Proteomics Study of COI1-regulated Proteins in Arabidopsis Flower

Jasmonates(JAs) are a new class of plant hormone that regulate expression of diverse genes to mediate various plant responses.The Arabidopsis F-box protein COI1 is required for plant defense and male fertility in JA signal pathway.To further investigate the regulatory role of COI1 in male fertility,we compared the proteomics profiles of Arabidopsis wild type(WT) flowers with coi1-1 mutan male-sterile flowers using two-dimensional difference gel electrophoresis coupled with matrix-assisted laser desoption/ionization-time-of-flight mass spectrometry.Sixteen proteins with potential function in specific biological processes such as metabolism processes and defense/stress responses were differentially expressed in WT and coi1-1 mutant flowers.Verification on a phi class glutathione transferase AtGSTF9,one out of these 16 identified proteins,revealed that the expression of AtGSTF9 was severely downregulated in flowers of coi1-1 mutant compared with that of WT.Further function analyses of these genes would provide new insights into the molecular basis of COI1-regulated male fertility.     

ω-Helices in Proteins

A modification of the α-helix, termed the ω-helix, has four residues in one turn of a helix. We searched the ω-helix in proteins by the HELFIT program which determines the helical parameters—pitch, residues per turn, radius, and handedness—and p = rmsd/(N ? 1)^1/2 estimating helical regularity, where “rmsd” is the root mean square deviation from the best fit helix and “N” is helix length. A total of 1,496 regular α-helices 6–9 residues long with p ≤ 0.10 Å were identified from 866 protein chains. The statistical analysis provides a strong evidence that the frequency distribution of helices versus n indicates the bimodality of typical α-helix and ω-helix. Sixty-two right handed ω-helices identified (7.2% of proteins) show non-planarity of the peptide groups. There is amino acid preference of Asp and Cys. These observations and analyses insist that the ω-helices occur really in proteins.     

Immobilized Metal Affinity Chromatography Co-Purifies TGF-β1 with Histidine-Tagged Recombinant Extracellular Proteins

Extracellular recombinant proteins are commonly produced using HEK293 cells as histidine-tagged proteins facilitating purification by immobilized metal affinity chromatography (IMAC). Based on gel analyses, this one-step purification typically produces proteins of high purity. Here, we analyzed the presence of TGF-β1 in such IMAC purifications using recombinant extracellular fibrillin-1 fragments as examples. Analysis of various purified recombinant fibrillin-1 fragments by ELISA consistently revealed the presence of picomolar concentrations of active and latent TGF-β1, but not of BMP-2. These quantities of TGF-β1 were not detectable by Western blotting and mass spectrometry. However, the amounts of TGF-β1 were sufficient to consistently trigger Smad2 phosphorylation in fibroblasts. The purification mechanism was analyzed to determine whether the presence of TGF-β1 in these protein preparations represents a specific or non-specific co-purification of TGF-β1 with fibrillin-1 fragments. Control purifications using conditioned medium from non-transfected 293 cells yielded similar amounts of TGF-β1 after IMAC. IMAC of purified TGF-β1 and the latency associated peptide showed that these proteins bound to the immobilized nickel ions. These data clearly demonstrate that TGF-β1 was co-purified by specific interactions with nickel, and not by specific interactions with fibrillin-1 fragments. Among various chromatographic methods tested for their ability to eliminate TGF-β1 from fibrillin-1 preparations, gel filtration under high salt conditions was highly effective. As various recombinant extracellular proteins purified in this fashion are frequently used for experiments that can be influenced by the presence of TGF-β1, these findings have far-reaching implications for the required chromatographic schemes and quality controls.     

Targeting Proteins for Degradation by Arabidopsis COP1： Teamwork Is What Matters

Arsbidopsis COP1 （Constitutive Photomorphogenic 1） defines a key repressor of photomorphogenesis in darkness by acting as an E3 ubiquitin Iigase in the nucleus, and is responsible for the targeted degradation of a number of photomorphogenesis-promoting factors, including phyA, HY5, LAF1, and HFR1. Light activation of multiple classes of photoreceptors （including both phytochromes and cryptochromes） inactivates COP1 and reduces its nuclear abundance, allowing the accumulation of these positively acting light signaling intermediates to promote photomorphogenic development. Recent studies suggest that Arabidopsis COP1 teams up with a family of SPA proteins （SPA1-SPA4） to form the physiologically active COP1-SPA E3 ubiquitin ligase complexes. These COP1-SPA complexes play overlapping and distinct functions in regulating seedling photomorphogenesis under different light conditions and adult plant growth. Further, the COP1-SPA complexes act In concert at a biochemical level with the CDD （COP10, DET1, and DDB1） complex and COP9 signalosome （CSN） to orchestrate the repression of photomorphogenesis.     

Cap-Gly Proteins at Microtubule Plus Ends: Is EB1 Detyrosination Involved?

Localization of CAP-Gly proteins such as CLIP170 at microtubule+ends results from their dual interaction with α-tubulin and EB1 through their C-terminal amino acids −EEY. Detyrosination (cleavage of the terminal tyrosine) of α-tubulin by tubulin-carboxypeptidase abolishes CLIP170 binding. Can detyrosination affect EB1 and thus regulate the presence of CLIP170 at microtubule+ends as well? We developed specific antibodies to discriminate tyrosinated vs detyrosinated forms of EB1 and detected only tyrosinated EB1 in fibroblasts, astrocytes, and total brain tissue. Over-expressed EB1 was not detyrosinated in cells and chimeric EB1 with the eight C-terminal amino acids of α-tubulin was only barely detyrosinated. Our results indicate that detyrosination regulates CLIPs interaction with α-tubulin, but not with EB1. They highlight the specificity of carboxypeptidase toward tubulin.     

W-motif Exchange Between β-Propeller Proteins

The similarity between the structural scaffold of PQQGDH and that of sialidase in the absence of any similarity in the primary structure, catalytic function and substrate recognition encouraged us to attempt a W-motif exchange between these enzymes. By substituting one W-motif in PQQGDH with one from sialidase, a chimeric PQQGDH was constructed, and its enzymatic properties were investigated. The overexpression of the chimeric enzyme resulted in the formation of an inclusion body. However, the refolding procedure resulted in a soluble chimeric enzyme with PQQGDH activity showing similar secondary-structure components as native PQQGDH. In contrast to native PQQGDH, the chimeric PQQGDH showed thermal instability and sensitivity to EDTA, ; this difference might have been due to the incomplete compatibility of the inserted W-motif. The potential of W-motif replacement was also discussed in view of the possible molecular evolution/engineering of β-propeller structures.     

Direct Association of Unfolded Proteins with Mammalian ER Stress Sensor,IRE1β

IRE1, an ER-localized transmembrane protein, plays a central role in the unfolded protein response (UPR). IRE1 senses the accumulation of unfolded proteins in its luminal domain and transmits a signal to the cytosolic side through its kinase and RNase domains. Although the downstream pathways mediated by two mammalian IRE1s, IRE1α and IRE1β, are well documented, their luminal events have not been fully elucidated. In particular, there have been no reports on how IRE1β senses the unfolded proteins. In this study, we performed a comparative analysis to clarify the luminal event mediated by the mammalian IRE1s. Confocal fluorescent microscopy using GFP-fused IRE1s revealed that IRE1β clustered into discrete foci upon ER stress. Also, fluorescence correlation spectroscopy (FCS) analysis in living cells indicated that the size of the IRE1β complex is robustly increased upon ER stress. Moreover, unlike IRE1α, the luminal domain of IRE1β showed anti-aggregation activity in vitro, and IRE1β was coprecipitated with the model unfolded proteins in cells. Strikingly, association with BiP was drastically reduced in IRE1β, while IRE1α was associated with BiP and dissociated upon ER stress. This is the first report indicating that, differently from IRE1α, the luminal event mediated by IRE1β involves direct interaction with unfolded proteins rather than association/dissociation with BiP, implying an intrinsic diversity in the sensing mechanism of mammalian sensors.     

α1B-Adrenergic Receptors Differentially Associate with Rab Proteins during Homologous and Heterologous Desensitization

Internalization of G protein-coupled receptors can be triggered by agonists or by other stimuli. The process begins within seconds of cell activation and contributes to receptor desensitization. The Rab GTPase family controls endocytosis, vesicular trafficking, and endosomal fusion. Among their remarkable properties is the differential distribution of its members on the surface of various organelles. In the endocytic pathway, Rab 5 controls traffic from the plasma membrane to early endosomes, whereas Rab 4 and Rab 11 regulate rapid and slow recycling from early endosomes to the plasma membrane, respectively. Moreover, Rab 7 and Rab 9 regulate the traffic from late endosomes to lysosomes and recycling to the trans-Golgi. We explore the possibility that α_1B-adrenergic receptor internalization induced by agonists (homologous) and by unrelated stimuli (heterologous) could involve different Rab proteins. This possibility was explored by Fluorescence Resonance Energy Transfer (FRET) using cells coexpressing α_1B-adrenergic receptors tagged with the red fluorescent protein, DsRed, and different Rab proteins tagged with the green fluorescent protein. It was observed that when α_1B-adrenergic receptors were stimulated with noradrenaline, the receptors interacted with proteins present in early endosomes, such as the early endosomes antigen 1, Rab 5, Rab 4, and Rab 11 but not with late endosome markers, such as Rab 9 and Rab 7. In contrast, sphingosine 1-phosphate stimulation induced rapid and transient α_1B-adrenergic receptor interaction of relatively small magnitude with Rab 5 and a more pronounced and sustained one with Rab 9; interaction was also observed with Rab 7. Moreover, the GTPase activity of the Rab proteins appears to be required because no FRET was observed when dominant-negative Rab mutants were employed. These data indicate that α_1B-adrenergic receptors are directed to different endocytic vesicles depending on the desensitization type (homologous vs. heterologous).     

Snake Venom C-Type Lectin Related Proteins Up-to-date

The research projects were supported by aresearch scholarship from the National Universityof Singapore,Singapore and by a fund from TheNational Bureau of Foreign Talents,China[2 0 0 1 -1 51 ] .1　 Introduction　　 L ectins are generally considered to be non-enzymatic proteins which selectively bind to spe-cific carbohydrate structure.Animal C- typelectins are a group of proteins which have carbo-hydrate- recognition domains (CRDs) in theirstructures and require calcium ion for their lig-…     

Drosophila Mechanotransduction—Linking Proteins and Functions

The sensation of touch, gravity, and sound all rely on dedicated ion channels that transduce mechanical stimulus forces into electrical response signals. The functional workings and molecular identities of these mechanotransducer channels are little understood. Recent work shows that the mechanotransducers for fly and vertebrate hearing share equivalent gating mechanisms, whereby this mechanism can be probed non-invasively in the mechanics of the Drosophila ear. Here, we describe how this mechanics can be used to evaluate the roles of identified proteins in the process of mechanosensation and, specifically, their contributions to mechanotransduction.     

Nuclear Functions for Plasma Membrane-Associated Proteins?

There are a growing number of observations that proteins, which were initially thought to perform a specific function in a given subcellular compartment, may also play additional roles in different locations within the cell. Proteins found in adhesion and endocytic structures of the plasma membrane and which also traffic to the nucleus perhaps represent the more spectacular examples of this phenomenon. The mechanisms involved in the transport of these molecules through the nuclear pores and their potential nuclear functions are discussed.     

Proteins binding to the 5''''-flanking regulatory elements of the human β-globin gene~1

The binding of nuclear proteins prepared from mouse erythroid tissue in different developmental stages to the 5'-flanking regulatory elements of human globin gene, two negative control regions(NCR1, -610 to -490 bp; NCR2,-338 to -233bp), was identified. Two stage specific protein factors corresponding to embryonic and fetal stages were found to be capable of binding to NCR2. These data provided evidence that the cis acting elements of the 5'-flanking region might be involved in the developmental control of globin gene and NCR2 might be responsible in part for the silence of globin gene in the embryonic and fetal stages.