miR-125a-5p/miR-125b-5p contributes to pathological activation of angiotensin II-AT1R in mouse distal convoluted tubule cells by the suppression of Atrap.

The renin-angiotensin system plays a crucial role in the regulation of blood pressure. Activation of the angiotensin II (Ang II)-Ang II type 1 receptor (AT1R) signaling pathway contributes to the pathogenesis of hypertension and subsequent organ damage. AT1R-associated protein (ATRAP) has been identified as an endogenous inhibitory protein of the AT1R pathological activation. We have shown that mouse Atrap (Atrap) represses various Ang II-AT1R-mediated pathologies, including hypertension in mice. The expression of human ATRAP (ATRAP)/Atrap can be altered in various pathological states in humans and mice, such as Ang II stimulation and serum starvation. However, the regulatory mechanisms of ATRAP/Atrap are not yet fully elucidated. miRNAs are 21 to 23 nucleotides of small RNAs that post-transcriptionally repress gene expression. Single miRNA can act on hundreds of target mRNAs, and numerous miRNAs have been identified as the Ang II-AT1R signaling-associated disease phenotype modulator, but nothing is known about the regulation of ATRAP/Atrap. In the present study, we identified miR-125a-5p/miR-125b-5p as the evolutionarily conserved miRNAs that potentially act on ATRAP/Atrap mRNA. Further analysis revealed that miR-125a-5p/miR-125b-5p can directly repress both ATRAP and Atrap. In addition, the inhibition of miR-125a-5p/miR-125b-5p resulted in the suppression of the Ang II-AT1R signaling in mouse distal convoluted tubule cells. Taken together, miR-125a-5p/miR-125b-5p activates Ang II-AT1R signaling by the suppression of ATRAP/Atrap. Our results provide new insights into the potential approaches for achieving the organ-protective effects by the repression of the miR-125 family associated with the enhancement of ATRAP/Atrap expression.

Extracellular vesicles DJ-1 derived from hypoxia-conditioned hMSCs alleviate cardiac hypertrophy by suppressing mitochondria dysfunction and preventing ATRAP degradation.

BACKGROUND: As a pathological myocardial remodeling process in a variety of cardiovascular diseases, cardiac hypertrophy still has no effective treatment. Human mesenchymal stem cells (hMSCs) derived extracellular vesicles (EVs) has been recognized as a promising treatment strategy for cardiac disease. METHODS: In this study, the inhibitory effects on cardiac hypertrophy are compared between normoxia-conditioned hMSC-derived EVs (Nor-EVs) and hypoxia-conditioned hMSC-derived EVs (Hypo-EVs) in neonatal rat cardiomyocytes (NRCMs) after angiotensin II (Ang II) stimulation and in a mouse model of transverse aortic constriction (TAC). RESULTS: We demonstrate that Hypo-EVs exert an increased inhibitory effect on cardiac hypertrophy compared with Nor-EVs. Parkinson disease protein 7 (PARK7/DJ-1) is identify as a differential protein between Nor-EVs and Hypo-EVs by quantitative proteomics analysis. Results show that DJ-1, which is rich in Hypo-EVs, alleviates mitochondrial dysfunction and excessive mitochondrial reactive oxygen species (mtROS) production as an antioxidant. Mechanistic studies demonstrate for the first time that DJ-1 may suppress cardiac hypertrophy by inhibiting the activity of proteasome subunit beta type 10 (PSMB10) through a direct physical interaction. This interaction can inhibit angiotensin II type 1 receptor (AT1R)-mediated signaling pathways resulting in cardiac hypertrophy through alleviating ubiquitination degradation of AT1R-associated protein (ATRAP). CONCLUSIONS: When taken together, our study suggests that Hypo-EVs have significant potential as a novel therapeutic agent for the treatment of cardiac hypertrophy.

Ablation of Immunoproteasome ß5i Subunit Suppresses Hypertensive Retinopathy by Blocking ATRAP Degradation in Mice.

Inflammation is associated with retinal diseases. Our recent data demonstrate that immunoproteasome catalytic subunit ß2i contributes to angiotensin II (Ang II)-induced retinopathy in mice. Here, we investigated the role of another catalytic subunit ß5i in regulating retinopathy and its underlying mechanisms. We induced a murine model of retinopathy by infusing Ang II (3,000 ng/kg/min) for 3 weeks into wild-type (WT) mice, ß5i-knockout (KO) mice, or WT mice injected with either adenovirus-expressing ß5i (Ad-ß5i) or angiotensin II type 1 receptor (AT1R)-associated protein (Ad-ATRAP), which inhibits AT1R. The ß5i expression and chymotrypsin-like activity were most significantly elevated in Ang II-infused retinas and serum from patients with hypertensive retinopathy. Moreover, Ang II infusion-induced retinopathy was markedly attenuated in ß5i-KO mice but aggravated in Ad-ß5i-injected mice. Accordingly, ß5i KO markedly restored Ang II-induced downregulation of ATRAP and activation of AT1R downstream mediators, which was further enhanced in Ad-ß5i-injected mice. Interestingly, overexpression of ATRAP significantly abrogated Ang II-induced retinopathy in Ad-ß5i-injected mice. This study found that ß5i promoted Ang II-induced retinopathy by promoting ATRAP degradation and activation of AT1R-mediated signals.

Silencing of AtRAP, a target gene of a bacteria-induced small RNA, triggers antibacterial defense responses through activation of LSU2 and down-regulation of GLK1.

Plants fine-tune their sophisticated immunity systems in response to pathogen infections. We previously showed that AtlsiRNA-1, a bacteria-induced plant endogenous small interfering RNA, silences the AtRAP gene, which encodes a putative RNA binding protein. In this study, we demonstrate that AtRAP functions as a negative regulator in plant immunity by characterizing molecular and biological responses of the knockout mutant and overexpression lines of AtRAP upon bacterial infection. AtRAP is localized in chloroplasts and physically interacts with Low Sulfur Upregulated 2 (LSU2), which positively regulates plant defense. Our results suggest that AtRAP negatively regulates defense responses by suppressing LSU2 through physical interaction. We also detected downregulation of the transcription factor GOLDEN2-LIKE 1 (GLK1) in atrap-1 using microarray analysis. The glk1 glk2 double mutant showed enhanced resistance to Pseudomonas syringae pv. tomato, which is consistent with a previous study showing enhanced resistance of a glk1 glk2 double mutant to Hyaloperonospora arabidopsidis. Taken together, our data suggest that silencing of AtRAP by AtlsiRNA-1 upon bacterial infection triggers defense responses through regulation of LSU2 and GLK1.

Melatonin inhibits angiotensin II-induced atrial fibrillation through preventing degradation of Ang II Type I Receptor-Associated Protein (ATRAP).

Angiotensin II (Ang II) induced Atrial fibrillation (AF) often accompanied with reduced ATRAP which is a negative modulator of Ang II type 1 receptor (AT1R). Melatonin can protect against AF, but the underlying molecular mechanism remains poorly understood. In this study, Ang II was used to induce AF, and AF inducibility and duration were documented telemetrically. Ang II-infused mice had a higher AF incidence, which was associated with atrial fibrosis, inflammation, and oxidative stress. Melatonin partially inhibited these effects, and enforced expression of siRNA-ATRAP in atria counteracted the beneficial role of melatonin. Specifically, melatonin inhibited expression of Ang II-induced proteasome and immunoproteasome subunits ß2, ß2i, ß5, and ß5i as well as their corresponding trypsin-like and chymotrypsin-like activities and blocked ATRAP degradation. In turn, this inhibited AT1R-mediated NF-κB signaling, transforming growth factor (TGF)-ß1/Smad signaling in the atria, and thereby affected atrial remodeling and AF. Melatonin receptor inhibition by the chemical inhibitor luzindole partially inhibited the inhibitory effects of melatonin on proteasome activity and also Ang II-induced pathological changes in the atria. Overall, our study demonstrates that melatonin protects against Ang II-induced AF by inhibiting proteasome activity and stabilizing ATRAP expression, and these effects are partially dependent on melatonin receptor activation.

USF1-ATRAP-PBX3 Axis Promote Breast Cancer Glycolysis and Malignant Phenotype by Activating AKT/mTOR Signaling.

Angiotensin II type 1 receptor-associated protein (ATRAP) is widely expressed in different tissues and organs, although its mechanistic role in breast cancer remains unclear. Here, we show that ATRAP is highly expressed in breast cancer tissues. Its aberrant upregulation promotes breast cancer aggressiveness and is positively correlated with poor prognosis. Functional assays revealed that ATRAP participates in promoting cell growth, metastasis, and aerobic glycolysis, while microarray analysis showed that ATRAP can activate the AKT/mTOR signaling pathway in cancer progression. In addition, ATRAP was revealed to direct Ubiquitin-specific protease 14 (USP14)-mediated deubiquitination and stabilization of Pre-B cell leukemia homeobox 3 (PBX3). Importantly, ATRAP is a direct target of Upstream stimulatory factor 1 (USF1), and that ATRAP overexpression reverses the inhibitory effects of USF1 knockdown. Our study demonstrates the broad contribution of the USF1/ATRAP/PBX3 axis to breast cancer progression and provides a strong potential therapeutic target.

The angiotensin receptor-associated protein Atrap is a stimulator of the cardiac Ca2+-ATPase SERCA2a.

AIMS: The angiotensin II type 1 receptor-associated protein (Atrap) is highly expressed in the heart, but its function in the heart is unknown. We hypothesized that cardiac Atrap may interact with proteins other than the AT1 receptor. METHODS AND RESULTS: To identify potential novel interacting partners of Atrap, pull-down assays were performed. Sequencing by MALDI-MS of the isolated complexes showed that Atrap interacts with the cardiac Ca(2+)-ATPase SERCA2a. The interaction between Atrap and SERCA2a was confirmed by co-immunoprecipitation and by surface plasmon resonance (SPR) spectroscopy. Atrap enhanced the SERCA-dependent Ca(2+) uptake in isolated SR membrane vesicles. Furthermore, sarcomere shortenings and [Ca(2+)]i transients (CaTs) were determined in ventricular myocytes isolated from Atrap-/- and wild-type (WT) mice. The amplitudes of CaTs and sarcomere shortenings were similar in Atrap-/- and WT myocytes. However, the CaT decay and sarcomere re-lengthening were prolonged in Atrap-/- myocytes. To further evaluate the functional relevance of the Atrap-SERCA2a interaction in vivo, left-ventricular function was assessed in WT and Atrap-/- mice. The heart rates (564 ± 10 b.p.m. vs. 560 ± 11 b.p.m.; P = 0.80) and ejection fractions (71.3 ± 1.3 vs. 72 ± 1.8%; P = 0.79) were similar in WT and Atrap-/- mice, respectively (n = 15 for each genotype). However, the maximum filling rate (dV/dtmax) was markedly decreased in Atrap-/- (725 ± 48 µL/s) compared with WT mice (1065 ± 122 µL/s; P = 0.01; n = 15). CONCLUSION: We identified Atrap as a novel regulatory protein of the cardiac Ca(2+)-ATPase SERCA2a. We suggest that Atrap enhances the activity of SERCA2a and, consequently, facilitates ventricular relaxation.

Strain differences and the role of AT(1) receptor expression in anxiety.

This study investigated strain specific differences to the anxiolytic response to losartan focusing on genetic variation that may influence such responses. This included: AT(1) receptor sequence variation, angiotensin II receptor associated protein (ATRAP) and receptor expression between strains. Sequencing of exon 3 of AT(1a)R revealed no differences between BKW mice (n=6) and C57 and DBA(2) strains (n=3). Comparisons of AT(1) expression do show significant differences, whereby BKW mice showed the highest levels of expression and DBA(2) mice intermediate levels when compared to the C57 strain. Sequencing of sections of the Angiotensin receptor associated protein (ATRAP) identified a non-synonymous point mutation- (T/C) transversion (position 109-161) (SNP id = rs13467517) resulting in a Valine → Alanine (V157A) amino acid change in the BKW and DBA(2) strains. Our results indicate that the previously reported strain dependent effects are not due to variation in AT(1a) receptor sequence. Differences in AT(1)gene expression levels between strains, which mirror their anxiety phenotype, are observed. This is coupled with a non-synonymous single nucleotide polymorphism in ATRAP, a negative regulator of AT(1) signalling.