Multilevel proteomics reveals host perturbations by SARS-CoV-2 and SARS-CoV.

The emergence and global spread of SARS-CoV-2 has resulted in the urgent need for an in-depth understanding of molecular functions of viral proteins and their interactions with the host proteome. Several individual omics studies have extended our knowledge of COVID-19 pathophysiology1-10. Integration of such datasets to obtain a holistic view of virus-host interactions and to define the pathogenic properties of SARS-CoV-2 is limited by the heterogeneity of the experimental systems. Here we report a concurrent multi-omics study of SARS-CoV-2 and SARS-CoV. Using state-of-the-art proteomics, we profiled the interactomes of both viruses, as well as their influence on the transcriptome, proteome, ubiquitinome and phosphoproteome of a lung-derived human cell line. Projecting these data onto the global network of cellular interactions revealed crosstalk between the perturbations taking place upon infection with SARS-CoV-2 and SARS-CoV at different levels and enabled identification of distinct and common molecular mechanisms of these closely related coronaviruses. The TGF-ß pathway, known for its involvement in tissue fibrosis, was specifically dysregulated by SARS-CoV-2 ORF8 and autophagy was specifically dysregulated by SARS-CoV-2 ORF3. The extensive dataset (available at https://covinet.innatelab.org ) highlights many hotspots that could be targeted by existing drugs and may be used to guide rational design of virus- and host-directed therapies, which we exemplify by identifying inhibitors of kinases and matrix metalloproteases with potent antiviral effects against SARS-CoV-2.

Correction to: HIV-1 IN/Pol recruits LEDGF/p75 into viral particles.

An amendment to this paper has been published and can be accessed via the original article.

A Novel LC System Embeds Analytes in Pre-formed Gradients for Rapid, Ultra-robust Proteomics.

To further integrate mass spectrometry (MS)-based proteomics into biomedical research and especially into clinical settings, high throughput and robustness are essential requirements. They are largely met in high-flow rate chromatographic systems for small molecules but these are not sufficiently sensitive for proteomics applications. Here we describe a new concept that delivers on these requirements while maintaining the sensitivity of current nano-flow LC systems. Low-pressure pumps elute the sample from a disposable trap column, simultaneously forming a chromatographic gradient that is stored in a long storage loop. An auxiliary gradient creates an offset, ensuring the re-focusing of the peptides before the separation on the analytical column by a single high-pressure pump. This simplified design enables robust operation over thousands of sample injections. Furthermore, the steps between injections are performed in parallel, reducing overhead time to a few minutes and allowing analysis of more than 200 samples per day. From fractionated HeLa cell lysates, deep proteomes covering more than 130,000 sequence unique peptides and close to 10,000 proteins were rapidly acquired. Using this data as a library, we demonstrate quantitation of 5200 proteins in only 21 min. Thus, the new system - termed Evosep One - analyzes samples in an extremely robust and high throughput manner, without sacrificing in depth proteomics coverage.

The Impact II, a Very High-Resolution Quadrupole Time-of-Flight Instrument (QTOF) for Deep Shotgun Proteomics.

Hybrid quadrupole time-of-flight (QTOF) mass spectrometry is one of the two major principles used in proteomics. Although based on simple fundamentals, it has over the last decades greatly evolved in terms of achievable resolution, mass accuracy, and dynamic range. The Bruker impact platform of QTOF instruments takes advantage of these developments and here we develop and evaluate the impact II for shotgun proteomics applications. Adaption of our heated liquid chromatography system achieved very narrow peptide elution peaks. The impact II is equipped with a new collision cell with both axial and radial ion ejection, more than doubling ion extraction at high tandem MS frequencies. The new reflectron and detector improve resolving power compared with the previous model up to 80%, i.e. to 40,000 at m/z 1222. We analyzed the ion current from the inlet capillary and found very high transmission (>80%) up to the collision cell. Simulation and measurement indicated 60% transfer into the flight tube. We adapted MaxQuant for QTOF data, improving absolute average mass deviations to better than 1.45 ppm. More than 4800 proteins can be identified in a single run of HeLa digest in a 90 min gradient. The workflow achieved high technical reproducibility (R2 > 0.99) and accurate fold change determination in spike-in experiments in complex mixtures. Using label-free quantification we rapidly quantified haploid against diploid yeast and characterized overall proteome differences in mouse cell lines originating from different tissues. Finally, after high pH reversed-phase fractionation we identified 9515 proteins in a triplicate measurement of HeLa peptide mixture and 11,257 proteins in single measurements of cerebellum-the highest proteome coverage reported with a QTOF instrument so far.

HIV-1 IN/Pol recruits LEDGF/p75 into viral particles.

BACKGROUND: The dynamic interaction between HIV and its host governs the replication of the virus and the study of the virus-host interplay is key to understand the viral lifecycle. The host factor lens epithelium-derived growth factor (LEDGF/p75) tethers the HIV preintegration complex to the chromatin through a direct interaction with integrase (IN). Small molecules that bind the LEDGF/p75 binding pocket of the HIV IN dimer (LEDGINs) block HIV replication through a multimodal mechanism impacting early and late stage replication including HIV maturation. Furthermore, LEDGF/p75 has been identified as a Pol interaction partner. This raised the question whether LEDGF/p75 besides acting as a molecular tether in the target cell, also affects late steps of HIV replication. RESULTS: LEDGF/p75 is recruited into HIV-1 particles through direct interaction with the viral IN (or Pol polyprotein) and is a substrate for HIV-1 protease. Incubation in the presence of HIV-1 protease inhibitors resulted in detection of full-length LEDGF/p75 in purified viral particles. We also demonstrate that inhibition of LEDGF/p75-IN interaction by specific mutants or LEDGINs precludes incorporation of LEDGF/p75 in virions, underscoring the specificity of the uptake. LEDGF/p75 depletion did however not result in altered LEDGIN potency. CONCLUSION: Together, these results provide evidence for an IN/Pol mediated uptake of LEDGF/p75 in viral particles and a specific cleavage by HIV protease. Understanding of the possible role of LEDGF/p75 or its cleavage fragments in the viral particle awaits further experimentation.

Accurate proteome-wide label-free quantification by delayed normalization and maximal peptide ratio extraction, termed MaxLFQ.

Protein quantification without isotopic labels has been a long-standing interest in the proteomics field. However, accurate and robust proteome-wide quantification with label-free approaches remains a challenge. We developed a new intensity determination and normalization procedure called MaxLFQ that is fully compatible with any peptide or protein separation prior to LC-MS analysis. Protein abundance profiles are assembled using the maximum possible information from MS signals, given that the presence of quantifiable peptides varies from sample to sample. For a benchmark dataset with two proteomes mixed at known ratios, we accurately detected the mixing ratio over the entire protein expression range, with greater precision for abundant proteins. The significance of individual label-free quantifications was obtained via a t test approach. For a second benchmark dataset, we accurately quantify fold changes over several orders of magnitude, a task that is challenging with label-based methods. MaxLFQ is a generic label-free quantification technology that is readily applicable to many biological questions; it is compatible with standard statistical analysis workflows, and it has been validated in many and diverse biological projects. Our algorithms can handle very large experiments of 500+ samples in a manageable computing time. It is implemented in the freely available MaxQuant computational proteomics platform and works completely seamlessly at the click of a button.

Minimal, encapsulated proteomic-sample processing applied to copy-number estimation in eukaryotic cells.

Mass spectrometry (MS)-based proteomics typically employs multistep sample-preparation workflows that are subject to sample contamination and loss. We report an in-StageTip method for performing sample processing, from cell lysis through elution of purified peptides, in a single, enclosed volume. This robust and scalable method largely eliminates contamination or loss. Peptides can be eluted in several fractions or in one step for single-run proteome analysis. In one day, we obtained the largest proteome coverage to date for budding and fission yeast, and found that protein copy numbers in these cells were highly correlated (R(2) = 0.78). Applying the in-StageTip method to quadruplicate measurements of a human cell line, we obtained copy-number estimates for 9,667 human proteins and observed excellent quantitative reproducibility between replicates (R(2) = 0.97). The in-StageTip method is straightforward and generally applicable in biological or clinical applications.

Mesenchymal stem cells and glioma cells form a structural as well as a functional syncytium in vitro.

The interaction of human mesenchymal stem cells (hMSCs) and tumor cells has been investigated in various contexts. HMSCs are considered as cellular treatment vectors based on their capacity to migrate towards a malignant lesion. However, concerns about unpredictable behavior of transplanted hMSCs are accumulating. In malignant gliomas, the recruitment mechanism is driven by glioma-secreted factors which lead to accumulation of both, tissue specific stem cells as well as bone marrow derived hMSCs within the tumor. The aim of the present work was to study specific cellular interactions between hMSCs and glioma cells in vitro. We show, that glioma cells as well as hMSCs differentially express connexins, and that they interact via gap-junctional coupling. Besides this so-called functional syncytium formation, we also provide evidence of cell fusion events (structural syncytium). These complex cellular interactions led to an enhanced migration and altered proliferation of both, tumor and mesenchymal stem cell types in vitro. The presented work shows that glioma cells display signs of functional as well as structural syncytium formation with hMSCs in vitro. The described cellular phenomena provide new insight into the complexity of interaction patterns between tumor cells and host cells. Based on these findings, further studies are warranted to define the impact of a functional or structural syncytium formation on malignant tumors and cell based therapies in vivo.

Tenascin-C enhances pancreatic cancer cell growth and motility and affects cell adhesion through activation of the integrin pathway.

BACKGROUND: Pancreatic cancer (PDAC) is characterized by an abundant fibrous tissue rich in Tenascin-C (TNC), a large ECM glycoprotein mainly synthesized by pancreatic stellate cells (PSCs). In human pancreatic tissues, TNC expression increases in the progression from low-grade precursor lesions to invasive cancer. Aim of this study was the functional characterization of the effects of TNC on biologic relevant properties of pancreatic cancer cells. METHODS: Proliferation, migration and adhesion assays were performed on pancreatic cancer cell lines treated with TNC or grown on a TNC-rich matrix. Stable transfectants expressing the large TNC splice variant were generated to test the effects of endogenous TNC. TNC-dependent integrin signaling was investigated by immunoblotting, immunofluorescence and pharmacological inhibition. RESULTS: Endogenous TNC promoted pancreatic cancer cell growth and migration. A TNC-rich matrix also enhanced migration as well as the adhesion to the uncoated growth surface of poorly differentiated cell lines. In contrast, adhesion to fibronectin was significantly decreased in the presence of TNC. The effects of TNC on cell adhesion were paralleled by changes in the activation state of paxillin and Akt. CONCLUSION: TNC affects proliferation, migration and adhesion of poorly differentiated pancreatic cancer cell lines and might therefore play a role in PDAC spreading and metastasis in vivo.

Nucleophosmin is overexpressed in thyroid tumors.

Nucleophosmin (NPM) is a protein that contributes to several cell functions. Depending on the context, it can act as an oncogene or tumor suppressor. No data are available on NPM expression in thyroid cells. In this work, we analyzed both NPM mRNA and protein levels in a series of human thyroid tumor tissues and cell lines. By using immunohistochemistry, NPM overexpression was detected in papillary, follicular, undifferentiated thyroid cancer, and also in follicular benign adenomas, indicating it as an early event during thyroid tumorigenesis. In contrast, various levels of NPM mRNA levels as detected by quantitative RT-PCR were observed in tumor tissues, suggesting a dissociation between protein and transcript expression. The same behavior was observed in the normal thyroid FRTL5 cell lines. In these cells, a positive correlation between NPM protein levels, but not mRNA, and proliferation state was detected. By using thyroid tumor cell lines, we demonstrated that such a post-mRNA regulation may depend on NPM binding to p-Akt, whose levels were found to be increased in the tumor cells, in parallel with reduction of PTEN. In conclusion, our present data demonstrate for the first time that nucleophosmin is overexpressed in thyroid tumors, as an early event of thyroid tumorigenesis. It seems as a result of a dysregulation occurring at protein and not transcriptional level related to an increase of p-Akt levels of transformed thyrocytes.

Detection of aromatic catabolic gene expression in heterogeneous organic matter used for reduction of volatile organic compounds (VOC) by biofiltration.

A qualitative procedure of purified DNA/RNA co-extraction from complex organic matter, used as biofilter support for removing volatile organic compounds, was set up and applied to detect xylene monooxygenase gene expression by RT-PCR. A DNA/RNA extraction protocol based on a combination of sample lyophilization pre-treatment and CTAB--phenol/chloroform extraction procedure was optimized for the recovery of purified nucleic acids [100-500 ng DNA (10 kb) and 0.5-2 microg of rRNA 16S from 100 mg matrix]. PCR and RT-PCR protocols were established to detect xylene monooxygenase gene expression starting from differentially induced organic matrices obtained by biofiltration technology. This work allowed the microbial degradation activities in heterogeneous organic solid media to be studied and suggests a rapid method to follow specific biological activities during solid and/or semisolid organic substrates biotransformation.

Proteomic analysis of liver tissues subjected to early ischemia/reperfusion injury during human orthotopic liver transplantation.

Knowledge of early molecular events occurring upon ischemia/reperfusion (I/R) during liver transplantation (LT) is of great importance to improve the therapeutic intervention of surgical treatment. However, nowadays, few data are available on early protein targets of I/R injury. To identify these proteins, we used a differential proteomics approach in the characterization human liver biopsies during I/R upon LT. Analyses were performed on nine donor livers during LT. By using 2-DE and MALDI-TOF MS, we identified 36 proteins which resulted significantly altered upon I/R injury. The majority of these proteins are functionally involved in lipid and energy metabolism, in different metabolic pathways, in redox signalling and in oxidative-stress response. Our data represent the first global approach in the study of I/R injury in liver.

Differential proteomic analysis of nuclear extracts from thyroid cell lines.

Nuclear proteins play a major role in controlling cell functions. Differential proteomic analysis of nuclear proteins by combined 2D gel electrophoresis (2D-E) and mass spectrometry procedures can provide useful information to understand the control of cell proliferation and differentiation. To identify proteins involved in dedifferentiation, we used a differential proteomics approach by comparing nuclear extracts from the differentiated rat thyroid cell line FRTL-5 and the derived undifferentiated Ki-mol cell line, obtained by transformation with the Ki-ras oncogene. Thirteen proteins were identified as differently expressed in the nuclear compartment between the two cell lines. RT-PCR analysis performed on seven differently expressed genes showed that only in two cases the difference may be ascribable to a transcriptional mechanism. Since one of the identified proteins, namely apurinic apyrimidinic endonuclease/redox effector factor-1 (APE1/Ref-1), is suspected to play a role in thyroid tumorigenesis, we used a glutathione S-transferase (GST)-pulldown assay coupled to a 2D electrophoretic/matrix assisted laser desorption ionization-time of flight (MALDI-TOF)-mass spectrometry (MS) analysis to detect and identify its interacting partners. We show here that beta-actin directly interacted with APE1/Ref-1, as confirmed by co-immunoprecipitation assays and that this interaction was enhanced by oxidative stress on FRTL-5 cells.

Overoxidation of peroxiredoxins as an immediate and sensitive marker of oxidative stress in HepG2 cells and its application to the redox effects induced by ischemia/reperfusion in human liver.

Oxidative stress is a major pathogenetic event occurring in several liver disorders and is a major cause of liver damage due to Ischemia/Reperfusion (I/R) during liver transplantation. While several markers of chronic oxidative stress are well known, early protein targets of oxidative injury are not well defined. In order to identify these proteins, we used a differential proteomics approach to HepG2 human liver cells treated for 10 min with 500 microM H(2)O(2). This dose was sufficient to induce a slight decrease of total GSH and total protein thiol content without affecting cell viability. By performing Differential Proteomic analysis, by means of two-dimensional gel electrophoresis and MALDI-TOF mass spectrometry, we identified four proteins which resulted sensitive to H(2)O(2) treatment. The main changes were due to post-translational modifications of native polypeptides. Three of these proteins belong to the Peroxiredoxin family of hydroperoxide scavengers, namely PrxI, PrxII and PrxVI, that showed changes in their pI as result of overoxidation. Mass mapping experiments demonstrated the specific modification of peroxiredoxins active site thiol into sulphinic and/or sulphonic acid, thus explaining the increase in negative charge measured for these proteins. The oxidation kinetic of all peroxiredoxins was extremely rapid and sensitive, occurring at H(2)O(2) doses unable to affect the common markers of cellular oxidative stress. Recovery experiments demonstrated a quite different behaviour between 1-Cys and 2-Cys containing Prxs as their retroreduction features is concerned, thus suggesting a functional difference between different class of Prxs. The in vivo relevance of our study is demonstrated by the finding that overoxidation of PrxI occurs during I/R upon liver transplantation and is dependent on the time of warm ischemia. Our present data could be of relevance in setting up more standardized procedures to preserve organs for transplantations.

Galectin-3 expression in non-small cell lung carcinoma.

Galectins are a family of animal beta-galactoside-binding lectins involved in malignant transformation and progression. The present study evaluated the immunohistochemical expression of galectin-3 in a consecutive series of 81 radically resected non-small cell lung carcinomas (NSCLCs). The main pattern of galectin-3 expression was cytoplasmic (median percentage of cells with cytoplasmic positivity: 80.0%). The median percentage of tumor cells with nuclear and cytoplasmic co-expression of galectin-3 was 3.5%. No cases with exclusive nuclear immunostaining were observed. Functional interaction between galectin-3 and the thyroid transcription factor-1 (TTF-1) was previously demonstrated by cotransfection experiments. In the present study, concomitant expression of nuclear galectin-3 and TTF-1 was independently associated with a worse clinical outcome (HR 2.0; P = 0.01).

Proteomic evaluation of core biopsy specimens from breast lesions.

Analysis of tumour samples by a proteomic technology, which combines two-dimensional gel electrophoresis and mass spectrometry analysis, is a promising approach for molecular characterization of cancer. Proteomic analysis of neoplasms is usually performed on surgical material. The possibility to perform proteomic analysis on pre-operative samples might be useful for diagnostic purposes or for determination of tumour sensitivity to therapy. In this study, we report how tissues from core biopsy of breast lesions can be routinely used to obtain accurate protein expression profiles by proteomic analysis. Protein profiles from fibroadenomas were compared to those from ductal infiltrating carcinomas. By using mass spectrometry, identification of proteins overexpressed in carcinomas with respect to fibroadenomas was obtained. Thus, our study provides a methodology to perform proteomic analysis on pre-operative samples of breast lesions.

A proteomic approach to identify early molecular targets of oxidative stress in human epithelial lens cells.

Oxidative stress is one of the most relevant contributors of cataractogenesis. To identify early protein targets of oxidative stress in lens cells, we used a differential proteomics approach to CD5A human epithelial lens cells treated with 500 microM H2O2 for 30 min. This dose of H2O2 was assayed to induce efficiently a block of cellular proliferation and to activate the oxidative stress-early inducible transcription factor EGR-1 (early growth response gene product 1), previously reported as stimulated factor in a model of cataractogenesis [Nakajima, Nakajima, Fukiage, Azuma and Shearer (2002) Exp. Eye Res. 74, 231-236]. We identified nine proteins, which sensitively reacted to H2O2 treatment by using two-dimensional gel electrophoresis and matrix-assisted laserdesorption ionization-time-of-flight-MS. In addition to cytoskeletal proteins (tubulin 1alpha and vimentin) and enzymes (phosphoglycerate kinase 1, ATP synthase beta, enolase alpha, nucleophosmin and heat-shock cognate 54 kDa protein), which presented quantitative differences in expression profiles, peroxiredoxin and glyceraldehyde 3-phosphate dehydrogenase showed changes in pI as a result of overoxidation. Mass-mapping experiments demonstrated the specific modification of peroxiredoxin I active-site cysteine into cysteic acid, thus providing an explanation for the increase in negative charge measured for this protein. With respect to other global differential approaches based on gene expression analysis, our results allowed us to identify novel molecular targets of oxidative stress in lens cells. These results indicate that a combination of different approaches is required for a complete functional understanding of the biological events triggered by oxidative stress.

Nuclear localization of Galectin-3 in transformed thyroid cells: a role in transcriptional regulation.

The differential proteomic approach (2D gel analysis coupled to MALDI-MS analysis) of nuclear proteins can provide an extremely useful tool to understand control of cell proliferation and differentiation. In order to identify possible markers of dedifferentiation between normal and cancerous thyroid cells, we used a differential proteomics approach by comparing nuclear extracts from the normal rat thyroid cell line FRTL-5 and the completely undifferentiated Ki-mol cell line, obtained by transformation with the Ki-ras oncogene. Galectin-3 (Gal-3) was identified as highly expressed, in the nuclear compartment, only in the transformed cell line. By using different human cancer cell lines, we showed that Gal-3 is maximally expressed in nuclei of papillary cancer cells. We focused on the functional relationship existing between Gal-3 and the thyroid-specific transcription factor TTF-1, whose expression is maintained in papillary cancer where it can contribute to the proliferating status. By using gel-retardation and transient tranfection assays, we demonstrate that Gal-3 upregulates the TTF-1 transcriptional activity. GST-pulldown experiments demonstrate the occurrence of interaction between Gal-3 and TTF-1 homeodomain. Since several lines of evidence suggest a role for Gal-3 in controlling proliferation and tumor progression in thyroid cancer, the stimulatory activity played by Gal-3 over TTF-1 would account for a possible molecular mechanism through which the galectin controls proliferation in thyroid cells.

Redox effector factor-1 regulates the activity of thyroid transcription factor 1 by controlling the redox state of the N transcriptional activation domain.

Thyroid transcription factor 1 (TTF-1) is a homeodomain-containing transcriptional regulator responsible for the activation of thyroid- and lung-specific genes. It has been demonstrated that its DNA binding activity is redox-regulated in vitro through the formation of dimers and oligomeric species. In this paper, we demonstrate that the redox regulation mainly involves a Cys residue (Cys(87)), which resides out of the DNA binding domain, belonging to the N-transactivation domain. In fact, the oxidized form of a truncated TTF-1 (containing the N-transactivation domain and the DNA-binding domain, here called TTF-1N-HD) looses specific DNA binding activity. Since most of the oxidized TTF-1N-HD is in a monomeric form, these data indicate that the redox state of Cys(87) may control the DNA-binding function of the homeodomain, suggesting that Cys(87) could play an important role in determining the correct folding of the homeodomain. By using gel retardation and transient transfection assays, we demonstrate that the redox effector factor-1 (Ref-1) mediates the redox effects on TTF-1N-HD binding and that it is able to modulate the TTF-1 transcriptional activity. Glutathione S-transferase pull-down experiments demonstrate the occurrence of interaction between Ref-1 and TTF-1N-HD. Having previously demonstrated that Ref-1 is able to modulate the transcriptional activity of another thyroid-specific transcription factor (Pax-8), our data suggest that Ref-1 plays a central role in the regulation of thyroid cells.