Crystal structure of the DENR-MCT-1 complex revealed zinc-binding site essential for heterodimer formation.

The density-regulated protein (DENR) and the malignant T cell-amplified sequence 1 (MCT-1/MCTS1) oncoprotein support noncanonical translation initiation, promote translation reinitiation on a specific set of mRNAs with short upstream reading frames, and regulate ribosome recycling. DENR and MCT-1 form a heterodimer, which binds to the ribosome. We determined the crystal structure of the heterodimer formed by human MCT-1 and the N-terminal domain of DENR at 2.0-Å resolution. The structure of the heterodimer reveals atomic details of the mechanism of DENR and MCT-1 interaction. Four conserved cysteine residues of DENR (C34, C37, C44, C53) form a classical tetrahedral zinc ion-binding site, which preserves the structure of the DENR's MCT-1-binding interface that is essential for the dimerization. Substitution of all four cysteines by alanine abolished a heterodimer formation. Our findings elucidate further the mechanism of regulation of DENR-MCT-1 activities in unconventional translation initiation, reinitiation, and recycling.

Supplementation of enzyme-treated soy protein saves dietary protein and promotes digestive and absorptive ability referring to TOR signaling in juvenile fish.

This study was conducted to evaluate the effect of enzyme-treated soy protein (ETSP) supplementation in the low-protein diet on growth performance, digestive and absorptive capacities, and related signaling molecules' gene expressions in juvenile Jian carp. The results showed that percent weight gain (PWG), specific growth rate (SGR), and feed intake (FI) were decreased by reducing dietary protein from 34 to 32% (P < 0.05). Supplying low-protein diet with optimal ETSP increased previously mentioned indices of juvenile Jian carp (P < 0.05), which also had no significant difference with the high-protein diet (34%CP) (P > 0.05). Compared with the low-protein diet, appropriate ETSP supplementation in the low-protein diet increased (P < 0.05) (1) the trypsin, lipase, and amylase activities in the hepatopancreas; (2) cholecystokinin concentration in the proximal intestine; (3) the γ-glutamyl transpeptidase (γ-GT), alkaline phosphatase (AKP), and Na+/K+-ATPase activities in all intestinal segments; and (4) the messenger RNA (mRNA) levels of trypsin, lipase, and amylase in hepatopancreas and γ-GT in the mid (MI) and distal (DI) intestine, alkaline phosphatase in MI, and Na+/K+-ATPase and target of rapamycin in all intestinal segments. At the same time, appropriate ETSP supplementation in the low-protein diet downregulated the mRNA levels of AKP in the DI and eIF4E-binding protein 2 in all intestinal segments (P < 0.05). In conclusion, adding 10 g ETSP/kg diet in the low-protein diet can restore the growth performance and digestive and absorptive abilities to the levels in group with 34% dietary protein. Supplementation of optimal ETSP in the low-protein diet enhanced the digestive and absorptive abilities and regulated the signaling molecules related to the TOR signaling pathway.

L-Arginine promotes protein synthesis and cell growth in brown adipocyte precursor cells via the mTOR signal pathway.

L-Arginine has been reported to enhance brown adipose tissue developments in fetal lambs of obese ewes, but the underlying mechanism is unknown. The present study tested the hypothesis that L-arginine stimulates growth and development of brown adipocyte precursor cells (BAPCs) through activation of mammalian target of rapamycin cell signaling. BAPCs isolated from fetal lambs at day 90 of gestation were incubated   for 6 h in arginine-free DMEM, and then cultured in DMEM with concentrations of 50, 100, 200, 500 or 1000 µmol L-arginine/L for 24-96 h. Cell proliferation, protein turnover, the mammalian target of rapamycin (mTOR) signaling pathway and pre-adipocyte differentiation markers were determined. L-arginine treatment enhanced (P < 0.05) BAPC growth and protein synthesis, while inhibiting proteolysis in a dose-dependent manner. Compared with 50 and 100 µmol/L (the concentrations of arginine in the maternal plasma of obese ewes), 200 µmol L-arginine/L (the concentrations of arginine in the maternal plasma of obese ewes receiving arginine supplementation) increased (P < 0.05) the abundances of phosphorylated mTOR, P70S6K and 4EBP1, as well as the abundances of PGC1α, UCP1, BMP7 and PRDM16. These novel findings indicate that increasing extra-cellular arginine concentration from 50 to 200 µmol/L activates mTOR cell signaling in BAPCs and enhances their growth and development in a dose-dependent manner. Our results provide a mechanism for arginine supplementation to enhance the development of brown adipose tissue in fetal lambs.

Possible involvement of eEF1A in Tomato spotted wilt virus RNA synthesis.

Tomato spotted wilt virus (TSWV) is a negative-strand RNA virus in the family Bunyaviridae and propagates in both insects and plants. Although TSWV can infect a wide range of plant species, host factors involved in viral RNA synthesis of TSWV in plants have not been characterized. In this report, we demonstrate that the cell-free extract derived from one of the host plants can activate mRNA transcriptional activity of TSWV. Based on activity-guided fractionation of the cell-free extract, we identified eukaryotic elongation factor (eEF) 1A as a possible host factor facilitating TSWV transcription and replication. The RNA synthesis-supporting activity decreased in the presence of an eEF1A inhibitor, suggesting that eEF1A plays an important role in RNA synthesis of TSWV.

An alternative splicing isoform of eukaryotic initiation factor 4H promotes tumorigenesis in vivo and is a potential therapeutic target for human cancer.

Deregulation of protein synthesis plays a critical role in cell transformation. Several translation initiation factors (eIFs) have been implicated in malignant transformation; thus, suppression of eIFs could be a potential cancer therapy if cancer cells are selectively killed without damaging healthy cells. One of the potential molecular targets is a cancer-specific splicing variant. We have previously shown that one of the splicing variants of eIF4H (isoform 1) was overexpressed in primary human colorectal cancer. Our study aimed to explore whether eIF4H isoform 1 contributes to carcinogenesis and could be an efficient molecular target for human cancer therapy. We found that its overexpression in immortalized mouse fibroblasts, NIH3T3 cells, generated tumors in nude mice. Conversely, suppression of eIF4H isoform 1 expression using specific siRNA inhibited the proliferation of colon cancer cells in vitro and subcutaneously implanted tumor in vivo. Strikingly, eIF4H isoform 1 specific siRNA showed no effect on the growth of immortalized human fibroblasts. More interestingly, ectopic expression of eIF4H isoform 1 greatly increased the cyclin D1 level. On the other hand, cyclin D1 decreased by shRNA-mediated suppression of eIF4H isoform 1. Moreover, cotransfection of eIF4H isoform 1 siRNA and cyclin D1 expression plasmid was able to reverse the growth suppression effect of eIF4H isoform 1 knockdown. These results suggest that eIF4H isoform 1 plays an important role in carcinogenesis through the activation of oncogenic signaling and could be a promising molecular target for cancer therapy.