Pyrrolysine is the 22nd amino acid. An unresolved question has been how this atypical genetically encoded residue is inserted into proteins, because all previously described naturally occurring aminoacyl-tRNA synthetases are specific for one of the 20 universally distributed amino acids. Here we establish that synthetic L-pyrrolysine is attached as a free molecule to tRNA(CUA) by PylS, an archaeal class II aminoacyl-tRNA synthetase. PylS activates pyrrolysine with ATP and ligates pyrrolysine to tRNA(CUA) in vitro in reactions specific for pyrrolysine. The addition of pyrrolysine to Escherichia coli cells expressing pylT (encoding tRNA(CUA)) and pylS results in the translation of UAG in vivo as a sense codon. This is the first example from nature of direct aminoacylation of a tRNA with a non-canonical amino acid and shows that the genetic code of E. coli can be expanded to include UAG-directed pyrrolysine incorporation into proteins.

Hydrophobicitya nalysesa pplied to databases of soluble andt ransmembrane( TM) proteins of known structure were used to resolvet otal genomic hydrophobicity profiles into (helical) TM sequences and mainly ''subhydrophobic''s oluble components.T his information wasu sed to define ar efined ''hydrophobicity''-typeT Ms equence predictions cale that should approach thet heoretical limit of accuracy.The refinement procedure involved adjusting scale values to eliminate differences between the average amino acid composition of populations TM and solubles equences of equalh ydrophobicity, ar equired property of as cale having maximum accuracy. Application of this procedure to different hydrophobicity scales caused them to collapse to essentiallyasingle TM tendency scale.A se xpected, whend ifferent scalesw ere compared,t he TM tendencys cale wast he most accuratea tp redicting TM sequences. It wasespecially highly correlated ( r ¼ 0.95)tothe biological hydrophobicity scale, derived experimentally from the percentT Mc onformationf ormed by artificial sequencesp assing though the translocon.I tw as also found that resolution of total genomic sequence data into TM ands oluble components could be used to define thep ercent probabilityt hat as equence with as pecific hydrophobicity valuef orms aT Ms egment. Application of theT Mt endencys cale to whole genomic data revealed an overlap of TM andsoluble sequences in the ''semihydrophobic''range. This raises thepossibilityt hat as ignificant number of proteins have sequences that can switch between TM and non-TM states. Such proteins may existinmoonlighting formshavingproperties very different from those of the predominant conformation.

During endoplasmic reticulum-associated degradation, the multifunctional AAA ATPase p97 is part of a protein degradation complex. p97 associates via its N-terminal domain with various cofactors to recruit ubiquitinated substrates. It also interacts with alternative substrate-processing cofactors, such as Ufd2, Ufd3, and peptide:N-glycanase (PNGase) in higher eukaryotes. These cofactors determine different fates of the substrates and they all bind outside of the N-terminal domain of p97. Here, we describe a cofactor-binding motif of p97 contained within the last 10 amino acid residues of the C terminus, which is both necessary and sufficient to mediate interactions of p97 with PNGase and Ufd3. The crystal structure of the N-terminal domain of PNGase in complex with this motif provides detailed insight into the interaction between p97 and its substrate-processing cofactors. Phosphorylation of p97's highly conserved penultimate tyrosine residue, which is the main phosphorylation site during T cell receptor stimulation, completely blocks binding of either PNGase or Ufd3 to p97. This observation suggests that phosphorylation of this residue modulates endoplasmic reticulum-associated protein degradation activity by discharging substrate-processing cofactors.PUB domain ͉ Ufd3 ͉ endoplasmic reticulum-associated protein degradation E ndoplasmic reticulum-associated protein degradation (ERAD) is a major component of the quality control system in the protein secretory pathway. It is responsible for the removal of misfolded proteins and unassembled protein subunits (1, 2). Once (glyco-)proteins are recognized for degradation in the endoplasmic reticulum, they are routed to the cytosol via the putative retrotranslocon, polyubiquitinated, and degraded by the ubiquitin-proteasome system (3). It has been proposed that ERAD substrates are threaded mechanically through the retrotranslocon by the chaperone-like protein p97 (CDC48 in yeast) (4), which is also known as VCP (valosin-containing protein). p97 belongs to the AAA ATPase family of proteins (5), and its sequence is highly conserved in eukaryotes. RNAi and mutagenesis experiments have confirmed the essential role of p97 in the proteasome-mediated degradation of misfolded proteins (6). Structural studies (7) revealed that p97 has an N-terminal domain (N), two AAA ATPase domains (D1 and D2), and a short disordered C-terminal region (C). In addition to its role in ERAD, p97 has been found to participate in other cellular processes, including DNA repair, cell cycle control, and membrane trafficking (4, 8). Structural and biochemical studies have suggested that p97 fulfills its various functions by interacting with a wide spectrum of cofactors (9-12).Most of its cofactors interact with the N-terminal domain of p97, with the exceptions of Ufd2, Ufd3, and peptide:N-glycanase (PNGase), which interact with the C-terminal region of p97 (9, 12, 13). The cofactors can be divided into substrate-recruiting cofactors, which regulate the substrate specificity of p97, and substrate-processi...

Highlights d In yeast, some cell-cycle activators increase in concentration as cells grow larger d Cell-cycle inhibitors decrease in concentration as cells grow larger d These effects can explain why cells divide at increased sizes d Reversal of this natural scaling of expression caused poor cell size control

miRNAs are associated with various types of cancer due to their ability to affect expression of genes that modulate tumorigenesis. In this study, we explored the role of miR-141 in pancreatic cancer. The analysis of clinical characteristics showed that miR-141 was significantly downregulated in tissues and cell lines of pancreatic cancer. Moreover, the decreased miR-141 level was significantly associated with tumor size and TNM stage, as well as lymph node and distant metastasis. Meanwhile, both Kaplan-Meier and multivariate survival analysis showed decreased miR-141 were associated with overall survival. Overexpression of miR-141 in pancreatic cancer cells inhibited cell proliferation, clonogenicity, and invasion; induced G 1 arrest and apoptosis; and enhanced chemosensitivity. To understand how miR-141 mediates the phenotype of pancreatic cancer cells, a bioinformatics tool was used to identify MAP4K4 as a potential target of miR-141. The DualLuciferase reporter gene assay showed that miR-141 binds directly to the 3 0 -untranslated region (3 0 UTR) of MAP4K4 to inhibit MAP4K4 expression. Western blot and quantitative real-time PCR (qRT-PCR) analyses revealed that MAP4K4 expression was inversely correlated with miR-141 expression both in pancreatic cancer samples and cell lines. Knockdown of MAP4K4 inhibited cell proliferation, clonogenicity, and invasion, induced G 1 arrest and apoptosis, and enhanced chemosensitivity. In a nude mouse xenograft model, both overexpression of miR-141 and knockdown of MAP4K4 significantly repressed pancreatic cancer cell growth. Therefore, we conclude that miR-141 targets MAP4K4, acts as a tumor suppressor in pancreatic cancer cells, and may serve as a novel therapeutic agent for miRNA-based pancreatic cancer therapy. Mol Cancer Ther; 12(11); 2569-80. Ó2013 AACR.