Developmentally Regulated Post-translational Modification of Nucleoplasmin Controls Histone Sequestration and Deposition

SummaryNucleoplasmin (Npm) is an abundant histone chaperone in vertebrate oocytes and embryos. During embryogenesis, regulation of Npm histone binding is critical for its function in storing and releasing maternal histones to establish and maintain the zygotic epigenome. Here, we demonstrate that Xenopus laevis Npm post-translational modifications (PTMs) specific to the oocyte and egg promote either histone deposition or sequestration, respectively. Mass spectrometry and Npm phosphomimetic mutations used in chromatin assembly assays identified hyperphosphorylation on the N-terminal tail as a critical regulator for sequestration. C-terminal tail phosphorylation and PRMT5-catalyzed arginine methylation enhance nucleosome assembly by promoting histone interaction with the second acidic tract of Npm. Electron microscopy reconstructions of Npm and TTLL4 activity toward the C-terminal tail demonstrate that oocyte- and egg-specific PTMs cause Npm conformational changes. Our results reveal that PTMs regulate Npm chaperoning activity by modulating Npm conformation and Npm-histone interaction, leading to histone sequestration in the egg

Developmentally Regulated Post-translational Modification of Nucleoplasmin Controls Histone Sequestration and Deposition

SummaryNucleoplasmin (Npm) is an abundant histone chaperone in vertebrate oocytes and embryos. During embryogenesis, regulation of Npm histone binding is critical for its function in storing and releasing maternal histones to establish and maintain the zygotic epigenome. Here, we demonstrate that Xenopus laevis Npm post-translational modifications (PTMs) specific to the oocyte and egg promote either histone deposition or sequestration, respectively. Mass spectrometry and Npm phosphomimetic mutations used in chromatin assembly assays identified hyperphosphorylation on the N-terminal tail as a critical regulator for sequestration. C-terminal tail phosphorylation and PRMT5-catalyzed arginine methylation enhance nucleosome assembly by promoting histone interaction with the second acidic tract of Npm. Electron microscopy reconstructions of Npm and TTLL4 activity toward the C-terminal tail demonstrate that oocyte- and egg-specific PTMs cause Npm conformational changes. Our results reveal that PTMs regulate Npm chaperoning activity by modulating Npm conformation and Npm-histone interaction, leading to histone sequestration in the egg

Anatomical-Molecular Distribution of EphrinA1 in Infarcted Mouse Heart Using MALDI Mass Spectrometry Imaging

EphrinA1 is a tyrosine kinase receptor localized in the cellular membrane of healthy cardiomyocytes, the expression of which is lost upon myocardial infarction (MI). Intra-cardiac injection of the recombinant form of ephrinA1 (ephrinA1-Fc) at the time of ligation in mice has shown beneficial effects by reducing infarct size and myocardial necrosis post-MI. To date, immunohistochemistry and Western blotting comprise the only experimental approaches utilized to localize and quantify relative changes of ephrinA1 in sections and homogenates of whole left ventricle, respectively. Herein, we used matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI) coupled with a time-of-flight mass spectrometer (MALDI/TOF MS) to identify intact as well as tryptic fragments of ephrinA1 in healthy controls and acutely infarcted murine hearts. The purpose of the present study was 3-fold: (1) to spatially resolve the molecular distribution of endogenous ephrinA1, (2) to determine the anatomical expression profile of endogenous ephrinA1 after acute MI, and (3) to identify molecular targets of ephrinA1-Fc action post-MI. The tryptic fragments detected were identified as the ephrinA1-isoform with 38% and 34% sequence coverage and Mascot scores of 25 for the control and MI hearts, respectively. By using MALDI-MSI, we have been able to simultaneously measure the distribution and spatial localization of ephrinA1, as well as additional cardiac proteins, thus offering valuable information for the elucidation of molecular partners, mediators, and targets of ephrinA1 action in cardiac muscle.Open Access Fun

Localized in Situ Hydrogel-Mediated Protein Digestion and Extraction Technique for on-Tissue Analysis

A simultaneous on-tissue proteolytic digestion and extraction method is described for the in situ analysis of proteins from spatially distinct areas of a tissue section. The digestion occurs on-tissue within a hydrogel network, and peptides extracted from this gel are identified with liquid chromatography tandem MS (LC-MS/MS). The hydrogels are compatible with solubility agents (e.g., chaotropes and detergents) known to improve enzymatic digestion of proteins. Additionally, digestions and extractions are compatible with imaging mass spectrometry (IMS) experiments. As an example application, an initial IMS experiment was conducted to profile lipid species using a traveling wave ion mobility mass spectrometer. On-tissue MS/MS was also performed on the same tissue section to identify lipid ions that showed spatial differences. Subsequently, the section underwent an on-tissue hydrogel digestion to reveal 96 proteins that colocalized to the rat brain cerebellum. Hematoxylin and eosin (H & E) staining was then performed to provide additional histological information about the tissue structure. This technology provides a versatile workflow that can be used to correlate multiple complementary analytical approaches in the analysis of a single tissue section

MALDI Imaging and in Situ Identification of Integral Membrane Proteins from Rat Brain Tissue Sections

Transmembrane proteins are greatly underrepresented in data generated by imaging mass spectrometry (IMS) because of analytical challenges related to their size and solubility. Here, we present the first example of MALDI IMS of two highly modified multitransmembrane domain proteins, myelin proteolipid protein (PLP, 30 kDa) and DM-20 (26 kDa), from various regions of rat brain, namely, the cerebrum, cerebellum, and medulla. We utilize a novel tissue pretreatment aimed at transmembrane protein enrichment to show the in situ distribution of fatty acylation of these proteins, particularly of post-translational palmitoylation. Additionally, we demonstrate the utility of protease-encapsulated hydrogels for spatially localized on-tissue protein digestion and peptide extraction for subsequent direct coupling to LC-MS/MS for protein identification