Combining in-cell NMR and X-ray fluorescence microscopy to reveal the intracellular maturation states of human superoxide dismutase 1

2015 ◽  
Vol 51 (3) ◽  
pp. 584-587 ◽  
Author(s):  
E. Luchinat ◽  
A. Gianoncelli ◽  
T. Mello ◽  
A. Galli ◽  
L. Banci
Keyword(s):  
Superoxide Dismutase ◽  
Nmr Spectroscopy ◽  
Fluorescence Microscopy ◽  
Superoxide Dismutase 1 ◽  
X Ray ◽  
Optical Fluorescence ◽  
Human Sod1

Combined in-cell NMR spectroscopy, X-ray fluorescence and optical fluorescence microscopies allow describing the intracellular maturation states of human SOD1.

scholarly journals Variation in the vulnerability of mice expressing human superoxide dismutase 1 to prion-like seeding: a study of the influence of primary amino acid sequence

2021 ◽  
Vol 9 (1) ◽  
Author(s):  
Jacob I. Ayers ◽  
Guilian Xu ◽  
Kristy Dillon ◽  
Qing Lu ◽  
Zhijuan Chen ◽  
...  
Keyword(s):  
Superoxide Dismutase ◽  
Motor Neuron ◽  
Motor Neuron Disease ◽  
Superoxide Dismutase 1 ◽  
Primary Sequence ◽  
Newborn Mice ◽  
Adult Mice ◽  
Ability To Act ◽  
Human Sod1

AbstractMisfolded forms of superoxide dismutase 1 (SOD1) with mutations associated with familial amyotrophic lateral sclerosis (fALS) exhibit prion characteristics, including the ability to act as seeds to accelerate motor neuron disease in mouse models. A key feature of infectious prion seeding is that the efficiency of transmission is governed by the primary sequence of prion protein (PrP). Isologous seeding, where the sequence of the PrP in the seed matches that of the host, is generally much more efficient than when there is a sequence mis-match. Here, we used paradigms in which mutant SOD1 seeding homogenates were injected intraspinally in newborn mice or into the sciatic nerve of adult mice, to assess the influence of SOD1 primary sequence on seeding efficiency. We observed a spectrum of seeding efficiencies depending upon both the SOD1 expressed by mice injected with seeds and the origin of the seed preparations. Mice expressing WT human SOD1 or the disease variant G37R were resistant to isologous seeding. Mice expressing G93A SOD1 were also largely resistant to isologous seeding, with limited success in one line of mice that express at low levels. By contrast, mice expressing human G85R-SOD1 were highly susceptible to isologous seeding but resistant to heterologous seeding by homogenates from paralyzed mice over-expressing mouse SOD1-G86R. In other seeding experiments with G85R SOD1:YFP mice, we observed that homogenates from paralyzed animals expressing the H46R or G37R variants of human SOD1 were less effective than seeds prepared from mice expressing the human G93A variant. These sequence mis-match effects were less pronounced when we used purified recombinant SOD1 that had been fibrilized in vitro as the seeding preparation. Collectively, our findings demonstrate diversity in the abilities of ALS variants of SOD1 to initiate or sustain prion-like propagation of misfolded conformations that produce motor neuron disease.

scholarly journals Cloning and high-level expression of monomeric human superoxide dismutase 1 (SOD1) and its interaction with pyrimidine analogs

PLoS ONE ◽  
2021 ◽  
Vol 16 (2) ◽  
pp. e0247684
Author(s):  
Marcia LeVatte ◽  
Matthias Lipfert ◽  
Dipankar Roy ◽  
Andriy Kovalenko ◽  
David Scott Wishart
Keyword(s):  
Superoxide Dismutase ◽  
Nmr Spectroscopy ◽  
Nmr Spectra ◽  
Expression System ◽  
Leader Peptide ◽  
Superoxide Dismutase 1 ◽  
H Nmr ◽  
Molecule Interactions ◽  
High Level ◽  
Pyrimidine Analogs

Superoxide dismutase 1 (SOD1) is known to be involved in the pathogenesis of Amyotrophic Lateral Sclerosis (ALS) and is therefore considered to be an important ALS drug target. Identifying potential drug leads that bind to SOD1 and characterizing their interactions by nuclear magnetic resonance (NMR) spectroscopy is complicated by the fact that SOD1 is a homodimer. Creating a monomeric version of SOD1 could alleviate these issues. A specially designed monomeric form of human superoxide dismutase (T2M4SOD1) was cloned into E. coli and its expression significantly enhanced using a number of novel DNA sequence, leader peptide and growth condition optimizations. Uniformly 15N-labeled T2M4SOD1 was prepared from minimal media using 15NH4Cl as the 15N source. The T2M4SOD1 monomer (both 15N labeled and unlabeled) was correctly folded as confirmed by 1H-NMR spectroscopy and active as confirmed by an in-gel enzymatic assay. To demonstrate the utility of this new SOD1 expression system for NMR-based drug screening, eight pyrimidine compounds were tested for binding to T2M4SOD1 by monitoring changes in their 1H NMR and/or 19F-NMR spectra. Weak binding to 5-fluorouridine (FUrd) was observed via line broadening, but very minimal spectral changes were seen with uridine, 5-bromouridine or trifluridine. On the other hand, 1H-NMR spectra of T2M4SOD1 with uracil or three halogenated derivatives of uracil changed dramatically suggesting that the pyrimidine moiety is the crucial binding component of FUrd. Interestingly, no change in tryptophan 32 (Trp32), the putative receptor for FUrd, was detected in the 15N-NMR spectra of 15N-T2M4SOD1 when mixed with these uracil analogs. Molecular docking and molecular dynamic (MD) studies indicate that interaction with Trp32 of SOD1 is predicted to be weak and that there was hydrogen bonding with the nearby aspartate (Asp96), potentiating the Trp32-uracil interaction. These studies demonstrate that monomeric T2M4SOD1 can be readily used to explore small molecule interactions via NMR.

scholarly journals Cloning, purification and enzymatic characterization of recombinant human Superoxide dismutase 1 expressed in Escherichia coli

2018 ◽  
Vol 65 (2) ◽  
pp. 235-240 ◽  
Author(s):  
LIN FENG ◽  
Yan Dan Dan ◽  
Chen Ya Wen ◽  
Fletcher Emmanuella E ◽  
Shi Hai Feng ◽  
...  
Keyword(s):  
Superoxide Dismutase ◽  
Expression Vector ◽  
Superoxide Dismutase 1 ◽  
Enzymatic Characterization ◽  
Activity Detection ◽  
Bivalent Cations ◽  
Bxpc3 Cell ◽  
Structure And Activity ◽  
Human Sod1

Superoxide dismutase 1 (SOD1) is a metalloenzyme that catalyzes disproportion-action superoxide into molecular oxygen and hydrogen peroxide. In this study, the human SOD1 (hSOD1) gene was cloned, expressed, and purified. The hSOD1 gene was amplified from a pool of Bxpc3 cell cDNAs by PCR and cloned into expression vector pET-28a (+). The recombinant soluble hSOD1 was expressed in E.coli BL21 (DE3) at 37°C and purified by Nickel column affinity chromatography. The soluble hSOD1 was produced with a yield of 5.9 ug/mL medium. Considering that metal ions have a certain influence on the structure and activity of protein, we researched the influences of different concentrations of Cu2+ and Zn2+ on hSOD1 activity at induction and the time of activity detection. The results implied Cu2+ and Zn2+ can’t enhance SOD1 expression, however can improve the catalytic activity at induction. Furthermore, most of bivalent cations have an improve effect on enzyme activity at the time of detection.

Scanning x-ray fluorescence microscopy and principal component analysis

1992 ◽  
Vol 50 (2) ◽  
pp. 1752-1753
Author(s):  
Brian Cross
Keyword(s):  
Principal Component Analysis ◽  
Fluorescence Microscopy ◽  
Spatial Resolution ◽  
High Speed ◽  
Image Acquisition ◽  
Principal Component ◽  
Fluorescence Microscope ◽  
Acquisition Rate ◽  
X Ray ◽  
Speed Up

A relatively new entry, in the field of microscopy, is the Scanning X-Ray Fluorescence Microscope (SXRFM). Using this type of instrument (e.g. Kevex Omicron X-ray Microprobe), one can obtain multiple elemental x-ray images, from the analysis of materials which show heterogeneity. The SXRFM obtains images by collimating an x-ray beam (e.g. 100 μm diameter), and then scanning the sample with a high-speed x-y stage. To speed up the image acquisition, data is acquired "on-the-fly" by slew-scanning the stage along the x-axis, like a TV or SEM scan. To reduce the overhead from "fly-back," the images can be acquired by bi-directional scanning of the x-axis. This results in very little overhead with the re-positioning of the sample stage. The image acquisition rate is dominated by the x-ray acquisition rate. Therefore, the total x-ray image acquisition rate, using the SXRFM, is very comparable to an SEM. Although the x-ray spatial resolution of the SXRFM is worse than an SEM (say 100 vs. 2 μm), there are several other advantages.

An unusual reaction of a bridged triterpenoid α-diketone with acetic anhydride

1991 ◽  
Vol 56 (12) ◽  
pp. 2917-2935 ◽  
Author(s):  
Eva Klinotová ◽  
Václav Křeček ◽  
Jiří Klinot ◽  
Miloš Buděšínský ◽  
Jaroslav Podlaha ◽  
...  
Keyword(s):  
Acetic Acid ◽  
Nmr Spectroscopy ◽  
Acetic Anhydride ◽  
Spectral Methods ◽  
X Ray Diffraction ◽  
X Ray ◽  
Mild Conditions ◽  
Condensation Products ◽  
Unsaturated Lactones ◽  
C Nmr

3β-Acetoxy-21,22-dioxo-18α,19βH-ursan-28,20β-olide (IIIa) reacts with acetic anhydride in pyridine under very mild conditions affording β-lactone IVa and γ-lactones Va and VIIa as condensation products. On reaction with pyridine, lactones Va and VIIa undergo elimination of acetic acid to give unsaturated lactones VIIIa and IXa, respectively. Similarly, the condensation of 20β,28-epoxy-21,22-dioxo-18α,19βH-ursan-3β-yl acetate (IIIb) with acetic anhydride leads to β-lactone IVb and γ-lactone Vb; the latter on heating with pyridine affords unsaturated lactone VIIIb and 21-methylene-22-ketone Xb. The structure of the obtained compounds was derived using spectral methods, particularly 1H and 13C NMR spectroscopy; structure of lactone IVa was confirmed by X-ray diffraction.

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