9 research outputs found
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
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