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Efficient transmission of herpesviruses is essential for dissemination in host populations; however, little is known about the viral genes that mediate transmission, mostly due to their close relationship to their natural host. Marek’s disease is a devastating herpesviral disease of chickens caused by Marek’s disease virus (MDV) and an excellent natural model to study skin- tropic herpesviruses and transmission. Similar to varicella zoster virus that causes chicken pox in humans, the only site where fully productive replication occurs is in epithelial skin cells and this is required for host to host transmission. Here, we enriched for actively replicating virus in feather follicle epithelial skin cells of live chickens to measure both viral transcription and protein expression using combined RNA sequencing and LC/MS-MS bottom-up proteomics. Enrichment produced a previously unseen breadth and depth of viral peptide sequencing. We confirmed protein translation for 84 viral genes at high confidence (1% FDR) and correlated relative protein abundance with RNA expression levels. Using a proteogenomic approach, we confirmed translation of most well-characterized spliced viral transcripts and identified a novel, abundant isoform of the 14 kDa transcript family via both intron-spanning sequencing reads as well as a high-quality junction-spanning peptide identification. We identified peptides representing alternative start codon usage in several genes and putative novel microORFs at the 5’ ends of two core herpesviral genes, pUL47 and ICP4, along with evidence of transcription and translation of the capsid scaffold protein pUL26.5. Using a natural animal host model system to examine viral gene expression provides a robust, efficient, and meaningful way of validating results gathered from cell culture systems.Author SummaryIn the natural host, the transcriptome and proteome of many herpesviruses are poorly defined. Here, we evaluated the viral transcriptome and proteome in feather follicle epithelial skin cells of chickens infected with Marek’s disease virus (MDV), an important poultry pathogen as well as an excellent model for skin-tropic human alphaherpesvirus replication in skin cells. Using fluorescently tagged virus, we significantly enriched the number of infected cells sampled from live chickens, greatly enhancing the detection of viral transcripts and proteins within a host background. Based on this, we could confirm the translation of most transcripts using deep MS/MS-based proteomics and identify novel expressed peptides supportive of an increasingly complex translational and regulatory viral landscape. The demonstrated deep peptide sequencing capability can serve as a template for future work in herpesviral proteomics.

ConservedHerpesviridaeprotein kinases (CHPK) are conserved among all members of theHerpesviridae. Herpesviruses lacking CHPK propagate in cell culture at varying degrees, depending on the virus and cell culture system. CHPK is dispensable for Marek’s disease herpesvirus (MDV) replication in cell culture and experimental infection in chickens; however, CHPK—particularly its kinase activity—is essential for horizontal transmission in chickens, also known as natural infection. To address the importance of CHPK during natural infection in chickens, we used liquid chromatography-tandem mass spectrometry (LC-MS/MS) based proteomics of samples collected from live chickens. Comparing modification of viral proteins in feather follicle epithelial (FFE) cells infected with wildtype or a CHPK-null virus, we identified the US10 protein (pUS10) as a potential target for CHPKin vivo. When expression of pUS10 was evaluated in cell culture and in FFE skin cells duringin vivoinfection, pUS10 was severely reduced or abrogated in cells infected with CHPK mutant or CHPK-null viruses, respectively, indicating a potential role for pUS10 in transmission. To test this hypothesis, US10 was deleted from the MDV genome, and the reconstituted virus was tested for replication, horizontal transmission, and disease induction. Our results showed that removal of US10 had no effect on the ability of MDV to transmit in experimentally infected chickens, but disease induction in naturally infected chickens was significantly reduced. These results show CHPK is necessary for pUS10 expression both in cell culture and in the host, and pUS10 is important for disease induction during natural infection.Author SummaryMarek’s disease herpesvirus (MDV) is an important pathogen in the poultry industry. Current vaccines reduce disease but do not protect chickens from infection. Understanding natural infection provides vital information for developing potential therapies to protect against Marek’s disease, while also providing a robust natural virus-host model to study herpesvirus pathogenesis. The conservedHerpesviridaeprotein kinase (CHPK) of MDV is essential for the initiation of natural infection, and we identified the virion protein US10 as a potential target for CHPK during natural infection. Our results showed that MDV CHPK was required for expression of pUS10 in both cell culture and in chickens. Although MDV lacking pUS10 replicated and spread in experimentally infected chickens, it was less virulent implicating an important role of pUS10 in natural infection of the host. These results emphasize the importance of studying specific viral proteins and their functions during infection of the natural host.

Mass spectrometry (MS)-based phosphoproteomics is a powerful tool for investigating cell signaling, yet it remains challenging to study plant phosphoproteomes due to the low yield of cell lysis and high complexity of plant lysate. Here we report a streamlined sample preparation workflow to analyze plant phosphoproteomes in a high-throughput manner. This workflow addresses the problem of low yield in the lysis step and eliminates the interferences of pigments and metabolites in plant lysate. Integrating chemical labeling and high pH reverse phase fractionation with this workflow achieves in-depth phosphoproteomic coverage. Notably, the scalability of this approach is demonstrated by systematically analyzing the effect of long-term cold stress in the perturbation of the tomato phosphoproteome. Identification of more than 30,000 phosphopeptides from tomato leaves and more than 5000 kinase-substrate pairs from Arabidopsis create the largest phosphoproteomic and signaling network resource to date.

Skeletal muscles contain heterogeneous myofibers that are different in size and contractile speed, with type IIb myofiber being the largest and fastest. Here, we identify methyltransferase-like 21e (Mettl21e), a member of newly classified nonhistone methyltransferases, as a gene enriched in type IIb myofibers. The expression of Mettl21e was strikingly up-regulated in hypertrophic muscles and during myogenic differentiation in vitro and in vivo. Knockdown (KD) of Mettl21e led to atrophy of cultured myotubes, and targeted mutation of Mettl21e in mice reduced the size of IIb myofibers without affecting the composition of myofiber types. Mass spectrometry and methyltransferase assay revealed that Mettl21e methylated valosin-containing protein (Vcp/p97), a key component of the ubiquitin-proteasome system. KD or knockout of Mettl21e resulted in elevated 26S proteasome activity, and inhibition of proteasome activity prevented atrophy of Mettl21e KD myotubes. These results demonstrate that Mettl21e functions to maintain myofiber size through inhibiting proteasome-mediated protein degradation.—Wang, C., Zhang, B., Ratliff, A. C., Arrington, J., Chen, J., Xiong, Y., Yue, F., Nie, Y., Hu, K., Jin, W., Tao, W. A., Hrycyna, C. A., Sun, X., Kuang, S. Methyltransferase-like 21e inhibits 26S proteasome activity to facilitate hypertrophy of type IIb myofibers.

Acquired and intrinsic resistance to receptor tyrosine kinase inhibitors (RTKi) represents a major hurdle in improving the management of clear cell renal cell carcinoma (ccRCC). Recent reports suggest that drug resistance is driven by tumor adaptation via epigenetic mechanisms that activate alternative survival pathways. The histone methyl transferase EZH2 is frequently altered in many cancers, including ccRCC. To evaluate its role in ccRCC resistance to RTKi, we established and characterized a spontaneously metastatic, patient-derived xenograft model that is intrinsically resistant to the RTKi sunitinib, but not to the VEGF therapeutic antibody bevacizumab. Sunitinib maintained its antiangiogenic and antimetastatic activity but lost its direct antitumor effects due to kinome reprogramming, which resulted in suppression of proapoptotic and cell-cycle–regulatory target genes. Modulating EZH2 expression or activity suppressed phosphorylation of certain RTKs, restoring the antitumor effects of sunitinib in models of acquired or intrinsically resistant ccRCC. Overall, our results highlight EZH2 as a rational target for therapeutic intervention in sunitinib-resistant ccRCC as well as a predictive marker for RTKi response in this disease. Cancer Res; 77(23); 6651–66. ©2017 AACR.

Protein kinases and their substrates comprise extensive signaling networks that regulate many diverse cellular functions. However, methods and techniques to systematically identify kinases directly responsible for specific phosphorylation events have remained elusive. Here we describe a novel proteomic strategy termed fluorescence complementation mass spectrometry (FCMS) to identify kinase–substrate pairs in high throughput. The FCMS strategy employs a specific substrate and a kinase library, both of which are fused with fluorescence complemented protein fragments. Transient and weak kinase–substrate interactions in living cells are stabilized by the association of fluorescence protein fragments. These kinase–substrate pairs are then isolated with high specificity and are identified and quantified by LC–MS. FCMS was applied to the identification of both known and novel kinases of the transcription factor, cAMP response element-binding protein (CREB). Novel CREB kinases were validated by in vitro kinase assays, and the phosphorylation sites were unambiguously located. These results uncovered possible new roles for CREB in multiple important signaling pathways and demonstrated the great potential of this new proteomic strategy., A high throughput strategy based on quantitative proteomics and fluorescent protein complementation is introduced to identify phosphoprotein’s upstream kinases

Group B streptococci (GBS; Streptococcus agalactiae ) are beta-hemolytic, Gram-positive bacteria that are common asymptomatic colonizers of healthy adults. However, these opportunistic bacteria also cause invasive infections in human newborns and in certain adult populations. To adapt to the various environments encountered during its disease cycle, GBS encodes a number of two-component signaling systems. Previous studies have indicated that the TCS comprising the sensor histidine kinase RgfC and the response regulator RgfA mediate GBS binding to extracellular matrix components, such as fibrinogen. However, in certain GBS clinical isolates, a point mutation in rgfA results in premature truncation of the response regulator. The truncated RgfA protein lacks the C-terminal DNA binding domain necessary for promoter binding and gene regulation. Here, we show that deletion of rgfC in GBS strains lacking a functional RgfA increased systemic infection. Furthermore, infection with the rgfC mutant increased induction of proinflammatory signaling pathways in vivo . Phosphoproteomic analysis revealed that 19 phosphopeptides corresponding to 12 proteins were differentially phosphorylated at aspartate, cysteine, serine, threonine, or tyrosine residues in the rgfC mutant. This included aspartate phosphorylation of a tyrosine kinase, CpsD, and a transcriptional regulator. Consistent with this observation, microarray analysis of the rgfC mutant indicated that >200 genes showed altered expression compared to the isogenic wild-type strain and included transcriptional regulators, transporters, and genes previously associated with GBS pathogenesis. Our observations suggest that in the absence of RgfA, nonspecific RgfC signaling affects the expression of virulence factors and GBS pathogenesis.

Phosphorylation has an incredible impact on the biological behavior of proteins, altering everything from intrinsic activity to cellular localization and complex formation. It is no surprise then that this post-translational modification has been the subject of intense study and that, with the advent of faster, more accurate instrumentation, the number of large-scale mass spectrometry-based phosphoproteomic studies has swelled over the past decade. Recent developments in sample preparation, phosphorylation enrichment, quantification, and data analysis strategies permit both targeted and ultra-deep phosphoproteome profiling, but challenges remain in pinpointing biologically relevant phosphorylation events. We describe here technological advances that have facilitated phosphoproteomic analysis of cells, tissues, and biofluids and note applications to neuropathologies in which the phosphorylation machinery may be dysregulated, much as it is in cancer.

Protein phosphorylation is one of the key events in the regulation of plant physiological responses to diverse environmental stimuli. As crucial regulators of phosphorylation, protein kinases have been linked to the control of seed germination, flowering, and stress responses. Identifying downstream substrates of kinases is important for dissecting kinase-substrate networks as well as delineating the underlying defense mechanisms in response to extracellular stimulation. Despite the fact that thousands of kinase-substrate networks have been identified in mammalian cells, the downstream substrates of important plant kinases are still elusive. Moreover, it remains challenging to identify bona fide kinase substrates from proteome-wide analyses. Thus, developing methodologies with high sensitivity and specificity is imperative for understanding plant kinase-substrate cascades. Here, we describe a proteomic strategy termed kinase assay-linked phosphoproteomics (KALIP) approach for large-scale identification of the direct substrates of plant kinases with high sensitivity and a low false-positive rate.

Myogenic regulatory factors (MRFs) including Myf5, MyoD and Myog are muscle-specific transcriptional factors orchestrating myogenesis. Although MRFs are essential for myogenic commitment and differentiation, timely repression of their activity is necessary for self-renewal and maintenance of muscle stem cells (satellite cells). Here we define a novel inhibitor of MRFs: the achaete-scute homologue 2 (Ascl2). During development, Ascl2 is transiently detected in a subpopulation of Pax7+MyoD+ progenitors (myoblasts) that become Pax7+MyoD− satellite cells prior to birth, but not detectable in postnatal satellite cells. Knockout of Ascl2 in embryonic myoblasts decreases both the number of Pax7+ cells and the proportion of Pax7+MyoD− cells. Conversely, overexpression of Ascl2 inhibits the proliferation and differentiation of cultured myoblasts, and impairs regeneration of injured muscles. At the molecular level, Ascl2 competes with MRFs for binding to E-boxes in the promoters of muscle genes, without activating gene transcription. Ascl2 also forms heterodimer with classical E-proteins to sequester their transcriptional activity on MRFs. Accordingly, MyoD or Myog expression rescues myogenic differentiation despite Ascl2 overexpression. Finally, Ascl2 expression is regulated by Notch signaling, a key governor of satellite cell self-renewal. These data together demonstrate that Ascl2 inhibits myogenic differentiation by targeting MRFs, and facilitates generation of postnatal satellite cells.

Dissection of direct kinase-substrate relationships provides invaluable information about phosphorylation pathways and can highlight both pathogenic mechanisms and possible drug targets for diseases in which abnormal kinase activity is linked to onset and progression. Here, we describe a mass spectrometry-based strategy to define the direct substrates of a kinase of interest. The kinase assay-linked phosphoproteomics approach examines putative kinase substrates both in vitro and in vivo to produce a list of highly confident substrates.

Protein phosphorylation is one of the key events in the regulation of plant physiological responses to diverse environmental stimuli. As crucial regulators of phosphorylation, protein kinases have been linked to the control of seed germination, flowering, and stress responses. Identifying downstream substrates of kinases is important for dissecting kinase-substrate networks as well as delineating the underlying defense mechanisms in response to extracellular stimulation. Despite the fact that thousands of kinase-substrate networks have been identified in mammalian cells, the downstream substrates of important plant kinases are still elusive. Moreover, it remains challenging to identify bona fide kinase substrates from proteome-wide analyses. Thus, developing methodologies with high sensitivity and specificity is imperative for understanding plant kinase-substrate cascades. Here, we describe a proteomic strategy termed kinase assay-linked phosphoproteomics (KALIP) approach for large-scale identification of the direct substrates of plant kinases with high sensitivity and a low false-positive rate.

Phosphoproteomics is the systematic study of one of the most common protein modifications in high throughput with the aim of providing detailed information of the control, response, and communication of biological systems in health and disease. Advances in analytical technologies and strategies, in particular the contributions of high-resolution mass spectrometers, efficient enrichments of phosphopeptides, and fast data acquisition and annotation, have catalyzed dramatic expansion of signaling landscapes in multiple systems during the past decade. While phosphoproteomics is an essential inquiry to map high-resolution signaling networks and to find relevant events among the apparently ubiquitous and widespread modifications of proteome, it presents tremendous challenges in separation sciences to translate it from discovery to clinical practice. In this mini-review, we summarize the analytical tools currently utilized for phosphoproteomic analysis (with focus on MS), progresses made on deciphering clinically relevant kinase-substrate networks, MS uses for biomarker discovery and validation, and the potential of phosphoproteomics for disease diagnostics and personalized medicine.

Protein phosphorylation plays an essential role in the regulation of various cellular functions. Dysregulation of phosphorylation is implicated in the pathogenesis of certain cancers, diabetes, cardiovascular diseases, and central nervous system disorders. As a result, protein kinases have become potential drug targets for treating a wide variety of diseases. Identification of kinase substrates is vital not only for dissecting signaling pathways, but also for understanding disease pathologies and identifying novel therapeutic targets. However, identification of bona fide kinase substrates has remained challenging, necessitating the development of new methods and techniques. The kinase assay linked phosphoproteomics (KALIP) approach integrates in vitro kinase assays with global phosphoproteomics experiments to identify the direct substrates of protein kinases. This strategy has demonstrated outstanding sensitivity and a low false-positive rate for kinase substrate screening.

Phosphorylation is a key posttranslational modification that regulates many signaling pathways, but quantifying changes in phosphorylation between samples can be challenging due to its low stoichiometry within cells. We have introduced a mass spectrometry-based label-free quantitation strategy termed LAXIC for the analysis of the phosphoproteome. This method uses a spiked-in synthetic peptide library designed to elute across the entire chromatogram for local normalization of phosphopeptides within complex samples. Normalization of phosphopeptides by library peptides that co-elute within a small time frame accounts for fluctuating ion suppression effects, allowing more accurate quantitation even when LC-MS performance varies. Here we explain the premise of LAXIC, the design of a suitable peptide library, and how the LAXIC algorithm can be implemented with software developed in-house.

Phosphoproteomics is the systematic study of one of the most common protein modifications in high throughput with the aim of providing detailed information of the control, response, and communication of biological systems in health and disease. Advances in analytical technologies and strategies, in particular the contributions of high-resolution mass spectrometers, efficient enrichments of phosphopeptides, and fast data acquisition and annotation, have catalyzed dramatic expansion of signaling landscapes in multiple systems during the past decade. While phosphoproteomics is an essential inquiry to map high-resolution signaling networks and to find relevant events among the apparently ubiquitous and widespread modifications of proteome, it presents tremendous challenges in separation sciences to translate it from discovery to clinical practice. In this mini-review, we summarize the analytical tools currently utilized for phosphoproteomic analysis (with focus on MS), progresses made on deciphering clinically relevant kinase-substrate networks, MS uses for biomarker discovery and validation, and the potential of phosphoproteomics for disease diagnostics and personalized medicine.

Phosphorylation is a key posttranslational modification that regulates many signaling pathways, but quantifying changes in phosphorylation between samples can be challenging due to its low stoichiometry within cells. We have introduced a mass spectrometry-based label-free quantitation strategy termed LAXIC for the analysis of the phosphoproteome. This method uses a spiked-in synthetic peptide library designed to elute across the entire chromatogram for local normalization of phosphopeptides within complex samples. Normalization of phosphopeptides by library peptides that co-elute within a small time frame accounts for fluctuating ion suppression effects, allowing more accurate quantitation even when LC-MS performance varies. Here we explain the premise of LAXIC, the design of a suitable peptide library, and how the LAXIC algorithm can be implemented with software developed in-house.