Biology subjects

Lopez-Gomez, J.

Publications and source records attributed to Lopez-Gomez, J..

3 recordsLinked to original sources

bioRxiv2024

Free Amino Acids Accelerate the Time-Dependent Inactivation of Rat Liver Nucleotide Pyrophosphatase / Phosphodiesterase Enpp3 elicited by EDTA

Nucleotide-pyrophosphatases/phosphodiesterases (NPP/PDE) are membrane or secreted Zn2+-metallohydrolases of nucleoside-5-monophosphate derivatives. They hydrolyze, for instance, ATP and 4-nitrophenyl-dTMP, and belong to the ecto-nucleotide pyrophosphatase/phosphodiesterase (ENPP) family that contains seven members (ENPP1-ENPP7). Earlier we had shown that an NPP/PDE activity solubilized and partially purified from rat liver membranes is inactivated by EDTA in a time-dependent fashion, an effect enhanced by glycine and blocked by the 4-nitrophenyl-dTMP. Here, we extended this observation to other free amino acids. Activity assays started after different incubation lengths with EDTA provided first-order, apparent inactivation constants (ki(ap)). With the exception of cysteine (a strong inhibitor) and histidine (itself evoking a time-dependent inactivation), free amino acids themselves did not affect activity but increased ki(ap). The results are compatible with a conformational change of NPP/PDE evoked by interaction with free amino acids. The enzyme preparation was analyzed to identify what ENPP family members were present. First, the hydrolytic activity on 2,3-cGAMP was assayed because until very recently ENPP1 was the only mammalian enzyme known to display it. 2,3-cGAMP hydrolase activity was clearly detected, but mass spectrometry data obtained by LC-MS/MS gave evidence that only rat Enpp3, Enpp4 and Enpp5 were present with low abundance. This finding coincided in time with a recent publication claiming that mouse Enpp3 hydrolyzes 2,3-cGAMP, and that Enpp1 and Enpp3 account for all the 2,3-cGAMP hydrolase activity in mice. So, our results are confirmatory of Enpp3 activity towards 2,3-cGAMP. Finally, the effect of amino acids could be relevant to NPP/PDE actions dependent on protein-protein interactions, like the known insulin-related effects of ENPP1 and possibly ENPP3.

biochemistry↗

bioRxiv2024

Quantification of histone H1 subtypes using targeted proteomics.

Histone H1 is involved in the regulation of chromatin structure. Human somatic cells express up to seven subtypes. The variability in the proportions of somatic H1s (H1 complement) is one evidence supporting their functional specificity. Alterations in the protein levels of different H1 subtypes have been observed in cancer, suggesting their potential as biomarkers and that they might play a role in disease development. We have developed a mass spectrometry based (MS) parallel reaction monitoring (PRM) assay suitable for the quantification of H1 subtypes. Our PRM method is based on the quantification of unique peptides for each subtype, providing high specificity. Evaluation of the PRM performance on three human cell lines showed high reproducibility and sensitivity. Quantification values agreed with the electrophoretic and Western blot data, indicating the accuracy of the method. We used PRM to quantify the H1 complement in peripheral blood samples of healthy individuals and chronic myeloid leukemia (CML) patients. Our preliminary data revealed differences in the H1 complement between responders and non-responder CML patients and suggest that the H1 content could help predicting imatinib response.

biochemistry↗

bioRxiv2023

Proteasome-dependent degradation of histone H1 subtypes is mediated by its C-terminal domain.

Histone H1 is involved in chromatin compaction and dynamics. In human cells, the H1 complement is formed by different amounts of somatic H1 subtypes, H1.0-H1.5 and H1X. The amount of each variant depends on the cell type, the cell cycle phase, and the time of development and can be altered in disease. However, the mechanisms regulating H1 protein levels have not been described. We have analyzed the contribution of the proteasome to the degradation of H1 subtypes in human cells using two different inhibitors: MG132 and bortezomib. H1 subtypes accumulate upon treatment with both drugs, indicating that the proteasome is involved in the regulation of H1 protein levels. Proteasome inhibition caused a global increase in cytoplasmatic H1, with slight changes in the composition of H1 bound to chromatin and chromatin accessibility and no alterations in the nucleosome repeat length. The analysis of the proteasome degradation pathway showed that H1 degradation is ubiquitin-independent, whereas the whole protein and its C-terminal domain can be degraded directly by the 20S proteasome. Our study shows that histone H1 protein levels are under tight regulation preventing its accumulation in the nucleus. We revealed a new regulatory mechanism for histone H1 degradation, where the C-terminal disordered domain is responsible for its targeting and degradation by the 20S proteasome. StatementHistone H1 subtypes are a family of proteins involved in the regulation of chromatin structure. This work describes the degradation mechanism controlling the levels of histone H1 subtypes and the region within these proteins involved in the initial recognition. This regulatory mechanism protects the cell nucleus from the damaging effects of its accumulation.

molecular biology↗