A Causal Model of Ion Interference Enables Assessment and Correction of Ratio Compression in Multiplex Proteomics.

Multiplex proteomics using isobaric labeling tags has emerged as a powerful tool for the simultaneous relative quantification of peptides and proteins across multiple experimental conditions. However, the quantitative accuracy of the approach is largely compromised by ion interference, a phenomenon that causes fold changes to appear compressed. The degree of compression is generally unknown, and the contributing factors are poorly understood. In this study, we thoroughly characterized ion interference at the MS2 level using a defined two-proteome experimental system with known ground-truth. We discovered remarkably poor agreement between the apparent precursor purity in the isolation window and the actual level of observed reporter ion interference in MS2 scans-a discrepancy that we found resolved by considering cofragmentation of peptide ions hidden within the spectral "noise" of the MS1 isolation window. To address this issue, we developed a regression modeling strategy to accurately predict reporter ion interference in any dataset. Finally, we demonstrate the utility of our procedure for improved fold change estimation and unbiased PTM site-to-protein normalization. All computational tools and code required to apply this method to any MS2 TMT dataset are documented and freely available.

GSE1 links the HDAC1/CoREST co-repressor complex to DNA damage.

Post-translational modifications of histones are important regulators of the DNA damage response (DDR). By using affinity purification mass spectrometry (AP-MS) we discovered that genetic suppressor element 1 (GSE1) forms a complex with the HDAC1/CoREST deacetylase/demethylase co-repressor complex. In-depth phosphorylome analysis revealed that loss of GSE1 results in impaired DDR, ATR signalling and γH2AX formation upon DNA damage induction. Altered profiles of ATR target serine-glutamine motifs (SQ) on DDR-related hallmark proteins point to a defect in DNA damage sensing. In addition, GSE1 knock-out cells show hampered DNA damage-induced phosphorylation on SQ motifs of regulators of histone post-translational modifications, suggesting altered histone modification. While loss of GSE1 does not affect the histone deacetylation activity of CoREST, GSE1 appears to be essential for binding of the deubiquitinase USP22 to CoREST and for the deubiquitination of H2B K120 in response to DNA damage. The combination of deacetylase, demethylase, and deubiquitinase activity makes the USP22-GSE1-CoREST subcomplex a multi-enzymatic eraser that seems to play an important role during DDR. Since GSE1 has been previously associated with cancer progression and survival our findings are potentially of high medical relevance.

Chemical Acetylation of Ligands and Two-Step Digestion Protocol for Reducing Codigestion in Affinity Purification-Mass Spectrometry.

We present an effective, fast, and user-friendly method to reduce codigestion of bead-bound ligands, such as antibodies or streptavidin, in affinity purification-mass spectrometry experiments. A short preincubation of beads with Sulfo-NHS-Acetate leads to chemical acetylation of lysine residues, making ligands insusceptible to Lys-C-mediated proteolysis. In contrast to similar approaches, our procedure offers the advantage of exclusively using nontoxic chemicals and employing mild chemical reaction conditions. After binding of bait proteins to Sulfo-NHS-Acetate treated beads, we employ a two-step digestion protocol with the sequential use of Lys-C protease for on-bead digestion followed by in-solution digestion of the released proteins with trypsin. The implementation of this protocol results in a strong reduction of contaminating ligand peptides, which allows significantly higher amounts of sample to be subjected to LC-MS analysis, improving sensitivity and quantitative accuracy.

Advances in RNA Labeling with Trifluoromethyl Groups.

Fluorine labeling of ribonucleic acids (RNA) in conjunction with 19 F NMR spectroscopy has emerged as a powerful strategy for spectroscopic analysis of RNA structure and dynamics, and RNA-ligand interactions. This study presents the first syntheses of 2'-OCF3 guanosine and uridine phosphoramidites, their incorporation into oligoribonucleotides by solid-phase synthesis and a comprehensive study of their properties. NMR spectroscopic analysis showed that the 2'-OCF3 modification is associated with preferential C2'-endo conformation of the U and G ribose in single-stranded RNA. When paired to the complementary strand, slight destabilization of the duplex caused by the modification was revealed by UV melting curve analysis. Moreover, the power of the 2'-OCF3 label for NMR spectroscopy is demonstrated by dissecting RNA pseudoknot folding and its binding to a small molecule. Furthermore, the 2'-OCF3 modification has potential for applications in therapeutic oligonucleotides. To this end, three 2'-OCF3 modified siRNAs were tested in silencing of the BASP1 gene which indicated enhanced performance for one of them. Importantly, together with earlier work, the present study completes the set of 2'-OCF3 nucleoside phosphoramidites to all four standard nucleobases (A, U, C, G) and hence enables applications that utilize the favorable properties of the 2'-OCF3 group without any restrictions in placing the modification into the RNA target sequence.

High Intrinsic Oncogenic Potential in the Myc-Box-Deficient Hydra Myc3 Protein.

The proto-oncogene myc has been intensively studied primarily in vertebrate cell culture systems. Myc transcription factors control fundamental cellular processes such as cell proliferation, cell cycle control and stem cell maintenance. Myc interacts with the Max protein and Myc/Max heterodimers regulate thousands of target genes. The genome of the freshwater polyp Hydra encodes four myc genes (myc1-4). Previous structural and biochemical characterization showed that the Hydra Myc1 and Myc2 proteins share high similarities with vertebrate c-Myc, and their expression patterns suggested a function in adult stem cell maintenance. In contrast, an additional Hydra Myc protein termed Myc3 is highly divergent, lacking the common N-terminal domain and all conserved Myc-boxes. Single cell transcriptome analysis revealed that the myc3 gene is expressed in a distinct population of interstitial precursor cells committed to nerve- and gland-cell differentiation, where the Myc3 protein may counteract the stemness actions of Myc1 and Myc2 and thereby allow the implementation of a differentiation program. In vitro DNA binding studies showed that Myc3 dimerizes with Hydra Max, and this dimer efficiently binds to DNA containing the canonical Myc consensus motif (E-box). In vivo cell transformation assays in avian fibroblast cultures further revealed an unexpected high potential for oncogenic transformation in the conserved Myc3 C-terminus, as compared to Hydra Myc2 or Myc1. Structure modeling of the Myc3 protein predicted conserved amino acid residues in its bHLH-LZ domain engaged in Myc3/Max dimerization. Mutating these amino acid residues in the human c-Myc (MYC) sequence resulted in a significant decrease in its cell transformation potential. We discuss our findings in the context of oncogenic transformation and cell differentiation, both relevant for human cancer, where Myc represents a major driver.

Clp-targeting BacPROTACs impair mycobacterial proteostasis and survival.

The ClpC1:ClpP1P2 protease is a core component of the proteostasis system in mycobacteria. To improve the efficacy of antitubercular agents targeting the Clp protease, we characterized the mechanism of the antibiotics cyclomarin A and ecumicin. Quantitative proteomics revealed that the antibiotics cause massive proteome imbalances, including upregulation of two unannotated yet conserved stress response factors, ClpC2 and ClpC3. These proteins likely protect the Clp protease from excessive amounts of misfolded proteins or from cyclomarin A, which we show to mimic damaged proteins. To overcome the Clp security system, we developed a BacPROTAC that induces degradation of ClpC1 together with its ClpC2 caretaker. The dual Clp degrader, built from linked cyclomarin A heads, was highly efficient in killing pathogenic Mycobacterium tuberculosis, with >100-fold increased potency over the parent antibiotic. Together, our data reveal Clp scavenger proteins as important proteostasis safeguards and highlight the potential of BacPROTACs as future antibiotics.

High-resolution structural information of membrane-bound α-synuclein provides insight into the MoA of the anti-Parkinson drug UCB0599.

α-synuclein (αS) is an intrinsically disordered protein whose functional ambivalence and protein structural plasticity are iconic. Coordinated protein recruitment ensures proper vesicle dynamics at the synaptic cleft, while deregulated oligomerization on cellular membranes contributes to cell damage and Parkinson's disease (PD). Despite the protein's pathophysiological relevance, structural knowledge is limited. Here, we employ NMR spectroscopy and chemical cross-link mass spectrometry on 14N/15N-labeled αS mixtures to provide for the first time high-resolution structural information of the membrane-bound oligomeric state of αS and demonstrate that in this state, αS samples a surprisingly small conformational space. Interestingly, the study locates familial Parkinson's disease mutants at the interface between individual αS monomers and reveals different oligomerization processes depending on whether oligomerization occurs on the same membrane surface (cis) or between αS initially attached to different membrane particles (trans). The explanatory power of the obtained high-resolution structural model is used to help determine the mode-of-actionof UCB0599. Here, it is shown that the ligand changes the ensemble of membrane-bound structures, which helps to explain the success this compound, currently being tested in Parkinson's disease patients in a phase 2 trial, has had in animal models of PD.

Mimicking Tumor Cell Heterogeneity of Colorectal Cancer in a Patient-derived Organoid-Fibroblast Model.

BACKGROUND & AIMS: Patient-derived organoid cancer models are generated from epithelial tumor cells and reflect tumor characteristics. However, they lack the complexity of the tumor microenvironment, which is a key driver of tumorigenesis and therapy response. Here, we developed a colorectal cancer organoid model that incorporates matched epithelial cells and stromal fibroblasts. METHODS: Primary fibroblasts and tumor cells were isolated from colorectal cancer specimens. Fibroblasts were characterized for their proteome, secretome, and gene expression signatures. Fibroblast/organoid co-cultures were analyzed by immunohistochemistry and compared with their tissue of origin, as well as on gene expression levels compared with standard organoid models. Bioinformatics deconvolution was used to calculate cellular proportions of cell subsets in organoids based on single-cell RNA sequencing data. RESULTS: Normal primary fibroblasts, isolated from tumor adjacent tissue, and cancer associated fibroblasts retained their molecular characteristics in vitro, including higher motility of cancer associated compared with normal fibroblasts. Importantly, both cancer-associated fibroblasts and normal fibroblasts supported cancer cell proliferation in 3D co-cultures, without the addition of classical niche factors. Organoids grown together with fibroblasts displayed a larger cellular heterogeneity of tumor cells compared with mono-cultures and closely resembled the in vivo tumor morphology. Additionally, we observed a mutual crosstalk between tumor cells and fibroblasts in the co-cultures. This was manifested by considerably deregulated pathways such as cell-cell communication and extracellular matrix remodeling in the organoids. Thrombospondin-1 was identified as a critical factor for fibroblast invasiveness. CONCLUSION: We developed a physiological tumor/stroma model, which will be vital as a personalized tumor model to study disease mechanisms and therapy response in colorectal cancer.

Strategies to target the cancer driver MYC in tumor cells.

The MYC oncoprotein functions as a master regulator of cellular transcription and executes non-transcriptional tasks relevant to DNA replication and cell cycle regulation, thereby interacting with multiple proteins. MYC is required for fundamental cellular processes triggering proliferation, growth, differentiation, or apoptosis and also represents a major cancer driver being aberrantly activated in most human tumors. Due to its non-enzymatic biochemical functions and largely unstructured surface, MYC has remained difficult for specific inhibitor compounds to directly address, and consequently, alternative approaches leading to indirect MYC inhibition have evolved. Nowadays, multiple organic compounds, nucleic acids, or peptides specifically interfering with MYC activities are in preclinical or early-stage clinical studies, but none of them have been approved so far for the pharmacological treatment of cancer patients. In addition, specific and efficient delivery technologies to deliver MYC-inhibiting agents into MYC-dependent tumor cells are just beginning to emerge. In this review, an overview of direct and indirect MYC-inhibiting agents and their modes of MYC inhibition is given. Furthermore, we summarize current possibilities to deliver appropriate drugs into cancer cells containing derailed MYC using viral vectors or appropriate nanoparticles. Finding the right formulation to target MYC-dependent cancers and to achieve a high intracellular concentration of compounds blocking or attenuating oncogenic MYC activities could be as important as the development of novel MYC-inhibiting principles.

Unexpected complexity of the ammonia monooxygenase in archaea.

Ammonia oxidation, as the first step of nitrification, constitutes a critical process in the global nitrogen cycle. However, fundamental knowledge of its key enzyme, the copper-dependent ammonia monooxygenase, is lacking, in particular for the environmentally abundant ammonia-oxidizing archaea (AOA). Here the structure of the enzyme is investigated by blue-native gel electrophoresis and proteomics from native membrane complexes of two AOA. Besides the known AmoABC subunits and the earlier predicted AmoX, two new protein subunits, AmoY and AmoZ, were identified. They are unique to AOA, highly conserved and co-regulated, and their genes are linked to other AMO subunit genes in streamlined AOA genomes. Modeling and in-gel cross-link approaches support an overall protomer structure similar to the distantly related bacterial particulate methane monooxygenase but also reveals clear differences in extracellular domains of the enzyme. These data open avenues for further structure-function studies of this ecologically important nitrification complex.

Analysis of Golgi Protein Acetylation Using In Vitro Assays and Parallel Reaction Monitoring Mass Spectrometry.

Acetylation is one of the most abundant post-translational protein modifications that regulates all cellular compartments ranging from chromatin to cytoskeleton and Golgi. The dynamic acetylation of the Golgi stacking protein GRASP55 was shown to regulate Golgi reassembly after mitosis. Here we provide a detailed protocol for the analysis of Golgi acetylation including in vitro assays to detect protein acetylation and mass spectrometry analysis to identify specific acetylation sites and their relative abundance.

amica: an interactive and user-friendly web-platform for the analysis of proteomics data.

BACKGROUND: Quantitative proteomics has become an increasingly prominent tool in the study of life sciences. A substantial hurdle for many biologists are, however, the intricacies involved in the associated high throughput data analysis. RESULTS: In order to facilitate this task for users with limited background knowledge, we have developed amica, a freely available open-source web-based software that accepts proteomic input files from different sources. amica provides quality control, differential expression, biological network and over-representation analysis on the basis of minimal user input. Scientists can use amica's query interface interactively to compare multiple conditions and rapidly identify enriched or depleted proteins. They can visualize their results using customized output graphics, and ultimately export the results in a tab-separated format that can be shared with collaborators. The code for the application, input data and documentation can be accessed online at https://github.com/tbaccata/amica and is also incorporated in the web application. CONCLUSIONS: The strong emphasis on dynamic user interactions, the integration of various databases and the option to download processed data, facilitate the analysis of complex proteomic data for both first-time users and experienced bioinformaticians. A freely available version of amica is available at https://bioapps.maxperutzlabs.ac.at/app/amica .

Robust synthesis of 2'-azido modified RNA from 2'-amino precursors by diazotransfer reaction.

Azides are versatile bioorthogonal reporter moieties that are commonly used for site-specific labeling and functionalization of RNA to probe its biology. The preparation of azido modified nucleic acids by solid-phase synthesis is problematic due to the inherent reactivity of P(III) species with azides according to the Staudinger reaction. Various strategies have been developed to bypass this limitation and are often time-consuming, low-yielding and labor-intensive. In particular, the synthesis of RNA with internal 2'-azido modifications is restricted to a single approach that employs P(V) chemistry instead of the widely used P(III) phosphoramidite chemistry. To fill this methodological gap, we present a novel convenient path toward 2'-azido RNA from readily accessible 2'-amino RNA through treatment with the diazotizing reagent fluorosulfuryl azide (FSO2N3). A diazotransfer reaction was established for oligoribonucleotides of different lengths and secondary structures. The robustness of the approach was further demonstrated for RNAs containing multiple 2'-azido moieties and for RNAs containing other sensitive modifications such as thiouridine or methylated nucleobases with a positive charge. The synthetic ease of generating 2'-azido RNA will pave the way for biotechnological applications, in particular for siRNA technologies and for referencing the growing number of RNA metabolic labeling approaches that rely on 2'-azido nucleosides.

A Proteomic Approach for the Quantification of Posttranslational Protein Lysine Acetylation in Candida albicans.

Candida albicans is a normal component of the human microflora that colonizes mucosal/epithelial surfaces and the gastrointestinal tract as a commensal organism. However, in an immunocompromised host, it can cause life-threatening infections of high mortality and morbidity. Virulence as well as antifungal drug resistance of C. albicans is often regulated by posttranslational modifications (PTM) of proteins via lysine acetylation by lysine acetyltransferases. Here, we report an experimental approach using tandem mass tag (TMT) labeling for the detection and quantification of lysine acetylation of histone and nonhistone proteins in C. albicans.

In Search of a Universal Method: A Comparative Survey of Bottom-Up Proteomics Sample Preparation Methods.

Robust, efficient, and reproducible protein extraction and sample processing is a key step for bottom-up proteomics analyses. While many sample preparation protocols for mass spectrometry have been described, selecting an appropriate method remains challenging since some protein classes may require specialized solubilization, precipitation, and digestion procedures. Here, we present a comprehensive comparison of the 16 most widely used sample preparation methods, covering in-solution digests, device-based methods, and commercially available kits. We find a remarkably good performance of the majority of the protocols with high reproducibility, little method dependency, and low levels of artifact formation. However, we revealed method-dependent differences in the recovery of specific protein features, which we summarized in a descriptive guide matrix. Our work thereby provides a solid basis for the selection of MS sample preparation strategies for a given proteomics project.

A toolbox for class I HDACs reveals isoform specific roles in gene regulation and protein acetylation.

The class I histone deacetylases are essential regulators of cell fate decisions in health and disease. While pan- and class-specific HDAC inhibitors are available, these drugs do not allow a comprehensive understanding of individual HDAC function, or the therapeutic potential of isoform-specific targeting. To systematically compare the impact of individual catalytic functions of HDAC1, HDAC2 and HDAC3, we generated human HAP1 cell lines expressing catalytically inactive HDAC enzymes. Using this genetic toolbox we compare the effect of individual HDAC inhibition with the effects of class I specific inhibitors on cell viability, protein acetylation and gene expression. Individual inactivation of HDAC1 or HDAC2 has only mild effects on cell viability, while HDAC3 inactivation or loss results in DNA damage and apoptosis. Inactivation of HDAC1/HDAC2 led to increased acetylation of components of the COREST co-repressor complex, reduced deacetylase activity associated with this complex and derepression of neuronal genes. HDAC3 controls the acetylation of nuclear hormone receptor associated proteins and the expression of nuclear hormone receptor regulated genes. Acetylation of specific histone acetyltransferases and HDACs is sensitive to inactivation of HDAC1/HDAC2. Over a wide range of assays, we determined that in particular HDAC1 or HDAC2 catalytic inactivation mimics class I specific HDAC inhibitors. Importantly, we further demonstrate that catalytic inactivation of HDAC1 or HDAC2 sensitizes cells to specific cancer drugs. In summary, our systematic study revealed isoform-specific roles of HDAC1/2/3 catalytic functions. We suggest that targeted genetic inactivation of particular isoforms effectively mimics pharmacological HDAC inhibition allowing the identification of relevant HDACs as targets for therapeutic intervention.

Depletion of endogenously biotinylated carboxylases enhances the sensitivity of TurboID-mediated proximity labeling in Caenorhabditis elegans.

Proximity-dependent protein labeling provides a powerful in vivo strategy to characterize the interactomes of specific proteins. We previously optimized a proximity labeling protocol for Caenorhabditis elegans using the highly active biotin ligase TurboID. A significant constraint on the sensitivity of TurboID is the presence of abundant endogenously biotinylated proteins that take up bandwidth in the mass spectrometer, notably carboxylases that use biotin as a cofactor. In C. elegans, these comprise POD-2/acetyl-CoA carboxylase alpha, PCCA-1/propionyl-CoA carboxylase alpha, PYC-1/pyruvate carboxylase, and MCCC-1/methylcrotonyl-CoA carboxylase alpha. Here, we developed ways to remove these carboxylases prior to streptavidin purification and mass spectrometry by engineering their corresponding genes to add a C-terminal His10 tag. This allows us to deplete them from C. elegans lysates using immobilized metal affinity chromatography. To demonstrate the method's efficacy, we use it to expand the interactome map of the presynaptic active zone protein ELKS-1. We identify many known active zone proteins, including UNC-10/RIM, SYD-2/liprin-alpha, SAD-1/BRSK1, CLA-1/CLArinet, C16E9.2/Sentryn, as well as previously uncharacterized potentially synaptic proteins such as the ortholog of human angiomotin, F59C12.3 and the uncharacterized protein R148.3. Our approach provides a quick and inexpensive solution to a common contaminant problem in biotin-dependent proximity labeling. The approach may be applicable to other model organisms and will enable deeper and more complete analysis of interactors for proteins of interest.

BacPROTACs mediate targeted protein degradation in bacteria.

Hijacking the cellular protein degradation system offers unique opportunities for drug discovery, as exemplified by proteolysis-targeting chimeras. Despite their great promise for medical chemistry, so far, it has not been possible to reprogram the bacterial degradation machinery to interfere with microbial infections. Here, we develop small-molecule degraders, so-called BacPROTACs, that bind to the substrate receptor of the ClpC:ClpP protease, priming neo-substrates for degradation. In addition to their targeting function, BacPROTACs activate ClpC, transforming the resting unfoldase into its functional state. The induced higher-order oligomer was visualized by cryo-EM analysis, providing a structural snapshot of activated ClpC unfolding a protein substrate. Finally, drug susceptibility and degradation assays performed in mycobacteria demonstrate in vivo activity of BacPROTACs, allowing selective targeting of endogenous proteins via fusion to an established degron. In addition to guiding antibiotic discovery, the BacPROTAC technology presents a versatile research tool enabling the inducible degradation of bacterial proteins.

Release of CHK-2 from PPM-1.D anchorage schedules meiotic entry.

Transition from the stem/progenitor cell fate to meiosis is mediated by several redundant posttranscriptional regulatory pathways in Caenorhabditis elegans. Interfering with all three branches causes tumorous germ lines. SCFPROM-1 comprises one branch and mediates a scheduled degradation step at entry into meiosis. prom-1 mutants show defects in the timely initiation of meiotic prophase I events, resulting in high rates of embryonic lethality. Here, we identify the phosphatase PPM-1.D/Wip1 as crucial substrate for PROM-1. We report that PPM-1.D antagonizes CHK-2 kinase, a key regulator for meiotic prophase initiation, including DNA double-strand breaks, chromosome pairing, and synaptonemal complex formation. We propose that PPM-1.D controls the amount of active CHK-2 via both catalytic and noncatalytic activities; notably, noncatalytic regulation seems to be crucial at meiotic entry. PPM-1.D sequesters CHK-2 at the nuclear periphery, and programmed SCFPROM-1-mediated degradation of PPM-1.D liberates the kinase and promotes meiotic entry.