Erik J. Soderblom

Identification and functional analysis of in vivo phosphorylation sites of the Arabidopsis BRASSINOSTEROID-INSENSITIVE1 receptor kinase.

Brassinosteroids (BRs) regulate multiple aspects of plant growth and development and require an active BRASSINOSTEROID-INSENSITIVE1 (BRI1) and BRI1-ASSOCIATED RECEPTOR KINASE1 (BAK1) for hormone perception and signal transduction. Many animal receptor kinases exhibit ligand-dependent oligomerization followed by autophosphorylation and activation of the intracellular kinase domain. To determine if early events in BR signaling share this mechanism, we used coimmunoprecipitation of epitope-tagged proteins to show that in vivo association of BRI1 and BAK1 was affected by endogenous and exogenous BR levels and that phosphorylation of both BRI1 and BAK1 on Thr residues was BR dependent. Immunoprecipitation of epitope-tagged BRI1 from Arabidopsis thaliana followed by liquid chromatography–tandem mass spectrometry (LC/MS/MS) identified S-838, S-858, T-872, and T-880 in the juxtamembrane region, T-982 in the kinase domain, and S-1168 in C-terminal region as in vivo phosphorylation sites of BRI1. MS analysis also strongly suggested that an additional two residues in the juxtamembrane region and three sites in the activation loop of kinase subdomain VII/VIII were phosphorylated in vivo. We also identified four specific BAK1 autophosphorylation sites in vitro using LC/MS/MS. Site-directed mutagenesis of identified and predicted BRI1 phosphorylation sites revealed that the highly conserved activation loop residue T-1049 and either S-1044 or T-1045 were essential for kinase function in vitro and normal BRI1 signaling in planta. Mutations in the juxtamembrane or C-terminal regions had only small observable effects on autophosphorylation and in planta signaling but dramatically affected phosphorylation of a peptide substrate in vitro. These findings are consistent with many aspects of the animal receptor kinase model in which ligand-dependent autophosphorylation of the activation loop generates a functional kinase, whereas phosphorylation of noncatalytic intracellular domains is required for recognition and/or phosphorylation of downstream substrates.

Convergent transcriptional specializations in the brains of humans and song-learning birds.

INTRODUCTION Vocal learning, the ability to imitate sounds, is a trait that has undergone convergent evolution in several lineages of birds and mammals, including song-learning birds and humans. This behavior requires cortical and striatal vocal brain regions, which form unique connections in vocal-learning species. These regions have been found to have specialized gene expression within some species, but the patterns of specialization across vocal-learning bird and mammal species have not been systematically explored. Identifying molecular brain similarities across species. Brain region gene expression specializations were hierarchically organized into specialization trees of each species (blue lines), including for circuits that control learned vocalizations (highlighted green, purple, and orange regions). A set of comparative genomic algorithms found the most similarly specialized regions between songbird and human (orange lines), some of which are convergently evolved. RATIONALE The sequencing of genomes representing all major vocal-learning and vocal-nonlearning avian lineages has allowed us to develop the genomic tools to measure anatomical gene expression across species. Here, we asked whether behavioral and anatomical convergence is associated with gene expression convergence in the brains of vocal-learning birds and humans. RESULTS We developed a computational approach that discovers homologous and convergent specialized anatomical gene expression profiles. This includes generating hierarchically organized gene expression specialization trees for each species and a dynamic programming algorithm that finds the optimal alignment between species brain trees. We applied this approach to brain region gene expression databases of thousands of samples and genes that we and others generated from multiple species, including humans and song-learning birds (songbird, parrot, and hummingbird) as well as vocal-nonlearning nonhuman primates (macaque) and birds (dove and quail). Our results confirmed the recently revised understanding of the relationships between avian and mammalian brains. We further found that songbird Area X, a striatal region necessary for vocal learning, was most similar to a part of the human striatum activated during speech production. The RA (robust nucleus of the arcopallium) analog of song-learning birds, necessary for song production, was most similar to laryngeal motor cortex regions in humans that control speech production. More than 50 genes contributed to their convergent specialization and were enriched in motor control and neural connectivity functions. These patterns were not found in vocal nonlearners, but songbird RA was similar to layer 5 of primate motor cortex for another set of genes, supporting previous hypotheses about the similarity of these cell types between bird and mammal brains. CONCLUSION Our approach can accurately and quantitatively identify functionally and molecularly analogous brain regions between species separated by as much as 310 million years from a common ancestor. We were able to identify analogous brain regions for song and speech between birds and humans, and broader homologous brain regions in which these specialized song and speech regions are located, for tens to hundreds of genes. These genes now serve as candidates involved in developing and maintaining the unique connectivity and functional properties of vocal-learning brain circuits shared across species. The finding that convergent neural circuits for vocal learning are accompanied by convergent molecular changes of multiple genes in species separated by millions of years from a common ancestor indicates that brain circuits for complex traits may have limited ways in which they could have evolved from that ancestor. Song-learning birds and humans share independently evolved similarities in brain pathways for vocal learning that are essential for song and speech and are not found in most other species. Comparisons of brain transcriptomes of song-learning birds and humans relative to vocal nonlearners identified convergent gene expression specializations in specific song and speech brain regions of avian vocal learners and humans. The strongest shared profiles relate bird motor and striatal song-learning nuclei, respectively, with human laryngeal motor cortex and parts of the striatum that control speech production and learning. Most of the associated genes function in motor control and brain connectivity. Thus, convergent behavior and neural connectivity for a complex trait are associated with convergent specialized expression of multiple genes.

The protein expression landscape of the Arabidopsis root

Because proteins are the major functional components of cells, knowledge of their cellular localization is crucial to gaining an understanding of the biology of multicellular organisms. We have generated a protein expression map of the Arabidopsis root providing the identity and cell type-specific localization of nearly 2,000 proteins. Grouping proteins into functional categories revealed unique cellular functions and identified cell type-specific biomarkers. Cellular colocalization provided support for numerous protein–protein interactions. With a binary comparison, we found that RNA and protein expression profiles are weakly correlated. We then performed peak integration at cell type-specific resolution and found an improved correlation with transcriptome data using continuous values. We performed GeLC-MS/MS (in-gel tryptic digestion followed by liquid chromatography-tandem mass spectrometry) proteomic experiments on mutants with ectopic and no root hairs, providing complementary proteomic data. Finally, among our root hair-specific proteins we identified two unique regulators of root hair development.

Survival Defects of Cryptococcus neoformans Mutants Exposed to Human Cerebrospinal Fluid Result in Attenuated Virulence in an Experimental Model of Meningitis

ABSTRACT Cryptococcus neoformans is a fungal pathogen that encounters various microenvironments during growth in the mammalian host, including intracellular vacuoles, blood, and cerebrospinal fluid (CSF). Because the CSF is isolated by the blood-brain barrier, we hypothesize that CSF presents unique stresses that C. neoformans must overcome to establish an infection. We assayed 1,201 mutants for survival defects in growth media, saline, and human CSF. We assessed CSF-specific mutants for (i) mutant survival in both human bronchoalveolar lavage (BAL) fluid and fetal bovine serum (FBS), (ii) survival in macrophages, and (iii) virulence using both Caenorhabditiselegans and rabbit models of cryptococcosis. Thirteen mutants exhibited significant survival defects unique to CSF. The mutations of three of these mutants were recreated in the clinical serotype A strain H99: deletions of the genes for a cation ATPase transporter (ena1Δ), a putative NEDD8 ubiquitin-like protein (rub1Δ), and a phosphatidylinositol 4-kinase (pik1Δ). Mutant survival rates in yeast media, saline, and BAL fluid were similar to those of the wild type; however, survival in FBS was reduced but not to the levels in CSF. These mutant strains also exhibited decreased intracellular survival in macrophages, various degrees of virulence in nematodes, and severe attenuation of survival in a rabbit meningitis model. We analyzed the CSF by mass spectrometry for candidate compounds responsible for the survival defect. Our findings indicate that the genes required for C. neoformans survival in CSF ex vivo are necessary for survival and infection in this unique host environment.

Quantitative Label-Free Phosphoproteomics Strategy for Multifaceted Experimental Designs

Protein phosphorylation is a critical regulator of signaling in nearly all eukaryotic cellular pathways and dysregulated phosphorylation has been implicated in an array of diseases. The majority of MS-based quantitative phosphorylation studies are currently performed from transformed cell lines because of the ability to generate large amounts of starting material with incorporated isotopically labeled amino acids during cell culture. Here we describe a general label-free quantitative phosphoproteomic strategy capable of directly analyzing relatively small amounts of virtually any biological matrix, including human tissue and biological fluids. The strategy utilizes a TiO2 enrichment protocol in which the selectivity and recovery of phosphopeptides were optimized by assessing a twenty-point condition matrix of binding modifier concentrations and peptide-to-resin capacity ratios. The quantitative reproducibility of the TiO2 enrichment was determined to be 16% RSD through replicate enrichments of a wild-type Danio rerio (zebrafish) lysate. Measured phosphopeptide fold-changes from alpha-casein spiked into wild-type zebrafish lysate backgrounds were within 5% of the theoretical value. Application to a morpholino induced knock-down of G protein-coupled receptor kinase 5 (GRK5) in zebrafish embryos resulted in the quantitation of 719 phosphorylated peptides corresponding to 449 phosphorylated proteins from 200 μg of zebrafish embryo lysates.

Identification of an elaborate complex mediating postsynaptic inhibition.

Inhibitory synapses dampen neuronal activity through postsynaptic hyperpolarization. The composition of the inhibitory postsynapse and the mechanistic basis of its regulation, however, remain poorly understood. We used an in vivo chemico-genetic proximity-labeling approach to discover inhibitory postsynaptic proteins. Quantitative mass spectrometry not only recapitulated known inhibitory postsynaptic proteins but also revealed a large network of new proteins, many of which are either implicated in neurodevelopmental disorders or are of unknown function. Clustered regularly interspaced short palindromic repeats (CRISPR) depletion of one of these previously uncharacterized proteins, InSyn1, led to decreased postsynaptic inhibitory sites, reduced the frequency of miniature inhibitory currents, and increased excitability in the hippocampus. Our findings uncover a rich and functionally diverse assemblage of previously unknown proteins that regulate postsynaptic inhibition and might contribute to developmental brain disorders.

Erythrocyte plasma membrane–bound ERK1/2 activation promotes ICAM-4–mediated sickle red cell adhesion to endothelium

The core pathology of sickle cell disease (SCD) starts with the erythrocyte (RBC). Aberration in MAPK/ERK1/2 signaling, which can regulate cell adhesion, occurs in diverse pathologies. Because RBCs contain abundant ERK1/2, we predicted that ERK1/2 is functional in sickle (SS) RBCs and promotes adherence, a hallmark of SCD. ERK1/2 remained active in SS but not normal RBCs. β(2)-adrenergic receptor stimulation by epinephrine can enhance ERK1/2 activity only in SS RBCs via PKA- and tyrosine kinase p72(syk)-dependent pathways. ERK signaling is implicated in RBC ICAM-4 phosphorylation, promoting SS RBC adhesion to the endothelium. SS RBC adhesion and phosphorylation of both ERK and ICAM-4 all decreased with continued cell exposure to epinephrine, implying that activation of ICAM-4-mediated SS RBC adhesion is temporally associated with ERK1/2 activation. Furthermore, recombinant ERK2 phosphorylated α- and β-adducins and dematin at the ERK consensus motif. Cytoskeletal protein 4.1 also showed dynamic phosphorylation but not at the ERK consensus motif. These results demonstrate that ERK activation induces phosphorylation of cytoskeletal proteins and the adhesion molecule ICAM-4, promoting SS RBC adhesion to the endothelium. Thus, blocking RBC ERK1/2 activation, such as that promoted by catecholamine stress hormones, could ameliorate SCD pathophysiology.

Phosphorylation of Calcineurin at a novel serine-proline rich region orchestrates hyphal growth and virulence in Aspergillus fumigatus.

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.

Characterization of a new family of protein kinases from Arabidopsis containing phosphoinositide 3/4-kinase and ubiquitin-like domains

At least two of the genes predicted to encode type II PI4K (phosphoinositide 4-kinase) in Arabidopsis thaliana (thale cress), namely AtPI4Kgamma4 and AtPI4Kgamma7, encode enzymes with catalytic properties similar to those of members of the PIKK (phosphoinositide kinase-related kinase) family. AtPI4Kgamma4 and AtPI4Kgamma7 undergo autophosphorylation and phosphorylate serine/threonine residues of protein substrates, but have no detectable lipid kinase activity. AtPI4Kgamma4 and AtPI4Kgamma7 are members of a subset of five putative AtPI4Ks that contain N-terminal UBL (ubiquitin-like) domains. In vitro analysis of AtPI4Kgamma4 indicates that it interacts directly with, and phosphorylates, two proteins involved in the ubiquitin-proteasome system, namely UFD1 (ubiquitin fusion degradation 1) and RPN10 (regulatory particle non-ATPase 10). On the basis of the present results, we propose that AtPI4Kgamma4 and AtPI4Kgamma7 should be designated UbDKgamma4 and UbDKgamma7 (ubiquitin-like domain kinases gamma4 and gamma7). These UBL-domain-containing AtPI4Ks correspond to a new PIKK subfamily of protein kinases. Furthermore, UFD1 and RPN10 phosphorylation represents an additional mechanism by which their function can be regulated.

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.