Heme oxygenase activates calcium release from the endoplasmic reticulum of bovine mammary epithelial cells to promote TFEB entry into the nucleus to reduce the intracellular load of Mycoplasma bovis.

Heme oxygenase HO-1 (HMOX) regulates cellular inflammation and apoptosis, but its role in regulation of autophagy in Mycoplasma bovis infection is unknown. The objective was to determine how the HO-1/CO- Protein kinase RNA-like endoplasmic reticulum kinase (PERK)-Ca2+- transcription factor EB (TFEB) signaling axis induces autophagy and regulates clearance of M. bovis by bovine mammary epithelial cells (bMECs). M. bovis inhibited autophagy and lysosomal biogenesis in bMECs and suppressed HO-1 protein and expression of related proteins, namely nuclear factor erythroid 2-related factor 2 (Nrf2) and Kelch-like ECH-associated protein 1 (keap1). Activation of HO-1 and its production of carbon monoxide (CO) were required for induction of autophagy and clearance of intracellular M. bovis. Furthermore, when HO-1 was deficient, CO sustained cellular autophagy. HO-1 activation increased intracellular calcium (Ca2+) and cytosolic localization activity of TFEB via PERK. Knockdown of PERK or chelation of intracellular Ca2+ inhibited HO-1-induced M. bovis autophagy and clearance. M. bovis infection affected nuclear localization of lysosomal TFEB in the MiT/TFE transcription factor subfamily, whereas activation of HO-1 mediated dephosphorylation and intranuclear localization of TFEB, promoting autophagy, lysosomal biogenesis and autophagic clearance of M. bovis. Nuclear translocation of TFEB in HO-1 was critical to induce M. bovis transport and survival of infected bMECs. Furthermore, the HO-1/CO-PERK-Ca2+-TFEB signaling axis induced autophagy and M. bovis clearance, providing a viable approach to treat persistent M. bovis infections.

Metabolite-based genome-wide association studies enable the dissection of the genetic bases of flavonoids, betaine and spermidine in wolfberry (Lycium).

Wolfberry is a plant with medicinal and food values. However, its bioactive ingredients and the corresponding genetic bases have not been determined. Here, we de novo generated a chromosome-level genome assembly for wolfberry, yielding a genome sequence of ~1.77 Gb with contig N50 of 50.55 Mb and 39 224 predicted gene models. A variation map, using 307 re-sequenced accessions, was called based on this genome assembly. Furthermore, the fruit metabolome of these accessions was profiled using 563 annotated metabolites, which separated Lycium barbarum L. and non-L. barbarum L. The flavonoids, coumarins, alkaloids and nicotinic acid contents were higher in the former than in the latter. A metabolite-based genome-wide association study mapped 156 164 significant single nucleotide polymorphisms corresponding to 340 metabolites. This included 19 219 unique lead single nucleotide polymorphisms in 1517 significant association loci, of which three metabolites, flavonoids, betaine and spermidine, were highlighted. Two candidate genes, LbUGT (evm.TU.chr07.2692) and LbCHS (evm.TU.chr07.2738), with non-synonymous mutations, were associated with the flavonoids content. LbCHS is a structural gene that interacts with a nearby MYB transcription factor (evm.TU.chr07.2726) both in L. barbarum and L. ruthenicum. Thus, these three genes might be involved in the biosynthesis/metabolism of flavonoids. LbSSADH (evm.TU.chr09.627) was identified as possibly participating in betaine biosynthesis/metabolism. Four lycibarbarspermidines (E-G and O) were identified, and only the lycibarbarspermidines O content was higher in L. barbarum varieties than in non-L. barbarum varieties. The evm.TU.chr07.2680 gene associated with lycibarbarspermidines O was annotated as an acetyl-CoA-benzylalcohol acetyltransferase, suggesting that it is a candidate gene for spermidine biosynthesis. These results provide novel insights into the specific metabolite profile of non-L. barbarum L. and the genetic bases of flavonoids, betaine and spermidine biosynthesis/metabolism.

Catalpol inhibits the proliferation, migration and metastasis of HCC cells by regulating miR­140­5p expression.

Hepatocellular carcinoma (HCC) is a frequent malignant tumor. Catalpol is a Chinese medicine extract with a number of pharmacologically active properties. The present study aimed to investigate the effects and mechanisms of catalpol in HCC. HCC cells were treated with catalpol in the presence or absence of microRNA (miR)­140­5p inhibitor, and assays to determine cell viability, proliferation, invasion and migration were performed. Reverse transcription­quantitative PCR and western blotting were performed to determine the mRNA and protein expression levels of miR­140­5p, vimentin, N­Cadherin and E­Cadherin. Moreover, cells were treated with catalpol in the absence or presence of transforming growth factor (TGF)­ß1, and the cell morphology was observed under a microscope. The results demonstrated that catalpol inhibited cell proliferation, invasion and migration, and decreased the expression levels of vimentin and N­cadherin, but increased the expression levels of E­cadherin and miR­140­5p. Catalpol inhibited morphological changes in epithelial­mesenchymal transformation (EMT) of cells induced by TGF­ß1. Following inhibition of miR­140­5p expression, the proliferation, invasion and migration of HCC cells were promoted, E­cadherin expression was decreased, and the levels of vimentin and N­cadherin were increased. The miR­140­5p inhibitor effectively reversed the inhibitory effect of catalpol on cell proliferation, invasion and migration. Thus, the results suggested that the antitumor potential of catalpol in HCC may be exerted by regulating the expression of miR­140­5p to inhibit proliferation, invasion, migration and EMT of HCC cells.

An RNF11: Smurf2 complex mediates ubiquitination of the AMSH protein.

RING-finger proteins play crucial roles in ubiquitination events involved in diverse cellular processes including signal transduction, differentiation and apoptosis. Most of the RING-finger proteins have E3-ubiquitin ligase activity. RNF11 is a small RING-finger protein and harbors a RING-H2 domain and a PY motif that could facilitate protein:protein interaction(s) involved in oncogenesis. To isolate RNF11 protein partners and determine its role in normal and cancer cells, we performed yeast two-hybrid screening. Among 18 in-frame positive clones, three were found to be ZBRK1, Eps15 and AMSH (associated molecule with the SH3 domain of STAM). ZBRK1 is a KRAB domain containing Zinc-finger protein and is known to repress target gene transcription in a BRCA1-dependent manner. Eps15 is monoubiquitinated and is part of an essential complex involved in the endocytosis of plasma membrane receptors via the clathrin-mediated internalization pathway. Recent studies have shown that AMSH protein is involved in BMP/TGF-beta signaling pathway by binding to Smad6 and Smad7. The association of RNF11 with these binding partners suggests that it would be involved in biological processes such as gene transcription, BMP/TGF-beta signaling and ubiquitination-associated events. Previously, we have shown that RNF11 interacts with the HECT-type E3 ligases AIP4 and Smurf2. Here, we show that RNF11 binds to AMSH in mammalian cells and that this interaction is independent of the RNF11 RING-finger domain and the PY motif. Our results also demonstrate that AMSH is ubiquitinated by Smurf2 E3 ligase in the presence of RNF11 and that a consequent reduction in its steady-state level requires both RNF11 and Smurf2. RNF11 therefore recruits AMSH to Smurf2 for ubiquitination, leading to its degradation by the 26S proteasome. The potential functions of RNF11-mediated degradation of AMSH in breast cancer are discussed.

Characterization of a novel human breast cancer associated gene (BCA3) encoding an alternatively spliced proline-rich protein.

As part of an integrated study of breast cancer gene expression, partial cDNAs were cloned from normal and tumor breast cells by subtractive-hybridization and differential display cloning. The DNA sequence for one of these breast cancer associated genes was used to construct the larger 1319 bp BCA3 cDNA sequence using ESTs without assigned names or functions. High-level BCA3 mRNA expression was found in breast and prostate tumor cell lines whereas normal breast and prostate tissues have low-level expression. Further analysis revealed possible functional domains and alternative splicing of BCA3 that we confirmed by RT-PCR analysis. Immunohistochemistry revealed that the protein is expressed in breast tumor cells in vivo, and not in surrounding stromal tissue.