Reversible covalent methylation of lysine residues on histone proteins constitutes a principal molecular mechanism that links chromatin states to diverse biological outcomes. Recently, lysine methylation has been observed on nonhistone proteins, suggesting broad cellular roles for the enzymes generating and removing methyl moieties. Here we report that the lysine methyltransferase enzyme SET8/PR-Set7 regulates the tumor suppressor protein p53. We find that SET8 specifically monomethylates p53 at lysine 382 (p53K382me1). This methylation event robustly suppresses p53-mediated transcription activation of highly responsive target genes but has little influence on weak targets. Further, depletion of SET8 augments the proapoptotic and checkpoint activation functions of p53, and accordingly, SET8 expression is downregulated upon DNA damage. Together, our study identifies SET8 as a p53-modifying enzyme, identifies p53K382me1 as a regulatory posttranslational modification of p53, and begins to dissect how methylation may contribute to a dynamic posttranslational code that modulates distinct p53 functions.
bSAMHD1 is a host protein responsible, at least in part, for the inefficient infection of dendritic, myeloid, and resting T cells by HIV-1. Interestingly, HIV-2 and SIVsm viruses are able to counteract SAMHD1 by targeting it for proteasomal degradation using their Vpx proteins. It has been proposed that SAMHD1 is a dGTP-dependent deoxynucleoside triphosphohydrolase (dNTPase) that restricts HIV-1 by reducing cellular dNTP levels to below that required for reverse transcription. However, nothing is known about SAMHD1 posttranslational modifications and their potential role in regulating SAMHD1 function. We used 32 P labeling and immunoblotting with phospho-specific antibodies to identify SAMHD1 as a phosphoprotein. Several amino acids in SAMHD1 were identified to be sites of phosphorylation using direct mass spectrometry. Mutation of these residues to alanine to prevent phosphorylation or to glutamic acid to mimic phosphorylation had no effect on the nuclear localization of SAMHD1 or its sensitivity to Vpx-mediated degradation. Furthermore, neither alanine nor glutamic acid substitutions had a significant effect on SAMHD1 dNTPase activity in an in vitro assay. Interestingly, however, we found that a T592E mutation, mimicking constitutive phosphorylation at a main phosphorylation site, severely affected the ability of SAMHD1 to restrict HIV-1 in a U937 cell-based restriction assay. In contrast, a T592A mutant was still capable of restricting HIV-1. These results indicate that SAMHD1 phosphorylation may be a negative regulator of SAMHD1 restriction activity. This conclusion is supported by our finding that SAMHD1 is hyperphosphorylated in monocytoid THP-1 cells under nonrestrictive conditions.
Currently, glycans are attracting attention from the scientific community as potential biomarkers or as posttranslational modifications (PTMs) of therapeutic proteins. However, structural characterization of glycoproteins and glycopeptides remains analytically challenging. Here, we report on the implementation of a novel acquisition strategy termed higher-energy collision dissociation-accurate mass-product-dependent electron transfer dissociation (HCD-PD-ETD) on a hybrid linear ion trap-orbitrap mass spectrometer. This acquisition strategy uses the complementary fragmentations of ETD and HCD for glycopeptides analysis in an intelligent fashion. Furthermore, the approach minimizes user input for optimizing instrumental parameters and enables straightforward detection of glycopeptides. ETD spectra are only acquired when glycan oxonium ions from MS/MS HCD are detected. The advantage of this approach is that it streamlines data analysis and improves dynamic range and duty cycle. Here, we present the benefits of HCD-PD-ETD relative to the traditional alternating HCD/ETD for a trainer set containing twelve-protein mixture with two glycoproteins: human serotransferrin, ovalbumin and contaminations of two other: bovine alpha 1 acid glycoprotein (bAGP) and bovine fetuin.
Discrepancy in synaptic structural plasticity is one of the earliest manifestations of the neurodegenerative state. In prion diseases, a reduction in synapses and dendritic spine densities is observed during preclinical disease in neurons of the cortex and hippocampus. The underlying molecular mechanisms of these alterations have not been identified but microRNAs (miRNAs), many of which are enriched at the synapse, likely regulate local protein synthesis in rapid response to stressors such as replicating prions. MiRNAs are therefore candidate regulators of these early neurodegenerative changes and may provide clues as to the molecular pathways involved. We therefore determined changes in mature miRNA abundance within synaptoneurosomes isolated from prion-infected, as compared to mock-infected animals, at asymptomatic and symptomatic stages of disease. During preclinical disease, miRNAs that are enriched in neurons including miR-124a-3p, miR-136-5p and miR-376a-3p were elevated. At later stages of disease we found increases in miRNAs that have previously been identified as deregulated in brain tissues of prion infected mice, as well as in Alzheimer's disease (AD) models. These include miR-146a-5p, miR-142-3p, miR-143-3p, miR-145a-5p, miR-451a, miR-let-7b, miR-320 and miR-150-5p. A number of miRNAs also decreased in abundance during clinical disease. These included almost all members of the related miR-200 family (miR-200a-3p, miR-200b-3p, miR-200c-3p, miR-141-3p, and miR-429-3p) and the 182 cluster (miR-182-5p and miR-183-5p).
The main objective of metabolomics is the analysis of all lowmolecular-weight compounds present in a particular living system. Metabolomics data is complementary to proteomics, genomics, and transcriptomics data and provides a better understanding of dynamic processes occurring in living systems.[1] The processes of sampling and sample preparation can significantly affect the composition of the measured metabolome, so the analytical results may not adequately reflect the true metabolome composition at the time of sampling. [2][3][4] This is due primarily to poor efficiency (or even complete omission) of metabolism quenching step and multistep handling procedures, which contribute to inadvertent metabolite loss and/or degradation.Herein we introduce in vivo solid-phase microextraction (SPME) as a new sample preparation method for global metabolomics studies of living systems using liquid chromatography-mass spectrometry (LC-MS). SPME is a nonexhaustive sample preparation procedure in which the amount of analyte extracted is governed by the distribution coefficient of the analyte between the SPME coating and sample matrix if the equilibrium is reached or the rate of mass transfer if a short sampling time is used.[5] In vivo SPME allows accurate extraction of the metabolome directly in the tissue or blood of freely moving animals without the need to withdraw a representative biological sample for analysis, under conditions of negligible depletion where the amount of analyte extracted by SPME is independent of the sample volume. [5][6][7] The blood-draw-free nature of the sampling method facilitates multiple sampling of the same living system and can capture unstable or short-lived metabolites.Large biomolecules are not extracted by the specially selected biocompatible SPME coating, so the need for a metabolism quenching step is eliminated. The amount of metabolites extracted is proportional to the biologically active unbound concentration. For metabolomics studies, in vivo SPME provides the simplest and most rapid sample preparation tool available to date to study living systems in a format directly compatible with LC-MS detection. Although SPME was successfully applied to metabolomics studies using GC-MS primarily in headspace mode, [8][9][10][11][12] its capability to provide instantaneous metabolism quenching directly during the sampling process to capture true metabolome of blood or tissue has not been previously evaluated.First, we developed a successful in vivo SPME workflow for direct sampling of metabolome, and applied it to mice as a model system (Figure 1). In this approach, a coated SPME fiber is housed inside hypodermic needle, [13] which is used to pierce the sampling interface containing circulating blood. The fiber is exposed to blood for a pre-set short sampling time of 2 min. During the sampling, analytes are extracted directly into the SPME coating. The key aspect of developing SPME device for metabolomics was selection of the chemical nature of the coating to ensure simultaneous extraction of both...
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