Biphasic oxygen kinetics of cellular respiration and linear oxygen dependence of antimycin A inhibited oxygen consumption

Oxygen kinetics in fibroblasts was biphasic. This was quantitatively explained by a major mitochondrial hyperbolic component in the low-oxygen range and a linear increase of rotenone-and antimycin A-inhibited oxygen consumption in the high-oxygen range. This suggests an increased production of reactive oxygen species and oxidative stress at elevated, air-level oxygen concentrations. The high oxygen affinity of mitochondrial respiration provides the basis for the maintenance of a high aerobic scope at physiological low-oxygen levels, whereas further pronounced depression of oxygen pressure induces energetic stress under hypoxia.     

Human papillomavirus type 16 E7 oncoprotein binds and inactivates growth-inhibitory insulin-like growth factor binding protein 3

The E7 protein encoded by human papillomavirus type 16 is one of the few viral genes that can immortalize primary human cells and thereby override cellular senescence. While it is generally assumed that this property of E7 depends on its interaction with regulators of the cell cycle, we show here that E7 targets insulin-like growth factor binding protein 3 (IGFBP-3), the product of a p53-inducible gene that is overexpressed in senescent cells. IGFBP-3 can suppress cell proliferation and induce apoptosis; we show here that IGFBP-3-mediated apoptosis is inhibited by E7, which binds to IGFBP-3 and triggers its proteolytic cleavage. Two transformation-deficient mutants of E7 failed to inactivate IGFBP-3, suggesting that inactivation of IGFBP-3 may contribute to cell transformation.     

The amyloid beta peptide abeta (25-35) induces apoptosis independent of p53

Apoptosis of neuronal cells apparently plays a role in Alzheimer's disease (AD). The amyloid beta (Abeta) peptide derived from beta-amyloid precursor protein is found in AD brain in vivo and can induce apoptosis in vitro. While p53 accumulates in cells of AD brain, it is not known if p53 plays an active role in Abeta-induced apoptosis. We show here that inactivation of p53 in two experimental cell lines, either by expression of the papillomavirus E6 protein or by a shift to restrictive temperature, does not affect apoptosis induction by Abeta (25-35), indicating that Abeta induces apoptosis in a p53-independent manner.     

Monitoring of Ubiquitin-proteasome Activity in Living Cells Using a Degron (dgn)-destabilized Green Fluorescent Protein (GFP)-based Reporter Protein

Proteasome is the main intracellular organelle involved in the proteolytic degradation of abnormal, misfolded, damaged or oxidized proteins ^{1, 2}. Maintenance of proteasome activity was implicated in many key cellular processes, like cell''s stress response ³, cell cycle regulation and cellular differentiation ⁴ or in immune system response ⁵. The dysfunction of the ubiquitin-proteasome system has been related to the development of tumors and neurodegenerative diseases ^{4, 6}. Additionally, a decrease in proteasome activity was found as a feature of cellular senescence and organismal aging ^{7, 8, 9, 10}. Here, we present a method to measure ubiquitin-proteasome activity in living cells using a GFP-dgn fusion protein. To be able to monitor ubiquitin-proteasome activity in living primary cells, complementary DNA constructs coding for a green fluorescent protein (GFP)–dgn fusion protein (GFP–dgn, unstable) and a variant carrying a frameshift mutation (GFP–dgnFS, stable ¹¹) are inserted in lentiviral expression vectors. We prefer this technique over traditional transfection techniques because it guarantees a very high transfection efficiency independent of the cell type or the age of the donor. The difference between fluorescence displayed by the GFP–dgnFS (stable) protein and the destabilized protein (GFP-dgn) in the absence or presence of proteasome inhibitor can be used to estimate ubiquitin-proteasome activity in each particular cell strain. These differences can be monitored by epifluorescence microscopy or can be measured by flow cytometry.     

Synergistic interplay of UV radiation and urban particulate matter induces impairment of autophagy and alters cellular fate in senescence-prone human dermal fibroblasts

Skin aging is a complex process influenced by intrinsic factors and environmental stressors, including ultraviolet (UV) radiation and air pollution, among others. In this study, we investigated the effects of UVA and UVB radiation, combined with urban particulate matter (UPM), on human dermal fibroblasts (HDF). We show here that treatment of HDF with a subcytotoxic dose of UVA/UVB results in a series of events leading to mitochondrial dysfunction, increased ROS levels, and DNA damage. These effects are known to trigger either cellular senescence or cell death, depending on the cells' ability to clear damage by activating autophagy. Whereas UPM treatment in isolation did not affect proliferation or survival of HDF, of note, simultaneous UPM treatment of UV-irradiated cells selectively inhibited autophagic flux, thereby changing cell fate of a fraction of the cell population from senescence to apoptotic cell death. Our findings highlight the synergistic effects of UV radiation and UPM on skin aging, emphasizing the need to consider these factors in assessing the impact of environmental stressors on human health and opening opportunities for developing comprehensive approaches to protect and preserve skin integrity in the face of growing environmental challenges.     

The Endoplasmic Reticulum Chaperone Calnexin Is a NADPH Oxidase NOX4 Interacting Protein

Within the family of NADPH oxidases, NOX4 is unique as it is predominantly localized in the endoplasmic reticulum, has constitutive activity, and generates hydrogen peroxide (H₂O₂). We hypothesize that these features are consequences of a so far unidentified NOX4-interacting protein. Two-dimensional blue native (BN) electrophorese combined with SDS-PAGE yielded NOX4 to reside in macromolecular complexes. Interacting proteins were screened by quantitative SILAC (stable isotope labeling of amino acids in cell culture) co-immunoprecipitation (Co-IP) in HEK293 cells stably overexpressing NOX4. By this technique, several interacting proteins were identified with calnexin showing the most robust interaction. Calnexin also resided in NOX4-containing complexes as demonstrated by complexome profiling from BN-PAGE. The calnexin NOX4 interaction could be confirmed by reverse Co-IP and proximity ligation assay, whereas NOX1, NOX2, or NOX5 did not interact with calnexin. Calnexin deficiency as studied in mouse embryonic fibroblasts from calnexin^−/− mice or in response to calnexin shRNA reduced cellular NOX4 protein expression and reactive oxygen species formation. Our results suggest that endogenous NOX4 forms macromolecular complexes with calnexin, which are needed for the proper maturation, processing, and function of NOX4 in the endoplasmic reticulum.