A mimetic peptide of ACE2 protects against SARS-CoV-2 infection and decreases pulmonary inflammation related to COVID-19.

Since human angiotensin-converting enzyme 2 (ACE2) serves as a primary receptor for SARS-CoV-2, characterizing ACE2 regions that allow SARS-CoV-2 to enter human cells is essential for designing peptide-based antiviral blockers and elucidating the pathogenesis of the virus. We identified and synthesized a 25-mer mimetic peptide (encompassing positions 22-46 of the ACE2 alpha-helix α1) implicated in the S1 receptor-binding domain (RBD)-ACE2 interface. The mimetic (wild-type, WT) ACE2 peptide significantly inhibited SARS-CoV-2 infection of human pulmonary Calu-3 cells in vitro. In silico protein modeling predicted that residues F28, K31, F32, F40, and Y41 of the ACE2 alpha-helix α1 are critical for the original, Delta, and Omicron strains of SARS-CoV-2 to establish the Spike RBD-ACE2 interface. Substituting these residues with alanine (A) or aspartic acid (D) abrogated the antiviral protective effect of the peptides, indicating that these positions are critical for viral entry into pulmonary cells. WT ACE2 peptide, but not the A or D mutated peptides, exhibited significant interaction with the SARS-CoV-2 S1 RBD, as shown through molecular dynamics simulations. Through identifying the critical amino acid residues of the ACE2 alpha-helix α1, which is necessary for the Spike RBD-ACE2 interface and mobilized during the in vitro viral infection of cells, we demonstrated that the WT ACE2 peptide protects susceptible K18-hACE2 mice against in vivo SARS-CoV-2 infection and is effective for the treatment of COVID-19.

The recombinant L-lysine α-oxidase from the fungus Trichoderma harzianum promotes apoptosis and necrosis of leukemia CD34 + hematopoietic cells.

BACKGROUND: In hematologic cancers, including leukemia, cells depend on amino acids for rapid growth. Anti-metabolites that prevent their synthesis or promote their degradation are considered potential cancer treatment agents. Amino acid deprivation triggers proliferation inhibition, autophagy, and programmed cell death. L-lysine, an essential amino acid, is required for tumor growth and has been investigated for its potential as a target for cancer treatment. L-lysine α-oxidase, a flavoenzyme that degrades L-lysine, has been studied for its ability to induce apoptosis and prevent cancer cell proliferation. In this study, we describe the use of L-lysine α-oxidase (LO) from the filamentous fungus Trichoderma harzianum for cancer treatment. RESULTS: The study identified and characterized a novel LO from T. harzianum and demonstrated that the recombinant protein (rLO) has potent and selective cytotoxic effects on leukemic cells by triggering the apoptotic cascade through mitochondrial dysfunction. CONCLUSIONS: The results support future translational studies using the recombinant LO as a potential drug for the treatment of leukemia.

Biological and therapeutic implications of RKIP in Gastrointestinal Stromal Tumor (GIST): an integrated transcriptomic and proteomic analysis.

BACKGROUND: Gastrointestinal stromal tumors (GIST) represent a significant clinical challenge due to their metastatic potential and limited treatment options. Raf kinase inhibitor protein (RKIP), a suppressor of the MAPK signaling pathway, is downregulated in various cancers and acts as a metastasis suppressor. Our previous studies demonstrated low RKIP expression in GIST and its association with poor outcomes. This study aimed to expand on the previous findings and investigate the biological and therapeutic implications of RKIP loss on GIST. METHODS: To validate the RKIP prognostic significance, its expression was evaluated by immunohistochemistry in 142 bona fide GIST cases. The functional role of RKIP was evaluated in vitro, using the GIST-T1 cell line, which was knocked out for RKIP. The biological and therapeutic implications of RKIP were evaluated by invasion, migration, apoptosis, and 2D / 3D viability assays. Additionally, the transcriptome and proteome of RKIP knockout cells were determined by NanoString and mass spectrometry, respectively. RESULTS: Immunohistochemical analysis revealed the absence of RKIP in 25.3% of GIST cases, correlating with a tendency toward poor prognosis. Functional assays demonstrated that RKIP knockout increased GIST cells' invasion and migration potential by nearly 60%. Moreover, we found that RKIP knockout cells exhibited reduced responsiveness to Imatinib treatment and higher cellular viability in 2D and 3D in vitro models, as assessed by apoptosis-related protein expression. Through comprehensive genetic and proteomic profiling of RKIP knockout cells, we identified several putative RKIP-regulated proteins in GIST, such as COL3A1. CONCLUSIONS: Using a multidimensional integrative analysis, we identified, for the first time in GIST, molecules and pathways modulated by RKIP that may potentially drive metastasis and, consequently, poor prognosis in this disease.

Proteomic analysis of exosomes secreted during the epithelial-mesenchymal transition and potential biomarkers of mesenchymal high-grade serous ovarian carcinoma.

BACKGROUND: The epithelial-mesenchymal transition (EMT) promotes cell signaling and morphology alterations, contributing to cancer progression. Exosomes, extracellular vesicles containing proteins involved in cell-cell communication, have emerged as a potential source of biomarkers for several diseases. METHODS: Our aim was to assess the proteome content of exosomes secreted after EMT-induction to identify potential biomarkers for ovarian cancer classification. EMT was induced in the ovarian cancer cell line CAOV3 by treating it with EGF (10 ng/mL) for 96 h following 24 h of serum deprivation. Subsequently, exosomes were isolated from the supernatant using selective centrifugation after decellularization, and their characteristics were determined. The proteins present in the exosomes were extracted, identified, and quantified using Label-Free-Quantification (LFQ) via Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS). To identify potential biomarkers, the obtained proteomic data was integrated with the TGGA database for mRNA expression using principal component analysis and a conditional inference tree. RESULTS: The exosomes derived from CAOV3 cells exhibited similar diameter and morphology, measuring approximately 150 nm, regardless of whether they were subjected to EMT stimulation or not. The proteomic analysis of proteins from CAOV3-derived exosomes revealed significant differential regulation of 157 proteins, with 100 showing upregulation and 57 downregulation upon EMT induction. Further comparison of the upregulated proteins with the TCGA transcriptomic data identified PLAU, LAMB1, COL6A1, and TGFB1 as potential biomarkers of the mesenchymal HGSOC subtype. CONCLUSIONS: The induction of EMT, the isolation of exosomes, and the subsequent proteomic analysis highlight potential biomarkers for an aggressive ovarian cancer subtype. Further investigation into the role of these proteins is warranted to enhance our understanding of ovarian cancer outcomes.

SARS-CoV-2 uses CD4 to infect T helper lymphocytes.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the agent of a major global outbreak of respiratory tract disease known as Coronavirus Disease 2019 (COVID-19). SARS-CoV-2 infects mainly lungs and may cause several immune-related complications, such as lymphocytopenia and cytokine storm, which are associated with the severity of the disease and predict mortality. The mechanism by which SARS-CoV-2 infection may result in immune system dysfunction is still not fully understood. Here, we show that SARS-CoV-2 infects human CD4+ T helper cells, but not CD8+ T cells, and is present in blood and bronchoalveolar lavage T helper cells of severe COVID-19 patients. We demonstrated that SARS-CoV-2 spike glycoprotein (S) directly binds to the CD4 molecule, which in turn mediates the entry of SARS- CoV-2 in T helper cells. This leads to impaired CD4 T cell function and may cause cell death. SARS-CoV-2-infected T helper cells express higher levels of IL-10, which is associated with viral persistence and disease severity. Thus, CD4-mediated SARS-CoV-2 infection of T helper cells may contribute to a poor immune response in COVID-19 patients.

What Do the Transcriptome and Proteome of Menstrual Blood-Derived Mesenchymal Stem Cells Tell Us about Endometriosis?

Given the importance of menstrual blood in the pathogenesis of endometriosis and the multifunctional roles of menstrual mesenchymal stem cells (MenSCs) in regenerative medicine, this issue has gained prominence in the scientific community. Moreover, recent reviews highlight how robust the integrated assessment of omics data are for endometriosis. To our knowledge, no study has applied the multi-omics approaches to endometriosis MenSCs. This is a case-control study at a university-affiliated hospital. MenSCs transcriptome and proteome data were obtained by RNA-seq and UHPLC-MS/MS detection. Among the differentially expressed proteins and genes, we emphasize ATF3, ID1, ID3, FOSB, SNAI1, NR4A1, EGR1, LAMC3, and ZFP36 genes and MT2A, TYMP, COL1A1, COL6A2, and NID2 proteins that were already reported in the endometriosis. Our functional enrichment analysis reveals integrated modulating signaling pathways such as epithelial-mesenchymal transition (↑) and PI3K signaling via AKT to mTORC1 (↓ in proteome), mTORC1 signaling, TGF beta signaling, TNFA signaling via NFkB, IL6 STAT3 signaling, and response to hypoxia via HIF1A targets (↑ in transcriptome). Our findings highlight primary changes in the endometriosis MenSCs, suggesting that the chronic inflammatory endometrial microenvironment can modulate these cells, providing opportunities for endometriosis etiopathogenesis. Moreover, they identify challenges for future research leveraging knowledge for regenerative and precision medicine in endometriosis.

The AIRE G228W mutation disturbs the interaction of AIRE with its partner molecule SIRT1.

The autoimmune regulator (AIRE) protein functions as a tetramer, interacting with partner proteins to form the "AIRE complex," which relieves RNA Pol II stalling in the chromatin of medullary thymic epithelial cells (mTECs). AIRE is the primary mTEC transcriptional controller, promoting the expression of a large set of peripheral tissue antigen genes implicated in the negative selection of self-reactive thymocytes. Under normal conditions, the SIRT1 protein temporarily interacts with AIRE and deacetylates K residues of the AIRE SAND domain. Once the AIRE SAND domain is deacetylated, the binding with SIRT1 is undone, allowing the AIRE complex to proceed downstream with the RNA Pol II to the elongation phase of transcription. Considering that the in silico and in vitro binding of the AIRE SAND domain with SIRT1 provides a powerful model system for studying the dominant SAND G228W mutation mechanism, which causes the autoimmune polyglandular syndrome-1, we integrated computational molecular modeling, docking, dynamics between the whole SAND domain with SIRT1, and surface plasmon resonance using a peptide harboring the 211 to 230 residues of the SAND domain, to compare the structure and energetics of binding/release between AIRE G228 (wild-type) and W228 (mutant) SAND domain to SIRT1. We observed that the G228W mutation in the SAND domain negatively influences the AIRE-SIRT1 interaction. The disturbed interaction might cause a disruption in the binding of the AIRE SAND domain with the SIRT1 catalytic site, impairing the AIRE complex to proceed downstream with RNA Pol II.

Multiple 9-1-1 complexes promote homolog synapsis, DSB repair, and ATR signaling during mammalian meiosis.

DNA damage response mechanisms have meiotic roles that ensure successful gamete formation. While completion of meiotic double-strand break (DSB) repair requires the canonical RAD9A-RAD1-HUS1 (9A-1-1) complex, mammalian meiocytes also express RAD9A and HUS1 paralogs, RAD9B and HUS1B, predicted to form alternative 9-1-1 complexes. The RAD1 subunit is shared by all predicted 9-1-1 complexes and localizes to meiotic chromosomes even in the absence of HUS1 and RAD9A. Here, we report that testis-specific disruption of RAD1 in mice resulted in impaired DSB repair, germ cell depletion, and infertility. Unlike Hus1 or Rad9a disruption, Rad1 loss in meiocytes also caused severe defects in homolog synapsis, impaired phosphorylation of ATR targets such as H2AX, CHK1, and HORMAD2, and compromised meiotic sex chromosome inactivation. Together, these results establish critical roles for both canonical and alternative 9-1-1 complexes in meiotic ATR activation and successful prophase I completion.

Phosphoproteomics of ATR signaling in mouse testes.

The phosphatidylinositol 3' kinase (PI3K)-related kinase ATR is crucial for mammalian meiosis. ATR promotes meiotic progression by coordinating key events in DNA repair, meiotic sex chromosome inactivation (MSCI), and checkpoint-dependent quality control during meiotic prophase I. Despite its central roles in meiosis, the ATR-dependent meiotic signaling network remains largely unknown. Here, we used phosphoproteomics to define ATR signaling events in testes from mice following chemical and genetic ablation of ATR signaling. Quantitative analysis of phosphoproteomes obtained after germ cell-specific genetic ablation of the ATR activating 9-1-1 complex or treatment with ATR inhibitor identified over 14,000 phosphorylation sites from testes samples, of which 401 phosphorylation sites were found to be dependent on both the 9-1-1 complex and ATR. Our analyses identified ATR-dependent phosphorylation events in crucial DNA damage signaling and DNA repair proteins including TOPBP1, SMC3, MDC1, RAD50, and SLX4. Importantly, we identified ATR and RAD1-dependent phosphorylation events in proteins involved in mRNA regulatory processes, including SETX and RANBP3, whose localization to the sex body was lost upon ATR inhibition. In addition to identifying the expected ATR-targeted S/T-Q motif, we identified enrichment of an S/T-P-X-K motif in the set of ATR-dependent events, suggesting that ATR promotes signaling via proline-directed kinase(s) during meiosis. Indeed, we found that ATR signaling is important for the proper localization of CDK2 in spermatocytes. Overall, our analysis establishes a map of ATR signaling in mouse testes and highlights potential meiotic-specific actions of ATR during prophase I progression.

COVID-19 diagnosis by SARS-CoV-2 Spike protein detection in saliva using an ultrasensitive magneto-assay based on disposable electrochemical sensor.

The outbreak of the COVID-19 pandemic, caused by Severe Acute Respiratory Syndrome of Coronavirus 2 (SARS-CoV-2), has fueled the search for diagnostic tests aiming at the control and reduction of the viral transmission. The main technique used for diagnosing the Coronavirus disease (COVID-19) is the reverse transcription-polymerase chain reaction (RT-PCR) technique. However, considering the high number of cases and the underlying limitations of the RT-PCR technique, especially with regard to accessibility and cost of the test, one does not need to overemphasize the need to develop new and less expensive testing techniques that can aid the early diagnosis of the disease. With that in mind, we developed an ultrasensitive magneto-assay using magnetic beads and gold nanoparticles conjugated to human angiotensin-converting enzyme 2 (ACE2) peptide (Gln24-Gln42) for the capturing and detection of SARS-CoV-2 Spike protein in human saliva. The technique applied involved the use of a disposable electrochemical device containing eight screen-printed carbon electrodes which allow the simultaneous analysis of eight samples. The magneto-assay exhibited an ultralow limit of detection of 0.35 ag mL-1 for the detection of SARS-CoV-2 Spike protein in saliva. The magneto-assay was tested in saliva samples from healthy and SARS-CoV-2-infected individuals. In terms of efficiency, the proposed technique - which presented a sensitivity of 100.0% and specificity of 93.7% for SARS-CoV-2 Spike protein-exhibited great similarity with the RT-PCR technique. The results obtained point to the application potential of this simple, low-cost magneto-assay for saliva-based point-of-care COVID-19 diagnosis.

The Expression of NTAL and Its Protein Interactors Is Associated With Clinical Outcomes in Acute Myeloid Leukemia.

Non-T cell activation linker (NTAL) membrane protein depletion from lipid rafts by alkylphospholipids or downregulation by shRNA knockdown decreases cell viability through regulation of the Akt/PI3K pathway in mantle cell lymphoma and acute promyelocytic leukemia cells. Here, we confirmed that the knockdown of NTAL in acute myeloid leukemia (AML) cell lines was associated with decreased cell proliferation and survival. Similarly, a xenograft model using AML cells transduced with NTAL-shRNA and transplanted into immunodeficient mice led to a 1.8-fold decrease in tumor burden. Using immunoprecipitation, LC-MS/MS analysis, and label-free protein quantification, we identified interactors of NTAL in two AML cell lines. By evaluating the gene expression signatures of the NTAL protein interactors using the PREdiction of Clinical Outcomes from Genomic Profiles database, we found that 12 NTAL interactors could predict overall survival in AML, in at least two independent cohorts. In addition, patients with AML exhibiting a high expression of NTAL and its interactors were associated with a leukemic granulocyte-macrophage progenitor-like state. Taken together, our data provide evidence that NTAL and its protein interactors are relevant to AML cell proliferation and survival and represent potential therapeutic targets for granulocyte-macrophage progenitor-like leukemias.

Schistosoma mansoni Heterochromatin Protein 1 (HP1) nuclear interactome in cercariae.

Schistosoma mansoni causes schistosomiasis, which affects 240 million people, and 700 million people are living at risk of infection. Epigenetic mechanisms are important for transcriptional control and are well-known conserved transcriptional co-regulators in evolution, already described in mammal, yeast, protozoa and S. mansoni, responsible for heterochromatization and gene silence mechanisms through the formation of complexes of transcriptional repression in chromatin. Previous results from another group have shown that HP1 (SmCBX) proteins form chromatin complexes with SmMDB2/3 proteins and regulate stem cells and oviposition in parasite adult worms. In addition, results from other groups have shown that cercariae are transcriptionally silent and epigenetic mechanisms are involved in the regulation of gene expression in this stage. In this work, our aim was to give insights into SmHP1 and proteins involved in transcriptional regulation in the cercariae stage. Using monoclonal anti-HP1 antibody for Western blotting, immunoprecipitation, and mass spectrometry, we preliminarily determined nuclear proteins that putatively interact with HP1 to form complexes to regulate gene expression, heterochromatin formation, and translational complexes in the cercariae stage. So far, our data is to give some insights into nuclear interactors in S. mansoni cercariae. SIGNIFICANCE: The significance of this original paper is the evidence for Heterochromatin Protein (HP1), interaction with nuclear proteins in the cercariae stage. Schistosoma mansoni cercariae are the infective stage of the human beings in endemic areas of schistosomiasis, a neglected disease, most prevalent in Brazil and Africa. While cercariae are waiting for a host, it does not feed, gene expression is silent and protein synthesis is stopped. These biochemical mechanisms are recovered when cercariae find a human host, but all proteins and mechanisms are not still elucidated. Until now, literature shows that these phenomena are regulated by epigenetics mechanisms, dependent of histone posttranslational modifications. But we have few pieces of evidence about the other proteins that participates in these processes and which are the co-regulators of expression.

Phosphoproteomics reveals a distinctive Mec1/ATR signaling response upon DNA end hyper-resection.

The Mec1/ATR kinase is crucial for genome maintenance in response to a range of genotoxic insults, but it remains unclear how it promotes context-dependent signaling and DNA repair. Using phosphoproteomic analyses, we uncovered a distinctive Mec1/ATR signaling response triggered by extensive nucleolytic processing (resection) of DNA ends. Budding yeast cells lacking Rad9, a checkpoint adaptor and an inhibitor of resection, exhibit a selective increase in Mec1-dependent phosphorylation of proteins associated with single-strand DNA (ssDNA) transactions, including the ssDNA-binding protein Rfa2, the translocase/ubiquitin ligase Uls1, and the Sgs1-Top3-Rmi1 (STR) complex that regulates homologous recombination (HR). Extensive Mec1-dependent phosphorylation of the STR complex, mostly on the Sgs1 helicase subunit, promotes an interaction between STR and the DNA repair scaffolding protein Dpb11. Fusion of Sgs1 to phosphopeptide-binding domains of Dpb11 strongly impairs HR-mediated repair, supporting a model whereby Mec1 signaling regulates STR upon hyper-resection to influence recombination outcomes. Overall, the identification of a distinct Mec1 signaling response triggered by hyper-resection highlights the multi-faceted action of this kinase in the coordination of checkpoint signaling and HR-mediated DNA repair.

Inhibition of Aurora kinase A activity enhances the antitumor response of beta-catenin blockade in human adrenocortical cancer cells.

Adrenocortical cancer (ACC) is a rare and aggressive type of endocrine tumor with high risk of recurrence and metastasis. The overall survival of patients diagnosed with ACC is low and treatment for metastatic stages remain limited to mitotane, which has low efficiency in advanced stages of the disease and is associated with high toxicity. Therefore, identification of new biological targets to improve ACC treatment is crucial. Blockade of the Wnt/beta-catenin pathway decreased adrenal steroidogenesis and increased apoptosis of NCI-H295 human ACC cells, in vitro and in a xenograft mouse model. Aurora kinases play important roles in cell division during the G1-M phase and their aberrant expression is correlated with a poor prognosis in different types of tumors. Hence, we hypothesized that inhibition of aurora kinases activity combined with the beta-catenin pathway blockade would improve the impairment of ACC cell growth in vitro. We studied the combinatorial effects of AMG 900, an aurora kinase inhibitor and PNU-74654, a beta-catenin pathway blocker, on proliferation, survival and tumor progression in multiple ACC cell lines: NCI-H295, CU-ACC1 and CU-ACC2. Exposure of ACC cells to the combination of AMG 900 with PNU-74654 decreased cell proliferation and viability compared to either treatment alone. In addition, AMG 900 inhibited cell invasion and clonogenesis compared to PNU-74654, and the combination showed no greater effects. In contrast, PNU-74654 was more effective in decreasing cortisol secretion. These data suggest that inhibition of aurora kinases activity combined with blockade of the beta-catenin pathway may provide a combinatorial approach for targeting ACC tumors.

A freeze-and-thaw-induced fragment of the microtubule-associated protein tau in rat brain extracts: implications for the biochemical assessment of neurotoxicity.

Tau is a microtubule-associated protein (MAP) responsible for controlling the stabilization of microtubules in neurons. Tau function is regulated by phosphorylation. However, in some neurological diseases Tau becomes aberrantly hyperphosphorylated, which contributes to the pathogenesis of neurological diseases, known as tauopathies. Western blotting (WB) has been widely employed to determine Tau levels in neurological disease models. However, Tau quantification by WB should be interpreted with care, as this approach has been recognized as prone to produce artifactual results if not properly performed. In the present study, our goal was to evaluate the influence of a freeze-and-thaw cycle, a common procedure preceding WB, to the integrity of Tau in brain homogenates from rats, 3xTg-AD mice and human samples. Homogenates were prepared in ice-cold RIPA buffer supplemented with protease/phosphatase inhibitors. Immediately after centrifugation, an aliquot of the extracts was analyzed via WB to quantify total and phosphorylated Tau levels. The remaining aliquots of the same extracts were stored for at least 2 weeks at either -20 or -80°C and then subjected to WB. Extracts from rodent brains submitted to freeze-and-thaw presented a ∼25 kDa fragment immunoreactive to anti-Tau antibodies. An in-gel digestion followed by mass spectrometry (MS) analysis in excised bands revealed this ∼25 kDa species corresponds to a Tau fragment. Freeze-and-thaw-induced Tau proteolysis was detected even when extracts were stored at -80°C. This phenomenon was not observed in human samples at any storage condition tested. Based on these findings, we strongly recommend the use of fresh extracts of brain samples in molecular analysis of Tau levels in rodents.

Analysis of ovarian cancer cell secretome during epithelial to mesenchymal transition reveals a protein signature associated with advanced stages of ovarian tumors.

Ovarian cancer (OvCA) is the most lethal neoplasia among gynecologic malignancies and faces high rates of new cases particularly in South America. In special, the High Grade Serous Ovarian Carcinoma (HGSC) presents very poor prognosis with deaths caused mainly by metastasis. Among several mechanisms involved in metastasis, the Epithelial to Mesenchymal Transition (EMT) molecular reprogramming represents a model for latest stages of cancer progression. EMT promotes important cellular changes in cellular adhesion and cell-cell communication, which particularly depends on the paracrine signaling from neighbor cells. Considering the importance of cellular communication during EMT and metastasis, here we analyzed the changes in the secretome of the ovarian cancer cell line Caov-3 induced to EMT by Epidermal Growth Factor (EGF). Using a combination of GEL-LC-MS/MS and stable isotopic metabolic labelling (SILAC), we identified up-regulated candidates during EMT as a starting point to identify relevant proteins for HGSC. Based on public databases, our candidate proteins were validated and prioritized for further analysis. Importantly, several of the protein candidates were associated with cellular vesicles, which are important to the cell-cell communication and metastasis. Furthermore, the association of candidate proteins with gene expression data uncovered a subset of proteins correlated with the mesenchymal subtype of ovarian cancer. Based on this relevant molecular signature for aggressive ovarian cancer, supported by protein and gene expression data, we developed a targeted proteomic method to evaluate individual OvCA clinical samples. The quantitative information obtained for 33 peptides, representative of 18 proteins, was able to segregate HGSC from other tumor types. Our study highlighted the richness of the secretome and EMT to reveal relevant proteins for HGSC, which could be used in further studies and larger patient cohorts as a potential stratification signature for ovarian cancer tumor that could guide clinical conduct for patient treatment.

In-depth and 3-dimensional exploration of the budding yeast phosphoproteome.

Phosphorylation is one of the most dynamic and widespread post-translational modifications regulating virtually every aspect of eukaryotic cell biology. Here, we assemble a dataset from 75 independent phosphoproteomic experiments performed in our laboratory using Saccharomyces cerevisiae. We report 30,902 phosphosites identified from cells cultured in a range of DNA damage conditions and/or arrested in distinct cell cycle stages. To generate a comprehensive resource for the budding yeast community, we aggregate our dataset with the Saccharomyces Genome Database and another recently published study, resulting in over 46,000 budding yeast phosphosites. With the goal of enhancing the identification of functional phosphorylation events, we perform computational positioning of phosphorylation sites on available 3D protein structures and systematically identify events predicted to regulate protein complex architecture. Results reveal hundreds of phosphorylation sites mapping to or near protein interaction interfaces, many of which result in steric or electrostatic "clashes" predicted to disrupt the interaction. With the advancement of Cryo-EM and the increasing number of available structures, our approach should help drive the functional and spatial exploration of the phosphoproteome.

Human and mouse melanoma cells recapitulate an EMT-like program in response to mesenchymal stromal cells secretome.

The mechanisms underlying the propensity of melanomas to metastasize are not completely understood. We hypothesized that melanoma cells are capable of promptly activating an epithelial-to-mesenchymal transition (EMT)-like profile in response to stroma-derived factors. Thus, we investigated the role of mesenchymal stromal cells (MSCs), a cell population considered as a precursor of tumor stroma, on the activation of an EMT-like profile and acquisition of metastatic traits in melanoma cells. After subcutaneous co-injection with mouse B16 melanoma cells, MSCs occupied perivascular sites within tumors and enhanced B16 metastasis to the lungs. In vitro, MSCs' secretome activated an EMT-like profile in B16 cells, reducing their avidity to fibronectin, and increasing their motility and invasiveness. These effects were abrogated upon blocking of MET phosphorylation in B16 cells using small molecule inhibitors. MSCs also activated an EMT-like profile in human melanoma cells from different stages of progression. Activation of EMT in human cells was associated with increased levels of p-STAT1 and p-STAT3. In conclusion, both mouse and human melanoma cells are equipped to activate an EMT-like program and acquire metastatic traits through the activation of distinct pathways by MSCs' secretome.

Maximized quantitative phosphoproteomics allows high confidence dissection of the DNA damage signaling network.

The maintenance of genomic stability relies on DNA damage sensor kinases that detect DNA lesions and phosphorylate an extensive network of substrates. The Mec1/ATR kinase is one of the primary sensor kinases responsible for orchestrating DNA damage responses. Despite the importance of Mec1/ATR, the current network of its identified substrates remains incomplete due, in part, to limitations in mass spectrometry-based quantitative phosphoproteomics. Phosphoproteomics suffers from lack of redundancy and statistical power for generating high confidence datasets, since information about phosphopeptide identity, site-localization, and quantitation must often be gleaned from a single peptide-spectrum match (PSM). Here we carefully analyzed the isotope label swapping strategy for phosphoproteomics, using data consistency among reciprocal labeling experiments as a central filtering rule for maximizing phosphopeptide identification and quantitation. We demonstrate that the approach allows drastic reduction of false positive quantitations and identifications even from phosphopeptides with a low number of spectral matches. Application of this approach identifies new Mec1/ATR-dependent signaling events, expanding our understanding of the DNA damage signaling network. Overall, the proposed quantitative phosphoproteomic approach should be generally applicable for investigating kinase signaling networks with high confidence and depth.

Host Retromer Protein Sorting Nexin 2 Interacts with Human Respiratory Syncytial Virus Structural Proteins and is Required for Efficient Viral Production.

Human respiratory syncytial virus (HRSV) envelope glycoproteins traffic to assembly sites through the secretory pathway, while nonglycosylated proteins M and N are present in HRSV inclusion bodies but must reach the plasma membrane, where HRSV assembly happens. Little is known about how nonglycosylated HRSV proteins reach assembly sites. Here, we show that HRSV M and N proteins partially colocalize with the Golgi marker giantin, and the glycosylated F and nonglycosylated N proteins are closely located in the trans-Golgi, suggesting their interaction in that compartment. Brefeldin A compromised the trafficking of HRSV F and N proteins and inclusion body sizes, indicating that the Golgi is important for both glycosylated and nonglycosylated HRSV protein traffic. HRSV N and M proteins colocalized and interacted with sorting nexin 2 (SNX2), a retromer component that shapes endosomes in tubular structures. Glycosylated F and nonglycosylated N HRSV proteins are detected in SNX2-laden aggregates with intracellular filaments projecting from their outer surfaces, and VPS26, another retromer component, was also found in inclusion bodies and filament-shaped structures. Similar to SNX2, TGN46 also colocalized with HRSV M and N proteins in filamentous structures at the plasma membrane. Cell fractionation showed enrichment of SNX2 in fractions containing HRSV M and N proteins. Silencing of SNX1 and 2 was associated with reduction in viral proteins, HRSV inclusion body size, syncytium formation, and progeny production. The results indicate that HRSV structural proteins M and N are in the secretory pathway, and SNX2 plays an important role in the traffic of HRSV structural proteins toward assembly sites.IMPORTANCE The present study contributes new knowledge to understand HRSV assembly by providing evidence that nonglycosylated structural proteins M and N interact with elements of the secretory pathway, shedding light on their intracellular traffic. To the best of our knowledge, the present contribution is important given the scarcity of studies about the traffic of HRSV nonglycosylated proteins, especially by pointing to the involvement of SNX2, a retromer component, in the HRSV assembly process.