Proximal Moiety Research Articles

Ultrasensitive detection of Staphylococcus aureus using a non-fluorescent cDNA-grafted dark BBQ®-650 chromophore integrated hydrophilic upconversion nanoparticles/aptamer system.

A highly structured fluorometric bioassay has been proposed for screening Staphylococcus aureus (S. aureus). The study exploits (i) the spectral attributes of the hexagonal NaYF4:Yb,Er upconversion nanoparticle (UCNP)-coated 3-aminopropyl)triethoxysilane; (ii) the intrinsic non-fluorescent quenching features of the highly stable dark blackberry (BBQ®-650) receptor; (iii) the aptamer (Apt-) biorecognition and binding affinity, and (iv) the complementary DNA hybridizer-linkage efficacy. The principle relied on the excited state energy transfer between the donor Apt-labeled NH2-UCNPs at the 3' end, and cDNA-grafted BBQ®-650 at the 5' end, as the effective receptors. The donor moieties in proximity (< 10.0nm) trigger hybridization with the cDNA-grafted dark BBQ®-650, as the receptors of energy from the 2F5/2 level of Yb3+ ions to initiate the Förster resonance energy transfer pathway. This was confirmed by the decline in the excited-state lifetimes from 223.52μs (τ1) to 179.26μs (τ2). The existence of the target S. aureus in the bioassay attracts the Apt- resulting in the detachment of the acceptor, and disintegration of the complex configuration via conformation reversal. The re-activated fluorescence monitored at λex/em = 980/652nm, as a function of the logarithmic concentration of S. aureus (42 to 4.2 × 108CFUmL-1), yielded an ultra-low detection response of 2.0CFUmL-1. The bioassay screening of S. aureus in real samples revealed satisfactory recoveries (92.44-107.82%) and validation results (p > 0.05). Hence, the comprehensive Apt-labeled NH2-UCNPs-cDNA-grafted dark BBQ®-650 bioassay offered fastand precise S. aureus screening in food and environmental settings.

High-Throughput Assay for Characterizing Rpn11 Deubiquitinase Activity.

Rpn11 is an essential metalloprotease responsible for the en bloc removal of ubiquitin chains from protein substrates that are targeted for degradation by the 26S proteasome. A unique feature of Rpn11 is that its deubiquitinase (DUB) activity is greatly stimulated by the mechanical translocation of the substrate into the proteasomal AAA+ (ATPase Associated with diverse cellular Activities) motor, which delivers the scissile isopeptide bond between a substrate lysine and the proximal moiety of an attached ubiquitin chain to the DUB catalytic active site. As a consequence, Rpn11 cleaves at the base of ubiquitin chains and lacks selectivity towards specific ubiquitin-chain linkage types, which is in contrast to other DUBs, including the related AMSH that selectively cleaves Lys63-linked chains. Prevention of Rpn11's deubiquitinase activity leads to inhibition of proteasomal degradation by stalling substrate translocation. With the proteasome as an approved anticancer target, Rpn11 is therefore an attractive point of attack for the development of new inhibitors, which requires robust biochemical assays to measure DUB activity. Here we describe a method for the purification of the Rpn8/Rpn11 heterodimer and ubiquitin-GC-TAMRA, a model substrate that can be used to characterize the DUB activity of Rpn11 in isolation without the need of purifying 26S proteasomes. This assay thus enables a high-throughput screening platform for Rpn11-targeted small-molecule discovery.

Conformational and functional characterization of artificially conjugated non-canonical ubiquitin dimers

Ubiquitylation is an eminent posttranslational modification referring to the covalent attachment of single ubiquitin molecules or polyubiquitin chains to a target protein dictating the fate of such labeled polypeptide chains. Here, we have biochemically produced artificially Lys11-, and Lys27-, and Lys63-linked ubiquitin dimers based on click-chemistry generating milligram quantities in high purity. We show that the artificial linkage used for the conjugation of two ubiquitin moieties represents a fully reliable surrogate of the natural isopeptide bond by acquiring highly resolved nuclear magnetic resonance (NMR) spectroscopic data including ligand binding studies. Extensive coarse grained and atomistic molecular dynamics (MD) simulations allow to extract structures representing the ensemble of domain-domain conformations used to verify the experimental data. Advantageously, this methodology does not require individual isotopic labeling of both ubiquitin moieties as NMR data have been acquired on the isotopically labeled proximal moiety and complementary MD simulations have been used to fully interpret the experimental data in terms of domain-domain conformation. This combined approach intertwining NMR spectroscopy with MD simulations makes it possible to describe the conformational space non-canonically Lys11-, and Lys27-linked ubiquitin dimers occupy in a solution averaged ensemble by taking atomically resolved information representing all residues in ubiquitin dimers into account.

Diverse fate of ubiquitin chain moieties: The proximal is degraded with the target, and the distal protects the proximal from removal and recycles

One of the enigmas in the ubiquitin (Ub) field is the requirement for a poly-Ub chain as a proteasomal targeting signal. The canonical chain appears to be longer than the distance between the two Ub-binding proteasomal receptors. Furthermore, genetic manipulation has shown that one receptor subunit is sufficient, which suggests that a single Ub can serve as a degradation signal. To shed light on this mystery, we chemically synthesized tetra-Ub, di-Ub (K48-based), and mono-Ub adducts of HA-α-globin, where the distal or proximal Ub moieties were tagged differentially with either Myc or Flag. When incubated in a crude cell extract, the distal Ub moiety in the tetra-Ub adduct was mostly removed by deubiquitinating enzymes (DUBs) and reconjugated to other substrates in the extract. In contrast, the proximal moiety was most likely degraded with the substrate. The efficacy of degradation was proportionate to the chain length; while tetra-Ub globin was an efficient substrate, with mono-Ub globin, we observed rapid removal of the Ub moiety with almost no degradation of the free globin. Taken together, these findings suggest that the proximal moieties are necessary for securing the association of the substrate with the proteasome along the proteolytic process, whereas the distal moieties are important in protecting the proximal moieties from premature deubiquitination. Interestingly, when the same experiment was carried out using purified 26S proteasome, mono- and tetra-Ub globin were similarly degraded, highlighting the roles of the entire repertoire of cellular DUBs in regulating the degradation of proteasomal substrates.

Superstructured metallocorroles for electrochemical CO2 reduction.

Cobalt and iron complexes of corroles with tyrosine-like proton-transfer-relay moieties in proximity to the metal center have been prepared and fully characterized. The (nitrosyl)iron complex performs very well as an electrocatalyst for the reduction of CO2 to CO.

Leucine-responsive regulatory protein Lrp and PapI homologues influence phase variation of CS31A fimbriae.

CS31A, a K88-related surface antigen specified by the clp operon, is a member of the type P family of adhesive factors and plays a key role in the establishment of disease caused by septicemic and enterotoxigenic Escherichia coli strains. Its expression is under the control of methylation-dependent transcriptional regulation, for which the leucine-responsive regulatory protein (Lrp) is essential. CS31A is preferentially in the OFF state and exhibits distinct regulatory features compared to the regulation of other P family members. In the present study, surface plasmon resonance and DNase I protection assays showed that Lrp binds to the distal moiety of the clp regulatory region with low micromolar affinity compared to its binding to the proximal moiety, which exhibits stronger, nanomolar affinity. The complex formation was also influenced by the addition of PapI or FooI, which increased the affinity of Lrp for the clp distal and proximal regions and was required to induce phase variation. The influence of PapI or FooI, however, was predominantly associated with a more complete shutdown of clp expression, in contrast to what has previously been observed with AfaF (a PapI ortholog). Taken together, these results suggest that the preferential OFF state observed in CS31A cells is mainly due to the weak interaction of the leucine-responsive regulatory protein with the clp distal region and that the PapI homolog favors the OFF phase. Within the large repertoire of fimbrial variants in the P family, our study illustrates that having a fimbrial operon that lacks its own PapI ortholog allows it to be more flexibly regulated by other orthologs in the cell.

A new crystal form of Lys48-linked diubiquitin

Lys48-linked polyubiquitin chains are recognized by the proteasome as a tag for the degradation of the attached substrates. Here, a new crystal form of Lys48-linked diubiquitin (Ub2) was obtained and the crystal structure was refined to 1.6 A resolution. The structure reveals an ordered isopeptide bond in a trans configuration. All three molecules in the asymmetric unit were in the same closed conformation, in which the hydrophobic patches of both the distal and the proximal moieties interact with each other. Despite the different crystallization conditions and different crystal packing, the new crystal structure of Ub2 is similar to the previously published structure of diubiquitin, but differences are observed in the conformation of the flexible isopeptide linkage.

Diastereoselective, Synergistic Dual-Mode Optical Switch with Integrated Chirochromic Helicene and Photochromic Bis-azobenzene Moieties

An unprecedented dual-mode optical switch by combining helicene and bis-azobenzene moieties in proximity allows for cooperative, highly diastereoselective chirochromic (92/8 to 3/97) and photochromic (>99/<1 to 19/81) switchings both in solution and in nematic LC with tunable pitches and handedness by permutation of four different metastable states.

The rubella virus E1 glycoprotein is arrested in a novel post-ER, pre-Golgi compartment.

Evidence is accumulating that a distinct compartment(s) exists in the secretory pathway interposed between the rough ER (RER) and the Golgi stack. In this study we have defined a novel post-RER, pre-Golgi compartment where unassembled subunits of rubella virus (RV) E1 glycoprotein accumulate. When RV E1 is expressed in CHO cells in the absence of E2 glycoprotein, transport of E1 to the Golgi complex is arrested. The compartment in which E1 accumulates consists of a tubular network of smooth membranes which is in continuity with the RER but has distinctive properties from either the RER, Golgi, or previously characterized intermediate compartments. It lacks RER and Golgi membrane proteins and is not disrupted by agents which disrupt either the RER (thapsigargin, ionomycin) or Golgi (nocodazole and brefeldin A). However, luminal ER proteins bearing the KDEL signal have access to this compartment. Kinetically the site of E1 arrest lies distal to or at the site where palmitylation occurs and proximal to the low temperature 15 degrees C block. Taken together the findings suggest that the site of E1 arrest corresponds to, or is located close to the exit site from the ER. This compartment could be identified morphologically because it is highly amplified in cells overexpressing unassembled E1 subunits, but it may have its counterpart among the transitional elements of non-transfected cells. We conclude that the site of E1 arrest may represent a new compartment or a differentiated proximal moiety of the intermediate compartment.

The nucleotide sequences of the tail fiber gene 36 of bacteriophage T2 and of genes 36 of the T-even type Escherichia coli phages K3 and Ox2.

Genes 36 have been cloned from phage T2 and the T-even type phages K3 and Ox2. The products of these genes are part of the long tail fibers of the phages, they form the proximal moiety of the distal half fiber. The genes have been sequenced, the nucleotide sequence of gene 36 of phage T4 is known (Oliver, D.B. & Crowther, R.A. (1981) J.Mol.Biol. 153, 545-568). Comparison of the deduced amino acid sequences of the four proteins revealed a surprising pattern. These sequences can be divided into two highly conserved and one very variable region. The former consist of about 60 NH2-terminal and 70 CO2H-terminal residues flanking the variable middle region of about 100 residues. Thus, an identical and unique morphology can be formed by a number of different primary structures. It is proposed that the conserved areas are involved in binding of the proteins to the neighboring products of genes 35 and 37 and that this function has put constraints on the variability of the primary protein structure. The overall amino acid composition of the proteins is rather similar; the codon usage is that known for phage T4. The intercistronic region between genes 35 and 36 consisting of 62 base pairs and containing a presumed terminator for g35 transcription and the 'late type' promoter for transcription of genes 36, 37, and 38, is almost completely identical in the four phages.

Biologically oriented organic sulfur chemistry. IV. Synthesis and properties of 1,2,5-trithiepane, a model for study of sulfide and disulfide moieties in proximity

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTBiologically oriented organic sulfur chemistry. IV. Synthesis and properties of 1,2,5-trithiepane, a model for study of sulfide and disulfide moieties in proximityLamar Field and Charles H. FosterCite this: J. Org. Chem. 1970, 35, 3, 749–752Publication Date (Print):March 1, 1970Publication History Published online1 May 2002Published inissue 1 March 1970https://doi.org/10.1021/jo00828a046RIGHTS & PERMISSIONSArticle Views138Altmetric-Citations29LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (608 KB) Get e-Alerts Get e-Alerts

Abnormalities of the muscular anatomy in the shoulder region of paralysed chick embryos

Chick embryos were paralysed with decamethonium during the time when the wing muscles were being formed by cleavage of the dorsal and ventral premuscle masses of the wing buds. Dosing with the drug was commenced at 5 days of incubation and was continued for periods ranging from 3 days to 1 week. Among the anatomical muscular abnormalities found in the paralysed embryos were various delays or failures of cleavage, a divergence of the proximal moiety of pars scapularis of the triceps away from the humerus, and a tendency for M. coracobrachialis posterior to be split into two portions. Although there were individual differences in details of the anomalies, a generally consistent syndrome of muscular aberrations prevailed throughout the experimental embryos. The results indicate that functional activity is important for the formation of a normal muscular anatomy. The abnormalities associated with paralysis suggest that tensile stresses arising from skeletal growth act in conjunction with those due to neurogenic contractions to influence the orientation of muscle fibres and their grouping to form individual muscles.