72 research outputs found
Genetic recoding to dissect the roles of site-specific protein O-GlcNAcylation
ISSN:1559-744XISSN:1559-743
The leading role of West Siberian Research and Geological Oil Exploration Institute in the development of the oil and gas potential of the West Siberian oil and gas province and the development of the country’s mineral resource base
The history of creation is given, the complex of studies carried out by the Tyumen branch of Siberian Research Institute of Geology, Geophysics and Mineral Resources (SNIIGGiMS) in 1960–1964 and by West Siberian Research and Geological Prospecting Oil Institute (ZapSibNIGNI) in 1964–1996 is analyzed. The role of the Institute in the substantiation of oil and gas potential and developing the resource base of the West Siberian oil and gas province is shown. The scientific forecasts and developments of the Institute’s employees, which influenced the increase in the efficiency of geological exploration are presented. In preparing the article, previously unpublished documents from the archive of the Directorate of ZapSibNIGNI were used
O-GlcNAcase Fragment Discovery with Fluorescence Polarimetry
The
attachment of the sugar N-acetyl-D-glucosamine (GlcNAc) to
specific serine and threonine residues on proteins is referred to
as protein O-GlcNAcylation. O-GlcNAc transferase (OGT) is the enzyme
responsible for carrying out the modification, while O-GlcNAcase (OGA)
reverses it. Protein O-GlcNAcylation has been implicated in a wide
range of cellular processes including transcription, proteostasis,
and stress response. Dysregulation of O-GlcNAc has been linked to
diabetes, cancer, and neurodegenerative and cardiovascular disease.
OGA has been proposed to be a drug target for the treatment of Alzheimer’s
and cardiovascular disease given that increased O-GlcNAc levels appear
to exert a protective effect. The search for specific, potent, and
drug-like OGA inhibitors with bioavailability in the brain is therefore
a field of active research, requiring orthogonal high-throughput assay
platforms. Here, we describe the synthesis of a novel probe for use
in a fluorescence polarization based assay for the discovery of inhibitors
of OGA. We show that the probe is suitable for use with both human
OGA, as well as the orthologous bacterial counterpart from <i>Clostridium perfringens</i>, <i>Cp</i>OGA, and the
lysosomal hexosaminidases HexA/B. We structurally characterize <i>Cp</i>OGA in complex with a ligand identified from a fragment
library screen using this assay. The versatile synthesis procedure
could be adapted for making fluorescent probes for the assay of other
glycoside hydrolases
Proteolysis of HCF-1 by Ser/Thr glycosylation-incompetent O-GlcNAc transferase:UDP-GlcNAc complexes
In complex with the cosubstrate UDP-N-acetylglucosamine (UDP-GlcNAc),O-linked-GlcNAc transferase (OGT) catalyzes Ser/ThrO-GlcNAcylation of many cellular proteins and proteolysis of the transcriptional coregulator HCF-1. Such a dual glycosyltransferase-protease activity, which occurs in the same active site, is unprecedented and integrates both reversible and irreversible forms of protein post-translational modification within one enzyme. Although occurring within the same active site, we show here that glycosylation and proteolysis occur through separable mechanisms. OGT consists of tetratricopeptide repeat (TPR) and catalytic domains, which, together with UDP-GlcNAc, are required for both glycosylation and proteolysis. Nevertheless, a specific TPR domain contact with the HCF-1 substrate is critical for proteolysis but not Ser/Thr glycosylation. In contrast, key catalytic domain residues and even a UDP-GlcNAc oxygen important for Ser/Thr glycosylation are irrelevant for proteolysis. Thus, from a dual glycosyltransferase-protease, essentially single-activity enzymes can be engineered both in vitro and in vivo. Curiously, whereas OGT-mediated HCF-1 proteolysis is limited to vertebrate species, invertebrate OGTs can cleave human HCF-1. We present a model for the evolution of HCF-1 proteolysis by OGT
GlcNAcstatin:a picomolar, selective O-GlcNAcase inhibitor that modulates intracellular O-glcNAcylation levels
The conserved threonine-rich region of the HCF-1PRO repeat activates promiscuous OGT:UDP-GlcNAc glycosylation and proteolysis activities
O-Linked GlcNAc transferase (OGT) possesses dual glycosyltransferase-protease activities. OGT thereby stably glycosylates serines and threonines of numerous proteins and, via a transient glutamate glycosylation, cleaves a single known substrate-the so-called HCF-1 <sub>PRO</sub> repeat of the transcriptional co-regulator host-cell factor 1 (HCF-1). Here, we probed the relationship between these distinct glycosylation and proteolytic activities. For proteolysis, the HCF-1 <sub>PRO</sub> repeat possesses an important extended threonine-rich region that is tightly bound by the OGT tetratricopeptide-repeat (TPR) region. We report that linkage of this HCF-1 <sub>PRO</sub> -repeat, threonine-rich region to heterologous substrate sequences also potentiates robust serine glycosylation with the otherwise poor R <sub>p</sub> -αS-UDP-GlcNAc diastereomer phosphorothioate and UDP-5S-GlcNAc OGT co-substrates. Furthermore, it potentiated proteolysis of a non-HCF-1 <sub>PRO</sub> -repeat cleavage sequence, provided it contained an appropriately positioned glutamate residue. Using serine- or glutamate-containing HCF-1 <sub>PRO</sub> -repeat sequences, we show that proposed OGT-based or UDP-GlcNAc-based serine-acceptor residue activation mechanisms can be circumvented independently, but not when disrupted together. In contrast, disruption of both proposed activation mechanisms even in combination did not inhibit OGT-mediated proteolysis. These results reveal a multiplicity of OGT glycosylation strategies, some leading to proteolysis, which could be targets of alternative molecular regulatory strategies
Thio-linked UDP-peptide conjugates as O-GlcNAc transferase inhibitors
O-GlcNAc
transferase (OGT) is an essential glycosyltransferase
that installs the O-GlcNAc post-translational modification on the
nucleocytoplasmic proteome. We report the development of S-linked
UDP–peptide conjugates as potent bisubstrate OGT inhibitors.
These compounds were assembled in a modular fashion by photoinitiated
thiol–ene conjugation of allyl-UDP and optimal acceptor peptides
in which the acceptor serine was replaced with cysteine. The conjugate
VTPVC(S-propyl-UDP)TA (<i>K</i><sub>i</sub> = 1.3 μM)
inhibits the OGT activity in HeLa cell lysates. Linear fusions of
this conjugate with cell penetrating peptides were explored as prototypes
of cell-penetrant OGT inhibitors. A crystal structure of human OGT
with the inhibitor revealed mimicry of the interactions seen in the
pseudo-Michaelis complex. Furthermore, a fluorophore-tagged derivative
of the inhibitor works as a high affinity probe in a fluorescence
polarimetry hOGT assay
A structural and biochemical model of processive chitin synthesis
Chitin synthases (CHS) produce chitin, an essential component of the fungal cell wall. The molecular mechanism of processive chitin synthesis is not understood, limiting the discovery of new inhibitors of this enzyme class. We identified the bacterial glycosyltransferase NodC as an appropriate model system to study the general structure and reaction mechanism of CHS. A high throughput screening-compatible novel assay demonstrates that a known inhibitor of fungal CHS also inhibit NodC. A structural model of NodC, on the basis of the recently published BcsA cellulose synthase structure, enabled probing of the catalytic mechanism by mutagenesis, demonstrating the essential roles of the DD and QXXRW catalytic motifs. The NodC membrane topology was mapped, validating the structural model. Together, these approaches give insight into the CHS structure and mechanism and provide a platform for the discovery of inhibitors for this antifungal target
Supplementary data for the article: Uraev, A. I.; Nefedov, S. E.; Lyssenko, K. A.; Vlasenko, V. G.; Ikorskii, V. N.; Garnovskii, D. A.; Makarova, N. I.; Levchenkov, S. I.; Shcherbakov, I. N.; Milenković, M. R.; Borodkin, G. S. Synthesis, Structure, Spectroscopic Studies and Magnetic Properties of Cu2N2O4-, Cu2N2O2(S2)-, Cu2N2S4-Chromophores Based on Aminomethylene Derivatives of Pyrazole-5-One(Thione). Polyhedron 2020, 188, 114623. https://doi.org/10.1016/j.poly.2020.114623
Supplementary material for: [https://doi.org/10.1016/j.poly.2020.114623]Related to published version: [https://cherry.chem.bg.ac.rs/handle/123456789/4221
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