Tumor suppressor Tsc1 is a new Hsp90 co‐chaperone that facilitates folding of kinase and non‐kinase clients

The tumor suppressors Tsc1 and Tsc2 form the tuberous sclerosis complex (TSC), a regulator of mTOR activity. Tsc1 stabilizes Tsc2; however, the precise mechanism involved remains elusive. The molecular chaperone heat‐shock protein 90 (Hsp90) is an essential component of the cellular homeostatic machinery in eukaryotes. Here, we show that Tsc1 is a new co‐chaperone for Hsp90 that inhibits its ATPase activity. The C‐terminal domain of Tsc1 (998–1,164 aa) forms a homodimer and binds to both protomers of the Hsp90 middle domain. This ensures inhibition of both subunits of the Hsp90 dimer and prevents the activating co‐chaperone Aha1 from binding the middle domain of Hsp90. Conversely, phosphorylation of Aha1‐Y223 increases its affinity for Hsp90 and displaces Tsc1, thereby providing a mechanism for equilibrium between binding of these two co‐chaperones to Hsp90. Our findings establish an active role for Tsc1 as a facilitator of Hsp90‐mediated folding of kinase and non‐kinase clients—including Tsc2—thereby preventing their ubiquitination and proteasomal degradation.     

ACE2‐EPC‐EXs protect ageing ECs against hypoxia/reoxygenation‐induced injury through the miR‐18a/Nox2/ROS pathway

Oxidative stress is one of the mechanisms of ageing‐associated vascular dysfunction. Angiotensin‐converting enzyme 2 (ACE2) and microRNA (miR)‐18a have shown to be down‐regulated in ageing cells. Our previous study has shown that ACE2‐primed endothelial progenitor cells (ACE2‐EPCs) have protective effects on endothelial cells (ECs), which might be due to their released exosomes (EXs). Here, we aimed to investigate whether ACE2‐EPC‐EXs could attenuate hypoxia/reoxygenation (H/R)‐induced injury in ageing ECs through their carried miR‐18a. Young and angiotensin II‐induced ageing ECs were subjected to H/R and co‐cultured with vehicle (medium), EPC‐EXs, ACE2‐EPCs‐EXs, ACE2‐EPCs‐EXs + DX600 or ACE2‐EPCs‐EXs with miR‐18a deficiency (ACE2‐EPCs‐EXs^{anti‐miR‐18a}). Results showed (1) ageing ECs displayed increased senescence, apoptosis and ROS production, but decreased ACE2 and miR‐18a expressions and tube formation ability; (2) under H/R condition, ageing ECs showed higher rate of apoptosis, ROS overproduction and nitric oxide reduction, up‐regulation of Nox2, down‐regulation of ACE2, miR‐18a and eNOS, and compromised tube formation ability; (3) compared with EPC‐EXs, ACE2‐EPC‐EXs had better efficiencies on protecting ECs from H/R‐induced changes; (4) The protective effects were less seen in ACE2‐EPCs‐EXs + DX600 and ACE2‐EPCs‐EXs^{anti‐miR‐18a} groups. These data suggest that ACE‐EPCs‐EXs have better protective effects on H/R injury in ageing ECs which could be through their carried miR‐18a and subsequently down‐regulating the Nox2/ROS pathway.     

CO2‐fixing liquid droplets: Towards a dissection of the microalgal pyrenoid

CO₂ enters the biosphere via the slow, oxygen‐sensitive carboxylase, Rubisco. To compensate, most microalgae saturate Rubisco with its substrate gas through a carbon dioxide concentrating mechanism. This strategy frequently involves compartmentalization of the enzyme in the pyrenoid, a non‐membrane enclosed compartment of the chloroplast stroma. Recently, tremendous advances have been achieved concerning the structure, physical properties, composition and in vitro reconstitution of the pyrenoid matrix from the green alga Chlamydomonas reinhardtii. The discovery of the intrinsically disordered multivalent Rubisco linker protein EPYC1 provided a biochemical framework to explain the subsequent finding that the pyrenoid resembles a liquid droplet in vivo. Reconstitution of the corresponding liquid‐liquid phase separation using pure Rubisco and EPYC1 allowed a detailed characterization of this process. Finally, a large high‐quality dataset of pyrenoidal protein‐protein interactions inclusive of spatial information provides ample substrate for rapid further functional dissection of the pyrenoid. Integrating and extending recent advances will inform synthetic biology efforts towards enhancing plant photosynthesis as well as contribute a versatile model towards experimentally dissecting the biochemistry of enzyme‐containing membraneless organelles.     

MicroRNA‐328 is involved in the effect of selenium on hydrogen peroxide‐induced injury in H9c2 cells

Oxidative stress induces apoptosis in cardiac cells, and antioxidants attenuate the injury. MicroRNAs (miRNAs) are also involved in cell death; therefore, this study aimed to investigate the role of miRNAs in the effect of selenium on oxidative stress‐induced apoptosis. The effects of sodium selenite were analyzed via cell viability, superoxide dismutase (SOD) activity, and malondialdehyde (MDA) concentration. Flow cytometry was used to evaluate cell apoptosis. Fura‐2AM was used to calculate intracellular Ca²⁺ concentration. Sodium selenite could ameliorate hydrogen peroxide (H₂O₂)‐induced cell apoptosis and improve expression levels of glutathione peroxidase and thioredoxin reductase. Pretreatment with sodium selenite improved SOD activity and reduced MDA concentration. Treatments with H₂O₂ or sodium selenite decreased miR‐328 levels. MiR‐328 overexpression enhanced cell apoptosis, reduced ATP2A2 levels, and increased intracellular Ca²⁺ concentration, while inhibition produced opposite effects. MiR‐328 might be involved in the effect of sodium selenite on H₂O₂‐induced cell death in H9c2 cells.     

The protection by heme oxygenase‐1 induction against thioacetamide‐induced liver toxicity is associated with changes in arginine and asymmetric dimethylarginine

This study was designed to investigate the role of HO‐1 induction in prevention of thioacetamide (TAA)‐induced oxidative stress, inflammation and liver damage. The changes in hepatic dimethylarginine dimethylaminohydrolase (DDAH) activity as well as plasma arginine and asymmetric dimethylarginine (ADMA) levels were also measured to evaluate nitric oxide (NO) bioavailability. Rats were divided into four groups as control, hemin, TAA and hemin + TAA groups. Hemin (50 mg kg^?1, i.p.) was injected to rats 18 h before TAA treatment to induce HO‐1 enzyme expression. Rats were given TAA (300 mg kg^?1, i.p.) and killed 24 h after treatment. Although TAA treatment produced severe hepatic injury, upregulation of HO‐1 ameliorated TAA‐induced liver damage up to some extent as evidence by decreased serum alanine transaminase, aspartate transaminase and arginase activities and histopathological findings. Induction of HO‐1 stimulated antioxidant system and decreased lipid peroxidation in TAA‐treated rats. Myeloperoxidase activity and inducible NO synthase protein expression were decreased, whereas DDAH activity was increased by hemin injection in TAA‐treated rats. Induction of HO‐1 was associated with increased arginine levels and decreased ADMA levels, being the main determinants of NO production, in plasma of TAA‐treated rats. In conclusion, our results indicate that HO‐1 induction alleviated increased oxidative stress and inflammatory reactions together with deterioration in NO production in TAA‐induced liver damage in rats. Copyright © 2012 John Wiley & Sons, Ltd.     

The D1‐D2 region of the large subunit ribosomal DNA as barcode for ciliates

Ciliates are a major evolutionary lineage within the alveolates, which are distributed in nearly all habitats on our planet and are an essential component for ecosystem function, processes and stability. Accurate identification of these unicellular eukaryotes through, for example, microscopy or mating type reactions is reserved to few specialists. To satisfy the demand for a DNA barcode for ciliates, which meets the standard criteria for DNA barcodes defined by the Consortium for the Barcode of Life (CBOL), we here evaluated the D1‐D2 region of the ribosomal DNA large subunit (LSU‐rDNA). Primer universality for the phylum Ciliophora was tested in silico with available database sequences as well as in the laboratory with 73 ciliate species, which represented nine of 12 ciliate classes. Primers tested in this study were successful for all tested classes. To test the ability of the D1‐D2 region to resolve conspecific and congeneric sequence divergence, 63 Paramecium strains were sampled from 24 mating species. The average conspecific D1‐D2 variation was 0.18%, whereas congeneric sequence divergence averaged 4.83%. In pairwise genetic distance analyses, we identified a D1‐D2 sequence divergence of <0.6% as an ideal threshold to discriminate Paramecium species. Using this definition, only 3.8% of all conspecific and 3.9% of all congeneric sequence comparisons had the potential of false assignments. Neighbour‐joining analyses inferred monophyly for all taxa but for two Paramecium octaurelia strains. Here, we present a protocol for easy DNA amplification of single cells and voucher deposition. In conclusion, the presented data pinpoint the D1‐D2 region as an excellent candidate for an official CBOL barcode for ciliated protists.     

Curcumin synergistically potentiates the protective effect of sitagliptin against chronic deltamethrin nephrotoxicity in rats: Impact on pro‐inflammatory cytokines and Nrf2/Ho‐1 pathway

Deltamethrin (DLM) is a synthesized organophosphorus acaricide and bug spray, broadly utilized for veterinary and farming purposes. Although its exposure to humans and animals causes toxicity in the kidney and other primary organs, our objective was to assess the defensive effects of sitagliptin (Sita) and additionally curcumin (Cur) in the DLM‐intoxicated rats’ kidney. DLM‐intoxicated rats revealed a huge increase of various biochemical parameters in serum identified with kidney damage: uric acid, urea, and creatinine. DLM intoxication altogether increased renal lipid peroxidation, and critically restrained antioxidative biomarkers including superoxide dismutase, glutathione, and glutathione peroxidase. Likewise, it increased the tumor necrosis factor‐α, interleukin 6 (IL‐6) and IL‐1β level in serum. Additionally, DLM intoxication diminished the outflow of the nuclear factor (erythroid‐derived 2)‐like 2 (Nrf2)/heme oxygenase‐1 (HO‐1) pathway in rats. Both Sita and Cur act against DLM‐prompted serum along with renal tissue biochemical parameters when utilized alone or in a mix alongside DLM intoxication. Besides this, both Sita and Cur delivered synergetic nephroprotective, antioxidative, and anti‐inflammatory impacts. Consequently, it could be presumed that Sita as well as Cur administration can limit the poisonous impacts of DLM by their free radical‐scavenging, strong antioxidant, and Nrf2/HO‐1 pathway upregulation activity.     

Effects of β‐carotene on antioxidant status in rats with chronic alcohol consumption

This study examined the effects of β‐carotene on antioxidant status in rats with chronic alcohol consumption. At the beginning of experiment (week 0), according to both the plasma aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities, rats (n = 24) were divided into 3 groups and fed with a standard diet (group C), a diet containing ethanol (group E), or a diet containing ethanol and β‐carotene (group E+B). After 10 weeks, plasma AST and ALT, fat accumulation in the liver, antioxidant enzyme activities in erythrocytes and the liver, malondialdehyde (MDA), and α‐tocopherol and retinol in plasma and hepatic samples were analyzed. The chronic alcohol diet significantly increased AST and ALT levels in plasma, and these changes were prevented by supplementing the diet with β‐carotene. Glutathione (GSH) in erythrocytes and in the liver was significantly elevated in rats fed with a diet containing β‐carotene. The results indicate that β‐carotene supplementation can prevent ethanol‐induced liver damage and increase GSH concentrations in erythrocytes and the liver. Copyright © 2009 John Wiley & Sons, Ltd.     

Increased Lipocalin‐2 in the retinal pigment epithelium of Cryba1 cKO mice is associated with a chronic inflammatory response

Although chronic inflammation is believed to contribute to the pathology of age‐related macular degeneration (AMD), knowledge regarding the events that elicit the change from para‐inflammation to chronic inflammation in the pathogenesis of AMD is lacking. We propose here that lipocalin‐2 (LCN2), a mammalian innate immunity protein that is trafficked to the lysosomes, may contribute to this process. It accumulates significantly with age in retinal pigment epithelial (RPE) cells of Cryba1 conditional knockout (cKO) mice, but not in control mice. We have recently shown that these mice, which lack βA3/A1‐crystallin specifically in RPE, have defective lysosomal clearance. The age‐related increase in LCN2 in the cKO mice is accompanied by increases in chemokine (C‐C motif) ligand 2 (CCL2), reactive gliosis, and immune cell infiltration. LCN2 may contribute to induction of a chronic inflammatory response in this mouse model with AMD‐like pathology.     

The role of chlorine atom on the binding between 2‐phenyl‐1H‐benzimidazole analogues and fat mass and obesity‐associated protein

In this work, nine 2‐phenyl‐1H‐benzimidazole structural analogues were screened for potential inhibitor of the fat mass and obesity‐associated protein (FTO) by isothermal titration calorimetry (ITC). The results show that the binding between 6‐chloro‐2‐phenyl‐1H‐benzimidazole (1d) and FTO was dominated by entropy. Results of enzymatic activity assays provided an IC₅₀ value of 24.65 μM for 1d. Our previous results and comparison of nine structural analogues indicated that the chlorine atom was crucial for the binding of small molecules with FTO. The identification of novel small molecules may provide information for the design of FTO inhibitors and the treatment of leukemia.     

The variant at TGFBRAP1 is significantly associated with type 2 diabetes mellitus and affects diabetes‐related miRNA expression

While the transforming growth factor‐β1 (TGF‐β1) regulates the growth and proliferation of pancreatic β‐cells, its receptors trigger the activation of Smad network and subsequently induce the insulin resistance. A case‐control was conducted to evaluate the associations of the polymorphisms of TGF‐β1 receptor‐associated protein 1 (TGFBRAP1) and TGF‐β1 receptor 2 (TGFBR2) with type 2 diabetes mellitus (T2DM), and its genetic effects on diabetes‐related miRNA expression. miRNA microarray chip was used to screen T2DM‐related miRNA and 15 differential expressed miRNAs were further validated in 75 T2DM and 75 normal glucose tolerance (NGT). The variation of rs2241797 (T/C) at TGFBRAP1 showed significant association with T2DM in case‐control study, and the OR (95% CI) of dominant model for cumulative effects was 1.204 (1.060‐1.370), Bonferroni corrected P < 0.05. Significant differences in the fast glucose and HOMA‐β indices were observed amongst the genotypes of rs2241797. The expression of has‐miR‐30b‐5p and has‐miR‐93‐5p was linearly increased across TT, TC, and CC genotypes of rs2241797 in NGT, P_trend values were 0.024 and 0.016, respectively. Our findings suggest that genetic polymorphisms of TGFBRAP1 may contribute to the genetic susceptibility of T2DM by mediating diabetes‐related miRNA expression.     

Differential responses to oxidative stress and calcium influx on expression of the transforming growth factor‐β family in myoblasts and myotubes

Changes in gene expression of TGF‐β family members and their receptors in response to treatment with H₂O₂ and a calcium ionophore, A23187, were examined in C2C12 myoblasts and myotubes. The expression of Myf5, an initial regulator of myogenesis, was increased by A23187, and H₂O₂ inhibited the up‐regulation of Myf5. Treatment with H₂O₂ decreased the expression of MHC IIb, a protein component of the myofibrils, irrespective of the presence of A23187, suggesting an inhibitory role of oxidative stress for myogenesis. Expression of ligands and receptors for the TGF‐β family was modulated in response to H₂O₂ and A23187. Treatment with H₂O₂ decreased expression of TGF‐β3, BMP‐4, ALK4, ALK5, and ActRIIB, and increased expression of inhibin α and inhibin βA in either the myoblast stage or the myotube stage, or both. A23187 potentiated down‐regulation of BMP‐4 and ALK4 expression, and up‐regulation of TGF‐β1, TGF‐β2, inhibin α, inhibin βA, ALK2, and ALK3 expression. These results indicate that oxidative stress and Ca²⁺ influx affect expression of the TGF‐β family in C2C12 myoblasts and myotubes. Copyright © 2009 John Wiley & Sons, Ltd.