The maintenance of skeletal muscle mass is essential for health and quality of life. It is well recognized that maximal-intensity contractions, such as those which occur during resistance exercise, promote an increase in muscle mass. Yet, the molecules that sense the mechanical information and convert it into the signalling events (e.g. phosphorylation) that drive the increase in muscle mass remain undefined. Here we describe a phosphoproteomics workflow to examine the effects of electrically evoked maximal-intensity contractions (MICs) on protein phosphorylation in mouse skeletal muscle. While a preliminary phosphoproteomics experiment successfully identified a number of MIC-regulated phosphorylation events, a large proportion of these identifications were present on highly abundant myofibrillar proteins. We subsequently incorporated a centrifugation-based fractionation step to deplete the highly abundant myofibrillar proteins and performed a second phosphoproteomics experiment. In total, we identified 5983 unique phosphorylation sites of which 663 were found to be regulated by MIC. GO term enrichment, phosphorylation motif analyses, and kinase-substrate predictions indicated that the MIC-regulated phosphorylation sites were chiefly modified by mTOR, as well as multiple isoforms of the MAPKs and CAMKs. Moreover, a high proportion of the regulated phosphorylation sites were found on proteins that are associated with the Z-disc, with over 74% of the Z-disc proteins experiencing robust changes in phosphorylation. Finally, our analyses revealed that the phosphorylation state of two Z-disc kinases (striated muscle-specific serine/threonine protein kinase and obscurin) was dramatically altered by MIC, and we propose ways these kinases could play a fundamental role in skeletal muscle mechanotransduction.

Negative-sense RNA viruses assemble large ribonucleoprotein (RNP) complexes that direct replication and transcription of the viral genome. Influenza virus RNPs contain the polymerase, genomic RNA and multiple copies of nucleoprotein (NP). During RNP assembly, monomeric NP oligomerizes along the length of the genomic RNA. Regulated assembly of the RNP is essential for virus replication, but how NP is maintained as a monomer that subsequently oligomerizes to form RNPs is poorly understood. Here we elucidate a mechanism whereby NP phosphorylation regulates oligomerization. We identified new evolutionarily conserved phosphorylation sites on NP and demonstrated that phosphorylation of NP decreased formation of higher-order complexes. Two phosphorylation sites were located on opposite sides of the NP:NP interface. In both influenza A and B virus, mutating or mimicking phosphorylation at these residues blocked homotypic interactions and drove NP towards a monomeric form. Highlighting the central role of this process during infection, these mutations impaired RNP formation, polymerase activity and virus replication. Thus, dynamic phosphorylation of NP regulates RNP assembly and modulates progression through the viral life cycle.

Influenza virus expresses transcripts early in infection and transitions towards genome replication at later time points. This process requires de novo assembly of the viral replication machinery, large ribonucleoprotein complexes (RNPs) composed of the viral polymerase, genomic RNA and oligomeric nucleoprotein (NP). Despite the central role of RNPs during infection, the factors dictating where and when they assemble are poorly understood. Here we demonstrate that human protein kinase C (PKC) family members regulate RNP assembly. Activated PKCδ interacts with the polymerase subunit PB2 and phospho-regulates NP oligomerization and RNP assembly during infection. Consistent with its role in regulating RNP assembly, knockout of PKCδ impairs virus infection by selectively disrupting genome replication. However, primary transcription from pre-formed RNPs deposited by infecting particles is unaffected. Thus, influenza virus exploits host PKCs to regulate RNP assembly, a step required for the transition from primary transcription to genome replication during the infectious cycle.

Protein kinase signaling along the kinetochore-centromere axis is crucial to assure mitotic fidelity, yet its spatial coordination is obscure. Here, we examined how pools of human Polo-like kinase 1 (Plk1) within this axis control signaling events to elicit mitotic functions. To do this, we restricted active Plk1 to discrete subcompartments within the kinetochore-centromere axis using chemical genetics and decoded functional and phosphoproteomic signatures of each. We observe distinct phosphoproteomic and functional roles, suggesting that Plk1 exists and functions in discrete pools along this axis. Deep within the centromere, Plk1 operates to assure proper chromosome alignment and segregation. Thus, Plk1 at the kinetochore is a conglomerate of an observable bulk pool coupled with additional functional pools below the threshold of microscopic detection/resolution. Although complex, this multiplicity of locales provides an opportunity to decouple functional and phosphoproteomic signatures for a comprehensive understanding of Plk1’s kinetochore functions.

SUMMARY Mechanical signals, such as those evoked by maximal-intensity contractions (MICs), can induce an increase in muscle mass. Rapamycin-sensitive signaling events are widely implicated in the regulation of this process; however, recent studies indicate that rapamycin-insensitive signaling events are also involved. Thus, to identify these events, we generate a map of the MIC-regulated and rapamycin-sensitive phosphoproteome. In total, we quantify more than 10,000 unique phosphorylation sites and find that more than 2,000 of these sites are significantly affected by MICs, but remarkably, only 38 of the MIC-regulated events are mediated through a rapamycin-sensitive mechanism. Further interrogation of the rapamycin-insensitive phosphorylation events identifies the S473 residue on Tripartite Motif-Containing 28 (TRIM28) as one of the most robust MIC-regulated phosphorylation sites, and extensive follow-up studies suggest that TRIM28 significantly contributes to the homeostatic regulation of muscle size and function as well as the hypertrophy that occurs in response to increased mechanical loading.

Peptides presented by the class-I major histocompatibility complex (MHC-I) are important targets for immunotherapy. The identification of these peptide targets greatly facilitates the generation of T-cell-based therapeutics. Herein, we report the capability of proteolysis targeting chimera (PROTAC) compounds to induce the presentation of specific MHC class-I peptides derived from endogenous cellular proteins. Using LC-MS/MS, we identified several BET-derived MHC-I peptides induced by treatment with three BET-directed PROTAC compounds. To understand our ability to tune this process, we measured the relative rate of presentation of these peptides under varying treatment conditions using label-free mass spectrometry quantification. We found that the rate of peptide presentation reflected the rate of protein degradation, indicating a direct relationship between PROTAC treatment and peptide presentation. We additionally analyzed the effect of PROTAC treatment on the entire immunopeptidome and found many new peptides that were displayed in a PROTAC-specific fashion: we determined that these identifications map to the BET pathway, as well as, potential off-target or unique-to-PROTAC pathways. This work represents the first evidence of the use of PROTAC compounds to induce the presentation of MHC-I peptides from endogenous cellular proteins, highlighting the capability of PROTAC compounds for the discovery and generation of new targets for immunotherapy.

Methods for the generation of substrates presenting biomolecules in a spatially controlled manner are enabling tools for applications in biosensor systems, microarray technologies, fundamental biological studies and biointerface science. We have implemented a method to create biomolecular patterns by using light to control the direct covalent immobilization of biomolecules onto benzophenone-modified glass substrates. We have generated substrates presenting up to three different biomolecules patterned in sequence, and demonstrate biomolecular photopatterning on corrugated substrates. The chemistry of the underlying monolayer was optimized to incorporate poly(ethylene glycol) to enable adhesive cell adhesion onto patterned extracellular matrix proteins. Substrates were characterized with contact angle goniometry, AFM, and immunofluorescence microscopy. Importantly, radioimmunoassays were performed to quantify the site density of immobilized biomolecules on photopatterned substrates. Retention of function of photopatterned proteins was demonstrated both by native ligand recognition and cell adhesion to photopatterned substrates, revealing that substrates generated with this method are suitable for probing specific cell receptor-ligand interactions. This molecularly general photochemical patterning method is an enabling tool that will allow the creation of substrates presenting both biochemical and topographical variation, which is an important feature of many native biointerfaces.

Label-free quantification is a powerful tool for the measurement of protein abundances by mass spectrometric methods. To maximize quantifiable identifications, MS1-based methods must balance the collection of survey scans and fragmentation spectra while maintaining reproducible extracted ion chromatograms (XIC). Here we present a method which increases the depth of proteome coverage over replicate data-dependent experiments without the requirement of additional instrument time or sample pre-fractionation. Sampling depth is increased by restricting precursor selection to a fraction of the full MS1 mass range for each replicate; collectively, the m/z segments of all replicates encompass the full MS1 range. Although selection windows are narrowed, full MS1 spectra are obtained throughout the method, enabling the collection of full mass range MS1 chromatograms such that label-free quantitation can be performed for any peptide in any experiment. We term this approach “binning” or “tiling” depending on the type of m/z window utilized. By combining the data obtained from each segment, we find that this approach increases the number of quantifiable yeast peptides and proteins by 31% and 52%, respectively, when compared to normal data-dependent experiments performed in replicate.