A Cross-Species Analysis Reveals a General Role for Piezo2 in Mechanosensory Specialization of Trigeminal Ganglia from Tactile Specialist Birds

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Systematic identification of SH3 domain-mediated human protein-protein interactions by peptide array target screening

Systematic identification of direct protein-protein interactions is often hampered by difficulties in expressing and purifying the corresponding full-length proteins. By taking advantage of the modular nature of many regulatory proteins, we attempted to simplify protein-protein interactions to the corresponding domain-ligand recognition and employed peptide arrays to identify such binding events. A group of 12 Src homology (SH) 3 domains from eight human proteins (Swiss-Prot ID: SRC, PLCG1, P85A, NCK1, GRB2, FYN, CRK) were used to screen a peptide target array composed of 1536 potential ligands, which led to the identification of 921 binary interactions between these proteins and 284 targets. To assess the efficiency of the peptide array target screening (PATS) method in identifying authentic protein-protein interactions, we examined a set of interactions mediated by the PLCgamma1 SH3 domain by coimmunoprecipitation and/or affinity pull-downs using full-length proteins and achieved a 75% success rate. Furthermore, we characterized a novel interaction between PLCgamma1 and hematopoietic progenitor kinase 1 (HPK1) identified by PATS and demonstrated that the PLCgamma1 SH3 domain negatively regulated HPK1 kinase activity. Compared to protein interactions listed in the online predicted human interaction protein database (OPHID), the majority of interactions identified by PATS are novel, suggesting that, when extended to the large number of peptide interaction domains encoded by the human genome, PATS should aid in the mapping of the human interactome.     

Dopamine modulates the plasticity of mechanosensory responses in Caenorhabditis elegans

Dopamine-modulated behaviors, including information processing and reward, are subject to behavioral plasticity. Disruption of these behaviors is thought to support drug addictions and psychoses. The plasticity of dopamine-mediated behaviors, for example, habituation and sensitization, are not well understood at the molecular level. We show that in the nematode Caenorhabditis elegans, a D1-like dopamine receptor gene (dop-1) modulates the plasticity of mechanosensory behaviors in which dopamine had not been implicated previously. A mutant of dop-1 displayed faster habituation to nonlocalized mechanical stimulation. This phenotype was rescued by the introduction of a wild-type copy of the gene. The dop-1 gene is expressed in mechanosensory neurons, particularly the ALM and PLM neurons. Selective expression of the dop-1 gene in mechanosensory neurons using the mec-7 promoter rescues the mechanosensory deficit in dop-1 mutant animals. The tyrosine hydroxylase-deficient C. elegans mutant (cat-2) also displays these specific behavioral deficits. These observations provide genetic evidence that dopamine signaling modulates behavioral plasticity in C. elegans.     

Using immobilized metal affinity chromatography, two-dimensional electrophoresis and mass spectrometry to identify hepatocellular proteins with copper-binding ability

To further our knowledge of intracellular copper transport, we used a proteomics strategy to search for hepatic proteins with copper-binding ability. Hep G2 cytosolic and microsomal fractions were applied to a copper(II)-loaded immobilized metal-affinity chromatography (IMAC) column. Protein identification was performed with 2-D gel electrophoresis and mass spectrometry. We identified 48 cytosolic proteins and 19 microsomal proteins displaying copper-binding ability. These proteins are diverse in function. Fifty-two of the 67 proteins contain putative metal-binding domains. We have identified many components of the Hep G2 copper metalloproteome including a large number of proteins not previously known to bind copper.     

Targeted ablation of gonadotrophs in transgenic mice depresses prolactin but not growth hormone gene expression at birth as measured by quantitative mRNA detection

We previously reported that transgenic ablation of gonadotrophs results in impaired development of cells immunostainable for prolactin (PRL) but not of cells immunostainable for growth hormone (GH) or proopiomelanocortin (POMC) in pituitary of newborn mice. The question remained whether this reduction in PRL protein is a reflection of reduced PRL mRNA expression, or whether this regulation is only situated at the translational level. We therefore generated a new series of transgenic mice in which gonadotrophs were ablated by diphtheria toxin A targeting, and analyzed hormone mRNA levels instead of hormone protein around the day of birth. Pituitary mRNA expression levels of luteinizing hormone- (LH), PRL and GH were quantified using real-time TaqMan RT-PCR. Of the 13 transgenic mice obtained, 8 showed a clear-cut reduction (ranging from 62 to 98%) in LH mRNA levels. PRL mRNA values were significantly reduced in the transgenic mice (p=0.0034), while GH mRNA expression was unaffected (p=0.93). An additional observation was that female newborn mice produce 5 times more LH mRNA than male mice whereas no sex difference was observed for expression levels of PRL and GH mRNA. Moreover, in the wild-type mice, LH mRNA expression was 20-fold higher than GH mRNA expression which in turn was 500- to 1,000-fold higher than PRL mRNA expression, suggesting a low expression level of the PRL gene at birth. In conclusion, the present data support the hypothesis that embryonic development of PRL gene expression is stimulated by gonadotrophs.     

Mass spectrometric investigation of the neuropeptide complement and release in the pericardial organs of the crab, Cancer borealis

The crustacean stomatogastric ganglion (STG) is modulated by both locally released neuroactive compounds and circulating hormones. This study presents mass spectrometric characterization of the complement of peptide hormones present in one of the major neurosecretory structures, the pericardial organs (POs), and the detection of neurohormones released from the POs. Direct peptide profiling of Cancer borealis PO tissues using matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) revealed many previously identified peptides, including proctolin, red pigment concentrating hormone (RPCH), crustacean cardioactive peptide (CCAP), several orcokinins, and SDRNFLRFamide. This technique also detected corazonin, a well-known insect hormone, in the POs for the first time. However, most mass spectral peaks did not correspond to previously known peptides. To characterize and identify these novel peptides, we performed MALDI postsource decay (PSD) and electrospray ionization (ESI) MS/MS de novo sequencing of peptides fractionated from PO extracts. We characterized a truncated form of previously identified TNRNFLRFamide, NRNFLRFamide. In addition, we sequenced five other novel peptides sharing a common C-terminus of RYamide from the PO tissue extracts. High K+ depolarization of isolated POs released many peptides present in this tissue, including several of the novel peptides sequenced in the current study.     

Beta-helix model for the filamentous haemagglutinin adhesin of Bordetella pertussis and related bacterial secretory proteins

Bordetella pertussis establishes infection by attaching to epithelial cells of the respiratory tract. One of its adhesins is filamentous haemagglutinin (FHA), a 500-A-long secreted protein that is rich in beta-structure and contains two regions, R1 and R2, of tandem 19-residue repeats. Two models have been proposed in which the central shaft is (i) a hairpin made up of a pairing of two long antiparallel beta-sheets; or (ii) a beta-helix in which the polypeptide chain is coiled to form three long parallel beta-sheets. We have analysed a truncated variant of FHA by electron microscopy (negative staining, shadowing and scanning transmission electron microscopy of unstained specimens): these observations support the latter model. Further support comes from detailed sequence analysis and molecular modelling studies. We applied a profile search method to the sequences adjacent to and between R1 and R2 and found additional "covert" copies of the same motifs that may be recognized in overt form in the R1 and R2 sequence repeats. Their total number is sufficient to support the tenet of the beta-helix model that the shaft domain--a 350 A rod--should consist of a continuous run of these motifs, apart from loop inserts. The N-terminus, which does not contain such repeats, was found to be weakly homologous to cyclodextrin transferase, a protein of known immunoglobulin-like structure. Drawing on crystal structures of known beta-helical proteins, we developed structural models of the coil motifs putatively formed by the R1 and R2 repeats. Finally, we applied the same profile search method to the sequence database and found several other proteins--all large secreted proteins of bacterial provenance--that have similar repeats and probably also similar structures.     

Origins of domestication and polyploidy in oca (Oxalis Tuberosa: Oxalidaceae). 2. Chloroplast-expressed glutamine synthetase data

In continuing study of the origins of the octoploid tuber crop oca, Oxalis tuberosa Molina, we used phylogenetic analysis of DNA sequences of the chloroplast-active (nuclear encoded) isozyme of glutamine synthetase (ncpGS) from cultivated oca, its allies in the "Oxalis tuberosa alliance," and other Andean Oxalis. Multiple ncpGS sequences found within individuals of both the cultigen and a yet unnamed wild tuber-bearing taxon of Bolivia were separated by molecular cloning, but some cloned sequences appeared to be artifacts of polymerase chain reaction (PCR) recombination and/or Taq error. Nonetheless, three classes of nonrecombinant sequences each joined a different part of the O. tuberosa alliance clade on the ncpGS gene tree. Octoploid oca shares two sequence classes with the Bolivian tuber-bearing taxon (of unknown ploidy level). Fixed heterozygosity of these two sequence classes in all ocas sampled suggests that they represent homeologous loci and that oca is allopolyploid. A third sequence class, found in eight of nine oca plants sampled, might represent a third homeologous locus, suggesting that oca may be autoallopolyploid, and is shared with another wild tuber-bearing species, tetraploid O. picchensis of southern Peru. Thus, ncpGS data identify these two taxa as the best candidates as progenitors of cultivated oca.