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Abstract One of the main drivers of autism spectrum disorder is risk alleles within hundreds of genes, which may interact within shared but unknown protein complexes. Here we develop a scalable genome-editing-mediated approach to target 14 high-confidence autism risk genes within the mouse brain for proximity-based endogenous proteomics, achieving the identification of high-specificity spatial proteomes. The resulting native proximity proteomes are enriched for human genes dysregulated in the brain of autistic individuals, and reveal proximity interactions between proteins from high-confidence risk genes with those of lower-confidence that may provide new avenues to prioritize genetic risk. Importantly, the datasets are enriched for shared cellular functions and genetic interactions that may underlie the condition. We test this notion by spatial proteomics and CRISPR-based regulation of expression in two autism models, demonstrating functional interactions that modulate mechanisms of their dysregulation. Together, these results reveal native proteome networks in vivo relevant to autism, providing new inroads for understanding and manipulating the cellular drivers underpinning its etiology.

One of the most extensively studied members of the Ras superfamily of small GTPases, Rac1 is an intracellular signal transducer that remodels actin and phosphorylation signaling networks. Previous studies have shown that Rac1-mediated signaling is associated with hippocampal-dependent working memory and longer-term forms of learning and memory and that Rac1 can modulate forms of both pre- and postsynaptic plasticity. How these different cognitive functions and forms of plasticity mediated by Rac1 are linked, however, is unclear. Here, we show that spatial working memory in mice is selectively impaired following the expression of a genetically encoded Rac1 inhibitor at presynaptic terminals, while longer-term cognitive processes are affected by Rac1 inhibition at postsynaptic sites. To investigate the regulatory mechanisms of this presynaptic process, we leveraged new advances in mass spectrometry to identify the proteomic and post-translational landscape of presynaptic Rac1 signaling. We identified serine/threonine kinases and phosphorylated cytoskeletal signaling and synaptic vesicle proteins enriched with active Rac1. The phosphorylated sites in these proteins are at positions likely to have regulatory effects on synaptic vesicles. Consistent with this, we also report changes in the distribution and morphology of synaptic vesicles and in postsynaptic ultrastructure following presynaptic Rac1 inhibition. Overall, this study reveals a previously unrecognized presynaptic role of Rac1 signaling in cognitive processes and provides insights into its potential regulatory mechanisms.

ObjectivesTo identify age-related plasma extracellular vehicle (EVs) phenotypes in healthy adults.MethodsEV proteomics by high-resolution mass spectrometry to evaluate EV protein stability and discover age-associated EV proteins (n=4 with 4 serial freeze-thaws each); validation by high-resolution flow cytometry and EV cytokine quantification by multiplex ELISA (n=28 healthy donors, aged 18-83 years); quantification of WI-38 fibroblast cell proliferation response to co-culture with PKH67-labeled young and old plasma EVs. The EV samples from these plasma specimens were previously characterized for bilayer structure, intra-vesicle mitochondria and cytokines, and hematopoietic cell-related surface markers.ResultsCompared with matched exo-EVs (EV-depleted supernatants), endo-EVs (EV-associated) had higher mean TNF-α and IL-27, lower mean IL-6, IL-11, IFN-γ, and IL-17A/F, and similar mean IL-1β, IL-21, and IL-22 concentrations. Some endo-EV and exo-EV cytokine concentrations were correlated, including TNF-α, IL-27, IL-6, IL-1β, and IFN-γ, but not IL-11, IL-17A/F, IL-21 or IL-22. Endo-EV IFN-γ and exo-EV IL-17A/F and IL-21 declined with age. By proteomics and confirmed by flow cytometry, we identified age-associated decline of fibrinogen (FGA, FGB and FGG) in EVs. Age-related EV proteins indicated predominant origins in the liver and innate immune system. WI-38 cells (>95%) internalized similar amounts of young and old plasma EVs, but cells that internalized PKH67-EVs, particularly young EVs, underwent significantly greater cell proliferation.ConclusionEndo-EV and exo-EV cytokines function as different biomarkers. The observed healthy aging EV phenotype reflected a downregulation of EV fibrinogen subpopulations consistent with the absence of a pro-coagulant and pro-inflammatory condition common with age-related disease.

Summary: An intriguing effect of short-term caloric restriction (CR) is the expansion of certain stem cell populations, including muscle stem cells (satellite cells), which facilitate an accelerated regenerative program after injury. Here, we utilized the MetRSL274G (MetRS) transgenic mouse to identify liver-secreted plasminogen as a candidate for regulating satellite cell expansion during short-term CR. Knockdown of circulating plasminogen prevents satellite cell expansion during short-term CR. Furthermore, loss of the plasminogen receptor KT (Plg-RKT) is also sufficient to prevent CR-related satellite cell expansion, consistent with direct signaling of plasminogen through the plasminogen receptor Plg-RKT/ERK kinase to promote proliferation of satellite cells. Importantly, we are able to replicate many of these findings in human participants from the CALERIE trial. Our results demonstrate that CR enhances liver protein secretion of plasminogen, which signals directly to the muscle satellite cell through Plg-RKT to promote proliferation and subsequent muscle resilience during CR.

Objective: To further validate a serum proteomics panel for predicting radiographic (structural) knee OA progression. Design: Serum peptides were targeted by multiple-reaction-monitoring mass spectrometry in the New York University cohort (n ​= ​104). Knee OA progression was defined as joint space narrowing ≥1 in the tibiofemoral compartment of one knee per study participant over a 24-month follow-up. The discriminative ability of an 11-peptide panel was evaluated by multivariable logistic regression and area under the receiver operating characteristic curve (AUC), without and with demographic characteristics of age, sex, and body mass index. The association of each peptide with OA progression was assessed by odds ratios (OR) in multivariable logistic regression models adjusted for demographics. Results: The cohort included 46 (44%) knee OA progressors. The panel of 11 peptides alone yielded AUC ​= ​0.66 (95% CI [0.55, 0.77]) for discriminating progressors from non-progressors; demographic traits alone yielded AUC ​= ​0.66 (95% CI [0.55, 0.77]). Together the 11 peptides and demographics yielded AUC ​= ​0.72 (95% CI [0.62, 0.83]). CRAC1 had the highest odds for predicting OA progression (OR 2.014, 95% CI [0.996, 4.296], p ​= ​0.058). Conclusions: We evaluated a parsimonious serum proteomic panel and found it to be a good discriminator of knee radiographic OA progression from non-progression. Since these biomarkers are quantifiable in serum, they could be deployed relatively easily to provide a simple, cost-effective strategy for identifying and monitoring individuals at high risk of knee OA progression.

Abstract The neurofilament (NF) cytoskeleton is critical for neuronal morphology and function. In particular, the neurofilament-light (NF-L) subunit is required for NF assembly in vivo and is mutated in subtypes of Charcot-Marie-Tooth (CMT) disease. NFs are highly dynamic, and the regulation of NF assembly state is incompletely understood. Here, we demonstrate that human NF-L is modified in a nutrient-sensitive manner by O-linked-β-N-acetylglucosamine (O-GlcNAc), a ubiquitous form of intracellular glycosylation. We identify five NF-L O-GlcNAc sites and show that they regulate NF assembly state. NF-L engages in O-GlcNAc-mediated protein-protein interactions with itself and with the NF component α-internexin, implying that O-GlcNAc may be a general regulator of NF architecture. We further show that NF-L O-GlcNAcylation is required for normal organelle trafficking in primary neurons. Finally, several CMT-causative NF-L mutants exhibit perturbed O-GlcNAc levels and resist the effects of O-GlcNAcylation on NF assembly state, suggesting a potential link between dysregulated O-GlcNAcylation and pathological NF aggregation. Our results demonstrate that site-specific glycosylation regulates NF-L assembly and function, and aberrant NF O-GlcNAcylation may contribute to CMT and other neurodegenerative disorders.

Organic anion transporting polypeptide (OATP) 1B1 and OATP1B3 are key determinants of drug–drug interactions (DDIs). Various drugs including the calcineurin inhibitor (CNI) cyclosporine A (CsA) exert preincubation-induced trans-inhibitory effects upon OATP1B1 and/or OATP1B3 (abbreviated as OATP1B1/3) by unknown mechanism(s). OATP1B1/3 are phosphoproteins; calcineurin, which dephosphorylates and regulates numerous phosphoproteins, has not previously been investigated in the context of preincubation-induced trans-inhibition of OATP1B1/3. Herein, we compare the trans-inhibitory effects exerted on OATP1B1 and OATP1B3 by CsA, the non-analogous CNI tacrolimus, and the non-CNI CsA analogue SCY-635 in transporter-overexpressing human embryonic kidney (HEK) 293 stable cell lines. Preincubation (10–60 min) with tacrolimus (1–10 µM) rapidly and significantly reduces OATP1B1- and OATP1B3-mediated transport up to 0.18 ± 0.03- and 0.20 ± 0.02-fold compared to the control, respectively. Both CsA and SCY-635 can trans-inhibit OATP1B1, with the inhibitory effects progressively increasing over a 60 min preincubation time. At each equivalent preincubation time, CsA has greater trans-inhibitory effects toward OATP1B1 than SCY-635. Preincubation with SCY-635 for 60 min yielded IC50 of 2.2 ± 1.4 µM against OATP1B1, which is ~18 fold greater than that of CsA (0.12 ± 0.04 µM). Furthermore, a proteomics-based screening for protein interactors was used to examine possible proteins and processes contributing to OATP1B1/3 regulation and preincubation-induced inhibition by CNIs and other drugs. A total of 861 and 357 proteins were identified as specifically associated with OATP1B1 and OATP1B3, respectively, including various protein kinases, ubiquitin-related enzymes, the tacrolimus (FK506)-binding proteins FKBP5 and FKBP8, and several known regulatory targets of calcineurin. The current study reports several novel findings that expand our understanding of impaired OATP1B1/3 function; these include preincubation-induced trans-inhibition of OATP1B1/3 by the CNI tacrolimus, greater preincubation-induced inhibition by CsA compared to its non-CNI analogue SCY-635, and association of OATP1B1/3 with various proteins relevant to established and candidate OATP1B1/3 regulatory processes.

The mechanisms underlying the activity of non-receptor tyrosine kinase, TNK1, in cancers are unclear. Here the authors show that MARK mediates 14-3-3 and TNK1 interaction which restrains TNK1 activity, while the release of TNK1 from 14-3-3 leads to TNK1 activation through its interaction with ubiquitin and thus results in TNK1-mediated tumor growth in vivo

Here, we present quantitative subcellular compartment-specific proteomic data from wildtype and DYT-TOR1A heterozygous mouse embryonic fibroblasts (MEFs) basally and following thapsigargin (Tg) treatment [1]. In this experiment, we generated MEFs from wild type (WT) and a heterozygous DYT-TOR1A mouse model of dystonia. Subsequently, these MEF cultures were treated with either 1 µM Tg or dimethylsulfoxide vehicle (Veh) for six hours. Following treatment, the cells were fractionated into nuclear and cytosolic fractions. Liquid chromatography, tandem mass spectrometry (LC/MS/MS)-based proteomic profiling identified 65,056 unique peptides and 4801 unique proteins across all samples. The data presented here provide subcellular compartment-specific proteomic information within a dystonia model system both basally and under cellular stress. These data can inform future experiments focused on studying the function of TorsinA, the protein encoded by TOR1A, and its potential role in nucleocytoplasmic transport and proteostasis. In addition, the information in this article can also inform future mechanistic studies investigating the relationship between DYT-TOR1A dystonia and the cellular stress response to advance understanding of the pathogenesis of dystonia.

In contrast to their postsynaptic counterparts, the contributions of activity-dependent cytoskeletal signaling to presynaptic plasticity remain controversial and poorly understood. To identify and evaluate these signaling pathways, we conducted a proteomic analysis of the presynaptic cytomatrix using in vivo biotin identification (iBioID). The resultant proteome was heavily enriched for actin cytoskeleton regulators, including Rac1, a Rho GTPase that activates the Arp2/3 complex to nucleate branched actin filaments. Strikingly, we find Rac1 and Arp2/3 are closely associated with synaptic vesicle membranes in adult mice. Using three independent approaches to alter presynaptic Rac1 activity (genetic knockout, spatially restricted inhibition, and temporal optogenetic manipulation), we discover that this pathway negatively regulates synaptic vesicle replenishment at both excitatory and inhibitory synapses, bidirectionally sculpting short-term synaptic depression. Finally, we use two-photon fluorescence lifetime imaging to show that presynaptic Rac1 activation is coupled to action potentials by voltage-gated calcium influx. Thus, this study uncovers a previously unrecognized mechanism of actin-regulated short-term presynaptic plasticity that is conserved across excitatory and inhibitory terminals. It also provides a new proteomic framework for better understanding presynaptic physiology, along with a blueprint of experimental strategies to isolate the presynaptic effects of ubiquitously expressed proteins.

The internal molecular mechanisms that underlie excitatory synaptogenesis remain poorly characterized. This study utilizes a chemogenetic approach, in vivo biotin identification (iBioID), to discover previously uncharacterized proteins at nascent synapses. CARMIL3 is identified as a cytoskeletal protein that facilitates spine maturation and AMPAR recruitment.

The rate of repair declines with age; however, exposure to young circulations can rejuvenate fracture repair, but how this is accomplished is unknown. Here, the authors identify proteins, including low-density lipoprotein receptor-related protein 1 (Lrp1), as being secreted from young macrophages and rejuvenating fracture repair in mice.

Mutation of the Wiskott–Aldrich syndrome protein and SCAR homology (WASH) complex subunit, SWIP, is implicated in human intellectual disability, but the cellular etiology of this association is unknown. We identify the neuronal WASH complex proteome, revealing a network of endosomal proteins. To uncover how dysfunction of endosomal SWIP leads to disease, we generate a mouse model of the human WASHC4c.3056C>G mutation. Quantitative spatial proteomics analysis of SWIPP1019R mouse brain reveals that this mutation destabilizes the WASH complex and uncovers significant perturbations in both endosomal and lysosomal pathways. Cellular and histological analyses confirm that SWIPP1019R results in endo-lysosomal disruption and uncover indicators of neurodegeneration. We find that SWIPP1019R not only impacts cognition, but also causes significant progressive motor deficits in mice. A retrospective analysis of SWIPP1019R patients reveals similar movement deficits in humans. Combined, these findings support the model that WASH complex destabilization, resulting from SWIPP1019R, drives cognitive and motor impairments via endo-lysosomal dysfunction in the brain.

ABSTRACT Protein kinase A (PKA) signaling plays a critical role in the growth and development of all eukaryotic microbes. However, few direct targets have been characterized in any organism. The fungus Aspergillus fumigatus is a leading infectious cause of death in immunocompromised patients, but the specific molecular mechanisms responsible for its pathogenesis are poorly understood. We used this important pathogen as a platform for a comprehensive and multifaceted interrogation of both the PKA-dependent whole proteome and phosphoproteome in order to elucidate the mechanisms through which PKA signaling regulates invasive microbial disease. Employing advanced quantitative whole-proteomic and phosphoproteomic approaches with two complementary phosphopeptide enrichment strategies, coupled to an independent PKA interactome analysis, we defined distinct PKA-regulated pathways and identified novel direct PKA targets contributing to pathogenesis. We discovered three previously uncharacterized virulence-associated PKA effectors, including an autophagy-related protein, Atg24; a CCAAT-binding transcriptional regulator, HapB; and a CCR4-NOT complex-associated ubiquitin ligase, Not4. Targeted mutagenesis, combined with in vitro kinase assays, multiple murine infection models, structural modeling, and molecular dynamics simulations, was employed to characterize the roles of these new PKA targets in growth, environmental and antimicrobial stress responses, and pathogenesis in a mammalian system. We also elucidated the molecular mechanisms of PKA regulation for these effectors by defining the functionality of phosphorylation at specific PKA target sites. We have comprehensively characterized the PKA-dependent phosphoproteome and validated PKA targets as direct regulators of infectious disease for the first time in any pathogen, providing new insights into PKA signaling and control over microbial pathogenesis. IMPORTANCE PKA is essential for the virulence of eukaryotic human pathogens. Understanding PKA signaling mechanisms is therefore fundamental to deciphering pathogenesis and developing novel therapies. Despite its ubiquitous necessity, specific PKA effectors underlying microbial disease remain unknown. To address this fundamental knowledge gap, we examined the whole-proteomic and phosphoproteomic impacts of PKA on the deadly fungal pathogen Aspergillus fumigatus to uncover novel PKA targets controlling growth and virulence. We also defined the functional consequences of specific posttranslational modifications of these target proteins to characterize the molecular mechanisms of pathogenic effector regulation by PKA. This study constitutes the most comprehensive analysis of the PKA-dependent phosphoproteome of any human pathogen and proposes new and complex roles played by PKA signaling networks in governing infectious disease.

Cellular recycling via autophagy-associated proteins is a key catabolic pathway critical to invasive fungal pathogen growth and virulence in the nutrient-limited host environment. Protein kinase A (PKA) is vital for the growth and virulence of numerous fungal pathogens. However, the underlying basis for its regulation of pathogenesis remains poorly understood in any species. Our Aspergillus fumigatus PKA-dependent whole proteome and phosphoproteome studies employing advanced mass spectroscopic approaches identified numerous previously undefined PKA-regulated proteins in catabolic pathways. Here, we demonstrate reciprocal inhibition of autophagy and PKA activity, and identify 16 autophagy-associated proteins as likely novel PKA-regulated effectors. We characterize the novel PKA-phosphoregulated sorting nexin Atg20, and demonstrate its importance for growth, cell wall stress response, and virulence of A. fumigatus in a murine infection model. Additionally, we identify physical and functional interaction of Atg20 with previously characterized sorting nexin Atg24. Furthermore, we demonstrate the importance of additional uncharacterized PKA-regulated putative autophagy-associated proteins to hyphal growth. Our data presented here indicate that PKA regulates the autophagy pathway much more extensively than previously known, including targeting of novel effector proteins with fungal-specific functions important for invasive disease.

Growth, molting, and hardening cuticle are intertwined processes for arthropods and share common protein systems to execute these functions. For barnacles, these processes are also tied to adhesion, which is vital to their survival and under great selection pressure. Unlike other crustaceans, barnacle growth, deposition of adhesive material and the curing of glue and cuticle, though associated, are spatially distinct from molting and occur at the interface of the calcareous baseplate and substrata. Here, we use an ancient system, the innate immune response as context and use proteomics and enzyme activity assays to test the hypothesis that chemistries associated with barnacle fluids and glue curing have their origins in wound healing and the innate immune response. Glue precursor material was extracted from barnacles and manipulated to accelerate known trypsin activity, curing, and the effects of sequential sampling. Using non-trypsin digests and searching for tryptic peptides, new products of endogenous trypsin were detected. We found extensive evidence of a prophenoloxidase system, including activating factors, serine proteases, and serine protease inhibitors. Through enzyme-specific substrate assays, phenoloxidase-like activity was found in glue and at the periphery of the base. The enzymatic nature of the phenoloxidase-like activity was confirmed through chemical inhibition and through heating the glue material to denature the enzyme. Further evidence for antimicrobial activity mediated by reactive oxygen species previously described during larval settlement and metamorphosis was the presence of peroxidases in glue precursor material. Beyond clotting, numerous extracellular matrix proteins were identified, suggesting glue curing may lead to stabilization of the initial clot and wound repair. We propose a model of the emerging hypothesis and find exciting potential for further testing innate immune components in this evolutionary approach to barnacle glue curing.

Transferrin receptor (Tfr1) is ubiquitously expressed, but its roles in non-hematopoietic cells are incompletely understood. We used a tissue-specific conditional knockout strategy to ask whether skeletal muscle required Tfr1 for iron uptake. We found that iron assimilation via Tfr1 was critical for skeletal muscle metabolism, and that iron deficiency in muscle led to dramatic changes, not only in muscle, but also in adipose tissue and liver. Inactivation of Tfr1 incapacitated normal energy production in muscle, leading to growth arrest and a muted attempt to switch to fatty acid β oxidation, using up fat stores. Starvation signals stimulated gluconeogenesis in the liver, but amino acid substrates became limiting and hypoglycemia ensued. Surprisingly, the liver was also iron deficient, and production of the iron regulatory hormone hepcidin was depressed. Our observations reveal a complex interaction between iron homeostasis and metabolism that has implications for metabolic and iron disorders.

O-linked β-N-acetylglucosamine (O-GlcNAc) is an abundant and essential intracellular form of protein glycosylation in animals and plants. In humans, dysregulation of O-GlcNAcylation occurs in a wide range of diseases, including cancer, diabetes, and neurodegeneration. Since its discovery more than 30 years ago, great strides have been made in understanding central aspects of O-GlcNAc signaling, including identifying thousands of its substrates and characterizing the enzymes that govern it. However, while many O-GlcNAcylated proteins have been reported, only a small subset of these change their glycosylation status in response to a typical stimulus or stress. Identifying the functionally important O-GlcNAcylation changes in any given signaling context remains a significant challenge in the field. To address this need, we leveraged chemical biology and quantitative mass spectrometry methods to create a new glycoproteomics workflow for profiling stimulus-dependent changes in O-GlcNAcylated proteins. In proof-of-principle experiments, we used this new workflow to interrogate changes in O-GlcNAc substrates in mammalian protein trafficking pathways. Interestingly, our results revealed dynamic O-GlcNAcylation of COPγ1, an essential component of the coat protein I (COPI) complex that mediates Golgi protein trafficking. Moreover, we detected 11 O-GlcNAc moieties on COPγ1 and found that this modification is reduced by a model secretory stress that halts COPI trafficking. Our results suggest that O-GlcNAcylation may regulate the mammalian COPI system, analogous to its previously reported roles in other protein trafficking pathways. More broadly, our glycoproteomics workflow is applicable to myriad systems and stimuli, empowering future studies of O-GlcNAc in a host of biological contexts.

ABSTRACT Invasive aspergillosis (IA), caused by the filamentous fungal pathogen Aspergillus fumigatus, is a major cause of death among immunocompromised patients. The cyclic AMP/protein kinase A (PKA) signaling pathway is essential for hyphal growth and virulence of A. fumigatus, but the mechanism of regulation of PKA remains largely unknown. Here, we discovered a novel mechanism for the regulation of PKA activity in A. fumigatus via phosphorylation of key residues within the major catalytic subunit, PkaC1. Phosphopeptide enrichment and tandem mass spectrometry revealed the phosphorylation of PkaC1 at four sites (S175, T331, T333, and T337) with implications for important and diverse roles in the regulation of A. fumigatus PKA. While the phosphorylation at one of the residues (T333) is conserved in other species, the identification of three other residues represents previously unknown PKA phosphoregulation in A. fumigatus. Site-directed mutagenesis of the phosphorylated residues to mimic or prevent phosphorylation revealed dramatic effects on kinase activity, growth, conidiation, cell wall stress response, and virulence in both invertebrate and murine infection models. Three-dimensional structural modeling of A. fumigatus PkaC1 substantiated the positive or negative regulatory roles for specific residues. Suppression of PKA activity also led to downregulation of PkaC1 protein levels in an apparent novel negative-feedback mechanism. Taken together, we propose a model in which PkaC1 phosphorylation both positively and negatively modulates its activity. These findings pave the way for future discovery of fungus-specific aspects of this key signaling network. IMPORTANCE Our understanding of signal transduction networks in pathogenic fungi is limited, despite the increase in invasive fungal infections and rising mortality rates in the immunosuppressed patient population. Because PKA is known to be essential for hyphal growth and virulence of A. fumigatus, we sought to identify fungus-specific regulatory mechanisms governing PKA activity. In this study, we identify, for the first time, a novel mechanism for the regulation of PKA signaling in which differential phosphorylation of the PkaC1 catalytic subunit can lead to either positive or negative regulation of activity. Furthermore, we show that inactivation of PKA signaling leads to downregulation of catalytic subunit protein levels in a negative-feedback mechanism distinct from expression patterns previously reported in the yeasts. Our findings represent a divergence in the regulation of PKA signaling in A. fumigatus, which could potentially be exploited as a target and also open the avenue for discovery of fungus-specific downstream effectors of PKA.

We asked whether the C-terminal repeat domain (CTD) kinase, CDK12/CyclinK, phosphorylates substrates in addition to the CTD of RPB1, using our CDK12analog-sensitive HeLa cell line to investigate CDK12 activity-dependent phosphorylation events in human cells. Characterizing the phospho-proteome before and after selective inhibition of CDK12 activity by the analog 1-NM-PP1, we identified 5,644 distinct phospho-peptides, among which were 50 whose average relative amount decreased more than 2-fold after 30 min of inhibition (none of these derived from RPB1). Half of the phospho-peptides actually showed >3-fold decreases, and a dozen showed decreases of 5-fold or more. As might be expected, the 40 proteins that gave rise to the 50 affected phospho-peptides mostly function in processes that have been linked to CDK12, such as transcription and RNA processing. However, the results also suggest roles for CDK12 in other events, notably mRNA nuclear export, cell differentiation and mitosis. While a number of the more-affected sites resemble the CTD in amino acid sequence and are likely direct CDK12 substrates, other highly-affected sites are not CTD-like, and their decreased phosphorylation may be a secondary (downstream) effect of CDK12 inhibition.

Calcineurin governs stress survival, sexual differentiation, and virulence of the human fungal pathogen Cryptococcus neoformans. Calcineurin is activated by increased Ca2+ levels caused by stress, and transduces signals by dephosphorylating protein substrates. Herein, we identified and characterized calcineurin substrates in C. neoformans by employing phosphoproteomic TiO2 enrichment and quantitative mass spectrometry. The identified targets include the transactivator Crz1 as well as novel substrates whose functions are linked to P-bodies/stress granules (PBs/SGs) and mRNA translation and decay, such as Pbp1 and Puf4. We show that Crz1 is a bona fide calcineurin substrate, and Crz1 localization and transcriptional activity are controlled by calcineurin. We previously demonstrated that thermal and other stresses trigger calcineurin localization to PBs/SGs. Several calcineurin targets localized to PBs/SGs, including Puf4 and Pbp1, contribute to stress resistance and virulence individually or in conjunction with Crz1. Moreover, Pbp1 is also required for sexual development. Genetic epistasis analysis revealed that Crz1 and the novel targets Lhp1, Puf4, and Pbp1 function in a branched calcineurin pathway that orchestrates stress survival and virulence. These findings support a model whereby calcineurin controls stress and virulence, at the transcriptional level via Crz1, and post-transcriptionally by localizing to PBs/SGs and acting on targets involved in mRNA metabolism. The calcineurin targets identified in this study share little overlap with known calcineurin substrates, with the exception of Crz1. In particular, the mRNA binding proteins and PBs/SGs residents comprise a cohort of novel calcineurin targets that have not been previously linked to calcineurin in mammals or in Saccharomyces cerevisiae. This study suggests either extensive evolutionary rewiring of the calcineurin pathway, or alternatively that these novel calcineurin targets have yet to be characterized as calcineurin targets in other organisms. These findings further highlight C. neoformans as an outstanding model to define calcineurin-responsive virulence networks as targets for antifungal therapy.

ABSTRACT Dengue virus is the most prevalent cause of arthropod-borne infection worldwide. Due to the limited coding capacity of the viral genome and the complexity of the viral life cycle, host cell proteins play essential roles throughout the course of viral infection. Host RNA-binding proteins mediate various aspects of virus replication through their physical interactions with viral RNA. Here we describe a technique designed to identify such interactions in the context of infected cells using UV cross-linking followed by antisense-mediated affinity purification and mass spectrometry. Using this approach, we identified interactions, several of them novel, between host proteins and dengue viral RNA in infected Huh7 cells. Most of these interactions were subsequently validated using RNA immunoprecipitation. Using small interfering RNA (siRNA)-mediated gene silencing, we showed that more than half of these host proteins are likely involved in regulating virus replication, demonstrating the utility of this method in identifying biologically relevant interactions that may not be identified using traditional in vitro approaches. IMPORTANCE Dengue virus is the most prevalent cause of arthropod-borne infection worldwide. Viral RNA molecules physically interact with cellular RNA-binding proteins (RBPs) throughout the course of infection; the identification of such interactions will lead to the elucidation of the molecular mechanisms of virus replication. Until now, the identification of host proteins bound to dengue viral RNA has been accomplished using in vitro strategies. Here, we used a method for the specific purification of dengue viral ribonucleoprotein (RNP) complexes from infected cells and subsequently identified the associated proteins by mass spectrometry. We then validated a functional role for the majority of these proteins in mediating efficient virus replication. This approach has broad relevance to virology and RNA biology, as it could theoretically be used to purify any viral RNP complex of interest.

HipA is a bacterial serine/threonine protein kinase that phosphorylates targets, bringing about persistence and multidrug tolerance. Autophosphorylation of residue Ser150 is a critical regulatory mechanism of HipA function. Intriguingly, Ser150 is not located on the activation loop, as are other kinases; instead, it is in the protein core, where it forms part of the ATP-binding “P loop motif.” How this buried residue is phosphorylated and regulates kinase activity is unclear. Here, we report multiple structures that reveal the P loop motif's exhibition of a remarkable “in-out” conformational equilibrium, which allows access to Ser150 and its intermolecular autophosphorylation. Phosphorylated Ser150 stabilizes the “out state,” which inactivates the kinase by disrupting the ATP-binding pocket. Thus, our data reveal a mechanism of protein kinase regulation that is vital for multidrug tolerance and persistence, as kinase inactivation provides the critical first step in allowing dormant cells to revert to the growth phenotype and to reinfect the host.

Photo-activation of psoralen with UVA irradiation, referred to as PUVA, is used in the treatment of proliferative skin disorders. The anti-proliferative effects of PUVA have been largely attributed to psoralen intercalation of DNA, which upon UV treatment, triggers the formation of interstrand DNA crosslinks (ICL) that inhibit transcription and DNA replication. Here, we show that PUVA exerts antitumor effects in models of human breast cancer that overexpress the ErbB2 receptor tyrosine kinase oncogene, through a new mechanism. Independent of ICL formation, the antitumor effects of PUVA in ErbB2+ breast cancer models can instead be mediated through inhibition of ErbB2 activation and signaling. Using a mass spectroscopy-based approach, we show for the first time that photo-activated 8MOP (8-methoxypsoralen) interacts with the ErbB2 catalytic autokinase domain. Furthermore, PUVA can reverse therapeutic resistance to lapatinib and other ErbB2 targeted therapies, including resistance mediated via expression of a phosphorylated, truncated form of ErbB2 (p85(ErbB2)) that is preferentially expressed in tumor cell nuclei. Current ErbB2 targeted therapies, small molecule kinase inhibitors or antibodies, do not block the phosphorylated, activated state of p85(ErbB2). Here we show that PUVA reduced p85(ErbB2) phosphorylation leading to tumor cell apoptosis. Thus, in addition to its effects on DNA and the formation of ICL, PUVA represents a novel ErbB2 targeted therapy for the treatment of ErbB2+ breast cancers, including those that have developed resistance to other ErbB2 targeted therapies.

The molecular differences between ischemic (IF) and non-ischemic (NIF) heart failure are poorly defined. A better understanding of the molecular differences between these two heart failure etiologies may lead to the development of more effective heart failure therapeutics. In this study extensive proteomic and phosphoproteomic profiles of myocardial tissue from patients diagnosed with IF or NIF were assembled and compared. Proteins extracted from left ventricular sections were proteolyzed and phosphopeptides were enriched using titanium dioxide resin. Gel- and label-free nanoscale capillary liquid chromatography coupled to high resolution accuracy mass tandem mass spectrometry allowed for the quantification of 4,436 peptides (corresponding to 450 proteins) and 823 phosphopeptides (corresponding to 400 proteins) from the unenriched and phospho-enriched fractions, respectively. Protein abundance did not distinguish NIF from IF. In contrast, 37 peptides (corresponding to 26 proteins) exhibited a ≥ 2-fold alteration in phosphorylation state (p

The fungus Aspergillus fumigatus is a leading infectious killer in immunocompromised patients. Calcineurin, a calmodulin (CaM)-dependent protein phosphatase comprised of calcineurin A (CnaA) and calcineurin B (CnaB) subunits, localizes at the hyphal tips and septa to direct A. fumigatus invasion and virulence. Here we identified a novel serine-proline rich region (SPRR) located between two conserved CnaA domains, the CnaB-binding helix and the CaM-binding domain, that is evolutionarily conserved and unique to filamentous fungi and also completely absent in human calcineurin. Phosphopeptide enrichment and tandem mass spectrometry revealed the phosphorylation of A. fumigatus CnaA in vivo at four clustered serine residues (S406, S408, S410 and S413) in the SPRR. Mutation of the SPRR serine residues to block phosphorylation led to significant hyphal growth and virulence defects, indicating the requirement of calcineurin phosphorylation at the SPRR for its activity and function. Complementation analyses of the A. fumigatus ΔcnaA strain with cnaA homologs from the pathogenic basidiomycete Cryptococcus neoformans, the pathogenic zygomycete Mucor circinelloides, the closely related filamentous fungi Neurospora crassa, and the plant pathogen Magnaporthe grisea, revealed filamentous fungal-specific phosphorylation of CnaA in the SPRR and SPRR homology-dependent restoration of hyphal growth. Surprisingly, circular dichroism studies revealed that, despite proximity to the CaM-binding domain of CnaA, phosphorylation of the SPRR does not alter protein folding following CaM binding. Furthermore, mutational analyses in the catalytic domain, CnaB-binding helix, and the CaM-binding domains revealed that while the conserved PxIxIT substrate binding motif in CnaA is indispensable for septal localization, CaM is required for its function at the hyphal septum but not for septal localization. We defined an evolutionarily conserved novel mode of calcineurin regulation by phosphorylation in filamentous fungi in a region absent in humans. These findings suggest the possibility of harnessing this unique SPRR for innovative antifungal drug design to combat invasive aspergillosis.

Many protein interaction domains bind short peptides based on canonical sequence consensus motifs. Here we report the development of a peptide array-based proteomics tool to identify proteins directly interacting with ligand peptides from cell lysates. Array-formatted bait peptides containing an amino acid-derived cross-linker are photo-induced to crosslink with interacting proteins from lysates of interest. Indirect associations are removed by high stringency washes under denaturing conditions. Covalently trapped proteins are subsequently identified by LC-MS/MS and screened by cluster analysis and domain scanning. We apply this methodology to peptides with different proline-containing consensus sequences and show successful identifications from brain lysates of known and novel proteins containing polyproline motif-binding domains such as EH, EVH1, SH3, WW domains. These results suggest the capacity of arrayed peptide ligands to capture and subsequently identify proteins by mass spectrometry is relatively broad and robust. Additionally, the approach is rapid and applicable to cell or tissue fractions from any source, making the approach a flexible tool for initial protein-protein interaction discovery.