CSN1 N-terminal-dependent activity is required for Arabidopsis development but not for Rub1/Nedd8 deconjugation of cullins: a structure-function study of CSN1 subunit of COP9 signalosome

The COP9 signalosome (CSN) is a multifunctional protein complex essential for arabidopsis development. One of its functions is to promote Rub1/Nedd8 deconjugation from the cullin subunit of the Skp1-cullin-F-box ubiquitin ligase. Little is known about the specific role of its eight subunits in deneddylation or any of the physiological functions of CSN. In the absence of CSN1 (the fus6 mutant), arabidopsis CSN complex cannot assemble, which destabilizes multiple CSN subunits and contributes, together with the loss of CSN1, to the phenotype of fus6. To distinguish CSN1-specific functions, we attempted to rescue the complex formation with deletion or point-mutation forms of CSN1 expressed as transgenes in fus6. We show that the central domain of CSN1 is critical for complex assembly, whereas the C-terminal domain has a supporting role. By expressing the C231 fragment, which contains the structural information but lacks the presumed functional domain located at the N terminus, we have rescued the complex formation and restored the Rub1/Nedd8 deconjugation activity on cullins (fus6/C231). Nonetheless, fus6/C231 exhibits pleiotropic phenotype, including photomorphogenic defects and growth arrest at seedling stage. We conclude that CSN1 N-terminal domain is not required for the Rub1/Nedd8 deconjugation activity of cullins, but contributes to a significant aspect of CSN functions that are essential for plant development.     

Influence of VIP on prolactinemia in turkey anterior pituitary cells: role of cAMP second messenger in VIP-induced prolactin gene expression

Vasoactive intestinal peptide (VIP) is the avian prolactin (PRL)-releasing factor. In the turkey, hypothalamic VIP immunoreactivity and mRNA content, as well as VIP levels in hypophyseal portal blood, are closely related to the state of prolactinemia and the reproductive stage. The present study investigated the role of VIP on prolactinemia in turkey anterior pituitary (AP) cells through PRL gene expression and the role of a cAMP second messenger system on VIP-induced PRL expression. In primary AP cells harvested from hens in different prolactinemic states, steady state promoter activities were positively correlated with secreted PRL levels. VIP increased PRL promoter activities in AP cells from hens with intermediate PRL levels (laying), but not in AP cells from hypoprolactinemic hens (nonphotostimulated reproductively quiescent). However, in AP cells from hyperprolactinemic hens (incubating), PRL promoter activity was down-regulated by VIP. PRL mRNA steady state levels were significantly decreased by the cAMP analogue, 8-bromo-cyclic adenosine monophosphate (8-Br-cAMP), and PRL secretion was down-regulated by the phosphodiesterase blocker, 3-isobutyl-1-methylxanthine (IBMX) in a dose-dependent manner, suggesting that the cAMP second messenger system might be involved in the inhibitory action of dopamine upon VIP-stimulated PRL secretion and gene expression at the pituitary level. In a study of VIP immediate and long-term effects on c-fos expression in relation to PRL expression, VIP dramatically induced c-fos mRNA expression within 5 min, suggesting that VIP-induced c-fos expression might be involved in VIP-stimulated PRL secretion and gene expression. These results provide additional evidence of the functional significance of VIP in PRL gene expression and suggest that changes in PRL promoter activity by VIP may be one of the important inductive mechanisms leading to prolactinemia.     

Studies of apoptosis and bcl-2 in experimental atherosclerosis in rabbit and influence of selenium supplementation

Apoptosis is a ubiquitous physiological mechanism of cell death regulating tissue mass and architecture. An attempt was made in the present study to see the occurrence of apoptotic cell death in three different treatment groups of rabbits viz. Control, HFD fed and HFD + Selenium fed. Apoptotic activity as checked by in situ DNA end labelling (TUNEL Assay) revealed excessive staining, mostly concentrated in plaque region both in fibrous as well as atheromatous plaque in HFD fed animals. However, in selenium (Se) supplemented animals, very little TUNEL staining could be seen, and even that confined to endothelial cells only. The control group on the other hand was totally devoid of any staining. Transmission Electron Microscopic (TEM) study also depicted the occurrence of apoptosis in aortic cells of HFD fed animals and very little in Se supplemented animals. Apoptotic activity has been discussed in relation to oxidative stress in HFD fed group. bcl-2, though an antiapoptotic oncoprotein, was found to be expressed more in atherogenic group as compared to control/HFD + Se treatment. On the whole, the study highlighted the occurrence of apoptotic process in atherosclerosis and the role of Se, a potent antioxidant, in inhibition of apoptotic process in HFD fed animals.     

Unlocking the secrets of syndecans: transgenic organisms as a potential key

Heparan sulfate proteoglycans are known to modulate the activity of a large number of extracellular ligands thereby having the potential to regulate a great diversity of biological processes. The long-term studies in our laboratory have focused on the syndecans, one of the major cell surface heparan sulfate proteoglycan families. Most early work on syndecans involved biochemical studies that provided initial information on their structure and putative biological roles. In recent years, the development of transgenic organisms has allowed a more complete understanding of syndecan function. Studies with transgenic syndecan-1 and syndecan-3 mice have demonstrated an unforeseen role for syndecans in the regulation of feeding behavior. Syndecan-1 knockout mice display a reduced susceptibility to both Wnt-induced tumorigenesis and microbial pathogenesis. Experiments with Drosophila show that syndecan is first expressed upon cellularization in the early embryo, and may play a role in the early developmental stages of the fly. This review focuses on these diverse functions of the syndecans that have been elucidated by the use of transgenic mice and Drosophila as model systems. Published in 2003.     

Crystal structures of unligated and CN-ligated Glycera dibranchiata monomer ferric hemoglobin components III and IV

Erythrocytes of the marine annelid, Glycera dibranchiata, contain a mixture of monomeric and polymeric hemoglobins. There are three major monomer hemoglobin components, II, III, IV (also called GMH2, 3, and 4), that have been highly purified and well characterized. We have now crystallized GMH3 and GMH4 and determined their structures to 1.4-1.8 A resolution. The structures were determined for these two monomer hemoglobins in the oxidized (Fe3+, ferric, or met-) forms in both the unligated and cyanide-ligated states. This work differs from two published, refined structures of a Glycera dibranchiata monomer hemoglobin, which has a sequence that is substantially different from any bona fide major monomer hemoglobins (GMH2, 3, or 4). The high-resolution crystal structures (presented here) and the previous NMR structure of CO-ligated GMH4, provide a basis for interpreting structure/function details of the monomer hemoglobins. These details include: (1) the strong correlation between temperature factor and NMR dynamics for respective protein forms; (2) the unique nature of the HisE7Leu primary sequence substitutions in GMH3 and GMH4 and their impact on cyanide ion binding kinetics; (3) the LeuB10Phe difference between GMH3 and GMH4 and its impact on ligand binding; and (4) elucidation of changes in the structural details of the distal and proximal heme pockets upon cyanide binding.     

Synaptosome-associated protein of 25 kilodaltons modulates Kv2.1 voltage-dependent K(+) channels in neuroendocrine islet beta-cells through an interaction with the channel N terminus

Insulin secretion is initiated by ionic events involving membrane depolarization and Ca(2+) entry, whereas exocytic SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins mediate exocytosis itself. In the present study, we characterize the interaction of the SNARE protein SNAP-25 (synaptosome-associated protein of 25 kDa) with the beta-cell voltage-dependent K(+) channel Kv2.1. Expression of Kv2.1, SNAP-25, and syntaxin 1A was detected in human islet lysates by Western blot, and coimmunoprecipitation studies showed that heterologously expressed SNAP-25 and syntaxin 1A associate with Kv2.1. SNAP-25 reduced currents from recombinant Kv2.1 channels by approximately 70% without affecting channel localization. This inhibitory effect could be partially alleviated by codialysis of a Kv2.1N-terminal peptide that can bind in vitro SNAP-25, but not the Kv2.1C-terminal peptide. Similarly, SNAP-25 blocked voltage-dependent outward K(+) currents from rat beta-cells by approximately 40%, an effect that was completely reversed by codialysis of the Kv2.1N fragment. Finally, SNAP-25 had no effect on outward K(+) currents in beta-cells where Kv2.1 channels had been functionally knocked out using a dominant-negative approach, indicating that the interaction is specific to Kv2.1 channels as compared with other beta-cell Kv channels. This study demonstrates that SNAP-25 can regulate Kv2.1 through an interaction at the channel N terminus and supports the hypothesis that SNARE proteins modulate secretion through their involvement in regulation of membrane ion channels in addition to exocytic membrane fusion.     

The Na(+)/H(+) exchanger regulatory factor 2 mediates phosphorylation of serum- and glucocorticoid-induced protein kinase 1 by 3-phosphoinositide-dependent protein kinase 1

The Na(+)/H(+) exchanger regulatory factor 2 (NHERF2/TKA-1/E3KARP) contains two PSD-95/Dlg/ZO-1 (PDZ) domains which interact with the PDZ docking motif (X-(S/T)-X-(V/L)) of proteins to mediate the assembly of transmembrane and cytosolic proteins into functional signal transduction complexes. One of the PDZ domains of NHERF2 interacts specifically with the DSLL, DSFL, and DTRL motifs present at the carboxy-termini of the 2-adrenergic receptor, the platelet-derived growth factor receptor, and the cystic fibrosis transmembrane conductance regulator, respectively. Serum- and glucocorticoid-induced protein kinase 1 (SGK1) also carries a putative PDZ-binding motif (D-S-F-L) at its carboxy tail, implicated in the specific interaction with NHERF2. There is a 3-phosphoinositide-dependent protein kinase 1 (PDK1) interacting fragment (PIF) in the tail of NHERF2. Using pull-down assays and co-transfection experiments, we demonstrated that the DSFL tail of SGK1 interacts with the first PDZ domain of NHERF2 and the PIF of NHERF2 binds to the PIF-binding pocket of PDK1 to form an SGK1-NHERF2-PDK1 complex. Formation of the protein complex promoted the phosphorylation and activation of SGK1 by PDK1. Thus, it was suggested that NHERF2 mediates the activation and phosphorylation of SGK1 by PDK1 through its first PDZ domain and PIF motif, as a novel SGK1 activation mechanism.     

NF-kappaB activates fibronectin gene expression in rat hepatocytes

Resveratrol (RSVL), an edible polyphenolic stilbene, claims a myriad of cardiovascular benefits. However, the molecular underpinnings of such actions are poorly understood. Currently, in sheep coronary arteries (SCA), RSVL markedly (threefold) enhanced cGMP formation (t(1/2): 6.5 min; EC(50): 3 microM). This response was not abrogated by the phosphodiesterase inhibitor (IBMX, 0.5 mM), but was partly sensitive (20-30%) to either removal of the endothelium, treatment with the nitric oxide synthase-inhibitor (L-NMMA, 10 microM), or with the soluble GC (sGC)-inhibitor (ODQ, 10 microM). In membrane preparations from denuded SCA, either RSVL or the pGC agonist atrial natriuretic peptide (ANP, 0.1-1 microM) activated GC in the particulate, but not in the soluble, membrane fraction. By contrast, the nitric oxide donor, sodium nitroprusside (SNP, 1-10 microM), stimulated GC only in the soluble fraction. Further, pretreatment with RSVL partly desensitized the ANP response, but was additive to that of SNP. In arterial tension studies, RSVL relaxed PGF(2alpha)-precontracted denuded rings in a concentration-dependent manner, a response that was markedly enhanced (approximately 18 fold) in the presence of IBMX. Conversely, precontraction with phorbol ester, which also desensitizes pGC, blunted relaxations to RSVL but not to forskolin or SNP. These findings demonstrate that RSVL increases cGMP in coronary arteries, mostly by activation of pGC. This pathway triggers vasorelaxant responses that remain effective in endothelium-disrupted arteries.     

Activity enhancement of Cel5Z from Pectobacterium chrysanthemi PY35 by removing C-terminal region

The phytopathogenic bacterium Pectobacteium chrysanthemi PY35 secretes Cel5Z endoglucanase belonging to the glycoside hydrolase family 5 of EC 3.2.1.4. The mutation of cel5Z::Omega gene was constructed by cloning the 2.0-kb SmaI fragment containing the streptomycin/spectinomycin-resistance gene of pHP45(Omega) into the BalI site of pPY100. The insertion of Omega fragment generated a new stop codon, removing the Ser/Thr-rich linker region and the cellulose binding domain (CBD) in the C-terminal region of cel5Z gene. By subsequent subcloning from this 4.9-kb fragment (pPY1001), a 1.0-kb (pPY1002) fragment was obtained and designated as cel5Z::Omega. The cel5Z::Omega gene had an open reading frame (ORF) of 1011 bp, encoding 336 amino acids, starting with an ATG codon and ending with a new TGA stop codon. The molecular mass of the Cel5Z::Omega protein in E. coli transformant appeared to be 32 kDa by SDS-PAGE analysis in the presence of carboxymethyl-cellulose (CMC). The Cel5Z::Omega protein hydrolyzed CMC with 1.7-fold higher activity than the intact Cel5Z cellulase.