scholarly journals Sorafenib as an Inhibitor of RUVBL2

Biomolecules ◽  
2020 ◽  
Vol 10 (4) ◽  
pp. 605 ◽  
Author(s):  
Nardin Nano ◽  
Francisca Ugwu ◽  
Thiago V. Seraphim ◽  
Tangzhi Li ◽  
Gina Azer ◽  
...  
Keyword(s):  
Atpase Activity ◽  
Competitive Inhibitor ◽  
Telomerase Activity ◽  
Size Exclusion ◽  
Scattering Data ◽  
Cellular Processes ◽  
Dna Damage Signaling ◽  
X Ray Scattering ◽  
Exclusion Chromatography ◽  
Aaa Atpases

RUVBL1 and RUVBL2 are highly conserved ATPases that belong to the AAA+ (ATPases Associated with various cellular Activities) superfamily and are involved in various complexes and cellular processes, several of which are closely linked to oncogenesis. The proteins were implicated in DNA damage signaling and repair, chromatin remodeling, telomerase activity, and in modulating the transcriptional activities of proto-oncogenes such as c-Myc and β-catenin. Moreover, both proteins were found to be overexpressed in several different types of cancers such as breast, lung, kidney, bladder, and leukemia. Given their various roles and strong involvement in carcinogenesis, the RUVBL proteins are considered to be novel targets for the discovery and development of therapeutic cancer drugs. Here, we describe the identification of sorafenib as a novel inhibitor of the ATPase activity of human RUVBL2. Enzyme kinetics and surface plasmon resonance experiments revealed that sorafenib is a weak, mixed non-competitive inhibitor of the protein’s ATPase activity. Size exclusion chromatography and small angle X-ray scattering data indicated that the interaction of sorafenib with RUVBL2 does not cause a significant effect on the solution conformation of the protein; however, the data suggested that the effect of sorafenib on RUVBL2 activity is mediated by the insertion domain in the protein. Sorafenib also inhibited the ATPase activity of the RUVBL1/2 complex. Hence, we propose that sorafenib could be further optimized to be a potent inhibitor of the RUVBL proteins.

scholarly journals Size-exclusion chromatography small-angle X-ray scattering of water soluble proteins on a laboratory instrument

2018 ◽  
Vol 51 (6) ◽  
pp. 1623-1632 ◽  
Author(s):  
Saskia Bucciarelli ◽  
Søren Roi Midtgaard ◽  
Martin Nors Pedersen ◽  
Søren Skou ◽  
Lise Arleth ◽  
...  
Keyword(s):  
Size Exclusion Chromatography ◽  
Small Angle ◽  
Size Exclusion ◽  
Scattering Data ◽  
X Ray ◽  
Laboratory Instrument ◽  
X Ray Scattering ◽  
Home Institution ◽  
Exclusion Chromatography ◽  
Ray Scattering

Coupling of size-exclusion chromatography with biological solution small-angle X-ray scattering (SEC-SAXS) on dedicated synchrotron beamlines enables structural analysis of challenging samples such as labile proteins and low-affinity complexes. For this reason, the approach has gained increased popularity during the past decade. Transportation of perishable samples to synchrotrons might, however, compromise the experiments, and the limited availability of synchrotron beamtime renders iterative sample optimization tedious and lengthy. Here, the successful setup of laboratory-based SEC-SAXS is described in a proof-of-concept study. It is demonstrated that sufficient quality data can be obtained on a laboratory instrument with small sample consumption, comparable to typical synchrotron SEC-SAXS demands. UV/vis measurements directly on the SAXS exposure cell ensure accurate concentration determination, crucial for direct molecular weight determination from the scattering data. The absence of radiation damage implies that the sample can be fractionated and subjected to complementary analysis available at the home institution after SEC-SAXS. Laboratory-based SEC-SAXS opens the field for analysis of biological samples at the home institution, thus increasing productivity of biostructural research. It may further ensure that synchrotron beamtime is used primarily for the most suitable and optimized samples.

scholarly journals Solution structures of long-acting insulin analogues and their complexes with albumin

2019 ◽  
Vol 75 (3) ◽  
pp. 272-282 ◽  
Author(s):  
Line A. Ryberg ◽  
Pernille Sønderby ◽  
Fabian Barrientos ◽  
Jens T. Bukrinski ◽  
Günther H. J. Peters ◽  
...  
Keyword(s):  
Solution Structure ◽  
Insulin Analogues ◽  
Size Exclusion ◽  
Life Extension ◽  
Once Daily ◽  
Solution Structures ◽  
X Ray Scattering ◽  
Exclusion Chromatography ◽  
Self Association ◽  
Long Acting

The lipidation of peptide drugs is one strategy to obtain extended half-lives, enabling once-daily or even less frequent injections for patients. The half-life extension results from a combination of self-association and association with human serum albumin (albumin). The self-association and association with albumin of two insulin analogues, insulin detemir and insulin degludec, were investigated by small-angle X-ray scattering (SAXS) and dynamic light scattering (DLS) in phenolic buffers. Detemir shows concentration-dependent self-association, with an equilibrium between hexamer, dihexamer, trihexamer and larger species, while degludec appears as a dihexamer independent of concentration. The solution structure of the detemir trihexamer has a bent shape. The stoichiometry of the association with albumin was studied using DLS. For albumin–detemir the molar stoichiometry was determined to be 1:6 (albumin:detemir ratio) and for albumin–degludec it was between 1:6 and 1:12 (albumin:degludec ratio). Batch SAXS measurements of a 1:6 albumin:detemir concentration series revealed a concentration dependence of complex formation. The data allowed the modelling of a complex between albumin and a detemir hexamer and a complex consisting of two albumins binding to opposite ends of a detemir dihexamer. Measurements of size-exclusion chromatography coupled to SAXS revealed a complex between a degludec dihexamer and albumin. Based on the results, equilibria for the albumin–detemir and albumin–degludec mixtures are proposed.

scholarly journals Solution scattering at the Life Science X-ray Scattering (LiX) beamline

2020 ◽  
Vol 27 (3) ◽  
pp. 804-812 ◽  
Author(s):  
Lin Yang ◽  
Stephen Antonelli ◽  
Shirish Chodankar ◽  
James Byrnes ◽  
Edwin Lazo ◽  
...  
Keyword(s):  
Data Analysis ◽  
Data Storage ◽  
User Interfaces ◽  
Life Science ◽  
Size Exclusion ◽  
Scattering Data ◽  
Batch Mode ◽  
X Ray ◽  
X Ray Scattering ◽  
Ray Scattering

This work reports the instrumentation and software implementation at the Life Science X-ray Scattering (LiX) beamline at NSLS-II in support of biomolecular solution scattering. For automated static measurements, samples are stored in PCR tubes and grouped in 18-position sample holders. Unattended operations are enabled using a six-axis robot that exchanges sample holders between a storage box and a sample handler, transporting samples from the PCR tubes to the X-ray beam for scattering measurements. The storage box has a capacity of 20 sample holders. At full capacity, the measurements on all samples last for ∼9 h. For in-line size-exclusion chromatography, the beamline-control software coordinates with a commercial high-performance liquid chromatography (HPLC) system to measure multiple samples in batch mode. The beamline can switch between static and HPLC measurements instantaneously. In all measurements, the scattering data span a wide q-range of typically 0.006–3.2 Å−1. Functionalities in the Python package py4xs have been developed to support automated data processing, including azimuthal averaging, merging data from multiple detectors, buffer scattering subtraction, data storage in HDF5 format and exporting the final data in a three-column text format that is acceptable by most data analysis tools. These functionalities have been integrated into graphical user interfaces that run in Jupyter notebooks, with hooks for external data analysis software.

LUTI: a double-function inhibitor isolated from naked flax seeds

2019 ◽  
Vol 51 (10) ◽  
pp. 989-996
Author(s):  
Lei Wang ◽  
Yinglu Chen ◽  
Feng Wu ◽  
Shasha Wu ◽  
Xiaojun Hu ◽  
...  
Keyword(s):  
Trypsin Inhibitor ◽  
Lactic Acid Production ◽  
Gel Filtration ◽  
Glucose Consumption ◽  
Competitive Inhibitor ◽  
Size Exclusion ◽  
Kinetic Experiment ◽  
Glucosidase Inhibitors ◽  
Exclusion Chromatography ◽  
Inhibitor Type

Abstract Acute glucose fluctuation during the postprandial period causes a risk for type 2 diabetes mellitus (T2DM). α-Glucosidase inhibitors have been approved as therapeutic agents for diabetes. In the present study, a protein with α-glucosidase inhibitory activity from Flax (Linum usitatissimum) seeds was isolated using a one-step purification with Q-Sepharose4B column, followed by Sephacryl S-200 size-exclusion chromatography. It was identified as a trypsin inhibitor, named L. usitatissimum trypsin inhibitor (LUTI). The half maximal inhibitory concentration (IC50) of LUTI was 113.92 μM for α-glucosidase and 6.17 μM for trypsin. Lineweaver–Burk kinetic experiment showed that the protein exhibited two distinct inhibitory modes, a competitive inhibitor type for α-glucosidase and a non-competitive type for trypsin. The interaction between LUTI and α-glucosidase was detected through gel filtration chromatography and dynamic light scattering. Increased glucose consumption and lactic acid production were also observed following LUTI treatment in Caco-2 and HepG2 cells. LUTI inhibits not only the activity of trypsin but also the activity of α-glucosidase. It is expected that LUTI will become an oral hypoglycemic polypeptide drug for T2DM.

Σύνθεση γραμμικών και αστεροειδών πολυμερών αποτελούμενα από συστάδες με υψηλή παράμετρο αλληλεπίδρασης Flory-Huggins χ

2020 ◽  
Author(s):  
Μαρία-Μαλβίνα Σταθουράκη
Keyword(s):  
Nuclear Magnetic Resonance ◽  
Magnetic Resonance ◽  
Magnetic Resonance Spectroscopy ◽  
Ring Opening ◽  
Ring Opening Polymerization ◽  
Size Exclusion ◽  
Resonance Spectroscopy ◽  
X Ray ◽  
X Ray Scattering ◽  
Exclusion Chromatography

Σκοπό της διατριβής αυτής αποτελεί η σύνθεση και η μελέτη της αυτοοργάνωσης γραμμικών και αστεροειδών συμπολυμερών με υψηλή παράμετρο αλληλεπιδρασης Flory-Huggins, χ. Τα πολυμερή αυτά, λόγω της μικρής αναμιξιμότητας που παρουσιάζουν τα συστατικά τους, έχουν την δυνατότητα σε μικρά μοριακά βάρη να μπορούν να σχηματίζουν πολύ μικρές και καλά καθορισμένες δομές κατά το μικροφασικό διαχωρισμό. Αρχικά, πραγματοποιήθηκε η σύνθεση των γραμμικών δισυσταδικών συμπολυμερών πολυ(2-βινυλοπυριδίνης)-b-πολυ(l-λακτιδίου) (P2VP-b-PLLA) και των τρισυσταδικών πολυ(l-λακτιδίου)-b-πολύ(διμεθυλοσιλοξάνη)-b-πολύ(l-λακτιδίου) (PLLA-b-PDMS-b-PLLA), καθώς και γραμμικών και αστεροειδών συμπολυμερών πολύ(στυρένιο)-b-πολυ(μονομεθακρυλική γλυκερόλη), PS-b-PGMA, (πολυστυρένιο)2(πολυ(μονομεθακρυλική γλυκερόλη)), (PS)2(PGMA), και (πολυστυρένιο)3(πολυ(μονομεθακρυλική γλυκερόλη)), (PS)3(PGMA), σε διάφορες αναλογίες μοριακών βαρών των συστατικών τους. Χρησιμοποιήθηκαν τεχνικές ζωντανού ανιοντικού πολυμερισμού για τη σύνθεση της P2VP, καθώς και για τη σύνθεση των αστεροειδών πολυμερών, ενώ η σύνθεση των PLLA πραγματοποιήθηκε με χρήση πολυμερισμού διάνοιξης δακτυλίου (Ring Opening Polymerization, ROP). Ο μοριακός χαρακτηρισμός των πολυμερών έγινε μέσω Χρωματογραφίας Αποκλεισμού Μεγεθών (Size Exclusion Chromatography, SEC) και Φασματοσκοπίας Πυρηνικού Μαγνητικού Συντονισμού Πρωτονίου (Nuclear Magnetic Resonance Spectroscopy, 1H-NMR). Τέλος, τίθενται τα αποτελέσματα που αφορούν τα γεωμετρικά χαρακτηριστικά (μέγεθος, μορφολογία) των περιοδικών νανοδομών που σχηματίζουν στο τήγμα τα συμπολυμερή, μέσω σκέδασης ακτίνων Χ σε μικρές γωνίες (Small-angle X-ray Scattering, SAXS).

scholarly journals Alpha-synuclein oligomers and fibrils originate in two distinct conformer pools: a small angle X-ray scattering and ensemble optimisation modelling study

2015 ◽  
Vol 11 (1) ◽  
pp. 190-196 ◽  
Author(s):  
Cyril C. Curtain ◽  
Nigel M. Kirby ◽  
Haydyn D. T. Mertens ◽  
Kevin J. Barnham ◽  
Robert B. Knott ◽  
...  
Keyword(s):  
Size Exclusion Chromatography ◽  
Small Angle ◽  
Alpha Synuclein ◽  
Size Exclusion ◽  
X Ray ◽  
X Ray Scattering ◽  
Exclusion Chromatography ◽  
Modelling Study ◽  
Ray Scattering

Size exclusion chromatography with small angle X-ray scattering and ensemble optimisation modelling reveals conformers in random pool of α-synuclein.

scholarly journals Distinguishing Between Monomeric scFv and Diabody in Solution Using Light and Small Angle X-ray Scattering

Antibodies ◽  
2019 ◽  
Vol 8 (4) ◽  
pp. 48
Author(s):  
Frank Lüdel ◽  
Sandra Bufe ◽  
Willem M. Bleymüller ◽  
Hugo de de Jonge ◽  
Luisa Iamele ◽  
...  
Keyword(s):  
Small Angle ◽  
Antibody Fragment ◽  
Correlation Spectroscopy ◽  
Size Exclusion ◽  
Single Chain ◽  
X Ray ◽  
X Ray Scattering ◽  
Single Chain Fv ◽  
Exclusion Chromatography ◽  
Ray Scattering

Depending on the linker length between the V H and the V L domain, single-chain Fv (scFv) antibody fragments form monomers, dimers (diabodies) or higher oligomers. We aimed at generating a diabody of the anti-MET antibody 3H3 to use it as crystallization chaperone to promote crystallization of the MET ectodomain through the introduction of a pre-formed twofold axis of symmetry. Size exclusion chromatography, however, suggested the protein to be monomeric. Hence, we used scattering techniques applied to solutions to further investigate its oligomerization state. The small angle X-ray scattering (SAXS) curve measured for our protein nicely fits to the scattering curve calculated from the known crystal structure of a diabody. In addition, concentration-dependent photon correlation spectroscopy (PCS) measurements revealed a hydrodynamic radius of 3.4 nm at infinite dilution and a negative interaction parameter k D , indicating attractive interactions that are beneficial for crystallization. Both SAXS and PCS measurements clearly suggest our antibody fragment to be a diabody in solution. Chemical cross-linking with glutaraldehyde and cell motility assays confirmed this conclusion.

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