Contrasting roles of MERS-CoV and SARS-CoV-2 internal proteins in pathogenesis in mice.

Betacoronaviruses encode an internal (I) gene via an alternative reading frame within the nucleocapsid gene, called ORF8b for Middle-East respiratory syndrome coronavirus (MERS-CoV) and ORF9b for severe acute respiratory syndrome coronavirus (SARS-CoV) and SARS-CoV-2. Previous reports suggested that proteins 8b and 9b are involved in evading multiple innate immune signaling pathways. However, their roles in mediating pathogenesis in infected animals have not been determined. In this study, we abrogated the expression of protein 8b in MERS-CoV and protein 9b in SARS-CoV-2. Using mouse models of MERS-CoV and SARS-CoV-2 infection, we found that MERS-CoV lacking protein 8b expression was more virulent, while SARS-CoV-2 lacking protein 9b expression was attenuated compared with the respective wild-type viruses. Upon further analysis, we detected increased levels of type I interferon and enhanced infiltration of immune cells to the lungs of mice infected with MERS-CoV lacking protein 8b expression. These data suggest that the I protein of MERS-CoV plays a role in limiting pathogenesis while that of SARS-CoV-2 enhances disease severity. IMPORTANCE The function of betacoronavirus internal protein has been relatively understudied. The earliest report on the internal protein of mouse hepatitis virus suggested that the internal protein is a structural protein without significant functions in virus replication and virulence. However, the internal proteins of severe acute respiratory syndrome coronavirus (SARS-CoV), Middle-East respiratory syndrome coronavirus, and SARS-CoV-2 have been shown to evade immune responses. Despite the reported functions of the internal protein in these highly pathogenic human coronaviruses, its role in mediating pathogenesis in experimentally infected animals has not been characterized. Our data indicated that despite the similar genomic location and expression strategy of these internal proteins, their effects on virulence are vastly different and virus specific, highlighting the complexity between host-virus interaction and disease outcome.

RLIP76 (RalBP1) is an R-Ras effector that mediates adhesion-dependent Rac activation and cell migration.

The Ras family of small GTPases regulates cell proliferation, spreading, migration and apoptosis, and malignant transformation by binding to several protein effectors. One such GTPase, R-Ras, plays distinct roles in each of these processes, but to date, identified R-Ras effectors were shared with other Ras family members (e.g., H-Ras). We utilized a new database of Ras-interacting proteins to identify RLIP76 (RalBP1) as a novel R-Ras effector. RLIP76 binds directly to R-Ras in a GTP-dependent manner, but does not physically associate with the closely related paralogues H-Ras and Rap1A. RLIP76 is required for adhesion-induced Rac activation and the resulting cell spreading and migration, as well as for the ability of R-Ras to enhance these functions. RLIP76 regulates Rac activity through the adhesion-induced activation of Arf6 GTPase and activation of Arf6 bypasses the requirement for RLIP76 in Rac activation and cell spreading. Thus, we identify a novel R-Ras effector, RLIP76, which links R-Ras to adhesion-induced Rac activation through a GTPase cascade that mediates cell spreading and migration.

Phosphorylation by casein kinase 2 regulates Nap1 localization and function.

In Saccharomyces cerevisiae, the evolutionarily conserved nucleocytoplasmic shuttling protein Nap1 is a cofactor for the import of histones H2A and H2B, a chromatin assembly factor and a mitotic factor involved in regulation of bud formation. To understand the mechanism by which Nap1 function is regulated, Nap1-interacting factors were isolated and identified by mass spectrometry. We identified several kinases among these proteins, including casein kinase 2 (CK2), and a new bud neck-associated protein, Nba1. Consistent with our identification of the Nap1-interacting kinases, we showed that Nap1 is phosphorylated in vivo at 11 sites and that Nap1 is phosphorylated by CK2 at three substrate serines. Phosphorylation of these serines was not necessary for normal bud formation, but mutation of these serines to either alanine or aspartic acid resulted in cell cycle changes, including a prolonged S phase, suggesting that reversible phosphorylation by CK2 is important for cell cycle regulation. Nap1 can shuttle between the nucleus and cytoplasm, and we also showed that CK2 phosphorylation promotes the import of Nap1 into the nucleus. In conclusion, our data show that Nap1 phosphorylation by CK2 appears to regulate Nap1 localization and is required for normal progression through S phase.

Performance characteristics of an FT MS-based workflow for label-free differential MS analysis of human plasma: standards, reproducibility, targeted feature investigation, and application to a model of controlled myocardial infarction.

Proteomics is undergoing a rapid transformation from a qualitative global peptide sequencing discipline into a quantitative, reproducibility-driven practice. Nowhere is this more evident than in the rapidly expanding field of protein biomarker discovery where the general goal is to uncover statistically robust patterns of differential expression between or among subjects/samples representing distinct biological/temporal states. This report presents the analytical characterization of a label-free LC FT-ICR-MS workflow for differential proteomics analysis of human plasma. The key elements discussed include (i) methodologies for performing properly replicated experiments with highly reproducible sample preparation and analysis, including the use of internal standards to quantify variance at different steps in the process, (ii) a new methodology for performing sample re-analysis that uses off-line targeted robotic acquisition of complementary spectral data (e.g. ECD and/or IRMPD) to enhance the identification of differentially expressed peptides/proteins, and (iii) data processing pipelines capable of integrating the automatic statistical analysis of the label-free (LC-) MS signal, together with the intuitive and highly interactive curation and annotation of differential features using the output from standard sequence database search programs. We illustrate the application of the complete sample-to-annotated-differential-peptides (-proteins) workflow by describing the acquisition and analysis of a large multidimensional dataset from patients undergoing a controlled myocardial infarction resulting in an experimental setup in which each patients serve as their own control. Furthermore, we discuss a couple illustrative examples of mid-level proteins observed in this study whose plasma concentrations change consistently within and across patients, in a treatment- and time-dependent fashion.

A novel model to identify interaction partners of the PTEN tumor suppressor gene in human bladder cancer.

Phosphatase and tensin homolog (PTEN), deleted on chromosome 10, is a potent tumor suppressor. PTEN expression is reduced in advanced bladder cancer and reduction correlates with disease stage. To gain insights into the function of PTEN in human bladder cancer by identifying its binding partners, we developed a novel IPTG inducible PTEN expression system and evaluated this system in the PTEN null UMUC-3 human bladder cancer xenograft model. In this model, induction of PTEN in vivo resulted in reduced tumor growth. We used mass spectrometry to identify PTEN interaction partners in these cells, which identified known interaction partners major vault protein (MVP) and paxillin as well as a novel interaction partner, TRK fused gene (TFG). In conclusion, using a biologically relevant model system to dissect PTEN tumor suppressor function in human bladder cancer, we identified three molecules important for many cellular functions in complex with PTEN.

An experimentally derived database of candidate Ras-interacting proteins.

We used a TAP-tag approach to identify candidate binding proteins for the related Ras family GTPases: H-Ras, R-Ras, and Rap1A. Protein complexes were isolated from mouse fibroblasts, and component proteins were identified by a combination of nanoflow HPLC and tandem mass spectrometry. H-Ras was found to associate with numerous cytoskeletal proteins including talin-1. R-Ras and Rap1A each associated with various signaling molecules, many of which are membrane-associated. Thus, we have established the first database of potential Ras interactors in mammalian cells.

Ultralow-volume fraction collection from NanoLC columns for mass spectrometric analysis of protein phosphorylation and glycosylation.

An ultralow volume fraction collection system referred to as nano fraction analysis chip technology (nanoFACT) is reported. The system collects 25-2500-nL fractions from 75-microm nanoLC columns into pipet tips at a user-defined, timed interval, typically one fraction every 15-120 s. Following collection, the fractions in the tip dry down naturally on their own in such a way as to create a concentrated band at the very end of the interior of the pipet tip. The fractions are then reconstituted directly in the pipet tips in approximately 250 nL of solvent prior to analysis. Because the chromatography and reconstitution solvent are independent, the reconstitution solvent can be selected to maximize ionization efficiency without compromising chromatography. In the infusion analysis of the nanoLC fractions, a low-flow electrospray chip is used which consists of 400 nozzles, each with an inner diameter of 2.5 microm and yielding flow rates of approximately 20 nL/min. Therefore, when reconstituted in 250 nL, each nanoLC fraction can be analyzed for over 10 min. This increase in analysis time allows for signal averaging, resulting in higher data quality, collision energy optimization, slower scanning techniques to be used, such as neutral loss and precursor ion scanning, higher resolution scans on FTMS instruments, and improved peptide quantitation. Furthermore, the nanoLC fractions could be archived in the pipet tips for analysis at a later date. Here, the advantages of nanoFACT are shown for phosphorylation analysis using bovine fetuin and glycosylation analysis using bovine ribonuclease B (RNase B). In the phosphorylation analysis, a comparison between conventional nanoLC and a nanoFACT analysis was performed. An MS/MS spectrum of a triply phosphorylated peptide, 313-HTFSGVApSVEpSpSSGEAFHVGK-333 could only be obtained using nanoFACT, not with nanoLC. Furthermore, spectral quality for the nanoFACT analysis was significantly improved over nanoLC. This was determined by comparing the number of diagnostic ions between the nanoFACT and nanoLC spectra, and it was found that the nanoFACT spectra contained a 19% or greater number of diagnostic ions for nonphosphorylated peptides and 55% or greater for phosphorylated peptides. For the glycosylation analysis, the glycosylation site of RNase B was fully characterized using 100 fmol of tryptic digest on a three-dimensional ion trap mass spectrometer.

Modifications of human histone H3 variants during mitosis.

Phosphorylation of histone H3 is a hallmark event in mitosis and is associated with chromosome condensation. Here, we use a combination of immobilized metal affinity chromatography and tandem mass spectrometry to characterize post-translational modifications associated with phosphorylation on the N-terminal tails of histone H3 variants purified from mitotically arrested HeLa cells. Modifications observed in vivo on lysine residues adjacent to phosphorylated Ser and Thr provide support for the existence of the "methyl/phos", binary-switch hypothesis [Fischle, W., Wang, Y., and Allis, C. D. (2003) Nature 425, 475-479]. ELISA with antibodies selective for H3 at Ser10, Ser28, and Thr3 show reduced activity when adjacent Lys residues are modified. When used together, mass spectrometry and immunoassay methods provide a powerful approach for elucidation of the histone code and identification of histone post-translational modifications that occur during mitosis and other specific cellular events.