Revealing the Arabidopsis AtGRP7 mRNA binding proteome by specific enhanced RNA interactome capture.

BACKGROUND: The interaction of proteins with RNA in the cell is crucial to orchestrate all steps of RNA processing. RNA interactome capture (RIC) techniques have been implemented to catalogue RNA- binding proteins in the cell. In RIC, RNA-protein complexes are stabilized by UV crosslinking in vivo. Polyadenylated RNAs and associated proteins are pulled down from cell lysates using oligo(dT) beads and the RNA-binding proteome is identified by quantitative mass spectrometry. However, insights into the RNA-binding proteome of a single RNA that would yield mechanistic information on how RNA expression patterns are orchestrated, are scarce. RESULTS: Here, we explored RIC in Arabidopsis to identify proteins interacting with a single mRNA, using the circadian clock-regulated Arabidopsis thaliana GLYCINE-RICH RNA-BINDING PROTEIN 7 (AtGRP7) transcript, one of the most abundant transcripts in Arabidopsis, as a showcase. Seedlings were treated with UV light to covalently crosslink RNA and proteins. The AtGRP7 transcript was captured from cell lysates with antisense oligonucleotides directed against the 5'untranslated region (UTR). The efficiency of RNA capture was greatly improved by using locked nucleic acid (LNA)/DNA oligonucleotides, as done in the enhanced RIC protocol. Furthermore, performing a tandem capture with two rounds of pulldown with the 5'UTR oligonucleotide increased the yield. In total, we identified 356 proteins enriched relative to a pulldown from atgrp7 mutant plants. These were benchmarked against proteins pulled down from nuclear lysates by AtGRP7 in vitro transcripts immobilized on beads. Among the proteins validated by in vitro interaction we found the family of Acetylation Lowers Binding Affinity (ALBA) proteins. Interaction of ALBA4 with the AtGRP7 RNA was independently validated via individual-nucleotide resolution crosslinking and immunoprecipitation (iCLIP). The expression of the AtGRP7 transcript in an alba loss-of-function mutant was slightly changed compared to wild-type, demonstrating the functional relevance of the interaction. CONCLUSION: We adapted specific RNA interactome capture with LNA/DNA oligonucleotides for use in plants using AtGRP7 as a showcase. We anticipate that with further optimization and up scaling the protocol should be applicable for less abundant transcripts.

Systematic analysis of RNA-binding proteins identifies targetable therapeutic vulnerabilities in osteosarcoma.

Osteosarcoma is the most common primary malignant bone tumor with a strong tendency to metastasize, limiting the prognosis of affected patients. Genomic, epigenomic and transcriptomic analyses have demonstrated the exquisite molecular complexity of this tumor, but have not sufficiently defined the underlying mechanisms or identified promising therapeutic targets. To systematically explore RNA-protein interactions relevant to OS, we define the RNA interactomes together with the full proteome and the transcriptome of cells from five malignant bone tumors (four osteosarcomata and one malignant giant cell tumor of the bone) and from normal mesenchymal stem cells and osteoblasts. These analyses uncover both systematic changes of the RNA-binding activities of defined RNA-binding proteins common to all osteosarcomata and individual alterations that are observed in only a subset of tumors. Functional analyses reveal a particular vulnerability of these tumors to translation inhibition and a positive feedback loop involving the RBP IGF2BP3 and the transcription factor Myc which affects cellular translation and OS cell viability. Our results thus provide insight into potentially clinically relevant RNA-binding protein-dependent mechanisms of osteosarcoma.

Structural basis for RAD18 regulation by MAGEA4 and its implications for RING ubiquitin ligase binding by MAGE family proteins.

MAGEA4 is a cancer-testis antigen primarily expressed in the testes but aberrantly overexpressed in several cancers. MAGEA4 interacts with the RING ubiquitin ligase RAD18 and activates trans-lesion DNA synthesis (TLS), potentially favouring tumour evolution. Here, we employed NMR and AlphaFold2 (AF) to elucidate the interaction mode between RAD18 and MAGEA4, and reveal that the RAD6-binding domain (R6BD) of RAD18 occupies a groove in the C-terminal winged-helix subdomain of MAGEA4. We found that MAGEA4 partially displaces RAD6 from the RAD18 R6BD and inhibits degradative RAD18 autoubiquitination, which could be countered by a competing peptide of the RAD18 R6BD. AlphaFold2 and cross-linking mass spectrometry (XL-MS) also revealed an evolutionary invariant intramolecular interaction between the catalytic RING and the DNA-binding SAP domains of RAD18, which is essential for PCNA mono-ubiquitination. Using interaction proteomics, we found that another Type-I MAGE, MAGE-C2, interacts with the RING ubiquitin ligase TRIM28 in a manner similar to the MAGEA4/RAD18 complex, suggesting that the MAGEA4 peptide-binding groove also serves as a ligase-binding cleft in other type-I MAGEs. Our data provide new insights into the mechanism and regulation of RAD18-mediated PCNA mono-ubiquitination.

Macromolecular crowding in human tenocyte and skin fibroblast cultures: A comparative analysis.

Although human tenocytes and dermal fibroblasts have shown promise in tendon engineering, no tissue engineered medicine has been developed due to the prolonged ex vivo time required to develop an implantable device. Considering that macromolecular crowding has the potential to substantially accelerate the development of functional tissue facsimiles, herein we compared human tenocyte and dermal fibroblast behaviour under standard and macromolecular crowding conditions to inform future studies in tendon engineering. Basic cell function analysis made apparent the innocuousness of macromolecular crowding for both cell types. Gene expression analysis of the without macromolecular crowding groups revealed expression of tendon related molecules in human dermal fibroblasts and tenocytes. Protein electrophoresis and immunocytochemistry analyses showed significantly increased and similar deposition of collagen fibres by macromolecular crowding in the two cell types. Proteomics analysis demonstrated great similarities between human tenocyte and dermal fibroblast cultures, as well as the induction of haemostatic, anti-microbial and tissue-protective proteins by macromolecular crowding in both cell populations. Collectively, these data rationalise the use of either human dermal fibroblasts or tenocytes in combination with macromolecular crowding in tendon engineering.

The RNA-binding protein landscapes differ between mammalian organs and cultured cells.

System-wide approaches have unveiled an unexpected breadth of the RNA-bound proteomes of cultured cells. Corresponding information regarding RNA-binding proteins (RBPs) of mammalian organs is still missing, largely due to technical challenges. Here, we describe ex vivo enhanced RNA interactome capture (eRIC) to characterize the RNA-bound proteomes of three different mouse organs. The resulting organ atlases encompass more than 1300 RBPs active in brain, kidney or liver. Nearly a quarter (291) of these had formerly not been identified in cultured cells, with more than 100 being metabolic enzymes. Remarkably, RBP activity differs between organs independent of RBP abundance, suggesting organ-specific levels of control. Similarly, we identify systematic differences in RNA binding between animal organs and cultured cells. The pervasive RNA binding of enzymes of intermediary metabolism in organs points to tightly knit connections between gene expression and metabolism, and displays a particular enrichment for enzymes that use nucleotide cofactors. We describe a generically applicable refinement of the eRIC technology and provide an instructive resource of RBPs active in intact mammalian organs, including the brain.

Unravelling metabolic cross-feeding in a yeast-bacteria community using 13 C-based proteomics.

Cross-feeding is fundamental to the diversity and function of microbial communities. However, identification of cross-fed metabolites is often challenging due to the universality of metabolic and biosynthetic intermediates. Here, we use 13 C isotope tracing in peptides to elucidate cross-fed metabolites in co-cultures of Saccharomyces cerevisiae and Lactococcus lactis. The community was grown on lactose as the main carbon source with either glucose or galactose fraction of the molecule labelled with 13 C. Data analysis allowing for the possible mass-shifts yielded hundreds of peptides for which we could assign both species identity and labelling degree. The labelling pattern showed that the yeast utilized galactose and, to a lesser extent, lactic acid shared by L. lactis as carbon sources. While the yeast provided essential amino acids to the bacterium as expected, the data also uncovered a complex pattern of amino acid exchange. The identity of the cross-fed metabolites was further supported by metabolite labelling in the co-culture supernatant, and by diminished fitness of a galactose-negative yeast mutant in the community. Together, our results demonstrate the utility of 13 C-based proteomics for uncovering microbial interactions.

The membrane-associated protein 17 (MAP17) is up-regulated in response to empagliflozin on top of RAS blockade in experimental diabetic nephropathy.

Sodium-glucose cotransporter 2 inhibitors (SGLT2i) have proven to delay diabetic kidney disease (DKD) progression on top of the standard of care with the renin-angiotensin system (RAS) blockade. The molecular mechanisms underlying the synergistic effect of SGLT2i and RAS blockers is poorly understood. We gave a SGLT2i (empagliflozin), an angiotensin-converting enzyme inhibitor (ramipril), or a combination of both drugs for 8 weeks to diabetic (db/db) mice. Vehicle-treated db/db and db/m mice were used as controls. At the end of the experiment, mice were killed, and the kidneys were saved to perform a differential high-throughput proteomic analysis by mass spectrometry using isobaric tandem mass tags (TMT labeling) that allow relative quantification of the identified proteins. The differential proteomic analysis revealed 203 proteins differentially expressed in one or more experimental groups (false discovery rate < 0.05 and Log2 fold change ≥ ±1). Fourteen were differentially expressed in the kidneys from the db/db mice treated with empagliflozin with ramipril. Among them, MAP17 was up-regulated. These findings were subsequently validated by Western blot. The combined therapy of empagliflozin and ramipril up-regulated MAP17 in the kidney of a diabetic mice model. MAP17 is a major scaffolding protein of the proximal tubular cells that places transporters together, namely SGLT2 and NHE3. Our results suggest that SGLT2i on top of RAS blockade may protect the kidney by boosting the inactivation of NHE3 via the up-regulation of key scaffolder proteins such as MAP17.

The augmin complex architecture reveals structural insights into microtubule branching.

In mitosis, the augmin complex binds to spindle microtubules to recruit the γ-tubulin ring complex (γ-TuRC), the principal microtubule nucleator, for the formation of branched microtubules. Our understanding of augmin-mediated microtubule branching is hampered by the lack of structural information on the augmin complex. Here, we elucidate the molecular architecture and conformational plasticity of the augmin complex using an integrative structural biology approach. The elongated structure of the augmin complex is characterised by extensive coiled-coil segments and comprises two structural elements with distinct but complementary functions in γ-TuRC and microtubule binding, linked by a flexible hinge. The augmin complex is recruited to microtubules via a composite microtubule binding site comprising a positively charged unordered extension and two calponin homology domains. Our study provides the structural basis for augmin function in branched microtubule formation, decisively fostering our understanding of spindle formation in mitosis.

Sumoylation of Cas9 at lysine 848 regulates protein stability and DNA binding.

CRISPR/Cas9 is a popular genome editing technology. Although widely used, little is known about how this prokaryotic system behaves in humans. An unwanted consequence of eukaryotic Cas9 expression is off-target DNA binding leading to mutagenesis. Safer clinical implementation of CRISPR/Cas9 necessitates a finer understanding of the regulatory mechanisms governing Cas9 behavior in humans. Here, we report our discovery of Cas9 sumoylation and ubiquitylation, the first post-translational modifications to be described on this enzyme. We found that the major SUMO2/3 conjugation site on Cas9 is K848, a key positively charged residue in the HNH nuclease domain that is known to interact with target DNA and contribute to off-target DNA binding. Our results suggest that Cas9 ubiquitylation leads to decreased stability via proteasomal degradation. Preventing Cas9 sumoylation through conversion of K848 into arginine or pharmacologic inhibition of cellular sumoylation enhances the enzyme's turnover and diminishes guide RNA-directed DNA binding efficacy, suggesting that sumoylation at this site regulates Cas9 stability and DNA binding. More research is needed to fully understand the implications of these modifications for Cas9 specificity.

CaMKK2 facilitates Golgi-associated vesicle trafficking to sustain cancer cell proliferation.

Calcium/calmodulin-dependent protein kinase kinase 2 (CaMKK2) regulates cell and whole-body metabolism and supports tumorigenesis. The cellular impacts of perturbing CAMKK2 expression are, however, not yet fully characterised. By knocking down CAMKK2 levels, we have identified a number of significant subcellular changes indicative of perturbations in vesicle trafficking within the endomembrane compartment. To determine how they might contribute to effects on cell proliferation, we have used proteomics to identify Gemin4 as a direct interactor, capable of binding CAMKK2 and COPI subunits. Prompted by this, we confirmed that CAMKK2 knockdown leads to concomitant and significant reductions in δ-COP protein. Using imaging, we show that CAMKK2 knockdown leads to Golgi expansion, the induction of ER stress, abortive autophagy and impaired lysosomal acidification. All are phenotypes of COPI depletion. Based on our findings, we hypothesise that CAMKK2 sustains cell proliferation in large part through effects on organelle integrity and membrane trafficking.

Global mapping of Salmonella enterica-host protein-protein interactions during infection.

Intracellular bacterial pathogens inject effector proteins to hijack host cellular processes and promote their survival and proliferation. To systematically map effector-host protein-protein interactions (PPIs) during infection, we generated a library of 32 Salmonella enterica serovar Typhimurium (STm) strains expressing chromosomally encoded affinity-tagged effectors and quantified PPIs in macrophages and epithelial cells. We identified 446 effector-host PPIs, 25 of which were previously described, and validated 13 by reciprocal co-immunoprecipitation. While effectors converged on the same host cellular processes, most had multiple targets, which often differed between cell types. We demonstrate that SseJ, SseL, and SifA modulate cholesterol accumulation at the Salmonella-containing vacuole (SCV) partially via the cholesterol transporter Niemann-Pick C1 protein. PipB recruits the organelle contact site protein PDZD8 to the SCV, and SteC promotes actin bundling by phosphorylating formin-like proteins. This study provides a method for probing host-pathogen PPIs during infection and a resource for interrogating STm effector mechanisms.

Structure and dynamics of the quaternary hunchback mRNA translation repression complex.

A key regulatory process during Drosophila development is the localized suppression of the hunchback mRNA translation at the posterior, which gives rise to a hunchback gradient governing the formation of the anterior-posterior body axis. This suppression is achieved by a concerted action of Brain Tumour (Brat), Pumilio (Pum) and Nanos. Each protein is necessary for proper Drosophila development. The RNA contacts have been elucidated for the proteins individually in several atomic-resolution structures. However, the interplay of all three proteins during RNA suppression remains a long-standing open question. Here, we characterize the quaternary complex of the RNA-binding domains of Brat, Pum and Nanos with hunchback mRNA by combining NMR spectroscopy, SANS/SAXS, XL/MS with MD simulations and ITC assays. The quaternary hunchback mRNA suppression complex comprising the RNA binding domains is flexible with unoccupied nucleotides functioning as a flexible linker between the Brat and Pum-Nanos moieties of the complex. Moreover, the presence of the Pum-HD/Nanos-ZnF complex has no effect on the equilibrium RNA binding affinity of the Brat RNA binding domain. This is in accordance with previous studies, which showed that Brat can suppress mRNA independently and is distributed uniformly throughout the embryo.

Structure of the mycobacterial ESX-5 type VII secretion system pore complex.

The ESX-5 type VII secretion system is a membrane-spanning protein complex key to the virulence of mycobacterial pathogens. However, the overall architecture of the fully assembled translocation machinery and the composition of the central secretion pore have remained unknown. Here, we present the high-resolution structure of the 2.1-megadalton ESX-5 core complex. Our structure captured a dynamic, secretion-competent conformation of the pore within a well-defined transmembrane section, sandwiched between two flexible protein layers at the cytosolic entrance and the periplasmic exit. We propose that this flexibility endows the ESX-5 machinery with large conformational plasticity required to accommodate targeted protein secretion. Compared to known secretion systems, a highly dynamic state of the pore may represent a fundamental principle of bacterial secretion machineries.

P38α-MAPK phosphorylates Snapin and reduces Snapin-mediated BACE1 transportation in APP-transgenic mice.

Amyloid ß peptide (Aß) is the major pathogenic molecule in Alzheimer's disease (AD). BACE1 enzyme is essential for the generation of Aß. Deficiency of p38α-MAPK in neurons increases lysosomal degradation of BACE1 and decreases Aß deposition in the brain of APP-transgenic mice. However, the mechanisms mediating effects of p38α-MAPK are largely unknown. In this study, we used APP-transgenic mice and cultured neurons and observed that deletion of p38α-MAPK specifically in neurons decreased phosphorylation of Snapin at serine, increased retrograde transportation of BACE1 in axons and reduced BACE1 at synaptic terminals, which suggests that p38α-MAPK deficiency promotes axonal transportation of BACE1 from its predominant locations, axonal terminals, to lysosomes in the cell body. In vitro kinase assay revealed that p38α-MAPK directly phosphorylates Snapin. By further performing mass spectrometry analysis and site-directed mutagenic experiments in SH-SY5Y cell lines, we identified serine residue 112 as a p38α-MAPK-phosphorylating site on Snapin. Replacement of serine 112 with alanine did abolish p38α-MAPK knockdown-induced reduction of BACE1 activity and protein level, and transportation to lysosomes in SH-SY5Y cells. Taken together, our study suggests that activation of p38α-MAPK phosphorylates Snapin and inhibits the retrograde transportation of BACE1 in axons, which might exaggerate amyloid pathology in AD brain.

Identification of dynamic RNA-binding proteins uncovers a Cpeb4-controlled regulatory cascade during pathological cell growth of cardiomyocytes.

RNA-binding proteins (RBPs) control critical aspects of cardiomyocyte function, but the repertoire of active RBPs in cardiomyocytes during the growth response is largely unknown. We define RBPs in healthy and diseased cardiomyocytes at a system-wide level by RNA interactome capture. This identifies 67 cardiomyocyte-specific RBPs, including several contractile proteins. Furthermore, we identify the cytoplasmic polyadenylation element-binding protein 4 (Cpeb4) as a dynamic RBP, regulating cardiac growth both in vitro and in vivo. We identify mRNAs bound to and regulated by Cpeb4 in cardiomyocytes. Cpeb4 regulates cardiac remodeling by differential expression of transcription factors. Among Cpeb4 target mRNAs, two zinc finger transcription factors (Zeb1 and Zbtb20) are discovered. We show that Cpeb4 regulates the expression of these mRNAs and that Cpeb4 depletion increases their expression. Thus, Cpeb4 emerges as a critical regulator of cardiomyocyte function by differential binding to specific mRNAs in response to pathological growth stimulation.

NMD inhibition by 5-azacytidine augments presentation of immunogenic frameshift-derived neoepitopes.

Frameshifted protein sequences elicit tumor-specific T cell-mediated immune responses in microsatellite-unstable (MSI) cancers if presented by HLA class I molecules. However, their expression and presentation are limited by nonsense-mediated RNA decay (NMD). We employed an unbiased immunopeptidomics workflow to analyze MSI HCT-116 cells and identified >10,000 HLA class I-presented peptides including five frameshift-derived InDel neoepitopes. Notably, pharmacological NMD inhibition with 5-azacytidine stabilizes frameshift-bearing transcripts and increases the HLA class I-mediated presentation of InDel neoepitopes. The frameshift mutation underlying one of the identified InDel neoepitopes is highly recurrent in MSI colorectal cancer cell lines and primary patient samples, and immunization with the corresponding neoepitope induces strong CD8+ T cell responses in an HLA-A∗02:01 transgenic mouse model. Our data show directly that pharmacological NMD inhibition augments HLA class I-mediated presentation of immunogenic frameshift-derived InDel neoepitopes thus highlighting the clinical potential of NMD inhibition in anti-cancer immunotherapy strategies.

Quality standards in proteomics research facilities: Common standards and quality procedures are essential for proteomics facilities and their users.

Proteomics research infrastructures and core facilities within the Core for Life alliance advocate for community policies for quality control to ensure high standards in proteomics services.

SARS-CoV-2 infection remodels the host protein thermal stability landscape.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a global threat to human health and has compromised economic stability. In addition to the development of an effective vaccine, it is imperative to understand how SARS-CoV-2 hijacks host cellular machineries on a system-wide scale so that potential host-directed therapies can be developed. In situ proteome-wide abundance and thermal stability measurements using thermal proteome profiling (TPP) can inform on global changes in protein activity. Here we adapted TPP to high biosafety conditions amenable to SARS-CoV-2 handling. We discovered pronounced temporal alterations in host protein thermostability during infection, which converged on cellular processes including cell cycle, microtubule and RNA splicing regulation. Pharmacological inhibition of host proteins displaying altered thermal stability or abundance during infection suppressed SARS-CoV-2 replication. Overall, this work serves as a framework for expanding TPP workflows to globally important human pathogens that require high biosafety containment and provides deeper resolution into the molecular changes induced by SARS-CoV-2 infection.

Aggregation and disaggregation features of the human proteome.

Protein aggregates have negative implications in disease. While reductionist experiments have increased our understanding of aggregation processes, the systemic view in biological context is still limited. To extend this understanding, we used mass spectrometry-based proteomics to characterize aggregation and disaggregation in human cells after non-lethal heat shock. Aggregation-prone proteins were enriched in nuclear proteins, high proportion of intrinsically disordered regions, high molecular mass, high isoelectric point, and hydrophilic amino acids. During recovery, most aggregating proteins disaggregated with a rate proportional to the aggregation propensity: larger loss in solubility was counteracted by faster disaggregation. High amount of intrinsically disordered regions were associated with faster disaggregation. However, other characteristics enriched in aggregating proteins did not correlate with the disaggregation rates. In addition, we analyzed changes in protein thermal stability after heat shock. Soluble remnants of aggregated proteins were more thermally stable compared with control condition. Therefore, our results provide a rich resource of heat stress-related protein solubility data and can foster further studies related to protein aggregation diseases.