Biology subjects

Nielsen, M. L.

Publications and source records attributed to Nielsen, M. L..

28 records · Page 2Linked to original sources

bioRxiv2023

Profiling ubiquitin signaling with UBIMAX reveals DNA damage- and SCFβTRCP-dependent ubiquitylation of the actin-organizing protein Dbn1

Ubiquitin widely modifies proteins, thereby regulating most cellular functions. The complexity of ubiquitin signalling necessitates unbiased methods enabling global detection of dynamic protein ubiquitylation. Here, we describe UBIMAX (UBiquitin target Identification by Mass spectrometry in Xenopus egg extracts), which enriches ubiquitin-conjugated proteins and quantifies regulation of protein ubiquitylation under precise and adaptable conditions. We benchmark UBIMAX by investigating DNA double-strand break-responsive ubiquitylation events, identifying previously known targets and revealing the actin-organising protein Dbn1 as a novel major target of DNA damage-induced ubiquitylation. We find that Dbn1 is targeted for proteasomal degradation by the SCF{beta}-Trcp1 ubiquitin ligase, in a conserved mechanism driven by ATM-mediated phosphorylation of a previously uncharacterized {beta}-Trcp1 degron containing an SQ motif. We further show that this degron is sufficient to induce DNA-damage dependent protein degradation of a model substrate. Collectively, we demonstrate UBIMAXs ability to identify novel targets of stimulus-regulated ubiquitylation and reveal an SCF{beta}-Trcp1-mediated ubiquitylation mechanism controlled directly by the apical DNA damage response kinases.

molecular biology↗

bioRxiv2022

A quantitative and site-specific atlas of the in vivo citrullinome reveals widespread existence of citrullination

Citrullination is the conversion of peptidyl-arginine into the non-coded amino acid citrulline. Despite its importance in physiology and disease, global identification of citrullinated proteins and precise modification sites has remained challenging. Here, we employed quantitative mass spectrometry-based proteomics to generate a comprehensive atlas of citrullination sites in a physiologically relevant cell type. Collectively, we identified 14.056 citrullination sites within 4.008 proteins and quantified their regulation upon inhibition of the citrullinating enzyme PADI4. Using this rich dataset, we uncover general mechanistic and cell biological principles of citrullination function, while providing site-specific and quantitative information on thousands of PAD4 substrates within cells. Our findings include signature histone marks and numerous modifications on transcriptional regulators and chromatin-related signaling effectors. Additionally, we identify precise citrullination sites on an extensive list of known autoantigens. Collectively, we describe systems attributes of the human citrullinome and provide a resource framework for understanding citrullinaiton at the mechanistic level.

molecular biology↗

bioRxiv2022

DAXX adds a de novo H3.3K9me3 deposition pathway to the histone chaperone network

A multitude of histone chaperones are required to protect histones after their biosynthesis until DNA deposition. They cooperate through the formation of co-chaperone complexes, but the crosstalk between nucleosome assembly pathways remains enigmatic. Using explorative interactomics approaches, we characterize the organization of the histone H3-H4 chaperones network and define the interplay between histone chaperone systems. We identify and validate several novel histone dependent complexes and predict the structure of the ASF1 and SPT2 co-chaperone complex, expanding the role of ASF1 in histone dynamics. We show that DAXX acts separately from the rest of the network, recruiting heterochromatin factors and promoting lysine 9 tri-methylation of new histone H3.3 prior to deposition onto DNA. With its functionality, DAXX provides a molecular mechanism for de novo heterochromatin assembly. Collectively, our findings provide a new framework for understanding how cells orchestrate histone supply and comply with chromatin dynamics throughout the cell cycle.

molecular biology↗

bioRxiv2021

Site-specific phosphorylation of PSD-95 dynamically regulates the post-synaptic density as observed by phase separation

Postsynaptic density protein 95 is a key scaffolding protein in the postsynaptic density of excitatory glutamatergic neurons, organizing signaling complexes primarily via its three PSD-95/Discs-large/Zona occludens domains. PSD-95 is regulated by phosphorylation, but technical challenges have limited studies of the molecular details. Here, we genetically introduced site-specific phosphorylations in single, tandem and full-length PSD-95 and generated a total of 11 phosphorylated protein variants. We examined how these phosphorylations affected binding to known interaction partners and the impact on phase separation of PSD-95 complexes, and identified two new phosphorylation sites with opposing effects. Phosphorylation of Ser78 inhibited phase separation with the glutamate receptor subunit GluN2B and the auxiliary protein stargazin, whereas phosphorylation of Ser116 induced phase separation with stargazin only. Thus, by genetically introducing phosphoserine site-specifically and exploring the impact on phase separation, we have provided new insights into the regulation of PSD-95 by phosphorylation and the dynamics of the PSD. Graphical Abstract: Phosphorylation of PSD-95. O_LIIllustration of the interaction between the PDZ domains of PSD-95 and the NMDA receptor (NMDAR) (PDB: 4PE5) at the plasma membrane. PSD-95 interacts with the AMPA receptor (AMPAR) (PDB: 3KG2) through Stargazin (PDB: 5KBU). Using the software Ranch, a model of FL PSD-95 was generated using known structural domains of PSD-95 connected via a fully-flexible linker (PDZ1-2, PDB: 3GSL; PDZ3, PDB: 5JXB; SH3-GK, PDB: 1KJW). C_LIO_LIIllustration of the effect of phosphorylation of PSD-95 on the formation of condensates at the PSD. PSD-95 can phase separate with receptors and intracellular proteins, and phosphorylation is can affect this interaction and lead to alterations, both negative and positive, in the formation of condensates. C_LI

biochemistry↗

bioRxiv2021

Waves of sumoylation support transcription dynamics during adipocyte differentiation

Tight control of gene expression networks required for adipose tissue formation and plasticity is essential for adaptation to energy needs and environmental cues. However, little is known about the mechanisms that orchestrate the dramatic transcriptional changes leading to adipocyte differentiation. We investigated the regulation of nascent transcription by the sumoylation pathway during adipocyte differentiation using SLAMseq and ChIPseq. We discovered that the sumoylation pathway has a dual function in differentiation; it supports the initial downregulation of pre-adipocyte-specific genes, while it promotes the establishment of the mature adipocyte transcriptional program. By characterizing sumoylome dynamics in differentiating adipocytes by mass spectrometry, we found that sumoylation of specific transcription factors like Ppar{gamma}/RXR and their co-factors is associated with the transcription of adipogenic genes. Our data demonstrate that the sumoylation pathway coordinates the rewiring of transcriptional networks required for formation of functional adipocytes. This study also provides an in-depth resource of gene transcription dynamics, SUMO-regulated genes and sumoylation sites during adipogenesis.

genomics↗

bioRxiv2021

The regulatory landscape of the human HPF1- and ARH3-dependent ADP-ribosylome

Despite the involvement of Poly(ADP-ribose) polymerase-1 (PARP1) in many important biological pathways, the target residues of PARP1-mediated ADP-ribosylation remain ambiguous. To explicate the ADP-ribosylation regulome, we analyzed human cells depleted for key regulators of PARP1 activity, histone PARylation factor 1 (HPF1) and ADP-ribosylhydrolase 3 (ARH3). Using quantitative proteomics, we quantified 1,596 ADPr sites, displaying a thousand-fold regulation across investigated knockout cells. We find that HPF1 and ARH3 inversely and homogenously regulate the serine ADP-ribosylome on a proteome-wide scale with consistent adherence to lysine-serine (KS)-motifs suggesting targeting is independent of HPF1 and ARH3. Our data reveal that ADPr globally exists as mono-ADP-ribosylation, and we detail a remarkable degree of histone co-modification with ADPr and other post-translational modifications. Strikingly, no HPF1-dependent target residue switch from serine to glutamate/aspartate was detectable in cells, which challenges current dogma related to PARP1 target residues. Collectively, we elucidate hitherto unappreciated processes related to cellular PARP1 activity.

molecular biology↗

bioRxiv2020

Mapping physiological ADP-ribosylation using Activated Ion Electron Transfer Dissociation (AI-ETD)

ADP-ribosylation (ADPr) is a post-translational modification that plays pivotal roles in a wide range of cellular processes. Mass spectrometry (MS)-based analysis of ADPr under physiological conditions, without relying on genetic or chemical perturbation, has been hindered by technical limitations. Here, we describe the applicability of Activated Ion Electron Transfer Dissociation (AI-ETD) for MS-based proteomics analysis of physiological ADPr using our unbiased Af1521 enrichment strategy. To benchmark AI-ETD, we profiled 9,000 ADPr peptides mapping to >5,000 unique ADPr sites from a limited number of cells exposed to oxidative stress, corresponding to 120% and 28% more ADPr peptides compared to contemporary strategies using ETD and EThcD, respectively. Under physiological conditions AI-ETD identified 450 ADPr sites on low-abundant proteins, including in vivo cysteine auto-modifications on PARP8 and tyrosine auto-modifications on PARP14, hinting at specialist enzymatic functions for these enzymes. Collectively, our data provides new insights into the physiological regulation of ADP-ribosylation.

molecular biology↗

bioRxiv2019

Interaction of Sox2 with RNA binding proteins in mouse embryonic stem cells

Sox2 is a master transcriptional regulator of embryonic development. In this study, we determined the protein interactome of Sox2 in the chromatin and nucleoplasm of mouse embryonic stem (mES) cells. Apart from canonical interactions with pluripotency-regulating transcription factors, we identified interactions with several chromatin modulators, including members of the heterochromatin protein 1 (HP1) family, suggesting a role of Sox2 in chromatin-mediated transcriptional repression. Sox2 was also found to interact with RNA binding proteins (RBPs), including proteins involved in RNA processing. RNA immunoprecipitation followed by sequencing revealed that Sox2 associates with different messenger RNAs, as well as small nucleolar RNA Snord34 and the non-coding RNA 7SK. 7SK has been shown to regulate transcription at regulatory regions, which could suggest a functional interaction with Sox2 for chromatin recruitment. Nevertheless, we found no evidence of Sox2 modulating recruitment of 7SK to chromatin when examining 7SK chromatin occupancy by Chromatin Isolation by RNA Purification (ChIRP) in Sox2 depleted mES cells. In addition, knockdown of 7SK in mES cells did not lead to any change in Sox2 occupancy at 7SK-regulated genes. Thus, our results show that Sox2 extensively interact with RBPs, and suggest that Sox2 and 7SK co-exist in a ribonucleoprotein complex whose function is not to regulate chromatin recruitment, but might rather regulate other processes in the nucleoplasm. Summary blurbSox2 interacts with RNA-binding proteins and diverse RNAs

molecular biology↗

bioRxiv2018

An advanced strategy for comprehensive profiling of ADP-ribosylation sites using mass spectrometry-based proteomics

ADP-ribosylation is a widespread post-translational modification (PTM) with crucial functions in many cellular processes. Here, we describe an in-depth ADP-ribosylome using our Af1521-based proteomics methodology for comprehensive profiling of ADP-ribosylation sites, by systematically assessing complementary proteolytic digestions and precursor fragmentation through application of electron-transfer higher-energy collisional dissociation (EThcD) and electron transfer dissociation (ETD), respectively. While ETD spectra yielded higher identification scores, EThcD generally proved superior to ETD in identification and localization of ADP-ribosylation sites regardless of protease employed. Notwithstanding, the propensities of complementary proteases and fragmentation methods expanded the detectable repertoire of ADP-ribosylation to an unprecedented depth. This system-wide profiling of the ADP-ribosylome in HeLa cells subjected to DNA damage uncovered >11,000 unique ADP-ribosylated peptides mapping to >7,000 ADP-ribosylation sites, in total modifying over one-third of the human nuclear proteome and highlighting the vast scope of this PTM. High-resolution MS/MS spectra enabled identification of dozens of proteins concomitantly modified by ADP-ribosylation and phosphorylation, revealing a considerable degree of crosstalk on histones. ADP-ribosylation was confidently localized to various amino acid residue types, including less abundantly modified residues, with hundreds of ADP-ribosylation sites pinpointed on histidine, arginine, and tyrosine residues. Functional enrichment analysis suggested modification of these specific residue types is directed in a spatial manner, with tyrosine ADP-ribosylation linked to the ribosome, arginine ADP-ribosylation linked to the endoplasmic reticulum, and histidine ADP-ribosylation linked to the mitochondrion.

molecular biology↗

bioRxiv2018

PADI2-mediated citrullination is required for efficient oligodendrocyte differentiation and myelination

Citrullination, the deimination of arginine residues into citrulline, has been implicated in the aetiology of several diseases. In multiple sclerosis (MS), citrullination is thought to be a major driver of pathology, through hypercitrullination and destabilization of myelin. As such, inhibition of citrullination has been suggested as a therapeutic strategy for MS. Here, in contrast, we show citrullination by peptidylarginine deiminase 2 (PADI2) is required for normal oligodendrocyte differentiation, myelination and motor function. We identify several targets for PADI2, including myelin-related proteins and chromatin-associated proteins, implicating PADI2 in epigenetic regulation. Accordingly, we observe that PADI2 inhibition and its knockdown affect chromatin accessibility and prevent the upregulation of oligodendrocyte differentiation genes. Moreover, mice lacking PADI2, display motor dysfunction and decreased number of myelinated axons in the corpus callosum. We conclude that citrullination is required for oligodendrocyte lineage progression and myelination and suggest its targeted activation in the oligodendrocyte lineage might be beneficial in the context of remyelination.

neuroscience↗