MAPK15 protects from oxidative stress‐dependent cellular senescence by inducing the mitophagic process

Mitochondria are the major source of reactive oxygen species (ROS), whose aberrant production by dysfunctional mitochondria leads to oxidative stress, thus contributing to aging as well as neurodegenerative disorders and cancer. Cells efficiently eliminate damaged mitochondria through a selective type of autophagy, named mitophagy. Here, we demonstrate the involvement of the atypical MAP kinase family member MAPK15 in cellular senescence, by preserving mitochondrial quality, thanks to its ability to control mitophagy and, therefore, prevent oxidative stress. We indeed demonstrate that reduced MAPK15 expression strongly decreases mitochondrial respiration and ATP production, while increasing mitochondrial ROS levels. We show that MAPK15 controls the mitophagic process by stimulating ULK1‐dependent PRKN Ser¹⁰⁸ phosphorylation and inducing the recruitment of damaged mitochondria to autophagosomal and lysosomal compartments, thus leading to a reduction of their mass, but also by participating in the reorganization of the mitochondrial network that usually anticipates their disposal. Consequently, MAPK15‐dependent mitophagy protects cells from accumulating nuclear DNA damage due to mitochondrial ROS and, consequently, from senescence deriving from this chronic DNA insult. Indeed, we ultimately demonstrate that MAPK15 protects primary human airway epithelial cells from senescence, establishing a new specific role for MAPK15 in controlling mitochondrial fitness by efficient disposal of old and damaged organelles and suggesting this kinase as a new potential therapeutic target in diverse age‐associated human diseases.     

Involvement of plasma membrane calcium influx in bacterial induction of the k/h and hypersensitive responses in tobacco

An early event in the hypersensitive response of tobacco to Pseudomonas syringae pv syringae is the initiation of a K⁺/H⁺ response characterized by specific plasma membrane K⁺ efflux, extracellular alkalinization, and intracellular acidification. We investigated the role of calcium in induction of these host responses. Suspension-cultured tobacco cells exhibited a baseline Ca²⁺ influx of 0.02 to 0.06 micromole per gram per hour as determined from ⁴⁵Ca²⁺ uptake. Following bacterial inoculation, uptake rates began to increase coincidently with onset of the K⁺/H⁺ response. Rates increased steadily for 2 to 3 hours, reaching 0.5 to 1 micromole per gram per hour. This increased Ca²⁺ influx was prevented by EGTA and calcium channel blockers such as La³⁺, Co²⁺, and Cd²⁺ but not by verapamil and nifedipine. Lanthanum, cobalt, cadmium, and EGTA inhibited the K⁺/H⁺ response in both suspension-cultured cells and leaf discs and prevented hypersensitive cell death in leaf discs. We conclude that increased plasmalemma Ca²⁺ influx is required for the K⁺/H⁺ and hypersensitive responses in tobacco.     

Sulfatases are determinants of alveolar formation

Alveolar formation or alveolarization is orchestrated by a finely regulated and complex interaction between growth factors and extracellular matrix proteins. The lung parenchyma contains various extracellular matrix proteins including proteoglycans, which are composed of glycosaminoglycans (GAGs) linked to a protein core. Although GAGs are known to regulate growth factor distribution and activity according to their degree of sulfation the role of sulfated GAG in the respiratory system is not well understood. The degree of sulfation of GAGs is regulated in part, by sulfatases that remove sulfate groups. In vertebrates, the enzyme Sulfatase-Modifying Factor 1 (Sumf1) activates all sulfatases. Here we utilized mice lacking Sumf1(-/-) to study the importance of proteoglycan desulfation in lung development. The Sumf1(-/-) mice have normal lungs up until the onset of alveolarization at post-natal day 5 (P5). We detected increased deposition of sulfated GAG throughout the lung parenchyma and a decrease in alveolar septa formation. Moreover, stereological analysis showed that the alveolar volume is 20% larger in Sumf1(-/-) as compared to wild type (WT) mice at P10 and P30. Additionally, pulmonary function test was consistent with increased alveolar volume. Genetic experiments demonstrate that in Sumf1(-/-) mice arrest of alveolarization is independent of fibroblast growth factor signaling. In turn, the Sumf1(-/-) mice have increased transforming growth factor β (TGFβ) signaling and in vivo injection of TGFβ neutralizing antibody leads to normalization of alveolarization. Thus, absence of sulfatase activity increases sulfated GAG deposition in the lungs causing deregulation of TGFβ signaling and arrest of alveolarization.     

p66(ShcA) and oxidative stress modulate myogenic differentiation and skeletal muscle regeneration after hind limb ischemia

Oxidative stress plays a pivotal role in ischemic injury, and p66(ShcA)ko mice exhibit both lower oxidative stress and decreased tissue damage following hind limb ischemia. Thus, it was investigated whether tissue regeneration following acute hind limb ischemia was altered in p66(ShcA)ko mice. Upon femoral artery dissection, muscle regeneration started earlier and was completed faster than in wild-type (WT) control. Moreover, faster regeneration was associated with decreased oxidative stress. Unlike ischemia, cardiotoxin injury induced similar skeletal muscle damage in both genotypes. However, p66(ShcA)ko mice regenerated faster, in agreement with the regenerative advantage upon ischemia. Since no difference between p66(ShcA)wt and knock-out (ko) mice was found in blood perfusion recovery after ischemia, satellite cells (SCs), a resident population of myogenic progenitors, were examined. Similar SCs numbers were present in WT and ko mice. However, in vitro cultured p66(ShcA)ko SCs displayed lower oxidative stress levels and higher proliferation rate and differentiated faster than WT. Furthermore, when exposed to sublethal H(2)O(2) doses, p66(ShcA)ko SCs were resistant to H(2)O(2)-induced inhibition of differentiation. Finally, myogenic conversion induced by MyoD overexpression was more efficient in p66(ShcA)ko fibroblasts compared with WT. The present work demonstrates that oxidative stress and p66(ShcA) play a crucial role in the regenerative pathways activated by acute ischemia.     

Synthesis and evaluation of diverse thio avarol derivatives as potential UVB photoprotective candidates

Semisynthesis of 13 new thio avarol derivatives (4-16) and in vitro evaluation on the photodamage response induced by UVB irradiation are described. Their ability to inhibit NF-kappaB activation and TNF-alpha generation in HaCaT cells as well as their antioxidant capacity in human neutrophils has also been studied. Among them we have identified two monophenyl thio avarol derivatives (4-5) lacking cytotoxicity which can be considered promising UVB photoprotective agents through the potent inhibition of NF-kappaB activation with a mild antioxidant pharmacological profile.     

A new mutant of bovine seminal ribonuclease with a reversed swapping propensity

Bovine seminal ribonuclease (BS-RNase) is made up of two identical subunits bridged through two disulfide bonds. In solution, it exists as a 2:1 equilibrium mixture between two forms, with (MxM) and without swapping (M=M) of the N-terminal arms. The swapping endows BS-RNase with some special biological functions, including antitumor activity, since MxM retains a dimeric structure even under reducing conditions, thus evading the cytosolic ribonuclease inhibitor. To investigate the structural basis of domain swapping in BS-RNase, we have obtained several mutants by replacing selected residues with the corresponding ones of its monomeric counterpart, bovine pancreatic ribonuclease (RNase A). We have already shown that, in contrast with all other cases of swapped proteins, the swapping propensity of BS-RNase does not depend on the specific sequence of the 16-22 hinge loop, which connects the main body to the dislocating arm. In this paper we report the design, the expression, and the structural characterization of two mutants obtained by replacing Arg80 with Ser either in BS-RNase or in the mutant already containing the 16-22 hinge sequence of RNase A. NMR and circular dichroism data indicate that, in the monomeric form of the latter mutant, Ser80 acts as a switch for the conformation of the hinge region. Accordingly, in the dimeric form of the same mutant the MxM:M=M equilibrium ratio is inverted to 1:2. Overall, these data suggest that the presence of Arg80 triggers the swapping of N-terminal ends and plays a relevant role in the stability of the swapped form of BS-RNase.     

Environmental conditions modulate the switch among different states of the hydrophobin Vmh2 from Pleurotus ostreatus

Fungal hydrophobins are amphipathic, highly surface-active, and self-assembling proteins. The class I hydrophobin Vmh2 from the basidiomycete fungus Pleurotus ostreatus seems to be the most hydrophobic hydrophobin characterized so far. Structural and functional properties of the protein as a function of the environmental conditions have been determined. At least three distinct phenomena can occur, being modulated by the environmental conditions: (1) when the pH increases or in the presence of Ca(2+) ions, an assembled state, β-sheet rich, is formed; (2) when the solvent polarity increases, the protein shows an increased tendency to reach hydrophobic/hydrophilic interfaces, with no detectable conformational change; and (3) when a reversible conformational change and reversible aggregation occur at high temperature. Modulation of the Vmh2 conformational/aggregation features by changing the environmental conditions can be very useful in view of the potential protein applications.