Extracellular Non-Coding RNAs in Cardiovascular Diseases.

Cardiovascular diseases (CVDs) remain the world's leading cause of death despite the best available healthcare and therapy. Emerging as a key mediator of intercellular and inter-organ communication in CVD pathogenesis, extracellular vesicles (EVs) are a heterogeneous group of membrane-enclosed nano-sized vesicles released by virtually all cells, of which their RNA cargo, especially non-coding RNAs (ncRNA), has been increasingly recognized as a promising diagnostic and therapeutic target. Recent evidence shows that ncRNAs, such as small ncRNAs, circular RNAs, and long ncRNAs, can be selectively sorted into EVs or other non-vesicular carriers and modulate various biological processes in recipient cells. In this review, we summarize recent advances in the literature regarding the origin, extracellular carrier, and functional mechanisms of extracellular ncRNAs with a focus on small ncRNAs, circular RNAs, and long ncRNAs. The pathophysiological roles of extracellular ncRNAs in various CVDs, including atherosclerosis, ischemic heart diseases, hypertension, cardiac hypertrophy, and heart failure, are extensively discussed. We also provide an update on recent developments and challenges in using extracellular ncRNAs as biomarkers or therapeutical targets in these CVDs.

Tead1 is essential for mitochondrial function in cardiomyocytes.

Mitochondrial dysfunction occurs in most forms of heart failure. We have previously reported that Tead1, the transcriptional effector of Hippo pathway, is critical for maintaining adult cardiomyocyte function, and its deletion in adult heart results in lethal acute dilated cardiomyopathy. Growing lines of evidence indicate that Hippo pathway plays a role in regulating mitochondrial function, although its role in cardiomyocytes is unknown. Here, we show that Tead1 plays a critical role in regulating mitochondrial OXPHOS in cardiomyocytes. Assessment of mitochondrial bioenergetics in isolated mitochondria from adult hearts showed that loss of Tead1 led to a significant decrease in respiratory rates, with both palmitoylcarnitine and pyruvate/malate substrates, and was associated with reduced electron transport chain complex I activity and expression. Transcriptomic analysis from Tead1-knockout myocardium revealed genes encoding oxidative phosphorylation, TCA cycle, and fatty acid oxidation proteins as the top differentially enriched gene sets. Ex vivo loss of function of Tead1 in primary cardiomyocytes also showed diminished aerobic respiration and maximal mitochondrial oxygen consumption capacity, demonstrating that Tead1 regulation of OXPHOS in cardiomyocytes is cell autonomous. Taken together, our data demonstrate that Tead1 is a crucial transcriptional node that is a cell-autonomous regulator, a large network of mitochondrial function and biogenesis related genes essential for maintaining mitochondrial function and adult cardiomyocyte homeostasis.NEW & NOTEWORTHY Mitochondrial dysfunction constitutes an important aspect of heart failure etiopathogenesis and progression. However, the molecular mechanisms are still largely unknown. Growing lines of evidence indicate that Hippo-Tead pathway plays a role in cellular bioenergetics. This study reveals the novel role of Tead1, the downstream transcriptional effector of Hippo pathway, as a novel regulator of mitochondrial oxidative phosphorylation and in vivo cardiomyocyte energy metabolism, thus providing a potential therapeutic target for modulating mitochondrial function and enhancing cytoprotection of cardiomyocytes.

Structure and regulation of human epithelial cell transforming 2 protein.

Epithelial cell transforming 2 (Ect2) protein activates Rho GTPases and controls cytokinesis and many other cellular processes. Dysregulation of Ect2 is associated with various cancers. Here, we report the crystal structure of human Ect2 and complementary mechanistic analyses. The data show the C-terminal PH domain of Ect2 folds back and blocks the canonical RhoA-binding site at the catalytic center of the DH domain, providing a mechanism of Ect2 autoinhibition. Ect2 is activated by binding of GTP-bound RhoA to the PH domain, which suggests an allosteric mechanism of Ect2 activation and a positive-feedback loop reinforcing RhoA signaling. This bimodal RhoA binding of Ect2 is unusual and was confirmed with Förster resonance energy transfer (FRET) and hydrogen-deuterium exchange mass spectrometry (HDX-MS) analyses. Several recurrent cancer-associated mutations map to the catalytic and regulatory interfaces, and dysregulate Ect2 in vitro and in vivo. Together, our findings provide mechanistic insights into Ect2 regulation in normal cells and under disease conditions.

99mTc-LHRH in tumor receptor imaging.

Detection of gonadotropin-releasing hormone (GnRH) also known as luteinizing hormone-releasing hormone (LHRH) in the relevant tumor tissue and normal tissues and organs in vivo expression was investigated. To examine the method of direct radio labeling of LHRH by 99mTc with relatively high radiochemical purity and stability, screening the best labeling conditions, to establish a simple and reliable method of preparation of 99mTc-LHRH was undertaken. The detection of radioisotope-labeled LHRH distribution in mice, LHRH receptor imaging for the study and treatment of cancer basis were evaluated. i) Immunohistochemical staining test was used in 23 patients with hepatocellular carcinoma (HCC), 20 patients with breast cancer, 10 patients with prostate cancer, 20 patients with lung cancer, 20 patients with endometrial cancer tumor cells and normal tissue LHRH-R De Biaoda levels; ii) pre-tin method use direct labeling of LHRH, marking completion of saline or human serum were added at room temperature, the chromatography was measured at different times, to calculate the rate of labeled product and the radiochemical purity of the label, in vivo observation of its stability, and comparative analysis of selected optimal condition; iii) rat pituitary cell membrane protein, the product of in vitro radio-receptor marker analysis, through the saturation and inhibition experiments, was used to test its receptor binding activity; iv) Ch-T method labeled 125I-LHRH, tail vein injection of normal mice at different times were sacrificed, blood and major organs were determined and calculated per gram organization percentage injected dose rate (%, ID/g). Detected by immunohistochemistry in 23 cases of HCC in the LHRH-positive rate was 82.61%, in the corresponding normal tissues, the positive rate was 15%; 20 cases of breast cancer positive rate of 95%, the corresponding normal tissues, the positive rate was 20%; 10 cases of prostate cancer positive rate of 70%, the corresponding normal tissues, the positive rate of 40%; 20 cases of lung cancer positive rate of 85%, the corresponding normal tissues, the positive rate of 15.79%; 20 cases of endometrial cancer positive rate of 80% in the corresponding normal tissues was 16.67% positive. 99mTc-LHRH mark was 97.9-100.0%, the radiochemical purity of 93.9-96.4%, marking the reaction gel content of <5%. Great product receptor marker analysis showed 99mTc-LHRH with saturable receptor binding characteristics and inhibition, and high affinity, RT = 23.2174 pmol, KD = 0.4348 nmol; intravenous injection of 131I-LHRH within 72 h after the mice rapidly cleared the blood radioactivity, the major radioactive accumulation in the liver and kidneys and by the liver, renal clearance, and other tissues and organs of the radioactivity gradually decreased with time. In conclusion, i) the liver, lung, breast, prostate, endometrial cancer exist in both LHRHR; ii) 99mTc-LHRH preparation is simple, rapid, radiochemical purity product obtained higher marks, better stability, no further purification; and iii) LHRH 99mTc labeled, still has a high receptor binding ability, biological activity; and has an ideal and realistic dynamics in animals, there is hope, as with the clinical value of imaging agent of GnRH receptors.

Crystal structure of a nuclear actin ternary complex.

Actin polymerizes and forms filamentous structures (F-actin) in the cytoplasm of eukaryotic cells. It also exists in the nucleus and regulates various nucleic acid transactions, particularly through its incorporation into multiple chromatin-remodeling complexes. However, the specific structure of actin and the mechanisms that regulate its polymeric nature inside the nucleus remain unknown. Here, we report the crystal structure of nuclear actin (N-actin) complexed with actin-related protein 4 (Arp4) and the helicase-SANT-associated (HSA) domain of the chromatin remodeler Swr1. The inner face and barbed end of N-actin are sequestered by interactions with Arp4 and the HSA domain, respectively, which prevents N-actin from polymerization and binding to many actin regulators. The two major domains of N-actin are more twisted than those of globular actin (G-actin), and its nucleotide-binding pocket is occluded, freeing N-actin from binding to and regulation by ATP. These findings revealed the salient structural features of N-actin that distinguish it from its cytoplasmic counterpart and provide a rational basis for its functions and regulation inside the nucleus.

Kinesin 1 Drives Autolysosome Tubulation.

Autophagic lysosome reformation (ALR) plays an important role in maintaining lysosome homeostasis. During ALR, lysosomes are reformed by recycling lysosomal components from autolysosomes. The most noticeable step of ALR is autolysosome tubulation, but it is currently unknown how the process is regulated. Here, using an approach combining in vivo studies and in vitro reconstitution, we found that the kinesin motor protein KIF5B is required for autolysosome tubulation and that KIF5B drives autolysosome tubulation by pulling on the autolysosomal membrane. Furthermore, we show that KIF5B directly interacts with PtdIns(4,5)P2. Kinesin motors are recruited and clustered on autolysosomes via interaction with PtdIns(4,5)P2 in a clathrin-dependent manner. Finally, we demonstrate that clathrin promotes formation of PtdIns(4,5)P2-enriched microdomains, which are required for clustering of KIF5B. Our study reveals a mechanism by which autolysosome tubulation was generated.

Mechanistic insights into the anchorage of the contractile ring by anillin and Mid1.

Anillins and Mid1 are scaffold proteins that play key roles in anchorage of the contractile ring at the cell equator during cytokinesis in animals and fungi, respectively. Here, we report crystal structures and functional analysis of human anillin and S. pombe Mid1. The combined data show anillin contains a cryptic C2 domain and a Rho-binding domain. Together with the tethering PH domain, three membrane-associating elements synergistically bind to RhoA and phospholipids to anchor anillin at the cleavage furrow. Surprisingly, Mid1 also binds to the membrane through a cryptic C2 domain. Dimerization of Mid1 leads to high affinity and preference for PI(4,5)P2, which stably anchors Mid1 at the division plane, bypassing the requirement for Rho GTPase. These findings uncover the unexpected general machinery and the divergent regulatory logics for the anchorage of the contractile ring through the anillin/Mid1 family proteins from yeast to humans.

Anillin Regulates Neuronal Migration and Neurite Growth by Linking RhoG to the Actin Cytoskeleton.

Neuronal migration and neurite growth are essential events in neural development, but it remains unclear how guidance cues are transduced through receptors to the actin cytoskeleton, which powers these processes. We report that a cytokinetic scaffold protein, Anillin, is redistributed to the leading edge of the C. elegans Q neuroblast during cell migration and neurite growth. To bypass the requirement for Anillin in cytokinesis, we used the somatic CRISPR-Cas9 technique to generate conditional mutations in Anillin. We demonstrate that Anillin regulates cell migration and growth cone extension by stabilizing the F-actin network at the leading edge. Our biochemical analysis shows that the actin-binding domain of Anillin is sufficient to stabilize F-actin by antagonizing the F-actin severing activity of Cofilin. We further uncover that the active form of RhoG/MIG-2 directly binds to Anillin and recruits it to the leading edge. Our results reveal a novel pathway in which Anillin transduces the RhoG signal to the actin cytoskeleton during neuronal migration and neurite growth.

Theranostic protein targeting ErbB2 for bioluminescence imaging and therapy for cancer.

A combination of molecular-targeted cancer imaging and therapy is an emerging strategy to improve cancer diagnosis and minimize the side effects of conventional treatments. Here, we generated a recombinant protein, EC1-GLuc-p53C, by fusing EC1 peptide, an artificial ligand of ErbB2, with Gaussia luciferase (GLuc) and a p53-activating peptide, p53C. EC1-GLuc-p53C was expressed and purified from E. coli BL21. In vitro experiments showed that EC1-GLuc-p53c was stable in luminescent activity and selectively targeted ErbB2-overexpressing BT474 cells for bioluminescence imaging. Moreover, the internalized EC1-GLuc-p53C in BT474 cells exerted its function to reactivate p53 and significantly inhibited cellular proliferation. In tumor-bearing mice, the ErbB2-targeted bioluminescence imaging and therapeutic effect of EC1-GLuc-p53C were also observed specifically in BT474 tumors but not in MCF7 tumors, which does not overexpress ErbB2. Thus, the present study demonstrates EC1-GLuc-p53C to be an effective theranostic reagent targeting ErbB2 for bioluminescence imaging and cancer therapy.

Potential therapeutic efficacy of curcumin in liver cancer.

PURPOSE: Liver cancer, one of the most common cancers in China, is reported to feature relatively high morbidity and mortality. Curcumin (Cum) is considered as a drug possessing anti-angiogenic, anti-inflammation and anti-oxidation effect. Previous research has demonstrated antitumor effects in a series of cancers. MATERIALS AND METHODS: In this study the in vitro cytotoxicity of Cum was measured by MTT assay and pro-apoptotic effects were assessed by DAPI staining and measurement of caspase-3 activity. In vivo anti-hepatoma efficacy of Cum was assessed with HepG2 xenografts. RESULTS: It is found that Cum dose-dependently inhibited cell growth in HepG2 cells with activation of apoptosis. Moreover, Cum delayed the growth of liver cancer in a dose-dependent manner in nude mice. CONCLUSIONS: Cum might be a promising phytomedicine in cancer therapy and further efforts are needed to explore this therapeutic strategy.