Epidermal fatty acid-binding protein is increased in rat lungs following in vivo treatment with keratinocyte growth factor

Exogenous application of keratinocyte growth factor protects the lung against a variety of injurious stimuli. KGF-treatment leads to pronounced hyperplasia of alveolar epithelial type II cells and to stabilization of surfactant homeostasis after lung injury. Epidermal fatty acid-binding protein is involved in the synthesis of surfactant phospholipids and acts as an antioxidant scavenging reactive lipids. We treated adult rats with recombinant human keratinocyte growth factor (Palifermin) via intratracheal instillation and analyzed the expression of epidermal fatty acid-binding protein mRNA and protein by quantitative RT-PCR, immunoblotting as well as immunohistochemistry. Keratinocyte growth factor-treatment in vivo leads to an increased expression of epidermal fatty acid-binding protein mRNA and protein in the total lung. Epidermal fatty acid-binding protein mRNA expression per alveolar epithelial type II cell remains constant as shown in isolated type II cells. Epidermal fatty acid-binding protein immunoreactivity is seen in most if not all hyperplastic alveolar epithelial type II cells, and is mainly localized to the cytoplasm. The increase in epidermal fatty acid-binding protein gene expression associated with type II cell hyperplasia might contribute to the molecular mechanisms mediating lung protection by keratinocyte growth factor.     

Long-term change of the littoral Cladocera in the Tatra Mountain lakes through a major acidification event

In our study, we focused on littoral Cladocera living and feeding in shallow shore parts of 46 mountain lakes in the Tatra Mountains (Slovakia and Poland). The studied lakes underwent a major acidification event in the 1980s and are now in the process of recovery. Lakes were divided into three categories based on their sensitivity to acidification: 5 extremely sensitive (ES), 11 acid sensitive (AS), and 30 non-sensitive (NS) lakes. In our study, we included historical data from the literature, and data from sediment core and littoral samples, which together represent the evolution of the littoral communities from a pre-industrial period up to the present. In total, 11 littoral species were found belonging to three cladoceran families. Most of the species were members of the family Chydoridae: Alona affinis, A. quadrangularis, A. rectangula, A. guttata, Acroperus harpae, Alonella excisa, A. nana, Chydorus sphaericus, and Eurycercus lamellatus. One species belonged to each family Daphniidae (Ceriodaphnia quadrangula) and Polyphemidae (Polyphemus pediculus). The most numerous littoral taxa were Alona affinis, Acroperus harpae, and Chydorus sphaericus. All species reacted to decreased pH levels during peak acidification in the 1980s by disappearing from most of the lakes of all categories; the only persisting species was Chydorus sphaericus. Most species returned to the lakes when pH started to increase in the 1990s, although their return was noticeably slower in AS lakes. Alona quadrangularis decreased its distribution range over the studied period; Polyphemus pediculus was mostly detected in the 1910s only. The number of species was highest in all lake categories when dwarf pine was present in the lake catchment. On the whole, the littoral community was richest in NS lakes.     

Biophysical properties and cellular toxicity of covalent crosslinked oligomers of α-synuclein formed by photoinduced side-chain tyrosyl radicals

Alpha-synuclein (αS), a 140 amino acid presynaptic protein, is the major component of the fibrillar aggregates (Lewy bodies) observed in dopaminergic neurons of patients affected by Parkinson's disease. It is currently believed that noncovalent oligomeric forms of αS, arising as intermediates in its aggregation, may constitute the major neurotoxic species. However, attempts to isolate and characterize such oligomers in vitro, and even more so in living cells, have been hampered by their transient nature, low concentration, polymorphism, and inherent instability. In this work, we describe the preparation and characterization of low molecular weight covalently bound oligomeric species of αS obtained by crosslinking via tyrosyl radicals generated by blue-light photosensitization of the metal coordination complex ruthenium (II) tris-bipyridine in the presence of ammonium persulfate. Numerous analytical techniques were used to characterize the αS oligomers: biochemical (anion-exchange chromatography, SDS-PAGE, and Western blotting); spectroscopic (optical: UV/Vis absorption, steady state, dynamic fluorescence, and dynamic light scattering); mass spectrometry; and electrochemical. Light-controlled protein oligomerization was mediated by formation of Tyr-Tyr (dityrosine) dimers through -C-C- bonds acting as covalent bridges, with a predominant involvement of residue Y39. The diverse oligomeric species exhibited a direct effect on the in vitro aggregation behavior of wild-type monomeric αS, decreasing the total yield of amyloid fibrils in aggregation assays monitored by thioflavin T (ThioT) fluorescence and light scattering, and by atomic force microscopy (AFM). Compared to the unmodified monomer, the photoinduced covalent oligomeric species demonstrated increased toxic effects on differentiated neuronal-like SH-SY5Y cells. The results highlight the importance of protein modification induced by oxidative stress in the initial molecular events leading to Parkinson's disease.     

Dual roles of the SUMO-interacting motif in the regulation of Srs2 sumoylation

The Srs2 DNA helicase of Saccharomyces cerevisiae affects recombination in multiple ways. Srs2 not only inhibits recombination at stalled replication forks but also promotes the synthesis-dependent strand annealing (SDSA) pathway of recombination. Both functions of Srs2 are regulated by sumoylation-sumoylated PCNA recruits Srs2 to the replication fork to disfavor recombination, and sumoylation of Srs2 can be inhibitory to SDSA in certain backgrounds. To understand Srs2 function, we characterize the mechanism of its sumoylation in vitro and in vivo. Our data show that Srs2 is sumoylated at three lysines, and its sumoylation is facilitated by the Siz SUMO ligases. We also show that Srs2 binds to SUMO via a C-terminal SUMO-interacting motif (SIM). The SIM region is required for Srs2 sumoylation, likely by binding to SUMO-charged Ubc9. Srs2's SIM also cooperates with an adjacent PCNA-specific interaction site in binding to sumoylated PCNA to ensure the specificity of the interaction. These two functions of Srs2's SIM exhibit a competitive relationship: sumoylation of Srs2 decreases the interaction between the SIM and SUMO-PCNA, and the SUMO-PCNA-SIM interaction disfavors Srs2 sumoylation. Our findings suggest a potential mechanism for the equilibrium of sumoylated and PCNA-bound pools of Srs2 in cells.     

The CDK5 repressor CDK5RAP1 is a methylthiotransferase acting on nuclear and mitochondrial RNA

The unusual cyclin-dependent protein kinase 5 (CDK5) was discovered based on its sequence homology to cell cycle regulating CDKs. CDK5 was found to be active in brain tissues, where it is not involved in cell cycle regulation but in the regulation of neuronal cell differentiation and neurocytoskeleton dynamics. An aberrant regulation of CDK5 leads to the development of various neurodegenerative diseases including Alzheimer's disease. Although CDK5 is not regulated by cyclins, its activity does depend on the association with a protein activator and the presence or absence of further inhibitory factors. Recently, CDK5RAP1 was discovered to inhibit the active CDK5 kinase. Here, we show that CDK5RAP1 is a radical SAM enzyme, which postsynthetically converts the RNA modification N6-isopentenyladenosine (i(6)A) into 2-methylthio-N6-isopentenyladenosine (ms(2)i(6)A). This conversion is surprisingly not limited to mitochondrial tRNA, where the modification was known to exist. Instead, CDK5RAP1 introduces the modification also into nuclear RNA species establishing a link between postsynthetic kinase-based protein modification and postsynthetic RNA modification.     

Angiotensin AT1 receptor-associated protein Arap1 in the kidney vasculature is suppressed by angiotensin II

Arap1 is a protein that interacts with angiotensin II type 1 (AT(1)) receptors and facilitates increased AT(1) receptor surface expression in vitro. In the present study, we assessed the tissue localization and regulation of Arap1 in vivo. Arap1 was found in various mouse organs, with the highest expression in the heart, kidney, aorta, and adrenal gland. Renal Arap1 protein was restricted to the vasculature and to glomerular mesangial cells and was absent from tubular epithelia. A similar localization was found in human kidneys. To test the hypothesis that angiotensin II may control renal Arap1 expression, mice were subjected to various conditions to alter the activity of the renin-angiotensin system. A high-salt diet (4% NaCl, 7 days) upregulated Arap1 expression in mice by 47% compared with controls (0.6% NaCl, P = 0.03). Renal artery stenosis (7 days) or water restriction (48 h) suppressed Arap1 levels compared with controls (-64 and -62% in the clipped and contralateral kidney, respectively; and -50% after water restriction, P < 0.01). Angiotensin II infusion (2 μg·kg(-1)·min(-1), 7 days) reduced Arap1 mRNA levels compared with vehicle by 29% (P < 0.01), whereas AT(1) antagonism (losartan, 30 mg·kg(-1)·day(-1), 7 days) enhanced Arap1 mRNA expression by 52% (P < 0.01); changes in mRNA were paralleled by Arap1 protein abundance. Experiments with hydralazine and epithelial nitric oxide synthase-/- mice further suggested that Arap1 expression changed in parallel with angiotensin II, rather than with blood pressure per se. Similar to in vivo, Arap1 mRNA and protein were suppressed by angiotensin II in a time- and dose-dependent manner in cultured mesangial cells. In summary, Arap1 is highly expressed in the renal vasculature, and its expression is suppressed by angiotensin II. Thus Arap1 may serve as a local modulator of vascular AT(1) receptor function in vivo.     

The small molecule inhibitor PR-619 of deubiquitinating enzymes affects the microtubule network and causes protein aggregate formation in neural cells: Implications for neurodegenerative diseases

A pathological hallmark of many neurodegenerative diseases is the aggregation of proteins. Protein aggregate formation may be linked to a failure of the ubiquitin proteasome system (UPS) and/or the autophagy pathway. The UPS involves the ubiquitination of proteins followed by proteasomal degradation. Deubiquitination of target proteins is performed by proteases called deubiquitinating proteins (DUBs). Inhibition of DUBs may lead to the dysregulation of homeostasis and have pathological consequences. To assess the effects of DUB-inhibition, we have used the oligodendroglial cell line, OLN-t40, stably expressing the longest human tau isoform. Cells were incubated with PR-619, a broad-range, reversible inhibitor of ubiquitin isopeptidases. Incubation with PR-619 led to morphological changes, the upregulation of heat shock proteins (HSP), including HSP70 and αB-crystallin, and to protein aggregates near the MTOC, containing ubiquitin, HSPs, and the ubiquitin binding protein p62, which may provide a link between the UPS and autophagy. Thus, inhibition of DUB activity caused stress responses and the formation of protein aggregates resembling pathological inclusions observed in aggregopathies. Furthermore, PR-619 led to the stabilization of the microtubule network, possibly through the modulation of tau phosphorylation, and small tau deposits assembled near the MTOC. Hence, organization and integrity of the cytoskeleton were affected, which is particularly important for the maintenance of the cellular architecture and intracellular transport processes, and essential for the functionality and survival of neural cells. Our data demonstrate that DUB inhibitors provide a useful tool to elucidate the manifold mechanisms of DUB functions in cells and their dysregulation in neurodegenerative diseases. This article is part of a Special Issue entitled: Ubiquitin Drug Discovery and Diagnostics.     

Hsp90 in non-mammalian metazoan model systems

The molecular chaperone Hsp90 has been discovered in the heat-shock response of the fruit fly more than 30years ago. Today, it is becoming clear that Hsp90 is in the middle of a regulatory system, participating in the modulation of many essential client proteins and signaling pathways. Exerting these activities, Hsp90 works together with about a dozen of cochaperones. Due to their organismal simplicity and the possibility to influence their genetics on a large scale, many studies have addressed the function of Hsp90 in several multicellular model systems. Defined pathways involving Hsp90 client proteins have been identified in the metazoan model systems of Caenorhabditis elegans, Drosophila melanogaster and the zebrafish Danio rerio. Here, we summarize the functions of Hsp90 during muscle maintenance, development of phenotypic traits and the involvement of Hsp90 in stress responses, all of which were largely uncovered using the model organisms covered in this review. These findings highlight the many specific and general actions of the Hsp90 chaperone machinery. This article is part of a Special Issue entitled: Heat Shock Protein 90 (HSP90).     

PPAR-alpha activation as a preconditioning-like intervention in rats in vivo confers myocardial protection against acute ischaemia-reperfusion injury: involvement of PI3K-Akt

Peroxisome proliferator-activated receptors (PPAR) regulate the expression of genes involved in lipid metabolism, energy production, and inflammation. Their role in ischaemia-reperfusion (I/R) is less clear, although research indicates involvement of PPARs in some forms of preconditioning. This study aimed to explore the effects of PPAR-α activation on the I/R injury and potential cardioprotective downstream mechanisms involved. Langendorff-perfused hearts of rats pretreated with the selective PPAR-α agonist WY-14643 (WY, pirinixic acid; 3 mg·(kg body mass)·day(-1); 5 days) were subjected to 30 min ischaemia - 2 h reperfusion with or without the phosphatidylinositol 3-kinase (PI3K)-Akt inhibitor wortmannin for the evaluation of functional (left ventricular developed pressure, LVDP) recovery, infarct size (IS), and reperfusion-induced arrhythmias. A 2-fold increase in baseline PPAR-α mRNA levels (qPCR) in the WY-treated group and higher post-I/R PPAR-α levels compared with those in untreated controls were accompanied by similar changes in the expression of PPAR-α target genes PDK4 and mCPT-1, regulating glucose and fatty acid metabolism, and by enhanced Akt phosphorylation. Post-ischaemic LVDP restoration in WY-treated hearts reached 60% ± 9% of the pre-ischaemic values compared with 24% ± 3% in the control hearts (P < 0.05), coupled with reduced IS and incidence of ventricular fibrillation that was blunted by wortmannin. Results indicate that PPAR-α up-regulation may confer preconditioning-like protection via metabolic effects. Downstream mechanisms of PPAR-α-mediated cardioprotection may involve PI3K-Akt activation.