Pharmacogenetics and Gender Association with Psychotic Episodes on Nortriptyline Lower Doses: Patient Cases

The variation in individual responses to psychotropic drug treatment remains a critical problem in the management of psychotic disorders. Although most patients will experience remission, some patients may develop drug-induced adverse effects that may range from troublesome to life threatening. Antidepressants are freely prescribed by general practitioners, and there should be constant awareness in the medical community about possible serious side effects. We describe two cases of adverse drug reactions on low dosage treatment that led to extreme psychotic episodes as examples of the potential for dangerous side effects. The patients developed adverse reactions on the normal recommended dosage of nortriptyline, a tricyclics antidepressant (TCA). Both were females, with no history of antidepressant treatment, unsocial behaviour, nor any family history of psychosis, but both experienced severe psychiatric symptoms. Pharmacogenetic tests can easily be performed and interpreted according to the likelihood of adverse reactions and should be included in toxicity interpretation

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

Ubiquitin Reference Technique and Its Use in Ubiquitin-Lacking Prokaryotes

In a pulse-chase assay, the in vivo degradation of a protein is measured through a brief labeling of cells with, for example, a radioactive amino acid, followed by cessation of labeling and analysis of cell extracts prepared at different times afterward (“chase”), using immunoprecipitation, electrophoresis and autoradiography of a labeled protein of interest. A conventional pulse-chase assay is fraught with sources of data scatter, as the efficacy of labeling and immunoprecipitation can vary, and sample volumes can vary as well. The ubiquitin reference technique (URT), introduced in 1996, addresses these problems. In eukaryotes, a DNA-encoded linear fusion of ubiquitin to another protein is cleaved by deubiquitylases at the ubiquitin-protein junction. A URT assay uses a fusion in which the ubiquitin moiety is located between a downstream polypeptide (test protein) and an upstream polypeptide (a long-lived reference protein). The cotranslational cleavage of a URT fusion by deubiquitylases after the last residue of ubiquitin produces, at the initially equimolar ratio, a test protein with a desired N-terminal residue and a reference protein containing C-terminal ubiquitin moiety. In addition to being more accurate than pulse-chases without a reference, URT makes it possible to detect and measure the degradation of a test protein during the pulse (before the chase). Because prokaryotes, including Gram-negative bacteria such as, for example, Escherichia coli and Vibrio vulnificus, lack the ubiquitin system, the use of URT in such cells requires ectopic expression of a deubiquitylase. We describe designs and applications of plasmid vectors that coexpress, in bacteria, both a URT-type fusion and Ubp1, a deubiquitylase of the yeast Saccharomyces cerevisiae. This single-plasmid approach extends the accuracy-enhancing URT assay to studies of protein degradation in prokaryotes

Neurodegeneration-Associated Protein Fragments as Short-Lived Substrates of the N-End Rule Pathway

Protein aggregates are a common feature of neurodegenerative syndromes. Specific protein fragments were found to be aggregated in disorders including Alzheimer’s disease, amyotrophic lateral sclerosis, and Parkinson’s disease. Here, we show that the natural C-terminal fragments of Tau, TDP43, and α-synuclein are short-lived substrates of the Arg/N-end rule pathway, a processive proteolytic system that targets proteins bearing “destabilizing” N-terminal residues. Furthermore, a natural TDP43 fragment is shown to be metabolically stabilized in Ate1−/− fibroblasts that lack the arginylation branch of the Arg/N-end rule pathway, leading to accumulation and aggregation of this fragment. We also found that a fraction of Aβ42, the Alzheimer’s disease-associated fragment of APP, is N-terminally arginylated in the brains of 5xFAD mice and is degraded by the Arg/N-end rule pathway. The discovery that neurodegeneration-associated natural fragments of TDP43, Tau, α-synuclein, and APP can be selectively destroyed by the Arg/N-end rule pathway suggests that this pathway counteracts neurodegeneration

Отголоски язычества в декоре уральской избы

Samples of a wooden carving of the XIX–XX centuries on Central Ural Mountains comprise basic symbolics of mythology of ancient Slavs. In the traditional dwelling elements of a mythological picture of the world are obviously traced. The myth represents model of a macrocosmos and the microcosm entered in it — the person. The traditional dwelling both in practical, and in symbolical aspect personifies the same relations. The mythological symbolics used as a decor had to fix, on the one hand, symbolical value of the house as center, Space as vital space of a sort, family, separately taken person. On the other hand, a number of symbols had to indicate specific preferences of some certain values of life of the owner of the house which, in turn, were caused by practical activity.Образцы деревянной резьбы XIX–XX веков на Урале представляют собой основную символику мифологии древних славян. Наиболее явно рудименты языческих культов были представлены в традиционном оформлении крестьянской избы. В избе прослеживаются элементы мифологической картины мира. Дом рассматривается и как центр пространства, и как элемент мироздания. Отсюда идет использование оберегающих знаков, защищающих проживающих в избе от разного вмешательства, идущего из внешнего мира

Analyzing N-terminal Arginylation Through the Use of Peptide Arrays and Degradation Assays

Nα-terminal arginylation (Nt-arginylation) of proteins is mediated by the Ate1 arginyltransferase (R-transferase), a component of the Arg/N-end rule pathway. This proteolytic system recognizes proteins containing N terminal degradation signals called N-degrons, polyubiquitylates these proteins, and thereby causes their degradation by the proteasome. The definitively identified (canonical) residues that are Nt-arginylated by R-transferase are N-terminal Asp, Glu and (oxidized) Cys. Over the last decade, several publications suggested (i) that Ate1 can also arginylate non-canonical N-terminal residues; (ii) that Ate1 is capable of arginylating not only alpha-amino groups of N-terminal residues but also gamma-carboxyl groups of internal (non-N-terminal) Asp and Glu; and (iii) that some isoforms of Ate1 are specific for substrates bearing N-terminal Cys residues. In the present study, we employed arrays of immobilized 11-residue peptides and pulse-chase assays to examine the substrate specificity of mouse R-transferase. We show that amino acid sequences immediately downstream of canonical (Nt-arginylatable) N-terminal residue of a substrate, particularly a residue at position 2, can affect the rate of Nt-arginylation by R-transferase and thereby the rate of degradation of a substrate protein. We also show that the four major isoforms of mouse R transferase have similar Nt-arginylation specificities in vitro, contrary to the claim about specificity of some Ate1 isoforms for N terminal Cys. In addition, we found no evidence for a significant activity of the Ate1 R-transferase toward previously invoked non-canonical N-terminal or internal amino acid residues. Together, our results raise technical concerns about earlier studies that invoked non-canonical arginylation specificities of Ate1

Glutamine-Specific N-Terminal Amidase, a Component of the N-End Rule Pathway

Deamidation of N-terminal Gln by Nt^Q-amidase, an N-terminal amidohydrolase, is a part of the N-end rule pathway of protein degradation. We detected the activity of Nt^Q-amidase, termed Ntaq1, in mouse tissues, purified Ntaq1 from bovine brains, identified its gene, and began analyzing this enzyme. Ntaq1 is highly conserved among animals, plants, and some fungi, but its sequence is dissimilar to sequences of other amidases. An earlier mutant in the Drosophila Cg8253 gene that we show here to encode Nt^Q-amidase has defective long-term memory. Other studies identified protein ligands of the uncharacterized human C8orf32 protein that we show here to be the Ntaq1 Nt^Q-amidase. Remarkably, “high-throughput” studies have recently solved the crystal structure of C8orf32 (Ntaq1). Our site-directed mutagenesis of Ntaq1 and its crystal structure indicate that the active site and catalytic mechanism of Nt^Q-amidase are similar to those of transglutaminases