Substrate-binding sites of UBR1, the ubiquitin ligase of the N-end rule pathway

Substrates of a ubiquitin-dependent proteolytic system called the N-end rule pathway include proteins with destabilizing N-terminal residues. N-recognins, the pathway’s ubiquitin ligases, contain three substrate-binding sites. The type-1 site is specific for basic N-terminal residues (Arg, Lys, His). The type-2 site is specific for bulky hydrophobic N-terminal residues (Trp, Phe, Tyr, Leu, Ile). We show here that the type-1/2 sites of UBR1, the sole N-recognin of the yeast Saccharomyces cerevisiae, are located in the first ~700 residues of the 1,950-residue UBR1. These sites are distinct in that they can be selectively inactivated by mutations, identified through a genetic screen. Mutations inactivating the type-1 site are in the previously delineated ~70 residue UBR motif characteristic of N-recognins. Fluorescence polarization and surface plasmon resonance were used to determine that UBR1 binds, with Kd of ~1 microM, to either type-1 or type-2 destabilizing N-terminal residues of reporter peptides, but does not bind to a stabilizing N-terminal residue such as Gly. A third substrate-binding site of UBR1 targets an internal degron of CUP9, a transcriptional repressor of peptide import. We show that the previously demonstrated in vivo dependence of CUP9 ubiquitylation on the binding of (cognate) dipeptides to the type-1/2 sites of UBR1 can be reconstituted in a completely defined in vitro system. We also found that purified UBR1 and CUP9 interact nonspecifically, and that specific binding (which involves, in particular, the binding by cognate dipeptides to the UBR1’s type-1/2 sites) can be restored either by a chaperone such as EF1A or through macromolecular crowding

An engineered Tetrahymena tRNA(Gln) for in vivo incorporation of unnatural amino acids into proteins by nonsense suppression

A new tRNA, THG73, has been designed and evaluated as a vehicle for incorporating unnatural amino acids site-specifically into proteins expressed in vivo using the stop codon suppression technique. The construct is a modification of tRNAGln(CUA) from Tetrahymena thermophila, which naturally recognizes the stop codon UAG. Using electrophysiological studies of mutations at several sites of the nicotinic acetylcholine receptor, it is established that THG73 represents a major improvement over previous nonsense suppressors both in terms of efficiency and fidelity of unnatural amino acid incorporation. Compared with a previous tRNA used for in vivo suppression, THG73 is as much as 100-fold less likely to be acylated by endogenous synthetases of the Xenopus oocyte. This effectively eliminates a major concern of the in vivo suppression methodology, the undesirable incorporation of natural amino acids at the suppression site. In addition, THG73 is 4-10-fold more efficient at incorporating unnatural amino acids in the oocyte system. Taken together, these two advances should greatly expand the range of applicability of the in vivo nonsense suppression methodology

An Essential Role of N-Terminal Arginylation in Cardiovascular Development

The enzymatic conjugation of arginine to the N-termini of proteins is a part of the ubiquitin-dependent N-end rule pathway of protein degradation. In mammals, three N-terminal residues—aspartate, glutamate, and cysteine—are substrates for arginylation. The mouseATE1 gene encodes a family of Arg-tRNA-protein transferases (R-transferases) that mediate N-terminal arginylation. We constructed ATE1-lacking mouse strains and found thatATE1 −/− embryos die with defects in heart development and in angiogenic remodeling of the early vascular plexus. Through biochemical analyses, we show that N-terminal cysteine, in contrast to N-terminal aspartate and glutamate, is oxidized before its arginylation by R-transferase, suggesting that the arginylation branch of the N-end rule pathway functions as an oxygen sensor

Selective Inhibition of Type III Secretion Activated Signaling by the Salmonella Effector AvrA

Salmonella enterica utilizes a type III secretion system (TTSS) encoded in its pathogenicity island 1 to mediate its initial interactions with intestinal epithelial cells, which are characterized by the stimulation of actin cytoskeleton reorganization and a profound reprogramming of gene expression. These responses result from the stimulation of Rho-family GTPases and downstream signaling pathways by specific effector proteins delivered by this TTSS. We show here that AvrA, an effector protein of this TTSS, specifically inhibits the Salmonella-induced activation of the JNK pathway through its interaction with MKK7, although it does not interfere with the bacterial infection-induced NF-κB activation. We also show that AvrA is phosphorylated at evolutionary conserved residues by a TTSS-effector-activated ERK pathway. This interplay between effector proteins delivered by the same TTSS highlights the remarkable complexity of these systems

Allosteric Activation of the Ubiquitin ligase UBR 1 by Short Peptides: Molecular Mechanisms and Physiological Functions

The N-end rule relates the in vivo half life of a protein to the identity of its N-terminal residue. UBR1, the E3 of the N-end rule pathway in Sacchnromzyces cerevisiae, targets proteins that bear destabilizing N-terminal residues for Ub-dependent, processive degradation. UBR1 binds protein substrates or dipetides through two distinct sites: the type 1 site, specific for basic residues, and the type 2 site, specific for bulky hydrophobic residues. UBR1 also recognizes an internal degradation signal of the 35 kDa homeodomain protein CUP9, a transcriptional repressor of the di- and tripeptide transporter PTR2. Here I report that the internal degradation signal of CUP9 is recognized by UBR1 through its third, distinct substrate-binding site. Occupation of the type 1 or type 2 sites of UBR1 by dipeptides allosterically stimulates the UBR1-dependent multi-ubiquitylation of CUP9 in an in vitro system, which consists of purified components of the yeast N-end rule pathway. UBR1 is the first E3 shown to be allosterically regulated by small compounds. This regulation underlies, in vivo, the accelerated UBR1-dependent degradation of CUP9 in the presence of dipeptides with destabilizing N-terminal residues. The result is a positive feedback circuit that controls the peptide import in S. cerevisiae. Specifically, the imported dipeptides bind to UBR1 and accelerate the UBR1-dependent degradation of CUP9, thereby derepressing the transcription of PTR2 and increasing the cell's capacity to import peptides. I also describe a new, autoinhibition-based molecular mechanism underlying the activation of UBR1 by dipeptides. UBR1 is an autoinhibited protein, in that the binding of dipeptides to the type 1 and type 2 sites of UBR1 enhances the dissociation of the C-terminal autoinhibitory domain of UBR1 from its substrate-binding N-terminal region. Moreover, this dissociation, which allows the interaction between UBRl and CUP9, is strongly increased only if both type 1 and type 2 sites of UBRl are occupied by dipeptides. An autoinhibitory mechanism discovered in the S. cerevisiae UBRl is likely to recur in metazoan homologs of UBRl, and may also be involved in controlling the activity of other Db-dependent pathways. </p

Study on Doppler characteristics of underwater bistatic reverberation

Active sonar has two typical types of geometry configuration respectively named monostatic and bistatic like a radar system. In the monostatic scenario, co-located underwater transducers transmit and receive sound energy, while in a bistatic situation the transducers are physically separated. Both object detection and identification can be significantly enhanced through utilization of the additional dimension provided by a bistatic geometry. So, more people care about bistatic scattering characteristics of underwater objects as well as bistatic reverberation in recent years. In this paper, Doppler characteristics of bistatic reverberation generated by moving transmitter and receiver are studied. Theoretical formulism for receiving frequency of bistatic reverberation is derived in case of a tone signal being transmitted. Further analysis shows bistatic reverberation is more complicated than monostatic reverberation when Doppler is concerned. In monostatic case, the Doppler frequency shift of reverberation relates to centre frequency of the tone signal, velocity of the transmitter as well as the arriving direction. While in bistatic situation, it varies not only with the above factors, but also the locations, moving directions, velocities of both transmitter and receiver, and also the arriving time of the reverberation, which makes extraction and utilization of Doppler information more difficult

Tensor‐based matched‐field processing applied to the SWellEx‐96 data

Abstract This study proposed a matched field source localization method based on tensor decomposition. By considering the advantages of tensors in multidimensional data processing, a three‐dimensional tensor signal model of space‐time‐frequency is constructed, and the signal subspace is estimated using high‐order singular value decomposition. The source position is estimated by matching the measured data tensor signal subspace with the replica field tensor signal subspace. The S5 event data of SWellEx‐96 is processed by the proposed tensor‐based matched‐field processing (TMFP). The comparison with the results of conventional matched field processing shows that TMFP has a better suppression effect on ambient noise under low SNR and better source localization performance

Testicular Transcriptome of Males and Pseudo-Males Provides Important New Insight into Sex Reversal of <i>Rana dybowskii</i>

Rana dybowskii (R. dybowskii) is an ecological species found in China, Japan, Korea, and Russia. Like most amphibians, R. dybowskii lacks heterotypic sex chromosomes, limiting the in-depth study of sex determination and sex reversal mechanisms. Previous studies have shown that certain environmental factors can modify R. dybowskii genotypic females into phenotypic males, but the mechanism is still unknown. Considering the difficulties in identifying and collecting sex reversal gonads at different stages of differentiation under natural conditions, testes from sexually mature wild adult R. dybowskii were taken in this study, and the genotypic sex of individuals and sex reversal were identified by two male-linked genetic markers reported in our most recent findings. Transcriptome sequencing was performed on testicular tissue from males and pseudo-males, as well as female ovary tissue. The results show that the gene expression patterns of pseudo-males’ testes were similar to those of the males but highly differed from females’ ovaries. One hundred and seventeen differentially expressed genes between testes of pseudo-males and males were found, and the up-regulation of doublesex and mab-3 related transcription factor 1 (Dmrt1) in testes of pseudo-males may play a key role in R. dybowskii sex reversal

Coloclyster of Red Peony Root Granules Alleviates Moderately Severe Acute Pancreatitis: A Double-Blinded, Placebo-Controlled, Randomized Clinical Trial

The red peony root derived from Paeonia lactiflora has been applied to treat human inflammatory diseases. To investigate its therapeutic potential in treating moderately severe acute pancreatitis (MSAP), which has been rarely studied, this study was designed as a double-blinded, placebo-controlled, randomized clinical trial. A total of 60 MSAP patients were enrolled and randomly divided into an experimental (n = 30) group and a control group (n = 30), who received a coloclyster of 15 g of red peony root or placebo granules dissolved in 150 mL of water, respectively. The patients’ demographic and clinical characteristics were recorded. The results showed that the experimental group had a shorter remission time of fever (p<0.05) and abdominal pain (p<0.01) and faster resumption of self-defecation (p<0.01) than did the control group. In addition, the coloclyster of red peony root decreased the modified Balthazar CT score as well as the serum interleukin-6 and tumor necrosis factor-alpha levels to a greater extent than did the placebo coloclyster (p<0.05). The remission times for the normalization of white blood cells and percentage of neutrophils and lymphocytes in the experimental group were also significantly shorter than those in the control group (p<0.05). In conclusion, a coloclyster of red peony root could help alleviate the clinical symptoms and shorten the course of MSAP by possibly attenuating systematic inflammation. This trial is registered with 14004664