| 1. |
- Beljantseva, Jelena, et al.
(author)
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Molecular mutagenesis of ppGpp : turning a RelA activator into an inhibitor
- 2017
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In: Scientific Reports. - : Nature Publishing Group. - 2045-2322. ; 7
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Journal article (peer-reviewed)abstract
- The alarmone nucleotide (p) ppGpp is a key regulator of bacterial metabolism, growth, stress tolerance and virulence, making (p) ppGpp-mediated signaling a promising target for development of antibacterials. Although ppGpp itself is an activator of the ribosome-associated ppGpp synthetase RelA, several ppGpp mimics have been developed as RelA inhibitors. However promising, the currently available ppGpp mimics are relatively inefficient, with IC50 in the sub-mM range. In an attempt to identify a potent and specific inhibitor of RelA capable of abrogating (p) ppGpp production in live bacterial cells, we have tested a targeted nucleotide library using a biochemical test system comprised of purified Escherichia coli components. While none of the compounds fulfilled this aim, the screen has yielded several potentially useful molecular tools for biochemical and structural work.
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| 2. |
- Mojr, Viktor, et al.
(author)
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Nonhydrolysable Analogues of (p)ppGpp and (p)ppApp Alarmone Nucleotides as Novel Molecular Tools
- 2021
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In: ACS Chemical Biology. - : American Chemical Society (ACS). - 1554-8929 .- 1554-8937. ; 16:9, s. 1680-1691
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Journal article (peer-reviewed)abstract
- While alarmone nudeotides guanosine-3',5'-bisdiphosphate (ppGpp) and guanosine-5'-triphosphate-3'-diphosphate (pppGpp) are archetypical bacterial second messengers, their adenosine analogues ppApp (adenosine-3',5'-bisdiphosphate) and pppApp (adenosine-5'-triphosphate-3'-diphosphate) are toxic effectors that abrogate bacterial growth. The alarmones are both synthesized and degraded by the members of the ReIA-SpoT Homologue (RSH) enzyme family. Because of the chemical and enzymatic liability of (p)ppGpp and (p)ppApp, these alarmones are prone to degradation during structural biology experiments. To overcome this limitation, we have established an efficient and straightforward procedure for synthesizing nonhydrolysable (p)ppNu(N)pp analogues starting from 3'-azido-3'-deoxyribonucleotides as key intermediates. To demonstrate the utility of (p)ppG(N)pp as a molecular tool, we show that (i) as an HD substrate mimic, ppG(N)pp competes with ppGpp to inhibit the enzymatic activity of human MESHI Small Alarmone Hyrolase, SAH; and (ii) mimicking the allosteric effects of (p)ppGpp, (p)ppG(N)pp acts as a positive regulator of the synthetase activity of long ribosome-associated RSHs Rel and ReIA. Finally, by solving the structure of the N-terminal domain region (NTD) of T. thermophilus Rel complexed with pppG(N)pp, we show that as an HD substrate mimic, the analogue serves as a bona fide orthosteric regulator that promotes the same intra-NTD structural rearrangements as the native substrate.
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| 3. |
- Pohl, Steffi, et al.
(author)
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Modelling method effects as individual causal effects
- 2008
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In: Journal of the Royal Statistical Society. - 0964-1998 .- 1467-985X. ; 171:1, s. 41-63
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Journal article (peer-reviewed)abstract
- Method effects often occur when different methods are used for measuring the same construct. We present a new approach for modelling this kind of phenomenon, consisting of a definition of method effects and a first model, the method effect model, that can be used for data analysis. This model may be applied to multitrait-multimethod data or to longitudinal data where the same construct is measured with at least two methods at all occasions. In this new approach, the definition of the method effects is based on the theory of individual causal effects by Neyman and Rubin. Method effects are accordingly conceptualized as the individual effects of applying measurement method j instead of k. They are modelled as latent difference scores in structural equation models. A reference method needs to be chosen against which all other methods are compared. The model fit is invariant to the choice of the reference method. The model allows the estimation of the average of the individual method effects, their variance, their correlation with the traits (and other latent variables) and the correlation of different method effects among each other. Furthermore, since the definition of the method effects is in line with the theory of causality, the method effects may (under certain conditions) be interpreted as causal effects of the method. The method effect model is compared with traditional multitrait-multimethod models. An example illustrates the application of the model to longitudinal data analysing the effect of negatively (such as 'feel bad') as compared with positively formulated items (such as 'feel good') measuring mood states.
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