Interfering with aggregated α-synuclein in advanced melanoma leads to a major upregulation of MHC class II proteins.

Melanoma is the most serious and deadly form of skin cancer and with progression to advanced melanoma, the intrinsically disordered protein α-synuclein is upregulated to high levels. While toxic to dopaminergic neurons in Parkinson's disease, α-synuclein is highly beneficial for primary and metastatic melanoma cells. To gain detailed insights into this exact opposite role of α-synuclein in advanced melanoma, we performed proteomic studies of high-level α-synuclein-expressing human melanoma cell lines that were treated with the diphenyl-pyrazole small-molecule compound anle138b, which binds to and interferes with the oligomeric structure of α-synuclein. We also performed proteomic and transcriptomic studies of human melanoma xenografts that were treated systemically with the anle138b compound. The results reveal that interfering with oligomerized α-synuclein in the melanoma cells in these tumor xenografts led to a substantial upregulation and expression of major histocompatibility complex proteins, which are pertinent to enhancing anti-melanoma immune responses.

Phosphatases modified by LH signaling in ovarian follicles: testing their role in regulating the NPR2 guanylyl cyclase†.

In response to luteinizing hormone (LH), multiple proteins in rat and mouse granulosa cells are rapidly dephosphorylated, but the responsible phosphatases remain to be identified. Because the phosphorylation state of phosphatases can regulate their interaction with substrates, we searched for phosphatases that might function in LH signaling by using quantitative mass spectrometry. We identified all proteins in rat ovarian follicles whose phosphorylation state changed detectably in response to a 30-min exposure to LH, and within this list, identified protein phosphatases or phosphatase regulatory subunits that showed changes in phosphorylation. Phosphatases in the phosphoprotein phosphatase (PPP) family were of particular interest because of their requirement for dephosphorylating the natriuretic peptide receptor 2 (NPR2) guanylyl cyclase in the granulosa cells, which triggers oocyte meiotic resumption. Among the PPP family regulatory subunits, PPP1R12A and PPP2R5D showed the largest increases in phosphorylation, with 4-10 fold increases in signal intensity on several sites. Although follicles from mice in which these phosphorylations were prevented by serine-to-alanine mutations in either Ppp1r12a or Ppp2r5d showed normal LH-induced NPR2 dephosphorylation, these regulatory subunits and others could act redundantly to dephosphorylate NPR2. Our identification of phosphatases and other proteins whose phosphorylation state is rapidly modified by LH provides clues about multiple signaling pathways in ovarian follicles.

FAT10 is phosphorylated by IKKß to inhibit the antiviral type-I interferon response.

IFN-I secretion provides a rapid host defense against infection with RNA viruses. Within the host cell, viral RNA triggers the activation of the RIG-I signaling pathway, leading to the production of IFN-I. Because an exaggerated IFN-I response causes severe tissue damage, RIG-I signaling is tightly regulated. One of the factors that control the IFN-I response is the ubiquitin-like modifier FAT10, which is induced by TNF and IFNγ and targets covalently FAT10-linked proteins for proteasomal degradation. However, the mechanism of how FAT10 modulates IFN-I secretion remains to be fully elucidated. Here, we provide strong evidence that FAT10 is phosphorylated by IκB kinase ß (IKKß) upon TNF stimulation and during influenza A virus infection on several serine and threonine residues. FAT10 phosphorylation increases the binding of FAT10 to the TRAF3-deubiquitylase OTUB1 and its FAT10-mediated activation. Consequently, FAT10 phosphorylation results in a low ubiquitylation state of TRAF3, which is unable to maintain interferon regulatory factor 3 phosphorylation and downstream induction of IFN-I. Taken together, we reveal a mechanism of how phosphorylation of FAT10 limits the production of tissue-destructive IFN-I in inflammation.

Metabolic switch from fatty acid oxidation to glycolysis in knock-in mouse model of Barth syndrome.

Mitochondria are central for cellular metabolism and energy supply. Barth syndrome (BTHS) is a severe disorder, due to dysfunction of the mitochondrial cardiolipin acyl transferase tafazzin. Altered cardiolipin remodeling affects mitochondrial inner membrane organization and function of membrane proteins such as transporters and the oxidative phosphorylation (OXPHOS) system. Here, we describe a mouse model that carries a G197V exchange in tafazzin, corresponding to BTHS patients. TAZG197V mice recapitulate disease-specific pathology including cardiac dysfunction and reduced oxidative phosphorylation. We show that mutant mitochondria display defective fatty acid-driven oxidative phosphorylation due to reduced levels of carnitine palmitoyl transferases. A metabolic switch in ATP production from OXPHOS to glycolysis is apparent in mouse heart and patient iPSC cell-derived cardiomyocytes. An increase in glycolytic ATP production inactivates AMPK causing altered metabolic signaling in TAZG197V . Treatment of mutant cells with AMPK activator reestablishes fatty acid-driven OXPHOS and protects mice against cardiac dysfunction.

A viral ADP-ribosyltransferase attaches RNA chains to host proteins.

The mechanisms by which viruses hijack the genetic machinery of the cells they infect are of current interest. When bacteriophage T4 infects Escherichia coli, it uses three different adenosine diphosphate (ADP)-ribosyltransferases (ARTs) to reprogram the transcriptional and translational apparatus of the host by ADP-ribosylation using nicotinamide adenine dinucleotide (NAD) as a substrate1,2. NAD has previously been identified as a 5' modification of cellular RNAs3-5. Here we report that the T4 ART ModB accepts not only NAD but also NAD-capped RNA (NAD-RNA) as a substrate and attaches entire RNA chains to acceptor proteins in an 'RNAylation' reaction. ModB specifically RNAylates the ribosomal proteins rS1 and rL2 at defined Arg residues, and selected E. coli and T4 phage RNAs are linked to rS1 in vivo. T4 phages that express an inactive mutant of ModB have a decreased burst size and slowed lysis of E. coli. Our findings reveal a distinct biological role for NAD-RNA, namely the activation of the RNA for enzymatic transfer to proteins. The attachment of specific RNAs to ribosomal proteins might provide a strategy for the phage to modulate the host's translation machinery. This work reveals a direct connection between RNA modification and post-translational protein modification. ARTs have important roles far beyond viral infections6, so RNAylation may have far-reaching implications.

Proteomic analysis of the human hippocampus identifies neuronal pentraxin 1 (NPTX1) as synapto-axonal target in late-stage Parkinson's disease.

Parkinson's disease (PD) affects a significant proportion of the population over the age of 60 years, and its prevalence is increasing. While symptomatic treatment is available for motor symptoms of PD, non-motor complications such as dementia result in diminished life quality for patients and are far more difficult to treat. In this study, we analyzed PD-associated alterations in the hippocampus of PD patients, since this brain region is strongly affected by PD dementia. We focused on synapses, analyzing the proteome of post-mortal hippocampal tissue from 16 PD cases and 14 control subjects by mass spectrometry. Whole tissue lysates and synaptosomal fractions were analyzed in parallel. Differential analysis combined with bioinformatic network analyses identified neuronal pentraxin 1 (NPTX1) to be significantly dysregulated in PD and interacting with proteins of the synaptic compartment. Modulation of NPTX1 protein levels in primary hippocampal neuron cultures validated its role in synapse morphology. Our analysis suggests that NPTX1 contributes to synaptic pathology in late-stage PD and represents a putative target for novel therapeutic strategies.

Phosphatases modified by LH signaling in ovarian follicles: testing their role in regulating the NPR2 guanylyl cyclase.

In response to luteinizing hormone, multiple proteins in rat and mouse granulosa cells are rapidly dephosphorylated, but the responsible phosphatases remain to be identified. Because the phosphorylation state of phosphatases can regulate their interaction with substrates, we searched for phosphatases that might function in LH signaling by using quantitative mass spectrometry. We identified all proteins in rat ovarian follicles whose phosphorylation state changed detectably in response to a 30-minute exposure to LH, and within this list, identified protein phosphatases or phosphatase regulatory subunits that showed changes in phosphorylation. Phosphatases in the PPP family were of particular interest because of their requirement for dephosphorylating the natriuretic peptide receptor 2 (NPR2) guanylyl cyclase in the granulosa cells, which triggers oocyte meiotic resumption. Among the PPP family regulatory subunits, PPP1R12A and PPP2R5D showed the largest increases in phosphorylation, with 4-10 fold increases in signal intensity on several sites. Although follicles from mice in which these phosphorylations were prevented by serine-to-alanine mutations in either Ppp1r12a or Ppp2r5d showed normal LH-induced NPR2 dephosphorylation, these regulatory subunits and others could act redundantly to dephosphorylate NPR2. Our identification of phosphatases and other proteins whose phosphorylation state is rapidly modified by LH provides clues about multiple signaling pathways in ovarian follicles. Summary sentence: Quantitative mass spectrometric analysis of phosphatases whose phosphorylation state is rapidly modified by luteinizing hormone provides clues about how LH signaling dephosphorylates NPR2 as well as a resource for future studies.

Npl3 functions in mRNP assembly by recruitment of mRNP components to the transcription site and their transfer onto the mRNA.

RNA-binding proteins (RBPs) control every RNA metabolic process by multiple protein-RNA and protein-protein interactions. Their roles have largely been analyzed by crude mutations, which abrogate multiple functions at once and likely impact the structural integrity of the large ribonucleoprotein particles (RNPs) these proteins function in. Using UV-induced RNA-protein crosslinking of entire cells, protein complex purification and mass spectrometric analysis, we identified >100 in vivo RNA crosslinks in 16 nuclear mRNP components in Saccharomyces cerevisiae. For functional analysis, we chose Npl3, which displayed crosslinks in its two RNA recognition motifs (RRMs) and in the connecting flexible linker region. Both RRM domains and the linker uniquely contribute to RNA recognition as revealed by NMR and structural analyses. Interestingly, mutations in these regions cause different phenotypes, indicating distinct functions of the different RNA-binding domains. Notably, an npl3-Linker mutation strongly impairs recruitment of several mRNP components to chromatin and incorporation of other mRNP components into nuclear mRNPs, establishing a so far unknown function of Npl3 in nuclear mRNP assembly. Taken together, our integrative analysis uncovers a specific function of the RNA-binding activity of the nuclear mRNP component Npl3. This approach can be readily applied to RBPs in any RNA metabolic process.

Extracellular vesicles derived from the choroid plexus trigger the differentiation of neural stem cells.

The choroid plexus secrets cerebrospinal fluid (CSF) composed of electrolytes, cytokines, growth factors, metabolites and extracellular vesicles (EVs) that flow through the interconnected brain ventricles. On their course, CSF components can act as signals that affect, for example, neural stem cells (NSCs) residing in niches of the ventricular wall. We studied EV-born CSF signals in an in vitro culture system. We purified EVs from the secretome of a choroid plexus cell line (Z310 cells), and from primary choroid plexus cultures and co-cultured those EVs with NSCs isolated from the niche of the lateral and the third ventricle. EVsZ310 and EVsCHP were purified by differential centrifugation. This yielded fractions of EVs of 50-150-nm diameter that induced a complex multicellular network formation and NSC differentiation. Both types of EV converted the round NSCs to cells that extended long processes that contacted nearby, alike-shaped cells. Mass spectrometry showed that the differentiation-inducing EVZ310 were enriched for membrane and membrane-associated proteins involved in cell differentiation, membrane trafficking, and membrane organization. We hypothesize that this type of EV Z310 cargo causes changes of stem cell morphology that leads to multicellular networks in the niches. This cell-shape transition may represent an initial step in NSC differentiation.

Colocalization of different neurotransmitter transporters on synaptic vesicles is sparse except for VGLUT1 and ZnT3.

Vesicular transporters (VTs) define the type of neurotransmitter that synaptic vesicles (SVs) store and release. While certain mammalian neurons release multiple transmitters, it is not clear whether the release occurs from the same or distinct vesicle pools at the synapse. Using quantitative single-vesicle imaging, we show that a vast majority of SVs in the rodent brain contain only one type of VT, indicating specificity for a single neurotransmitter. Interestingly, SVs containing dual transporters are highly diverse (27 types) but small in proportion (2% of all SVs), excluding the largest pool that carries VGLUT1 and ZnT3 (34%). Using VGLUT1-ZnT3 SVs, we demonstrate that the transporter colocalization influences the SV content and synaptic quantal size. Thus, the presence of diverse transporters on the same vesicle is bona fide, and depending on the VT types, this may act to regulate neurotransmitter type, content, and release in space and time.

Low-Expressing Synucleinopathy Mouse Models Based on Oligomer-Forming Mutations and C-Terminal Truncation of α-Synuclein.

α-synuclein (αSyn) is the main protein component of Lewy bodies, intracellular inclusions found in the brain of Parkinson's disease (PD) patients. Neurotoxic αSyn species are broadly modified post-translationally and, in patients with genetic forms of PD, carry genetically encoded amino acid substitutions. Mutations and C-terminal truncation can increase αSyn oligomerization and fibrillization. Although several genetic mouse models based on αSyn mutations and/or truncations exist, there is still a lack of mouse models for synucleinopathies not relying on overexpression. We report here two synucleinopathy mouse models, which are based on a triple alanine to proline mutation and a C-terminal truncation of αSyn, but do not overexpress the mutant protein when compared to the endogenous mouse protein. We knocked hαSyn TP or hαSynΔ119 (h stands for "human") into the murine αSyn locus. hαSynTP is a structure-based mutant with triple alanine to proline substitutions that favors oligomers, is neurotoxic and evokes PD-like symptoms in Drosophila melanogaster. hαSynΔ119 lacks 21 amino acids at the C-terminus, favors fibrillary aggregates and occurs in PD. Knocking-in of hαSyn TP or hαSynΔ119 into the murine αSyn locus places the mutant protein under the control of the endogenous regulatory elements while simultaneously disrupting the mαSyn gene. Mass spectrometry revealed that hαSyn TP and hαSynΔ119 mice produced 12 and 10 times less mutant protein, compared to mαSyn in wild type mice. We show phenotypes in 1 and 1.5 years old hαSyn TP and hαSynΔ119 mice, despite the lower levels of hαSynTP and hαSynΔ119 expression. Direct comparison of the two mouse models revealed many commonalities but also aspects unique to each model. Commonalities included strong immunoactive state, impaired olfaction and motor coordination deficits. Neither model showed DAergic neuronal loss. Impaired climbing abilities at 1 year of age and a deviant gait pattern at 1.5 years old were specific for hαSynΔ119 mice, while a compulsive behavior was exclusively detected in hαSyn TP mice starting at 1 year of age. We conclude that even at very moderate levels of expression the two αSyn variants evoke measurable and progressive deficiencies in mutant mice. The two transgenic mouse models can thus be suitable to study αSyn-variant-based pathology in vivo and test new therapeutic approaches.

Evaluation and Optimization of High-Field Asymmetric Waveform Ion-Mobility Spectrometry for Multiplexed Quantitative Site-Specific N-Glycoproteomics.

The heterogeneity and complexity of glycosylation hinder the depth of site-specific glycoproteomics analysis. High-field asymmetric-waveform ion-mobility spectrometry (FAIMS) has been shown to improve the scope of bottom-up proteomics. The benefits of FAIMS for quantitative N-glycoproteomics have not been investigated yet. In this work, we optimized FAIMS settings for N-glycopeptide identification, with or without the tandem mass tag (TMT) label. The optimized FAIMS approach significantly increased the identification of site-specific N-glycopeptides derived from the purified immunoglobulin M (IgM) protein or human lymphoma cells. We explored in detail the changes in FAIMS mobility caused by N-glycopeptides with different characteristics, including TMT labeling, charge state, glycan type, peptide sequence, glycan size, and precursor m/z. Importantly, FAIMS also improved multiplexed N-glycopeptide quantification, both with the standard MS2 acquisition method and with our recently developed Glyco-SPS-MS3 method. The combination of FAIMS and Glyco-SPS-MS3 methods provided the highest quantitative accuracy and precision. Our results demonstrate the advantages of FAIMS for improved mass spectrometry-based qualitative and quantitative N-glycoproteomics.

Relative Quantification of Phosphorylated and Glycosylated Peptides from the Same Sample Using Isobaric Chemical Labelling with a Two-Step Enrichment Strategy.

Post-translational modifications (PTMs) are essential for the regulation of all cellular processes. The interplay of various PTMs on a single protein or different proteins comprises a complexity that we are far from understanding in its entirety. Reliable strategies for the enrichment and accurate quantification of PTMs are needed to study as many PTMs on proteins as possible. In this protocol we present a liquid chromatography-tandem mass spectrometry (LC/MS/MS)-based workflow that enables the enrichment and quantification of phosphorylated and N-glycosylated peptides from the same sample. After extraction and digestion of proteins, we label the peptides with stable isotope-coded tandem mass tags (TMTs) and enrich N-glycopeptides and phosphopeptides by using zwitterionic hydrophilic interaction chromatography (ZIC-HILIC) and titanium dioxide (TiO2) beads, respectively. Labelled and enriched N-glycopeptides and phosphopeptides are further separated by high pH (basic) reversed-phase chromatography and analyzed by LC/MS/MS. The enrichment strategies, together with quantification of two different PTM types from the same sample, allow investigation of the interplay of those two PTMs, which are important for signal transduction inside the cell (phosphorylation), as well as for messaging between cells through decoration of the cellular surface (glycosylation).

Protein Phosphorylation in Depolarized Synaptosomes: Dissecting Primary Effects of Calcium from Synaptic Vesicle Cycling.

Synaptic transmission is mediated by the regulated exocytosis of synaptic vesicles. When the presynaptic membrane is depolarized by an incoming action potential, voltage-gated calcium channels open, resulting in the influx of calcium ions that triggers the fusion of synaptic vesicles (SVs) with the plasma membrane. SVs are recycled by endocytosis. Phosphorylation of synaptic proteins plays a major role in these processes, and several studies have shown that the synaptic phosphoproteome changes rapidly in response to depolarization. However, it is unclear which of these changes are directly linked to SV cycling and which might regulate other presynaptic functions that are also controlled by calcium-dependent kinases and phosphatases. To address this question, we analyzed changes in the phosphoproteome using rat synaptosomes in which exocytosis was blocked with botulinum neurotoxins (BoNTs) while depolarization-induced calcium influx remained unchanged. BoNT-treatment significantly alters the response of the synaptic phoshoproteome to depolarization and results in reduced phosphorylation levels when compared with stimulation of synaptosomes by depolarization with KCl alone. We dissect the primary Ca2+-dependent phosphorylation from SV-cycling-dependent phosphorylation and confirm an effect of such SV-cycling-dependent phosphorylation events on syntaxin-1a-T21/T23, synaptobrevin-S75, and cannabinoid receptor-1-S314/T322 on exo- and endocytosis in cultured hippocampal neurons.

Post-translational modifications soften vimentin intermediate filaments.

The mechanical properties of biological cells are determined by the cytoskeleton, a composite biopolymer network consisting of microtubules, actin filaments and intermediate filaments (IFs). By differential expression of cytoskeletal proteins, modulation of the network architecture and interactions between the filaments, cell mechanics may be adapted to varying requirements on the cell. Here, we focus on the intermediate filament protein vimentin and introduce post-translational modifications as an additional, much faster mechanism for mechanical modulation. We study the impact of phosphorylation on filament mechanics by recording force-strain curves using optical traps. Partial phosphorylation softens the filaments. We show that binding of the protein 14-3-3 to phosphorylated vimentin IFs further enhances this effect and speculate that in the cell 14-3-3 may serve to preserve the softening and thereby the altered cell mechanics. We explain our observation by the additional charges introduced during phosphorylation.

A streamlined pipeline for multiplexed quantitative site-specific N-glycoproteomics.

Regulation of protein N-glycosylation is essential in human cells. However, large-scale, accurate, and site-specific quantification of glycosylation is still technically challenging. We here introduce SugarQuant, an integrated mass spectrometry-based pipeline comprising protein aggregation capture (PAC)-based sample preparation, multi-notch MS3 acquisition (Glyco-SPS-MS3) and a data-processing tool (GlycoBinder) that enables confident identification and quantification of intact glycopeptides in complex biological samples. PAC significantly reduces sample-handling time without compromising sensitivity. Glyco-SPS-MS3 combines high-resolution MS2 and MS3 scans, resulting in enhanced reporter signals of isobaric mass tags, improved detection of N-glycopeptide fragments, and lowered interference in multiplexed quantification. GlycoBinder enables streamlined processing of Glyco-SPS-MS3 data, followed by a two-step database search, which increases the identification rates of glycopeptides by 22% compared with conventional strategies. We apply SugarQuant to identify and quantify more than 5,000 unique glycoforms in Burkitt's lymphoma cells, and determine site-specific glycosylation changes that occurred upon inhibition of fucosylation at high confidence.