Chemico-genetic discovery of astrocytic control of inhibition in vivo.

Perisynaptic astrocytic processes are an integral part of central nervous system synapses1,2; however, the molecular mechanisms that govern astrocyte-synapse adhesions and how astrocyte contacts control synapse formation and function are largely unknown. Here we use an in vivo chemico-genetic approach that applies a cell-surface fragment complementation strategy, Split-TurboID, and identify a proteome that is enriched at astrocyte-neuron junctions in vivo, which includes neuronal cell adhesion molecule (NRCAM). We find that NRCAM is expressed in cortical astrocytes, localizes to perisynaptic contacts and is required to restrict neuropil infiltration by astrocytic processes. Furthermore, we show that astrocytic NRCAM interacts transcellularly with neuronal NRCAM coupled to gephyrin at inhibitory postsynapses. Depletion of astrocytic NRCAM reduces numbers of inhibitory synapses without altering glutamatergic synaptic density. Moreover, loss of astrocytic NRCAM markedly decreases inhibitory synaptic function, with minor effects on excitation. Thus, our results present a proteomic framework for how astrocytes interface with neurons and reveal how astrocytes control GABAergic synapse formation and function.

Signal transduction at GPCRs: Allosteric activation of the ERK MAPK by ß-arrestin.

ß-arrestins are multivalent adaptor proteins that bind active phosphorylated G protein-coupled receptors (GPCRs) to inhibit G protein signaling, mediate receptor internalization, and initiate alternative signaling events. ß-arrestins link agonist-stimulated GPCRs to downstream signaling partners, such as the c-Raf-MEK1-ERK1/2 cascade leading to ERK1/2 activation. ß-arrestins have been thought to transduce signals solely via passive scaffolding by facilitating the assembly of multiprotein signaling complexes. Recently, however, ß-arrestin 1 and 2 were shown to activate two downstream signaling effectors, c-Src and c-Raf, allosterically. Over the last two decades, ERK1/2 have been the most intensely studied signaling proteins scaffolded by ß-arrestins. Here, we demonstrate that ß-arrestins play an active role in allosterically modulating ERK kinase activity in vitro and within intact cells. Specifically, we show that ß-arrestins and their GPCR-mediated active states allosterically enhance ERK2 autophosphorylation and phosphorylation of a downstream ERK2 substrate, and we elucidate the mechanism by which ß-arrestins do so. Furthermore, we find that allosteric stimulation of dually phosphorylated ERK2 by active-state ß-arrestin 2 is more robust than by active-state ß-arrestin 1, highlighting differential capacities of ß-arrestin isoforms to regulate effector signaling pathways downstream of GPCRs. In summary, our study provides strong evidence for a new paradigm in which ß-arrestins function as active "catalytic" scaffolds to allosterically unlock the enzymatic activity of signaling components downstream of GPCR activation.

Serum proteomic biomarkers diagnostic of knee osteoarthritis.

OBJECTIVE: To better understand the pathogenesis of knee osteoarthritis (OA) through identification of serum diagnostics. DESIGN: We conducted multiple reaction monitoring mass spectrometry analysis of 107 peptides in baseline sera of two cohorts: the Foundation for National Institutes of Health (NIH) (n = 596 Kellgren-Lawrence (KL) grade 1-3 knee OA participants); and the Johnston County Osteoarthritis Project (n = 127 multi-joint controls free of radiographic OA of the hands, hips, knees (bilateral KL=0), and spine). Data were split into (70%) training and (30%) testing sets. Diagnostic peptide and clinical data predictors were selected by random forest (RF); selection was based on association (p < 0.05) with OA status in multivariable logistic regression models. Model performance was based on area under the curve (AUC) of receiver operating characteristic (ROC) and precision-recall (PR) curves. RESULTS: RF selected 23 peptides (19 proteins) and body mass index (BMI) as diagnostic of OA. BMI weakly diagnosed OA (ROC-AUC 0.57, PR-AUC 0.812) and only symptomatic OA cases. ACTG was the strongest univariable predictor (ROC-AUC 0.705, PR-AUC 0.897). The final model (8 serum peptides) was highly diagnostic (ROC-AUC 0.833, 95% confidence interval [CI] 0.751, 0.905; PR-AUC 0.929, 95% CI 0.876, 0.973) in the testing set and equally diagnostic of non-symptomatic and symptomatic cases (AUCs 0.830-0.835), and not significantly improved with addition of BMI. The STRING database predicted multiple high confidence interactions of the 19 diagnostic OA proteins. CONCLUSIONS: No more than 8 serum protein biomarkers were required to discriminate knee OA from non-OA. These biomarkers lend strong support to the involvement and cross-talk of complement and coagulation pathways in the development of OA.

Glypican-4 regulated actin cytoskeletal reorganization in glucocorticoid treated trabecular meshwork cells and involvement of Wnt/PCP signaling.

A common adverse response to the clinical use of glucocorticoids (GCs) is elevated intraocular pressure (IOP) which is a major risk factor for glaucoma. Elevated IOP arises due to impaired outflow of aqueous humor (AH) through the trabecular meshwork (TM). Although GC-induced changes in actin cytoskeletal dynamics, contractile characteristics, and cell adhesive interactions of TM cells are believed to influence AH outflow and IOP, the molecular mechanisms mediating changes in these cellular characteristics are poorly understood. Our studies focused on evaluating changes in the cytoskeletal and cytoskeletal-associated protein (cytoskeletome) profile of human TM cells treated with dexamethasone (Dex) using label-free mass spectrometric quantification, identified elevated levels of specific proteins known to regulate actin stress fiber formation, contraction, actin networks crosslinking, cell adhesion, and Wnt signaling, including LIMCH1, ArgBP2, CNN3, ITGBL1, CTGF, palladin, FAT1, DIAPH2, EPHA4, SIPA1L1, and GPC4. Several of these proteins colocalized with the actin cytoskeleton and underwent alterations in distribution profile in TM cells treated with Dex, and an inhibitor of Abl/Src kinases. Wnt/Planar Cell Polarity (PCP) signaling agonists-Wnt5a and 5b were detected prominently in the cytoskeletome fraction of TM cells, and studies using siRNA to suppress expression of glypican-4 (GPC4), a known modulator of the Wnt/PCP pathway revealed that GPC4 deficiency impairs Dex induced actin stress fiber formation, and activation of c-Jun N-terminal Kinase (JNK) and Rho kinase. Additionally, while Dex augmented, GPC4 deficiency suppressed the formation of actin stress fibers in TM cells in the presence of Dex and Wnt5a. Taken together, these results identify the GPC4-dependent Wnt/PCP signaling pathway as one of the crucial upstream regulators of Dex induced actin cytoskeletal reorganization and cell adhesion in TM cells, opening an opportunity to target the GPC4/Wnt/PCP pathway for treatment of ocular hypertension in glaucoma.

Comprehensive characterization of pathogenic synovial fluid extracellular vesicles from knee osteoarthritis.

Synovial fluid (SF) extracellular vesicles (EVs) play a pathogenic role in osteoarthritis (OA). However, the surface markers, cell and tissue origins, and effectors of these EVs are largely unknown. We found that SF EVs contained 692 peptides that were positively associated with knee radiographic OA severity; 57.4% of these pathogenic peptides were from 46 proteins of the immune system, predominantly the innate immune system. CSPG4, BGN, NRP1, and CD109 are the major surface markers of pathogenic SF EVs. Genes encoding surface marker CSPG4 and CD109 were highly expressed by chondrocytes from damaged cartilage, while VISG4, MARCO, CD163 and NRP1 were enriched in the synovial immune cells. The frequency of CSPG4+ and VSIG4+ EV subpopulations in OA SF was high. We conclude that pathogenic SF EVs carry knee OA severity-associated proteins and specific surface markers, which could be developed as a new source of diagnostic biomarkers or therapeutic targets in OA.

BioID reveals an ATG9A interaction with ATG13-ATG101 in the degradation of p62/SQSTM1-ubiquitin clusters.

ATG9A, the only multi-pass transmembrane protein among core ATG proteins, is an essential regulator of autophagy, yet its regulatory mechanisms and network of interactions are poorly understood. Through quantitative BioID proteomics, we identify a network of ATG9A interactions that includes members of the ULK1 complex and regulators of membrane fusion and vesicle trafficking, including the TRAPP, EARP, GARP, exocyst, AP-1, and AP-4 complexes. These interactions mark pathways of ATG9A trafficking through ER, Golgi, and endosomal systems. In exploring these data, we find that ATG9A interacts with components of the ULK1 complex, particularly ATG13 and ATG101. Using knockout/reconstitution and split-mVenus approaches to capture the ATG13-ATG101 dimer, we find that ATG9A interacts with ATG13-ATG101 independently of ULK1. Deletion of ATG13 or ATG101 causes a shift in ATG9A distribution, resulting in an aberrant accumulation of ATG9A at stalled clusters of p62/SQSTM1 and ubiquitin, which can be rescued by an ULK1 binding-deficient mutant of ATG13. Together, these data reveal ATG9A interactions in vesicle-trafficking and autophagy pathways, including a role for an ULK1-independent ATG13 complex in regulating ATG9A.

Cnksr2 Loss in Mice Leads to Increased Neural Activity and Behavioral Phenotypes of Epilepsy-Aphasia Syndrome.

Epilepsy Aphasia Syndromes (EAS) are a spectrum of childhood epileptic, cognitive, and language disorders of unknown etiology. CNKSR2 is a strong X-linked candidate gene implicated in EAS; however, there have been no studies of genetic models to dissect how its absence may lead to EAS. Here we develop a novel Cnksr2 KO mouse line and show that male mice exhibit increased neural activity and have spontaneous electrographic seizures. Cnksr2 KO mice also display significantly increased anxiety, impaired learning and memory, and a progressive and dramatic loss of ultrasonic vocalizations. We find that Cnksr2 is expressed in cortical, striatal, and cerebellar regions and is localized at both excitatory and inhibitory postsynapses. Proteomics analysis reveals Cnksr2 anchors key binding partners at synapses, and its loss results in significant alterations of the synaptic proteome, including proteins implicated in epilepsy disorders. Our results validate that loss of CNKSR2 leads to EAS and highlights the roles of Cnksr2 in synaptic organization and neuronal network activity.SIGNIFICANCE STATEMENT Epilepsy Aphasia Syndromes (EAS) are at the severe end of a spectrum of cognitive-behavioral symptoms seen in childhood epilepsies, and they remain an inadequately understood disorder. The prognosis of EAS is frequently poor, and patients have life-long language and cognitive disturbances. Here we describe a genetic mouse model of EAS, based on the KO of the EAS risk gene Cnksr2 We show that these mice exhibit electrophysiological and behavioral phenotypes similar to those of patients, providing an important new model for future studies of EAS. We also provide insights into the molecular disturbances downstream of Cnksr2 loss by using in vivo quantitative proteomics tools.

Structural basis of O-GlcNAc recognition by mammalian 14-3-3 proteins.

O-GlcNAc is an intracellular posttranslational modification that governs myriad cell biological processes and is dysregulated in human diseases. Despite this broad pathophysiological significance, the biochemical effects of most O-GlcNAcylation events remain uncharacterized. One prevalent hypothesis is that O-GlcNAc moieties may be recognized by "reader" proteins to effect downstream signaling. However, no general O-GlcNAc readers have been identified, leaving a considerable gap in the field. To elucidate O-GlcNAc signaling mechanisms, we devised a biochemical screen for candidate O-GlcNAc reader proteins. We identified several human proteins, including 14-3-3 isoforms, that bind O-GlcNAc directly and selectively. We demonstrate that 14-3-3 proteins bind O-GlcNAc moieties in human cells, and we present the structures of 14-3-3ß/α and γ bound to glycopeptides, providing biophysical insights into O-GlcNAc-mediated protein-protein interactions. Because 14-3-3 proteins also bind to phospho-serine and phospho-threonine, they may integrate information from O-GlcNAc and O-phosphate signaling pathways to regulate numerous physiological functions.

Calcineurin-dependent dephosphorylation of the transcription factor CrzA at specific sites controls conidiation, stress tolerance, and virulence of Aspergillus fumigatus.

Calcium signaling through calcineurin and its major transcription factor (TF), CrzA, is integral to hyphal growth, stress response and virulence of the pathogenic fungus Aspergillus fumigatus, the leading etiology of invasive aspergillosis. Dephosphorylation of CrzA by calcineurin activates the TF, but the specific phosphorylation sites and their roles in the activation/inactivation mechanism are unknown. Mass spectroscopic analysis identified 20 phosphorylation sites, the majority of which were specific to filamentous fungi and distributed throughout the CrzA protein, with particular concentration in a serine-rich region N-terminal to the conserved DNA-binding domain (DBD). Site-directed mutagenesis of phosphorylated residues revealed that CrzA activity during calcium stimulation can only be suppressed by a high degree of phosphorylation in multiple regions of the protein. Our findings further suggest that this regulation is not solely accomplished through control of CrzA nuclear import. Additionally, we demonstrate the importance of the CrzA phosphorylation state in regulating growth, conidiation, calcium and cell wall stress tolerance, and virulence. Finally, we identify two previously undescribed nuclear localization sequences in the DBD. These findings provide novel insight into the phosphoregulation of CrzA which may be exploited to selectively target A. fumigatus.

The tail domain of the Aspergillus fumigatus class V myosin MyoE orchestrates septal localization and hyphal growth.

Myosins are critical motor proteins that contribute to the secretory pathway, polarized growth, and cytokinesis. The globular tail domains of class V myosins have been shown to be important for cargo binding and actin cable organization. Additionally, phosphorylation plays a role in class V myosin cargo choice. Our previous studies on the class V myosin MyoE in the fungal pathogen Aspergillus fumigatus confirmed its requirement for normal morphology and virulence. However, the domains and molecular mechanisms governing the functions of MyoE remain unknown. Here, by analyzing tail mutants, we demonstrate that the tail is required for radial growth, conidiation, septation frequency and MyoE's location at the septum. Furthermore, MyoE is phosphorylated at multiple residues in vivo; however, alanine substitution mutants revealed that no single phosphorylated residue was critical. Importantly, in the absence of the phosphatase calcineurin, an additional residue was phosphorylated in its tail domain. Mutation of this tail residue led to mislocalization of MyoE from the septa. This work reveals the importance of the MyoE tail domain and its phosphorylation/dephosphorylation in the growth and morphology of A. fumigatus.

Dynamic Glycosylation Governs the Vertebrate COPII Protein Trafficking Pathway.

The COPII coat complex, which mediates secretory cargo trafficking from the endoplasmic reticulum, is a key control point for subcellular protein targeting. Because misdirected proteins cannot function, protein sorting by COPII is critical for establishing and maintaining normal cell and tissue homeostasis. Indeed, mutations in COPII genes cause a range of human pathologies, including cranio-lenticulo-sutural dysplasia (CLSD), which is characterized by collagen trafficking defects, craniofacial abnormalities, and skeletal dysmorphology. Detailed knowledge of the COPII pathway is required to understand its role in normal cell physiology and to devise new treatments for disorders in which it is disrupted. However, little is known about how vertebrates dynamically regulate COPII activity in response to developmental, metabolic, or pathological cues. Several COPII proteins are modified by O-linked ß-N-acetylglucosamine (O-GlcNAc), a dynamic form of intracellular protein glycosylation, but the biochemical and functional effects of these modifications remain unclear. Here, we use a combination of chemical, biochemical, cellular, and genetic approaches to demonstrate that site-specific O-GlcNAcylation of COPII proteins mediates their protein-protein interactions and modulates cargo secretion. In particular, we show that individual O-GlcNAcylation sites of SEC23A, an essential COPII component, are required for its function in human cells and vertebrate development, because mutation of these sites impairs SEC23A-dependent in vivo collagen trafficking and skeletogenesis in a zebrafish model of CLSD. Our results indicate that O-GlcNAc is a conserved and critical regulatory modification in the vertebrate COPII-dependent trafficking pathway.

Scanning Quadrupole Data-Independent Acquisition, Part A: Qualitative and Quantitative Characterization.

A novel data-independent acquisition (DIA) method incorporating a scanning quadrupole in front of a collision cell and orthogonal acceleration time-of-flight mass analyzer is described. The method has been characterized for the qualitative and quantitative label-free proteomic analysis of complex biological samples. The principle of the scanning quadrupole DIA method is discussed, and analytical instrument characteristics, such as the quadrupole transmission width, scan/integration time, and chromatographic separation, have been optimized in relation to sample complexity for a number of different model proteomes of varying complexity and dynamic range including human plasma, cell lines, and bacteria. In addition, the technological merits over existing DIA approaches are described and contrasted. The qualitative and semiquantitative performance of the method is illustrated for the analysis of relatively simple protein digest mixtures and a well-characterized human cell line sample using untargeted and targeted search strategies. Finally, the results from a human cell line were compared against publicly available data that used similar chromatographic conditions but were acquired with DDA technology and alternative mass analyzer systems. Qualitative comparison showed excellent concordance of results with >90% overlap of the detected proteins.

Scanning Quadrupole Data-Independent Acquisition, Part B: Application to the Analysis of the Calcineurin-Interacting Proteins during Treatment of Aspergillus fumigatus with Azole and Echinocandin Antifungal Drugs.

Calcineurin is a critical cell-signaling protein that orchestrates growth, stress response, virulence, and antifungal drug resistance in several fungal pathogens. Blocking calcineurin signaling increases the efficacy of several currently available antifungals and suppresses drug resistance. We demonstrate the application of a novel scanning quadrupole DIA method for the analysis of changes in the proteins coimmunoprecipitated with calcineurin during therapeutic antifungal drug treatments of the deadly human fungal pathogen Aspergillus fumigatus. Our experimental design afforded an assessment of the precision of the method as demonstrated by peptide- and protein-centric analysis from eight replicates of the study pool QC samples. Two distinct classes of clinically relevant antifungal drugs that are guideline recommended for the treatment of invasive "aspergillosis" caused by Aspergillus fumigatus, the azoles (voriconazole) and the echinocandins (caspofungin and micafungin), which specifically target the fungal plasma membrane and the fungal cell wall, respectively, were chosen to distinguish variations occurring in the proteins coimmunoprecipitated with calcineurin. Novel potential interactors were identified in response to the different drug treatments that are indicative of the possible role for calcineurin in regulating these effectors. Notably, treatment with voriconazole showed increased immunoprecipitation of key proteins involved in membrane ergosterol biosynthesis with calcineurin. In contrast, echinocandin (caspofungin or micafungin) treatments caused increased immunoprecipitation of proteins involved in cell-wall biosynthesis and septation. Furthermore, abundant coimmunoprecipitation of ribosomal proteins with calcineurin occurred exclusively in echinocandins treatment, indicating reprogramming of cellular growth mechanisms during different antifungal drug treatments. While variations in the observed calcineurin immunoprecipitated proteins may also be due to changes in their expression levels under different drug treatments, this study suggests an important role for calcineurin-dependent cellular mechanisms in response to antifungal treatment of A. fumigatus that warrants future studies.

Kin1 kinase localizes at the hyphal septum and is dephosphorylated by calcineurin but is dispensable for septation and virulence in the human pathogen Aspergillus fumigatus.

Studies in yeasts have implicated the importance of Kin1 protein kinase, a member of the eukaryotic PAR1/MARK/MELK family, in polarized growth, cell division and septation through coordinated activity with the phosphatase, calcineurin. Kin1 is also required for virulence of the fungal pathogens Cryptococcus neoformans and Fusarium graminearum. Here we show that kin1 deletion in the human fungal pathogen Aspergillus fumigatus does not affect hyphal growth and septation but results in differential susceptibility to antifungals targeting the cell wall and cell membrane. The Δkin1 strain remained virulent in a Galleria mellonella model of invasive aspergillosis. Expression of Kin1 tagged to GFP or RFP showed its stable localization at the septum. Co-localization experiments revealed calcineurin (CnaA) localization on either side of Kin1 at the septum suggesting possible interaction. Bimolecular fluorescence complementation assay confirmed the interaction of Kin1 with CnaA at the hyphal tips and septa in the presence of the antifungal caspofungin. Furthermore, phosphoproteomic analyses for the first time revealed Kin1 as a substrate of calcineurin providing novel insight into Kin1 regulation through calcineurin-mediated dephosphorylation mechanism.

Calcineurin Targets Involved in Stress Survival and Fungal Virulence.

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.

Vagal nerve stimulation modifies neuronal activity and the proteome of excitatory synapses of amygdala/piriform cortex.

Vagal Nerve Stimulation (VNS) Therapy® is a United States Food and Drug Administration approved neurotherapeutic for medically refractory partial epilepsy and treatment-resistant depression. The molecular mechanisms underlying its beneficial effects are unclear. We hypothesized that one mechanism involves neuronal activity-dependent modifications of central nervous system excitatory synapses. To begin to test this hypothesis, we asked whether VNS modifies the activity of neurons in amygdala and hippocampus. Neuronal recordings from adult, freely moving rats revealed that activity in both amygdala and hippocampus was modified by VNS immediately after its application, and changes were detected following 1 week of stimulation. To investigate whether VNS modifies the proteome of excitatory synapses, we established a label-free, quantitative liquid chromatography-tandem mass spectrometry workflow that enables global analysis of the constituents of the postsynaptic density (PSD) proteome. PSD proteins were biochemically purified from amygdala/piriform cortex of VNS- or dummy-treated rats following 1-week stimulation, and individual PSD protein levels were quantified by liquid chromatography-tandem mass spectrometry analysis. We identified 1899 unique peptides corresponding to 425 proteins in PSD fractions, of which expression levels of 22 proteins were differentially regulated by VNS with changes greater than 150%. Changes in a subset of these proteins, including significantly increased expression of neurexin-1α, cadherin 13 and voltage-dependent calcium channel α2δ1, the primary target of the antiepileptic drug gabapentin, and decreased expression of voltage-dependent calcium channel γ3, were confirmed by western blot analysis of PSD samples. These results demonstrate that VNS modulates excitatory synapses through regulating a subset of the PSD proteome. Our study reveals molecular targets of VNS and point to possible mechanisms underlying its beneficial effects, including activity-dependent formation of excitatory synapses.

Caspofungin exposure alters the core septin AspB interactome of Aspergillus fumigatus.

Aspergillus fumigatus, the main etiological agent of invasive aspergillosis, is a leading cause of death in immunocompromised patients. Septins, a conserved family of GTP-binding proteins, serve as scaffolding proteins to recruit enzymes and key regulators to different cellular compartments. Deletion of the A. fumigatus septin aspB increases susceptibility to the echinocandin antifungal caspofungin. However, how AspB mediates this response to caspofungin is unknown. Here, we characterized the AspB interactome under basal conditions and after exposure to a clinically relevant concentration of caspofungin. While A. fumigatus AspB interacted with 334 proteins, including kinases, cell cycle regulators, and cell wall synthesis-related proteins under basal growth conditions, caspofungin exposure altered AspB interactions. A total of 69 of the basal interactants did not interact with AspB after exposure to caspofungin, and 54 new interactants were identified following caspofungin exposure. We generated A. fumigatus deletion strains for 3 proteins (ArpB, Cyp4, and PpoA) that only interacted with AspB following exposure to caspofungin that were previously annotated as induced after exposure to antifungal agents, yet only PpoA was implicated in the response to caspofungin. Taken together, we defined how the septin AspB interactome is altered in the presence of a clinically relevant antifungal.

Hydrolytic function of Exo1 in mammalian mismatch repair.

Genetic and biochemical studies have previously implicated exonuclease 1 (Exo1) in yeast and mammalian mismatch repair, with results suggesting that function of the protein in the reaction depends on both its hydrolytic activity and its ability to interact with other components of the repair system. However, recent analysis of an Exo1-E109K knockin mouse has concluded that Exo1 function in mammalian mismatch repair is restricted to a structural role, a conclusion based on a prior report that N-terminal His-tagged Exo1-E109K is hydrolytically defective. Because Glu-109 is distant from the nuclease hydrolytic center, we have compared the activity of untagged full-length Exo1-E109K with that of wild type Exo1 and the hydrolytically defective active site mutant Exo1-D173A. We show that the activity of Exo1-E109K is comparable to that of wild type enzyme in a conventional exonuclease assay and that in contrast to a D173A active site mutant, Exo1-E109K is fully functional in mismatch-provoked excision and repair. We conclude that the catalytic function of Exo1 is required for its participation in mismatch repair. We also consider the other phenotypes of the Exo1-E109K mouse in the context of Exo1 hydrolytic function.

Quantitative proteomics of bronchoalveolar lavage fluid in idiopathic pulmonary fibrosis.

The proteomic analysis of bronchoalveolar lavage fluid (BALF) can give insight into pulmonary disease pathology and response to therapy. Here, we describe the first gel-free quantitative analysis of BALF in idiopathic pulmonary fibrosis (IPF), a chronic and fatal scarring lung disease. We utilized two-dimensional reversed-phase liquid chromatography and ion-mobility-assisted data-independent acquisition (HDMSE) for quantitation of >1000 proteins in immunodepleted BALF from the right middle and lower lobes of normal controls and patients with IPF. Among the analytes that were increased in IPF were well-described mediators of pulmonary fibrosis (osteopontin, MMP7, CXCL7, CCL18), eosinophil- and neutrophil-derived proteins, and proteins associated with fibroblast foci. For additional discovery and targeted validation, BALF was also screened by multiple reaction monitoring (MRM), using the JPT Cytokine SpikeMix library of >400 stable isotope-labeled peptides. A refined MRM assay confirmed the robust expression of osteopontin, and demonstrated, for the first time, upregulation of the pro-fibrotic cytokine, CCL24, in BALF in IPF. These results show the utility of BALF proteomics for the molecular profiling of fibrotic lung diseases and the targeted quantitation of soluble markers of IPF. More generally, this study addresses critical quality control measures that should be widely applicable to BALF profiling in pulmonary disease.

Metabolic control of Ca2+/calmodulin-dependent protein kinase II (CaMKII)-mediated caspase-2 suppression by the B55ß/protein phosphatase 2A (PP2A).

High levels of metabolic activity confer resistance to apoptosis. Caspase-2, an apoptotic initiator, can be suppressed by high levels of nutrient flux through the pentose phosphate pathway. This metabolic control is exerted via inhibitory phosphorylation of the caspase-2 prodomain by activated Ca(2+)/calmodulin-dependent protein kinase II (CaMKII). We show here that this activation of CaMKII depends, in part, on dephosphorylation of CaMKII at novel sites (Thr(393)/Ser(395)) and that this is mediated by metabolic activation of protein phosphatase 2A in complex with the B55ß targeting subunit. This represents a novel locus of CaMKII control and also provides a mechanism contributing to metabolic control of apoptosis. These findings may have implications for metabolic control of the many CaMKII-controlled and protein phosphatase 2A-regulated physiological processes, because both enzymes appear to be responsive to alterations in glucose metabolized via the pentose phosphate pathway.