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.

Molecular profiling of sponge deflation reveals an ancient relaxant-inflammatory response.

A hallmark of animals is the coordination of whole-body movement. Neurons and muscles are central to this, yet coordinated movements also exist in sponges that lack these cell types. Sponges are sessile animals with a complex canal system for filter-feeding. They undergo whole-body movements resembling "contractions" that lead to canal closure and water expulsion. Here, we combine live 3D optical coherence microscopy, pharmacology, and functional proteomics to elucidate the sequence and detail of shape changes, the tissues and molecular physiology involved, and the control of these movements. Morphometric analysis and targeted perturbation suggest that the movement is driven by the relaxation of actomyosin stress fibers in epithelial canal cells, which leads to whole-body deflation via collapse of the incurrent and expansion of the excurrent canal system. Thermal proteome profiling and quantitative phosphoproteomics confirm the control of cellular relaxation by an Akt/NO/PKG/PKA pathway. Agitation-induced deflation leads to differential phosphorylation of proteins forming epithelial cell junctions, implying their mechanosensitive role. Unexpectedly, untargeted metabolomics detect a concomitant decrease in antioxidant molecules during deflation, reflecting an increase in reactive oxygen species. Together with the secretion of proteinases, cytokines, and granulin, this indicates an inflammation-like state of the deflating sponge reminiscent of vascular endothelial cells experiencing oscillatory shear stress. These results suggest the conservation of an ancient relaxant-inflammatory response of perturbed fluid-carrying systems in animals and offer a possible mechanism for whole-body coordination through diffusible paracrine signals and mechanotransduction.

STPP-UP: An alternative method for drug target identification using protein thermal stability.

Thermal proteome profiling (TPP) has significantly advanced the field of drug discovery by facilitating proteome-wide identification of drug targets and off-targets. However, TPP has not been widely applied for high-throughput drug screenings, since the method is labor intensive and requires a lot of measurement time on a mass spectrometer. Here, we present Single-tube TPP with Uniform Progression (STPP-UP), which significantly reduces both the amount of required input material and measurement time, while retaining the ability to identify drug targets for compounds of interest. By using incremental heating of a single sample, changes in protein thermal stability across a range of temperatures can be assessed, while alleviating the need to measure multiple samples heated to different temperatures. We demonstrate that STPP-UP is able to identify the direct interactors for anticancer drugs in both human and mice cells. In summary, the STPP-UP methodology represents a useful tool to advance drug discovery and drug repurposing efforts.

Redox Proteomic Profile of Tirapazamine-Resistant Murine Hepatoma Cells.

3-Amino-1,2,4-benzotriazine-1,4-dioxide (tirapazamine, TPZ) and other heteroaromatic N-oxides (ArN→O) exhibit tumoricidal, antibacterial, and antiprotozoal activities. Their action is attributed to the enzymatic single-electron reduction to free radicals that initiate the prooxidant processes. In order to clarify the mechanisms of aerobic mammalian cytotoxicity of ArN→O, we derived a TPZ-resistant subline of murine hepatoma MH22a cells (resistance index, 5.64). The quantitative proteomic of wild-type and TPZ-resistant cells revealed 5818 proteins, of which 237 were up- and 184 down-regulated. The expression of the antioxidant enzymes aldehyde- and alcohol dehydrogenases, carbonyl reductases, catalase, and glutathione reductase was increased 1.6-5.2 times, whereas the changes in the expression of glutathione peroxidase, superoxide dismutase, thioredoxin reductase, and peroxiredoxins were less pronounced. The expression of xenobiotics conjugating glutathione-S-transferases was increased by 1.6-2.6 times. On the other hand, the expression of NADPH:cytochrome P450 reductase was responsible for the single-electron reduction in TPZ and for the 2.1-fold decrease. These data support the fact that the main mechanism of action of TPZ under aerobic conditions is oxidative stress. The unchanged expression of intranuclear antioxidant proteins peroxiredoxin, glutaredoxin, and glutathione peroxidase, and a modest increase in the expression of DNA damage repair proteins, tend to support non-site-specific but not intranuclear oxidative stress as a main factor of TPZ aerobic cytotoxicity.

The Mechanism of Dendrite Formation in a Solid-State Transformation of High Aluminum Fe-Al Alloys.

The mechanism of solid-state dendrite formation in high-aluminum Fe-Al alloys is not clear. Applying an in-situ observation technique, the real-time formation and growth of FeAl solid-state dendrites during the eutectoid decomposition of the high-temperature phase Fe5Al8 is visualized. In-situ experiments by HT-CSLM reveal that proeutectoid FeAl usually does not preferentially nucleate at grain boundaries regardless of rapid or slow cooling conditions. The critical radii for generating morphological instability are 1.2 µm and 0.9 µm for slow and rapid cooling, respectively. The morphology after both slow and rapid cooling exhibits dendrites, while there are differences in the size and critical instability radius Rc, which are attributed to the different supersaturation S and the number of protrusions l. The combination of crystallographic and thermodynamic analysis indicates that solid-state dendrites only exist on the hypoeutectoid side in high-aluminum Fe-Al alloys. A large number of lattice defects in the parent phase provides an additional driving force for nucleation, leading to coherent nucleation from the interior of the parent phase grains based on the orientation relationship {3¯30}Fe5Al8//{1¯10}FeAl, <111¯>Fe5Al8//<111¯>FeAl. The maximum release of misfit strain energy leads to the preferential growth of the primary arm of the nucleus along <111¯> {1¯10}. During the rapid cooling process, a large supersaturation is induced in the matrix, driving the Al atoms to undergo unstable uphill diffusion and causing variations in the concentration gradient as well as generating constitutional undercooling, ultimately leading to morphological instability and the growth of secondary arms.

Independent human mesenchymal stromal cell-derived extracellular vesicle preparations differentially attenuate symptoms in an advanced murine graft-versus-host disease model.

BACKGROUND AIMS: Extracellular vesicles (EVs) harvested from conditioned media of human mesenchymal stromal cells (MSCs) suppress acute inflammation in various disease models and promote regeneration of damaged tissues. After successful treatment of a patient with acute steroid-refractory graft-versus-host disease (GVHD) using EVs prepared from conditioned media of human bone marrow-derived MSCs, this study focused on improving the MSC-EV production for clinical application. METHODS: Independent MSC-EV preparations all produced according to a standardized procedure revealed broad immunomodulatory differences. Only a proportion of the MSC-EV products applied effectively modulated immune responses in a multi-donor mixed lymphocyte reaction (mdMLR) assay. To explore the relevance of such differences in vivo, at first a mouse GVHD model was optimized. RESULTS: The functional testing of selected MSC-EV preparations demonstrated that MSC-EV preparations revealing immunomodulatory capabilities in the mdMLR assay also effectively suppress GVHD symptoms in this model. In contrast, MSC-EV preparations, lacking such in vitro activities, also failed to modulate GVHD symptoms in vivo. Searching for differences of the active and inactive MSC-EV preparations, no concrete proteins or miRNAs were identified that could serve as surrogate markers. CONCLUSIONS: Standardized MSC-EV production strategies may not be sufficient to warrant manufacturing of MSC-EV products with reproducible qualities. Consequently, given this functional heterogeneity, every individual MSC-EV preparation considered for the clinical application should be evaluated for its therapeutic potency before administration to patients. Here, upon comparing immunomodulating capabilities of independent MSC-EV preparations in vivo and in vitro, we found that the mdMLR assay was qualified for such analyses.

Mitochondrial dysfunction rapidly modulates the abundance and thermal stability of cellular proteins.

Cellular functionality relies on a well-balanced, but highly dynamic proteome. Dysfunction of mitochondrial protein import leads to the cytosolic accumulation of mitochondrial precursor proteins which compromise cellular proteostasis and trigger a mitoprotein-induced stress response. To dissect the effects of mitochondrial dysfunction on the cellular proteome as a whole, we developed pre-post thermal proteome profiling. This multiplexed time-resolved proteome-wide thermal stability profiling approach with isobaric peptide tags in combination with a pulsed SILAC labelling elucidated dynamic proteostasis changes in several dimensions: In addition to adaptations in protein abundance, we observed rapid modulations of the thermal stability of individual cellular proteins. Different functional groups of proteins showed characteristic response patterns and reacted with group-specific kinetics, allowing the identification of functional modules that are relevant for mitoprotein-induced stress. Thus, our new pre-post thermal proteome profiling approach uncovered a complex response network that orchestrates proteome homeostasis in eukaryotic cells by time-controlled adaptations of the abundance and the conformation of proteins.

Extended N-Terminal Acetyltransferase Naa50 in Filamentous Fungi Adds to Naa50 Diversity.

Most eukaryotic proteins are N-terminally acetylated by a set of Nα acetyltransferases (NATs). This ancient and ubiquitous modification plays a fundamental role in protein homeostasis, while mutations are linked to human diseases and phenotypic defects. In particular, Naa50 features species-specific differences, as it is inactive in yeast but active in higher eukaryotes. Together with NatA, it engages in NatE complex formation for cotranslational acetylation. Here, we report Naa50 homologs from the filamentous fungi Chaetomium thermophilum and Neurospora crassa with significant N- and C-terminal extensions to the conserved GNAT domain. Structural and biochemical analyses show that CtNaa50 shares the GNAT structure and substrate specificity with other homologs. However, in contrast to previously analyzed Naa50 proteins, it does not form NatE. The elongated N-terminus increases Naa50 thermostability and binds to dynein light chain protein 1, while our data suggest that conserved positive patches in the C-terminus allow for ribosome binding independent of NatA. Our study provides new insights into the many facets of Naa50 and highlights the diversification of NATs during evolution.

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.

Increased levels of mitochondrial import factor Mia40 prevent the aggregation of polyQ proteins in the cytosol.

The formation of protein aggregates is a hallmark of neurodegenerative diseases. Observations on patient samples and model systems demonstrated links between aggregate formation and declining mitochondrial functionality, but causalities remain unclear. We used Saccharomyces cerevisiae to analyze how mitochondrial processes regulate the behavior of aggregation-prone polyQ protein derived from human huntingtin. Expression of Q97-GFP rapidly led to insoluble cytosolic aggregates and cell death. Although aggregation impaired mitochondrial respiration only slightly, it considerably interfered with the import of mitochondrial precursor proteins. Mutants in the import component Mia40 were hypersensitive to Q97-GFP, whereas Mia40 overexpression strongly suppressed the formation of toxic Q97-GFP aggregates both in yeast and in human cells. Based on these observations, we propose that the post-translational import of mitochondrial precursor proteins into mitochondria competes with aggregation-prone cytosolic proteins for chaperones and proteasome capacity. Mia40 regulates this competition as it has a rate-limiting role in mitochondrial protein import. Therefore, Mia40 is a dynamic regulator in mitochondrial biogenesis that can be exploited to stabilize cytosolic proteostasis.

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.

Stroma Transcriptomic and Proteomic Profile of Prostate Cancer Metastasis Xenograft Models Reveals Prognostic Value of Stroma Signatures.

Resistance acquisition to androgen deprivation treatment and metastasis progression are a major clinical issue associated with prostate cancer (PCa). The role of stroma during disease progression is insufficiently defined. Using transcriptomic and proteomic analyses on differentially aggressive patient-derived xenografts (PDXs), we investigated whether PCa tumors predispose their microenvironment (stroma) to a metastatic gene expression pattern. RNA sequencing was performed on the PCa PDXs BM18 (castration-sensitive) and LAPC9 (castration-resistant), representing different disease stages. Using organism-specific reference databases, the human-specific transcriptome (tumor) was identified and separated from the mouse-specific transcriptome (stroma). To identify proteomic changes in the tumor (human) versus the stroma (mouse), we performed human/mouse cell separation and subjected protein lysates to quantitative Tandem Mass Tag labeling and mass spectrometry. Tenascin C (TNC) was among the most abundant stromal genes, modulated by androgen levels in vivo and highly expressed in castration-resistant LAPC9 PDX. The tissue microarray of primary PCa samples (n = 210) showed that TNC is a negative prognostic marker of the clinical progression to recurrence or metastasis. Stroma markers of osteoblastic PCa bone metastases seven-up signature were induced in the stroma by the host organism in metastatic xenografts, indicating conserved mechanisms of tumor cells to induce a stromal premetastatic signature. A 50-gene list stroma signature was identified based on androgen-dependent responses, which shows a linear association with the Gleason score, metastasis progression and progression-free survival. Our data show that metastatic PCa PDXs, which differ in androgen sensitivity, trigger differential stroma responses, which show the metastasis risk stratification and prognostic biomarker potential.

ACLY is the novel signaling target of PIP2/PIP3 and Lyn in acute myeloid leukemia.

A fundamental feature of tumor progression is reprogramming of metabolic pathways. ATP citrate lyase (ACLY) is a key metabolic enzyme that catalyzes the generation of Acetyl-CoA and is upregulated in cancer cells and required for their growth. The phosphoinositide 3-kinase (PI3K) and Src-family kinase (SFK) Lyn are constitutively activate in many cancers. We show here, for the first time, that both the substrate and product of PI3K, phosphatidylinositol-(4,5)-bisphosphate (PIP2) and phosphatidylinositol-(3,4,5)-trisphosphate (PIP3), respectively, bind to ACLY in Acute Myeloid Leukemia (AML) patient-derived, but not normal donor-derived cells. We demonstrate the binding of PIP2 to the CoA-binding domain of ACLY and identify the six tyrosine residues of ACLY that are phosphorylated by Lyn. Three of them (Y682, Y252, Y227) can be also phosphorylated by Src and they are located in catalytic, citrate binding and ATP binding domains, respectively. PI3K and Lyn inhibitors reduce the ACLY enzyme activity, ACLY-mediated Acetyl-CoA synthesis, phospholipid synthesis, histone acetylation and cell growth. Thus, PIP2/PIP3 binding and Src tyrosine kinases-mediated stimulation of ACLY links oncogenic pathways to Acetyl-CoA-dependent pro-growth and survival metabolic pathways in cancer cells. These results indicate a novel function for Lyn, as a regulator of Acetyl-CoA-mediated metabolic pathways.

Plasticity of nuclear and cytoplasmic stress responses of RNA-binding proteins.

Cellular stress causes multifaceted reactions to trigger adaptive responses to environmental cues at all levels of the gene expression pathway. RNA-binding proteins (RBP) are key contributors to stress-induced regulation of RNA fate and function. Here, we uncover the plasticity of the RNA interactome in stressed cells, differentiating between responses in the nucleus and in the cytoplasm. We applied enhanced RNA interactome capture (eRIC) analysis preceded by nucleo-cytoplasmic fractionation following arsenite-induced oxidative stress. The data reveal unexpectedly compartmentalized RNA interactomes and their responses to stress, including differential responses of RBPs in the nucleus versus the cytoplasm, which would have been missed by whole cell analyses.

The Hsp90 isoforms from S. cerevisiae differ in structure, function and client range.

The molecular chaperone Hsp90 is an important regulator of proteostasis. It has remained unclear why S. cerevisiae possesses two Hsp90 isoforms, the constitutively expressed Hsc82 and the stress-inducible Hsp82. Here, we report distinct differences despite a sequence identity of 97%. Consistent with its function under stress conditions, Hsp82 is more stable and refolds more efficiently than Hsc82. The two isoforms also differ in their ATPases and conformational cycles. Hsc82 is more processive and populates closed states to a greater extent. Variations in the N-terminal ATP-binding domain modulate its dynamics and conformational cycle. Despite these differences, the client interactomes are largely identical, but isoform-specific interactors exist both under physiological and heat shock conditions. Taken together, changes mainly in the N-domain create a stress-specific, more resilient protein with a shifted activity profile. Thus, the precise tuning of the Hsp90 isoforms preserves the basic mechanism but adapts it to specific needs.

Effect of Sec61 interaction with Mpd1 on endoplasmic reticulum-associated degradation.

Proteins that misfold in the endoplasmic reticulum (ER) are transported back to the cytosol for ER-associated degradation (ERAD). The Sec61 channel is one of the candidates for the retrograde transport conduit. Channel opening from the ER lumen must be triggered by ERAD factors and substrates. Here we aimed to identify new lumenal interaction partners of the Sec61 channel by chemical crosslinking and mass spectrometry. In addition to known Sec61 interactors we detected ERAD factors including Cue1, Ubc6, Ubc7, Asi3, and Mpd1. We show that the CPY* ERAD factor Mpd1 binds to the lumenal Sec61 hinge region. Deletion of the Mpd1 binding site reduced the interaction between both proteins and caused an ERAD defect specific for CPY* without affecting protein import into the ER or ERAD of other substrates. Our data suggest that Mpd1 binding to Sec61 is a prerequisite for CPY* ERAD and confirm a role of Sec61 in ERAD of misfolded secretory proteins.

Discovery of RNA-binding proteins and characterization of their dynamic responses by enhanced RNA interactome capture.

Following the realization that eukaryotic RNA-binding proteomes are substantially larger than anticipated, we must now understand their detailed composition and dynamics. Methods such as RNA interactome capture (RIC) have begun to address this need. However, limitations of RIC have been reported. Here we describe enhanced RNA interactome capture (eRIC), a method based on the use of an LNA-modified capture probe, which yields numerous advantages including greater specificity and increased signal-to-noise ratios compared to existing methods. In Jurkat cells, eRIC reduces the rRNA and DNA contamination by >10-fold compared to RIC and increases the detection of RNA-binding proteins. Due to its low background, eRIC also empowers comparative analyses of changes of RNA-bound proteomes missed by RIC. For example, in cells treated with dimethyloxalylglycine, which inhibits RNA demethylases, eRIC identifies m6A-responsive RNA-binding proteins that escape RIC. eRIC will facilitate the unbiased characterization of RBP dynamics in response to biological and pharmacological cues.

TRRAP is essential for regulating the accumulation of mutant and wild-type p53 in lymphoma.

Tumors accumulate high levels of mutant p53 (mutp53), which contributes to mutp53 gain-of-function properties. The mechanisms that underlie such excessive accumulation are not fully understood. To discover regulators of mutp53 protein accumulation, we performed a large-scale RNA interference screen in a Burkitt lymphoma cell line model. We identified transformation/transcription domain-associated protein (TRRAP), a constituent of several histone acetyltransferase complexes, as a critical positive regulator of both mutp53 and wild-type p53 levels. TRRAP silencing attenuated p53 accumulation in lymphoma and colon cancer models, whereas TRRAP overexpression increased mutp53 levels, suggesting a role for TRRAP across cancer entities and p53 mutations. Through clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 screening, we identified a 109-amino-acid region in the N-terminal HEAT repeat region of TRRAP that was crucial for mutp53 stabilization and cell proliferation. Mass spectrometric analysis of the mutp53 interactome indicated that TRRAP silencing caused degradation of mutp53 via the MDM2-proteasome axis. This suggests that TRRAP is vital for maintaining mutp53 levels by shielding it against the natural p53 degradation machinery. To identify drugs that alleviated p53 accumulation similarly to TRRAP silencing, we performed a small-molecule drug screen and found that inhibition of histone deacetylases (HDACs), specifically HDAC1/2/3, decreased p53 levels to a comparable extent. In summary, here we identify TRRAP as a key regulator of p53 levels and link acetylation-modifying complexes to p53 protein stability. Our findings may provide clues for therapeutic targeting of mutp53 in lymphoma and other cancers.

A single-cell model of PIP3 dynamics using chemical dimerization.

Most cellular processes are driven by simple biochemical mechanisms such as protein and lipid phosphorylation, but the sum of all these conversions is exceedingly complex. Hence, intuition alone is not enough to discern the underlying mechanisms in the light of experimental data. Toward this end, mathematical models provide a conceptual and numerical framework to formally evaluate the plausibility of biochemical processes. To illustrate the use of these models, here we built a mechanistic computational model of PI3K (phosphatidylinositol 3-kinase) activity, to determine the kinetics of lipid metabolizing enzymes in single cells. The model is trained to data generated upon perturbation with a reversible small-molecule based chemical dimerization system that allows for the very rapid manipulation of the PIP3 (phosphatidylinositol 3,4,5-trisphosphate) signaling pathway, and monitored with live-cell microscopy. We find that the rapid relaxation system used in this work decreased the uncertainty of estimating kinetic parameters compared to methods based on in vitro assays. We also examined the use of Bayesian parameter inference and how the use of such a probabilistic method gives information on the kinetics of PI3K and PTEN activity.