Mitochondrial NAD+ Controls Nuclear ARTD1-Induced ADP-Ribosylation.

In addition to its role as an electron transporter, mitochondrial nicotinamide adenine dinucleotide (NAD+) is an important co-factor for enzymatic reactions, including ADP-ribosylation. Although mitochondria harbor the most intra-cellular NAD+, mitochondrial ADP-ribosylation remains poorly understood. Here we provide evidence for mitochondrial ADP-ribosylation, which was identified using various methodologies including immunofluorescence, western blot, and mass spectrometry. We show that mitochondrial ADP-ribosylation reversibly increases in response to respiratory chain inhibition. Conversely, H2O2-induced oxidative stress reciprocally induces nuclear and reduces mitochondrial ADP-ribosylation. Elevated mitochondrial ADP-ribosylation, in turn, dampens H2O2-triggered nuclear ADP-ribosylation and increases MMS-induced ARTD1 chromatin retention. Interestingly, co-treatment of cells with the mitochondrial uncoupler FCCP decreases PARP inhibitor efficacy. Together, our results suggest that mitochondrial ADP-ribosylation is a dynamic cellular process that impacts nuclear ADP-ribosylation and provide evidence for a NAD+-mediated mitochondrial-nuclear crosstalk.

PCfun: a hybrid computational framework for systematic characterization of protein complex function.

In molecular biology, it is a general assumption that the ensemble of expressed molecules, their activities and interactions determine biological function, cellular states and phenotypes. Stable protein complexes-or macromolecular machines-are, in turn, the key functional entities mediating and modulating most biological processes. Although identifying protein complexes and their subunit composition can now be done inexpensively and at scale, determining their function remains challenging and labor intensive. This study describes Protein Complex Function predictor (PCfun), the first computational framework for the systematic annotation of protein complex functions using Gene Ontology (GO) terms. PCfun is built upon a word embedding using natural language processing techniques based on 1 million open access PubMed Central articles. Specifically, PCfun leverages two approaches for accurately identifying protein complex function, including: (i) an unsupervised approach that obtains the nearest neighbor (NN) GO term word vectors for a protein complex query vector and (ii) a supervised approach using Random Forest (RF) models trained specifically for recovering the GO terms of protein complex queries described in the CORUM protein complex database. PCfun consolidates both approaches by performing a hypergeometric statistical test to enrich the top NN GO terms within the child terms of the GO terms predicted by the RF models. The documentation and implementation of the PCfun package are available at https://github.com/sharmavaruns/PCfun. We anticipate that PCfun will serve as a useful tool and novel paradigm for the large-scale characterization of protein complex function.

Diagnostics and correction of batch effects in large-scale proteomic studies: a tutorial.

Advancements in mass spectrometry-based proteomics have enabled experiments encompassing hundreds of samples. While these large sample sets deliver much-needed statistical power, handling them introduces technical variability known as batch effects. Here, we present a step-by-step protocol for the assessment, normalization, and batch correction of proteomic data. We review established methodologies from related fields and describe solutions specific to proteomic challenges, such as ion intensity drift and missing values in quantitative feature matrices. Finally, we compile a set of techniques that enable control of batch effect adjustment quality. We provide an R package, "proBatch", containing functions required for each step of the protocol. We demonstrate the utility of this methodology on five proteomic datasets each encompassing hundreds of samples and consisting of multiple experimental designs. In conclusion, we provide guidelines and tools to make the extraction of true biological signal from large proteomic studies more robust and transparent, ultimately facilitating reliable and reproducible research in clinical proteomics and systems biology.

Reproducible Determination of High-Density Lipoprotein Proteotypes.

High-density lipoprotein (HDL) is a heterogeneous mixture of blood-circulating multimolecular particles containing many different proteins, lipids, and RNAs. Recent advancements in mass spectrometry-based proteotype analysis show promise for the analysis of proteoforms across large patient cohorts. In order to create the required spectral libraries enabling these data-independent acquisition (DIA) strategies, HDL was isolated from the plasma of more than 300 patients with a multiplicity of physiological HDL states. HDL proteome spectral libraries consisting of 296 protein groups and more than 786 peptidoforms were established, and the performance of the DIA strategy was benchmarked for the detection of HDL proteotype differences between healthy individuals and a cohort of patients suffering from diabetes mellitus type 2 and/or coronary heart disease. Bioinformatic interrogation of the data using the generated spectral libraries showed that the DIA approach enabled robust HDL proteotype determination. HDL peptidoform analysis enabled by using spectral libraries allowed for the identification of post-translational modifications, such as in APOA1, which could affect HDL functionality. From a technical point of view, data analysis further shows that protein and peptide quantities are currently more discriminative between different HDL proteotypes than peptidoforms without further enrichment. Together, DIA-based HDL proteotyping enables the robust digitization of HDL proteotypes as a basis for the analysis of larger clinical cohorts.

Statistical control of peptide and protein error rates in large-scale targeted data-independent acquisition analyses.

Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) is the main method for high-throughput identification and quantification of peptides and inferred proteins. Within this field, data-independent acquisition (DIA) combined with peptide-centric scoring, as exemplified by the technique SWATH-MS, has emerged as a scalable method to achieve deep and consistent proteome coverage across large-scale data sets. We demonstrate that statistical concepts developed for discovery proteomics based on spectrum-centric scoring can be adapted to large-scale DIA experiments that have been analyzed with peptide-centric scoring strategies, and we provide guidance on their application. We show that optimal tradeoffs between sensitivity and specificity require careful considerations of the relationship between proteins in the samples and proteins represented in the spectral library. We propose the application of a global analyte constraint to prevent the accumulation of false positives across large-scale data sets. Furthermore, to increase the quality and reproducibility of published proteomic results, well-established confidence criteria should be reported for the detected peptide queries, peptides and inferred proteins.

The SysteMHC Atlas project.

Mass spectrometry (MS)-based immunopeptidomics investigates the repertoire of peptides presented at the cell surface by major histocompatibility complex (MHC) molecules. The broad clinical relevance of MHC-associated peptides, e.g. in precision medicine, provides a strong rationale for the large-scale generation of immunopeptidomic datasets and recent developments in MS-based peptide analysis technologies now support the generation of the required data. Importantly, the availability of diverse immunopeptidomic datasets has resulted in an increasing need to standardize, store and exchange this type of data to enable better collaborations among researchers, to advance the field more efficiently and to establish quality measures required for the meaningful comparison of datasets. Here we present the SysteMHC Atlas (https://systemhcatlas.org), a public database that aims at collecting, organizing, sharing, visualizing and exploring immunopeptidomic data generated by MS. The Atlas includes raw mass spectrometer output files collected from several laboratories around the globe, a catalog of context-specific datasets of MHC class I and class II peptides, standardized MHC allele-specific peptide spectral libraries consisting of consensus spectra calculated from repeat measurements of the same peptide sequence, and links to other proteomics and immunology databases. The SysteMHC Atlas project was created and will be further expanded using a uniform and open computational pipeline that controls the quality of peptide identifications and peptide annotations. Thus, the SysteMHC Atlas disseminates quality controlled immunopeptidomic information to the public domain and serves as a community resource toward the generation of a high-quality comprehensive map of the human immunopeptidome and the support of consistent measurement of immunopeptidomic sample cohorts.

Protein degradation and dynamic tRNA thiolation fine-tune translation at elevated temperatures.

Maintenance of protein quality control has implications in various processes such as neurodegeneration and ageing. To investigate how environmental insults affect this process, we analysed the proteome of yeast continuously exposed to mild heat stress. In agreement with previous transcriptomics studies, amongst the most marked changes, we found up-regulation of cytoprotective factors; a shift from oxidative phosphorylation to fermentation; and down-regulation of translation. Importantly, we also identified a novel, post-translationally controlled, component of the heat shock response. The abundance of Ncs2p and Ncs6p, two members of the URM1 pathway responsible for the thiolation of wobble uridines in cytoplasmic tRNAs tK(UUU), tQ(UUG) and tE(UUC), is down-regulated in a proteasomal dependent fashion. Using random forests we show that this results in differential translation of transcripts with a biased content for the corresponding codons. We propose that the role of this pathway in promoting catabolic and inhibiting anabolic processes, affords cells with additional time and resources needed to attain proper protein folding under periods of stress.

tRNA tKUUU, tQUUG, and tEUUC wobble position modifications fine-tune protein translation by promoting ribosome A-site binding.

tRNA modifications are crucial to ensure translation efficiency and fidelity. In eukaryotes, the URM1 and ELP pathways increase cellular resistance to various stress conditions, such as nutrient starvation and oxidative agents, by promoting thiolation and methoxycarbonylmethylation, respectively, of the wobble uridine of cytoplasmic (tK(UUU)), (tQ(UUG)), and (tE(UUC)). Although in vitro experiments have implicated these tRNA modifications in modulating wobbling capacity and translation efficiency, their exact in vivo biological roles remain largely unexplored. Using a combination of quantitative proteomics and codon-specific translation reporters, we find that translation of a specific gene subset enriched for AAA, CAA, and GAA codons is impaired in the absence of URM1- and ELP-dependent tRNA modifications. Moreover, in vitro experiments using native tRNAs demonstrate that both modifications enhance binding of tK(UUU) to the ribosomal A-site. Taken together, our data suggest that tRNA thiolation and methoxycarbonylmethylation regulate translation of genes with specific codon content.

Activation of the canonical IKK complex by K63/M1-linked hybrid ubiquitin chains.

Polyubiquitin (pUb) chains formed between the C terminus of ubiquitin and lysine 63 (K63) or methionine 1 (M1) of another ubiquitin have been implicated in the activation of the canonical IκB kinase (IKK) complex. Here, we demonstrate that nearly all of the M1-pUb chains formed in response to interleukin-1, or the Toll-Like Receptors 1/2 agonist Pam3CSK4, are covalently attached to K63-pUb chains either directly as K63-pUb/M1-pUb hybrids or indirectly by attachment to the same protein. Interleukin-1 receptor (IL-1R)-associated kinase (IRAK) 1 is modified first by K63-pUb chains to which M1-pUb linkages are added subsequently, and myeloid differentiation primary response gene 88 (MyD88) and IRAK4 are also modified by both K63-pUb and M1-pUb chains. We show that the heme-oxidized IRP2 ubiquitin ligase 1 interacting protein (HOIP) component of the linear ubiquitin assembly complex catalyzes the formation of M1-pUb chains in response to interleukin-1, that the formation of K63-pUb chains is a prerequisite for the formation of M1-pUb chains, and that HOIP interacts with K63-pUb but not M1-pUb linkages. These findings identify K63-Ub oligomers as a major substrate of HOIP in cells where the MyD88-dependent signaling network is activated. The TGF-beta-activated kinase 1 (TAK1)-binding protein (TAB) 2 and TAB3 components of the TAK1 complex and the NFκB Essential Modifier (NEMO) component of the canonical IKK complex bind to K63-pUb chains and M1-pUb chains, respectively. The formation of K63/M1-pUb hybrids may therefore provide an elegant mechanism for colocalizing both complexes to the same pUb chain, facilitating the TAK1-catalyzed activation of IKKα and IKKß. Our study may help to resolve the debate about the relative importance of K63-pUb and M1-pUb chains in activating the canonical IKK complex.

PGE(2) induces macrophage IL-10 production and a regulatory-like phenotype via a protein kinase A-SIK-CRTC3 pathway.

The polarization of macrophages into a regulatory-like phenotype and the production of IL-10 plays an important role in the resolution of inflammation. We show in this study that PGE(2), in combination with LPS, is able to promote an anti-inflammatory phenotype in macrophages characterized by high expression of IL-10 and the regulatory markers SPHK1 and LIGHT via a protein kinase A-dependent pathway. Both TLR agonists and PGE(2) promote the phosphorylation of the transcription factor CREB on Ser(133). However, although CREB regulates IL-10 transcription, the mutation of Ser(133) to Ala in the endogenous CREB gene did not prevent the ability of PGE(2) to promote IL-10 transcription. Instead, we demonstrate that protein kinase A regulates the phosphorylation of salt-inducible kinase 2 on Ser(343), inhibiting its ability to phosphorylate CREB-regulated transcription coactivator 3 in cells. This in turn allows CREB-regulated transcription coactivator 3 to translocate to the nucleus where it serves as a coactivator with the transcription factor CREB to induce IL-10 transcription. In line with this, we find that either genetic or pharmacological inhibition of salt-inducible kinases mimics the effect of PGE(2) on IL-10 production.

Ubiquitin-related modifier Urm1 acts as a sulphur carrier in thiolation of eukaryotic transfer RNA.

Ubiquitin-like proteins (UBLs) can change protein function, localization or turnover by covalent attachment to lysine residues. Although UBLs achieve this conjugation through an intricate enzymatic cascade, their bacterial counterparts MoaD and ThiS function as sulphur carrier proteins. Here we show that Urm1p, the most ancient UBL, acts as a sulphur carrier in the process of eukaryotic transfer RNA (tRNA) modification, providing a possible evolutionary link between UBL and sulphur transfer. Moreover, we identify Uba4p, Ncs2p, Ncs6p and Yor251cp as components of this conserved pathway. Using in vitro assays, we show that Ncs6p binds to tRNA, whereas Uba4p first adenylates and then directly transfers sulphur onto Urm1p. Finally, functional analysis reveals that the thiolation function of Urm1p is critical to regulate cellular responses to nutrient starvation and oxidative stress conditions, most likely by increasing translation fidelity.

Phosphorylation of CRTC3 by the salt-inducible kinases controls the interconversion of classically activated and regulatory macrophages.

Macrophages acquire strikingly different properties that enable them to play key roles during the initiation, propagation, and resolution of inflammation. Classically activated (M1) macrophages produce proinflammatory mediators to combat invading pathogens and respond to tissue damage in the host, whereas regulatory macrophages (M2b) produce high levels of anti-inflammatory molecules, such as IL-10, and low levels of proinflammatory cytokines, like IL-12, and are important for the resolution of inflammatory responses. A central problem in this area is to understand how the formation of regulatory macrophages can be promoted at sites of inflammation to prevent and/or alleviate chronic inflammatory and autoimmune diseases. Here, we demonstrate that the salt-inducible kinases (SIKs) restrict the formation of regulatory macrophages and that their inhibition induces striking increases in many of the characteristic markers of regulatory macrophages, greatly stimulating the production of IL-10 and other anti-inflammatory molecules. We show that SIK inhibitors elevate IL-10 production by inducing the dephosphorylation of cAMP response element-binding protein (CREB)-regulated transcriptional coactivator (CRTC) 3, its dissociation from 14-3-3 proteins and its translocation to the nucleus where it enhances a gene transcription program controlled by CREB. Importantly, the effects of SIK inhibitors on IL-10 production are lost in macrophages that express a drug-resistant mutant of SIK2. These findings identify SIKs as a key molecular switch whose inhibition reprograms macrophages to an anti-inflammatory phenotype. The remarkable effects of SIK inhibitors on macrophage function suggest that drugs that target these protein kinases may have therapeutic potential for the treatment of inflammatory and autoimmune diseases.

RNAi-based screening identifies the Mms22L-Nfkbil2 complex as a novel regulator of DNA replication in human cells.

Cullin 4 (Cul4)-based ubiquitin ligases emerged as critical regulators of DNA replication and repair. Over 50 Cul4-specific adaptors (DNA damage-binding 1 (Ddb1)-Cul4-associated factors; DCAFs) have been identified and are thought to assemble functionally distinct Cul4 complexes. Using a live-cell imaging-based RNAi screen, we analysed the function of DCAFs and Cul4-linked proteins, and identified specific subsets required for progression through G1 and S phase. We discovered C6orf167/Mms22-like protein (Mms22L) as a putative human orthologue of budding yeast Mms22, which, together with cullin Rtt101, regulates genome stability by promoting DNA replication through natural pause sites and damaged templates. Loss of Mms22L function in human cells results in S phase-dependent genomic instability characterised by spontaneous double-strand breaks and DNA damage checkpoint activation. Unlike yeast Mms22, human Mms22L does not stably bind to Cul4, but is degraded in a Cul4-dependent manner and upon replication stress. Mms22L physically and functionally interacts with the scaffold-like protein Nfkbil2 that co-purifies with histones, several chromatin remodelling and DNA replication/repair factors. Together, our results strongly suggest that the Mms22L-Nfkbil2 complex contributes to genome stability by regulating the chromatin state at stalled replication forks.

Hydrophilic strong anion exchange (hSAX) chromatography for highly orthogonal peptide separation of complex proteomes.

Due to its compatibility and orthogonality to reversed phase (RP) liquid chromatography (LC) separation, ion exchange chromatography, and mainly strong cation exchange (SCX), has often been the first choice in multidimensional LC experiments in proteomics. Here, we have tested the ability of three strong anion exchanger (SAX) columns differing in their hydrophobicity to fractionate RAW264.7 macrophage cell lysate. IonPac AS24, a strong anion exchange material with ultralow hydrophobicity, demonstrated to be superior to other materials by fractionation and separation of tryptic peptides from both a mixture of 6 proteins as well as mouse cell lysate. The chromatography displayed very high orthogonality and high robustness depending on the hydrophilicity of column chemistry, which we termed hydrophilic strong anion exchange (hSAX). Mass spectrometry analysis of 34 SAX fractions from RAW264.7 macrophage cell lysate digest resulted in an identification of 9469 unique proteins and 126318 distinct peptides in one week of instrument time. Moreover, when compared to an optimized high pH/low pH RP separation approach, the method presented here raised the identification of proteins and peptides by 10 and 28%, respectively. This novel hSAX approach provides robust, reproducible, and highly orthogonal separation of complex protein digest samples for deep coverage proteome analysis.

Integrated multi-omics reveals anaplerotic rewiring in methylmalonyl-CoA mutase deficiency.

Methylmalonic aciduria (MMA) is an inborn error of metabolism with multiple monogenic causes and a poorly understood pathogenesis, leading to the absence of effective causal treatments. Here we employ multi-layered omics profiling combined with biochemical and clinical features of individuals with MMA to reveal a molecular diagnosis for 177 out of 210 (84%) cases, the majority (148) of whom display pathogenic variants in methylmalonyl-CoA mutase (MMUT). Stratification of these data layers by disease severity shows dysregulation of the tricarboxylic acid cycle and its replenishment (anaplerosis) by glutamine. The relevance of these disturbances is evidenced by multi-organ metabolomics of a hemizygous Mmut mouse model as well as through identification of physical interactions between MMUT and glutamine anaplerotic enzymes. Using stable-isotope tracing, we find that treatment with dimethyl-oxoglutarate restores deficient tricarboxylic acid cycling. Our work highlights glutamine anaplerosis as a potential therapeutic intervention point in MMA.

Proteogenetic drug response profiling elucidates targetable vulnerabilities of myelofibrosis.

Myelofibrosis is a hematopoietic stem cell disorder belonging to the myeloproliferative neoplasms. Myelofibrosis patients frequently carry driver mutations in either JAK2 or Calreticulin (CALR) and have limited therapeutic options. Here, we integrate ex vivo drug response and proteotype analyses across myelofibrosis patient cohorts to discover targetable vulnerabilities and associated therapeutic strategies. Drug sensitivities of mutated and progenitor cells were measured in patient blood using high-content imaging and single-cell deep learning-based analyses. Integration with matched molecular profiling revealed three targetable vulnerabilities. First, CALR mutations drive BET and HDAC inhibitor sensitivity, particularly in the absence of high Ras pathway protein levels. Second, an MCM complex-high proliferative signature corresponds to advanced disease and sensitivity to drugs targeting pro-survival signaling and DNA replication. Third, homozygous CALR mutations result in high endoplasmic reticulum (ER) stress, responding to ER stressors and unfolded protein response inhibition. Overall, our integrated analyses provide a molecularly motivated roadmap for individualized myelofibrosis patient treatment.

An improved SUMmOn-based methodology for the identification of ubiquitin and ubiquitin-like protein conjugation sites identifies novel ubiquitin-like protein chain linkages.

Ubiquitin (Ub) and the ubiquitin-like proteins (Ubls) comprise a remarkable assortment of polypeptides that are covalently conjugated to target proteins (or other biomolecules) to modulate their intracellular localization, half-life, and/or activity. Identification of Ub/Ubl conjugation sites on a protein of interest can thus be extremely important for understanding how it is regulated. While MS has become a powerful tool for the study of many classes of PTMs, the identification of Ub/Ubl conjugation sites presents a number of unique challenges. Here, we present an improved Ub/Ubl conjugation site identification strategy, utilizing SUMmOn analysis and an additional protease (lysyl endopeptidase C), as a complement to standard approaches. As compared with standard trypsin proteolysis-database search protocols alone, the addition of SUMmOn analysis can (i) identify Ubl conjugation sites that are not detected by standard database searching methods, (ii) better preserve Ub/Ubl conjugate identity, and (iii) increase the number of identifications of Ub/Ubl modifications in lysine-rich protein regions. Using this methodology, we characterize for the first time a number of novel Ubl linkages and conjugation sites, including alternative yeast (K54) and mammalian small ubiquitin-related modifier (SUMO) chain (SUMO-2 K42, SUMO-3 K41) assemblies, as well as previously unreported NEDD8 chain (K27, K33, and K54) topologies.

Urm1 at the crossroad of modifications. 'Protein Modifications: Beyond the Usual Suspects' Review Series.

The ubiquitin-like protein Urm1 can be covalently conjugated to other proteins, such as the yeast thioredoxin peroxidase protein Ahp1p, through a mechanism involving the ubiquitin E1-like enzyme Uba4. Recent findings have revealed a second function of Urm1 as a sulphur carrier in the thiolation of eukaryotic cytoplasmic transfer RNAs (tRNAs). Interestingly, this new role of Urm1 is similar to the sulphur-carrier activity of its prokaryotic counterparts, strengthening the hypothesis that Urm1 is a molecular fossil of the ubiquitin-like protein family. Here, we discuss the function of Urm1 in light of its dual role in protein and RNA modification.

A high-quality catalog of the Drosophila melanogaster proteome.

Understanding how proteins and their complex interaction networks convert the genomic information into a dynamic living organism is a fundamental challenge in biological sciences. As an important step towards understanding the systems biology of a complex eukaryote, we cataloged 63% of the predicted Drosophila melanogaster proteome by detecting 9,124 proteins from 498,000 redundant and 72,281 distinct peptide identifications. This unprecedented high proteome coverage for a complex eukaryote was achieved by combining sample diversity, multidimensional biochemical fractionation and analysis-driven experimentation feedback loops, whereby data collection is guided by statistical analysis of prior data. We show that high-quality proteomics data provide crucial information to amend genome annotation and to confirm many predicted gene models. We also present experimentally identified proteotypic peptides matching approximately 50% of D. melanogaster gene models. This library of proteotypic peptides should enable fast, targeted and quantitative proteomic studies to elucidate the systems biology of this model organism.

Review of Batch Effects Prevention, Diagnostics, and Correction Approaches.

Systematic technical variation in high-throughput studies consisting of the serial measurement of large sample cohorts is known as batch effects. Batch effects reduce the sensitivity of biological signal extraction and can cause significant artifacts. The systematic bias in the data caused by batch effects is more common in studies in which logistical considerations restrict the number of samples that can be prepared or profiled in a single experiment, thus necessitating the arrangement of subsets of study samples in batches. To mitigate the negative impact of batch effects, statistical approaches for batch correction are used at the stage of experimental design and data processing. Whereas in genomics batch effects and possible remedies have been extensively discussed, they are a relatively new challenge in proteomics because methods with sufficient throughput to systematically measure through large sample cohorts have only recently become available. Here we provide general recommendations to mitigate batch effects: we discuss the design of large-scale proteomic studies, review the most commonly used tools for batch effect correction and overview their application in proteomics.