2.5 (3 μg/mL) with/without 5 mM NALC, TGFβ inhibitor (SB431542, 10 μM), or siRNA-NOX4 for 24 h. The results demonstrated that PM2.5 induced evident collagen deposition and elevated expression of fibrosis biomarkers (Col1a1 & Col3a1). Significant systemic inflammatory response and cardiac oxidative stress were triggered by PM2.5. PM2.5 increased the protein expression of TGFβ1, NOX4, and P38 MAPK. Notably, the increased effects of PM2.5 could be suppressed by SB431542, siRNA-NOX4 in vitro or apocynin in vivo, and NALC. The reverse verification experiments further supported the involvement of the TGFβ/NOX4/ROS/P38 MAPK signaling pathway in the myocardial fibrosis induced by PM2.5. In summary, the current study provided evidence that PM2.5 challenge led to cardiac fibrosis through oxidative stress, systemic inflammation, and subsequent TGFβ/NOX4/ROS/P38 MAPK pathway and may offer new therapeutic targets in cardiac fibrosis.

Keywords: Cardiac; Fibrosis; Fine particulate matter; Molecular mechanism

DOI: https://doi.org/10.1016/j.scitotenv.2022.155558

Cell Commun Signal. 2022 May 06. 20(1): 59

AIP1 suppresses neovascularization by inhibiting the NOX4-induced NLRP3/NLRP6 imbalance in a murine corneal alkali burn model.

Qingyu Li, Xia Hua, Liangpin Li, Xueyan Zhou, Ye Tian, Yang Deng, Min Zhang, Xiaoyong Yuan, Wei Chi.

BACKGROUND: Apoptosis signal-regulating kinase 1-interacting protein 1 (AIP1) participates in inflammatory neovascularization induction. NADPH oxidase 4 (NOX4) produces reactive oxygen species (ROS), leading to an imbalance in nucleotide-binding oligomerization domain-like receptor family pyrin domain containing 3 (NLRP3) and NLR family pyrin domain containing 6 (NLRP6) expression. The mechanisms of AIP1, NOX4, ROS and inflammasomes in corneal neovascularization were studied herein.METHODS: C57BL/6 and AIP1-knockout mice were used in this study. The alkali burn procedure was performed on the right eye. Adenovirus encoding AIP1 plus green fluorescence protein (GFP) (Ad-AIP1-GFP) or GFP alone was injected into the right anterior chamber, GLX351322 was applied as a NOX4 inhibitor, and then corneal neovascularization was scored. The expression of related genes was measured by quantitative real-time polymerase chain reaction, western blotting and immunofluorescence staining. 2',7'-Dichlorofluorescin diacetate staining was used to determine the ROS levels.
RESULTS: The expression of AIP1 was decreased, while that of cleaved interleukin-1β (clv-IL-1β) and vascular endothelial growth factor A (VEGFa) was increased after alkali burn injury. NOX4 expression was increased, the imbalance in NLRP3/NLRP6 was exacerbated, and corneal neovascularization was increased significantly in AIP1-knockout mice compared with those in C57BL/6 mice after alkali burns. These effects were reversed by AIP1 overexpression. NLRP3/NLRP6 expression was imbalanced after alkali burns. GLX351322 reversed the imbalance in NLRP3/NLRP6 by reducing the ROS levels. This treatment also reduced the expression of clv-IL-1β and VEGFa, suppressing neovascularization.
CONCLUSIONS: AIP1 and NOX4 can regulate corneal inflammation and neovascularization after alkali burn injury. Based on the pathogenesis of corneal neovascularization, these findings are expected to provide new therapeutic strategies for patients. Corneal alkali burn injury is a common type of ocular injury that is difficult to treat in the clinic. The cornea is a clear and avascular tissue. Corneal neovascularization after alkali burn injury is a serious complication; it not only seriously affects the patient's vision but also is the main reason for failed corneal transplantation. Corneal neovascularization affects approximately 1.4 million patients a year. We show for the first time that AIP1 and NOX4 can regulate corneal inflammation and neovascularization after alkali burns. The expression of AIP1 was decreased, while that of clv-IL-1β and VEGFa was increased after alkali burns. We tried to elucidate the specific molecular mechanisms by which AIP1 regulates corneal neovascularization. NOX4 activation was due to decreased AIP1 expression in murine corneas with alkali burns. NOX4 expression was increased, the imbalance in NLRP3/NLRP6 was exacerbated, and corneal neovascularization was increased significantly in AIP1-knockout mice compared with those in C57BL/6 mice after alkali burns. These effects were reversed by AIP1 overexpression. Additionally, NLRP3/NLRP6 expression was unbalanced, with NLRP3 activation and NLRP6 suppression in the corneal alkali burn murine model. Eye drops containing GLX351322, a NOX4 inhibitor, reversed the imbalance in NLRP3/NLRP6 by reducing ROS expression. This treatment also reduced the expression of clv-IL-1β and VEGFa, reducing neovascularization. Therefore, we provide new gene therapeutic strategies for patients. With the development of neovascularization therapy, we believe that in addition to corneal transplantation, new drug or gene therapies can achieve better results. Video Abstract.

Keywords: AIP1; Alkali burn; Corneal neovascularization; NADPH oxidase; NLRP3; NLRP6; ROS

DOI: https://doi.org/10.1186/s12964-022-00877-5

Curr Pharm Des. 2022 May 06.

Novel Insights into the Molecular Mechanisms Involved in the Neuroprotective Effects of C-Phycocyanin Against Brain Ischemia in Rats.

Javier Marín-Prida, José Luiz Liberato, Alexey Llópiz-Arzuaga, Karina Stringhetta-Padovani, Nancy Pavón-Fuentes, Andréia Machado Leopoldino, Osmany Guirola Cruz, Ignacio Hernández González, Mariela León Pérez, Antoni Camins Espuny, Wagner Ferreira Dos Santos Dos Santos, Sergio Akira Uyemura, Gilberto L Pardo-Andreu, Giselle Pentón-Rol.

BACKGROUND: Ischemic stroke produces a large health impact worldwide, with scarce therapeutic options.OBJECTIVE: This study aimed to reveal the role of NADPH oxidase and neuroinflammatory genes on the cerebral anti-ischemic effects of C-Phycocyanin (C-PC), the chief biliprotein of Spirulina platensis.
METHODS: Rats with either focal cerebral ischemia/reperfusion (I/R) or acute brain hypoperfusion, received C-PC at different doses, or a vehicle, for up to 6 h post-stroke. Neurological, behavioral and histochemical parameters were assessed in I/R rats at 24 h. Cerebral gene expression and hippocampal neuron viability were evaluated in hypoperfused rats at acute (24 h) or chronic phases (30 days), respectively. A molecular docking analysis between NOX2 and C-PC-derived Phycocyanobilin (PCB) was also performed.
RESULTS: C-PC, obtained with a purity of 4.342, significantly reduced the infarct volume and neurologic deficit in a dose-dependent manner, and improved the exploratory activity of the I/R rats. This biliprotein inhibited NOX2 expression, a crucial NAPDH oxidase isoform in the brain, and the superoxide increase produced by the ischemic event. Moreover, C-PC-derived PCB showed a high binding affinity in silico with NOX2. C-PC downregulated the expression of pro-inflammatory genes (IFN-γ, IL-6, IL-17A, CD74, CCL12) and upregulated immune suppressive genes (Foxp3, IL-4, TGF-β) in hypoperfused brain areas. This compound also decreased chronic neuronal death in the hippocampus of hypoperfused rats.
CONCLUSION: These results suggest that the inhibition of cerebral NADPH oxidase and the improvement of neuroinflammation are key mechanisms mediating the neuroprotective actions of C-PC against brain ischemia.

Keywords: C-Phycocyanin; NADPH oxidase; brain ischemia; ischemic stroke; neuroinflammation; neuroprotection

DOI: https://doi.org/10.2174/1381612828666220506145542

Blood Adv. 2022 May 02. pii: bloodadvances.2021005776. [Epub ahead of print]

IFNγ Regulates NAD+ Metabolism to Promote the Respiratory Burst in Human Monocytes.

Katelyn J McCann, Stephen M Christensen, Devon H Colby, Peter J McGuire, Ian A Myles, Christa S Zerbe, Clifton L Dalgard, Gauthaman Sukumar, Warren J Leonard, Beth A McCormick, Steven M Holland.

IFNγ is an essential and pleiotropic activator of human monocytes, but little is known about the changes in cellular metabolism required for IFNγ-induced activation. We sought to elucidate the mechanisms by which IFNγ reprograms monocyte metabolism to support its immunologic activities. We found that IFNγ increased oxygen consumption rates (OCR) in monocytes, indicative of reactive oxygen species generation by both mitochondria and NADPH oxidase. Transcriptional profiling revealed that this oxidative phenotype was driven by IFNγ-induced reprogramming of NAD+ metabolism, which is dependent on nicotinamide phosphoribosyltransferase (NAMPT)-mediated NAD+ salvage to generate NADH and NADPH for oxidation by mitochondrial complex I and NADPH oxidase, respectively. Consistent with this pathway, monocytes from patients with gain-of-function mutations in STAT1 demonstrated higher than normal OCR. Whereas chemical or genetic disruption of mitochondrial complex I (rotenone treatment or Leigh Syndrome patient monocytes) or NADPH oxidase (DPI treatment or chronic granulomatous disease (CGD) patient monocytes) reduced OCR. Interestingly, inhibition of NAMPT in healthy monocytes completely abrogated the IFNγ-induced oxygen consumption, comparable to levels observed in CGD monocytes. These data identify an IFNγ-induced, NAMPT-dependent, NAD+ salvage pathway that is critical for IFNγ activation of human monocytes.

DOI: https://doi.org/10.1182/bloodadvances.2021005776

J Cell Biol. 2022 Jun 06. pii: e202105112. [Epub ahead of print]221(6):

V-ATPase is a universal regulator of LC3-associated phagocytosis and non-canonical autophagy.

Kirsty M Hooper, Elise Jacquin, Taoyingnan Li, Jonathan M Goodwin, John H Brumell, Joanne Durgan, Oliver Florey.

Non-canonical autophagy is a key cellular pathway in immunity, cancer, and neurodegeneration, characterized by conjugation of ATG8 to endolysosomal single membranes (CASM). CASM is activated by engulfment (endocytosis, phagocytosis), agonists (STING, TRPML1), and infection (influenza), dependent on K490 in the ATG16L1 WD40-domain. However, factors associated with non-canonical ATG16L1 recruitment and CASM induction remain unknown. Here, using pharmacological inhibitors, we investigate a role for V-ATPase during non-canonical autophagy. We report that increased V0-V1 engagement is associated with, and sufficient for, CASM activation. Upon V0-V1 binding, V-ATPase recruits ATG16L1, via K490, during LC3-associated phagocytosis (LAP), STING- and drug-induced CASM, indicating a common mechanism. Furthermore, during LAP, key molecular players, including NADPH oxidase/ROS, converge on V-ATPase. Finally, we show that LAP is sensitive to Salmonella SopF, which disrupts the V-ATPase-ATG16L1 axis and provide evidence that CASM contributes to the Salmonella host response. Together, these data identify V-ATPase as a universal regulator of CASM and indicate that SopF evolved in part to evade non-canonical autophagy.

DOI: https://doi.org/10.1083/jcb.202105112