| 1. |
- Scrima, Simone, et al.
(author)
-
Comparison of force fields to study the zinc-finger containing protein NPL4, a target for disulfiram in cancer therapy
- 2023
-
In: Biochimica et Biophysica Acta - Proteins and Proteomics. - 1570-9639. ; 1871:4
-
Journal article (peer-reviewed)abstract
- Molecular dynamics (MD) simulations are a powerful approach to studying the structure and dynamics of proteins related to health and disease. Advances in the MD field allow modeling proteins with high accuracy. However, modeling metal ions and their interactions with proteins is still challenging. NPL4 is a zinc-binding protein and works as a cofactor for p97 to regulate protein homeostasis. NPL4 is of biomedical importance and has been proposed as the target of disulfiram, a drug recently repurposed for cancer treatment. Experimental studies proposed that the disulfiram metabolites, bis-(diethyldithiocarbamate)‑copper and cupric ions, induce NPL4 misfolding and aggregation. However, the molecular details of their interactions with NPL4 and consequent structural effects are still elusive. Here, biomolecular simulations can help to shed light on the related structural details. To apply MD simulations to NPL4 and its interaction with copper the first important step is identifying a suitable force field to describe the protein in its zinc-bound states. We examined different sets of non-bonded parameters because we want to study the misfolding mechanism and cannot rule out that the zinc may detach from the protein during the process and copper replaces it. We investigated the force-field ability to model the coordination geometry of the metal ions by comparing the results from MD simulations with optimized geometries from quantum mechanics (QM) calculations using model systems of NPL4. Furthermore, we investigated the performance of a force field including bonded parameters to treat copper ions in NPL4 that we obtained based on QM calculations.
|
|
| 2. |
- Brix, Ditte Marie, et al.
(author)
-
Release of transcriptional repression via ErbB2-induced, SUMO-directed phosphorylation of myeloid zinc finger-1 serine 27 activates lysosome redistribution and invasion
- 2019
-
In: Oncogene. - : Springer Science and Business Media LLC. - 0950-9232 .- 1476-5594. ; 38:17, s. 3170-3184
-
Journal article (peer-reviewed)abstract
- HER2/ErbB2 activation turns on transcriptional processes that induce local invasion and lead to systemic metastasis. The early transcriptional changes needed for ErbB2-induced invasion are poorly understood. Here, we link ErbB2 activation to invasion via ErbB2-induced, SUMO-directed phosphorylation of a single serine residue, S27, of the transcription factor myeloid zinc finger-1 (MZF1). Utilizing an antibody against MZF1-pS27, we show that the phosphorylation of S27 correlates significantly (p < 0.0001) with high-level expression of ErbB2 in primary invasive breast tumors. Phosphorylation of MZF1-S27 is an early response to ErbB2 activation and results in increased transcriptional activity of MZF1. It is needed for the ErbB2-induced expression of MZF1 target genes CTSB and PRKCA, and invasion of single-cells from ErbB2-expressing breast cancer spheroids. The phosphorylation of MZF1-S27 is preceded by poly-SUMOylation of K23, which can make S27 accessible to efficient phosphorylation by PAK4. Based on our results, we suggest for an activation mechanism where phosphorylation of MZF1-S27 triggers MZF1 dissociation from its transcriptional repressors such as the CCCTC-binding factor (CTCF). Our findings increase understanding of the regulation of invasive signaling in breast cancer by uncovering a detailed biological mechanism of how ErbB2 activation can rapidly lead to its invasion-promoting target gene expression and invasion.
|
|
| 3. |
- Hatos, Andras, et al.
(author)
-
DisProt : intrinsic protein disorder annotation in 2020
- 2020
-
In: Nucleic Acids Research. - : Oxford University Press (OUP). - 0305-1048 .- 1362-4962. ; 48:D1, s. D269-D276
-
Journal article (peer-reviewed)abstract
- The Database of Protein Disorder (DisProt, URL:https://disprot.org) provides manually curated annotations of intrinsically disordered proteins from the literature. Here we report recent developments with DisProt (version 8), including the doubling of protein entries, a new disorder ontology, improvements of the annotation format and a completely new website. The website includes a redesigned graphical interface, a better search engine, a clearer API for programmatic access and a new annotation interface that integrates text mining technologies. The new entry format provides a greater flexibility, simplifies maintenance and allows the capture of more information from the literature. The new disorder ontology has been formalized and made interoperable by adopting the OWL format, as well as its structure and term definitions have been improved. The new annotation interface has made the curation process faster and more effective. We recently showed that new DisProt annotations can be effectively used to train and validate disorder predictors. We believe the growth of DisProt will accelerate, contributing to the improvement of function and disorder predictors and therefore to illuminate the 'dark' proteome.
|
|
| 4. |
- Lambrughi, Matteo, et al.
(author)
-
Conformational gating in ammonia lyases
- 2020
-
In: Biochimica et Biophysica Acta - General Subjects. - : Elsevier BV. - 1872-8006 .- 0304-4165. ; 1864:7
-
Journal article (peer-reviewed)abstract
- Background: Ammonia lyases are enzymes of industrial and biomedical interest. Knowledge of structure-dynamics-function relationship in ammonia lyases is instrumental for exploiting the potential of these enzymes in industrial or biomedical applications. Methods: We investigated the conformational changes in the proximity of the catalytic pocket of a 3-methylaspartate ammonia lyase (MAL) as a model system. At this scope, we used microsecond all-atom molecular dynamics simulations, analyzed with dimensionality reduction techniques, as well as in terms of contact networks and correlated motions. Results: We identify two regulatory elements in the MAL structure, i.e., the β5-α2 loop and the helix-hairpin-loop subdomain. These regulatory elements undergo conformational changes switching from ‘occluded’ to ‘open’ states. The rearrangements are coupled to changes in the accessibility of the active site. The β5-α2 loop and the helix-hairpin-loop subdomain modulate the formation of tunnels from the protein surface to the catalytic site, making the active site more accessible to the substrate when they are in an open state. Conclusions: Our work pinpoints a sequential mechanism, in which the helix-hairpin-loop subdomain of MAL needs to break a subset of intramolecular interactions first to favor the displacement of the β5-α2 loop. The coupled conformational changes of these two elements contribute to modulate the accessibility of the catalytic site. General significance: Similar molecular mechanisms can have broad relevance in other ammonia lyases with similar regulatory loops. Our results also imply that it is important to account for protein dynamics in the design of variants of ammonia lyases for industrial and biomedical applications.
|
|
| 5. |
- Lambrughi, Matteo, et al.
(author)
-
DEAMINATION REACTIONS AS PART OF THE METABOLIC PATHWAY FOR THE PRODUCTION OF ADIPIC ACID
- 2018
-
Conference paper (other academic/artistic)abstract
- Deamination of lysine, β-lysine or 2-aminoadipic acid are enzymatic reactions that have great biotechnological interest as part of the metabolic pathways for the production of adipic acid. The enzymatic activity necessary to catalyze the mentioned deamination reactions is defined as ammonia lyase (EC 4.3.1.-) and cleaves the bond between the amine group and adjacent carbon. Whereas ammonia lyases able to act on the target substrates lysine, β-lysine or 2-aminoadipic acid have not been identified so far, ammonia lyases able to act on other amino acids have long been known. We selected 3-methylaspartate-ammonia lyase (MAL, EC 4.3.1.2) as enzyme that potentially could be made active on the target substrates. Three MAL were recombinantly expressed and purified and the activity tested towards the substrates, but no activity was observed. Different MAL single mutant variants potentially able to catalyze the desired reactions were designed using a computational approach, but unfortunately, no activity towards the target substrates was detected. In order to better understand the substrate scope and catalytic mechanism of MAL, different compunds with similar structure to the natural substrate were tried as substrates. MAL showed activity towards aspartic acid, but no towards the other substrates indicating the narrow specificity of the enzyme. Inhibition studies showed that β-lysine was a competitive inhibitor suggesting that the amino group of the substrate need to be in the β-carbon for the binding. 2-aminoadipic acid was shown to be a non-competitive inhibitor. Finally, docking experiments were carried out to understand if the target substrates fit in the catalytic pocket. The study provides a deeper knowledge of the substrate scope and inhibitors of MAL and analyze if and how the target substrates could be deaminated by MAL. Moreover, the study establishes reliable methods for the detection of deamination activity of lysine, β-lysine and 2-aminoadipic acid.
|
|
| 6. |
- Quaglia, Federica, et al.
(author)
-
DisProt in 2022 : improved quality and accessibility of protein intrinsic disorder annotation
- 2022
-
In: Nucleic Acids Research. - : Oxford University Press (OUP). - 0305-1048 .- 1362-4962. ; 50:D1, s. D480-D487
-
Journal article (peer-reviewed)abstract
- The Database of Intrinsically Disordered Proteins (DisProt, URL: https://disprot.org) is the major repository of manually curated annotations of intrinsically disordered proteins and regions from the literature. We report here recent updates of DisProt version 9, including a restyled web interface, refactored Intrinsically Disordered Proteins Ontology (IDPO), improvements in the curation process and significant content growth of around 30%. Higher quality and consistency of annotations is provided by a newly implemented reviewing process and training of curators. The increased curation capacity is fostered by the integration of DisProt with APICURON, a dedicated resource for the proper attribution and recognition of biocuration efforts. Better interoperability is provided through the adoption of the Minimum Information About Disorder (MIADE) standard, an active collaboration with the Gene Ontology (GO) and Evidence and Conclusion Ontology (ECO) consortia and the support of the ELIXIR infrastructure.
|
|
| 7. |
- Saez Jimenez, Veronica, 1985, et al.
(author)
-
Directed Evolution of (R)-2-Hydroxyglutarate Dehydrogenase Improves 2-Oxoadipate Reduction by 2 Orders of Magnitude
- 2022
-
In: ACS Synthetic Biology. - : American Chemical Society (ACS). - 2161-5063. ; 11:8, s. 2779-2790
-
Journal article (peer-reviewed)abstract
- Pathway engineering is commonly employed to improve the production of various metabolites but may incur in bottlenecks due to the low catalytic activity of a particular reaction step. The reduction of 2-oxoadipate to (R)-2-hydroxyadipate is a key reaction in metabolic pathways that exploit 2-oxoadipate conversion via α-reduction to produce adipic acid, an industrially important platform chemical. Here, we engineered (R)-2-hydroxyglutarate dehydrogenase from Acidaminococcus fermentans (Hgdh) with the aim of improving 2-oxoadipate reduction. Using a combination of computational analysis, saturation mutagenesis, and random mutagenesis, three mutant variants with a 100-fold higher catalytic efficiency were obtained. As revealed by rational analysis of the mutations found in the variants, this improvement could be ascribed to a general synergistic effect where mutation A206V played a key role since it boosted the enzyme's activity by 4.8-fold. The Hgdh variants with increased activity toward 2-oxoadipate generated within this study pave the way for the bio-based production of adipic acid.
|
|
| 8. |
- Saez Jimenez, Veronica, 1985, et al.
(author)
-
Engineering ammonia-lyases for lysine transformation: first steps to green production of adipic acid
- 2017
-
Conference paper (other academic/artistic)abstract
- Adipic acid is one of the most important dicarboxylic acid for commercial purposes, mainly used as building block for nylon polymers. The current chemical production process has serious consequences for the environment. Therefore, the implementation of a by a bio-based process using renewable feedstocks would be highly beneficial for the society and the environment. The construction of a microbial metabolic pathway to produce adipic acid using L-lysine as precursor is a potential alternative. The first step of the proposed pathway converts L-lysine into 6-aminohex-2-enoic acid (6-AHEA) via removal of the α-amino group. Chemical methods for this deamination reaction are known; however, no enzymes able to carry out this reaction on L-lysine have been disclosed yet. The main goal of the current research is the generation a novel enzyme activity for the conversion of L-lysine to 6-AHEA. The enzymatic activity necessary to catalyze the required deamination is defined as ammonia lyase and results in the removal of the α-amino group. Histidine ammonia-lyase (HAL) and 3-methylaspartate-ammonia-lyase (MAL), enzymes acting on histidine and 3-methylaspartate, respectively, were selected to be engineered to catalyze the deamination of lysine. HAL from Pseudomonas putida and MAL from Clostridium tetanomorphum and Carboxydothermus hydrogenoformans were expressed in E.coli and purified. The capability of the enzymes to deaminate lysine was tested. No deamination activity was observed, while the inhibitory effect of L-lysine on HAL activity was shown. Computational structural biology methodologies were applied on MAL and combined with protein engineering techniques in order to design mutant enzyme variants potentially active on L-lysine. In-silico saturation mutagenesis tools were used to model all the possible mutations in the active site or its surroundings that are expected to increase affinity for the L-lysine substrate. Following the results obtained, the residues C361, M389 and L384 were mutagenized. The mutant variants were produced and purified, and the activity on L-lysine tested by monitoring the production of 6-AHEA and the release of ammonia.
|
|
| 9. |
- Saez Jimenez, Veronica, 1985, et al.
(author)
-
Structure-function investigation of 3-methylaspartate ammonia lyase reveals substrate molecular determinants for the deamination reaction
- 2020
-
In: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203 .- 1932-6203. ; 15:5
-
Journal article (peer-reviewed)abstract
- The enzymatic reactions leading to the deamination of β-lysine, lysine, or 2-aminoadipic acid are of great interest for the metabolic conversion of lysine to adipic acid. Enzymes able to carry out these reactions are not known, however ammonia lyases (EC 4.3.1.-) perform deamination on a wide range of substrates. We have studied 3-methylaspartate ammonia lyase (MAL, EC 4.3.1.2) as a potential candidate for protein engineering to enable deamination towards β-lysine, that we have shown to be a competitive inhibitor of MAL. We have characterized MAL activity, binding and inhibition properties on six different compounds that would allow to define the molecular determinants necessary for MAL to deaminate our substrate of interest. Docking calculations showed that β-lysine as well as the other compounds investigated could fit spatially into MAL catalytic pocket, although they probably are weak or very transient binders and we identified molecular determinants involved in the binding of the substrate. The hydrophobic interactions formed by the methyl group of 3-methylaspartic acid, together with the presence of the amino group on carbon 2, play an essential role in the appropriate binding of the substrate. The results showed that β-lysine is able to fit and bind in MAL catalytic pocket and can be potentially converted from inhibitor to substrate of MAL upon enzyme engineering. The characterization of the binding and inhibition properties of the substrates tested here provide the foundation for future and more extensive studies on engineering MAL that could lead to a MAL variant able to catalyse this challenging deamination reaction.
|
|
