FHA domain boundaries of the Dun1p and Rad53p cell cycle checkpoint kinases

Dun1p and Rad53p of the budding yeast Saccharomyces cerevisiae are members of a conserved family of cell cycle checkpoint protein kinases that contain forkhead-associated (FHA) domains. Here, we demonstrate that these FHA domains contain 130-140 residues, and are thus considerably larger than previously predicted by sequence comparisons (55-75 residues), In vivo, expression of the proteolytically defined Dun1p FHA domain, but not a fragment containing only the predicted domain boundaries, inhibited the transcriptional induction of repair genes following replication blocks, This indicates that the non-catalytic FI-IA domain plays an important role in the transcriptional function of the Dun1p protein kinase. (C) 2000 Federation of European Biochemical Societies.

In vivo protein cyclization promoted by a circularly permuted Synechocystis sp PCC6803 DnaB mini-intein

A synthetic Synechocystis sp. PCC6803 DnaB split mini-intein gene was constructed for the in vivo cyclization of recombinant proteins expressed in Escherichia coli. The system was used to cyclize the NH2-terminal domain of E. coli DnaB, the structure of which had been determined previously by NMR spectroscopy. Cyclization was found to proceed efficiently, with little accumulation of precursor, and the product was purified in high yield. The solution structure of cyclic DnaB-N is not significantly different from that of linear DnaB-N and it unfolds reversibly at temperatures similar to14 degreesC higher. Improved hydrogen bonding was observed in the first and last helices, and the length of the last helix was increased, while the 9-amino acid linker used to join the NH2 and COOH termini was found to be highly mobile. The measured thermodynamic stabilization of the structure (DeltaDeltaG approximate to 2 kcal/mol) agrees well with the value estimated from the reduced conformational entropy in the unfolded form. Simple polymer theory can be used to predict likely free energy changes resulting from protein cyclization and how the stabilization depends on the size of the protein and the length of the linker used to connect the termini.

Myb-binding protein 1a augments AhR-dependent gene expression

We have studied the mechanism by which an acidic domain (amino acids 515-583) of the aromatic hydrocarbon receptor (AhR) transactivates a target gene. Studies with glutathione S-transferase fusion proteins demonstrate that the wild-type acidic domain associates in vitro with Myb-binding protein la, whereas a mutant domain (F542A, 1569A) does not. AhR-defective cells reconstituted with an AhR containing the wild-type acidic domain exhibit normal AhR function; however, cells reconstituted with an AhR containing the mutant acidic domain do not function normally. Transient transfection of Myb-binding protein la into mouse hepatoma cells is associated with augmentation of AhR-dependent gene expression. Such augmentation does not occur when Myb-binding protein la is transfected into AhR-defective cells that have been reconstituted with an AhR that lacks the acidic domain. We infer that 1) Myb-binding protein la associates with AhR, thereby enhancing transactivation, and 2) the presence of AhR's acidic domain is both necessary and sufficient for Myb-binding protein la to increase AhR-dependent gene expression.

Novel gene containing multiple epidermal growth factor-like motifs transiently expressed in the papillae of the ascidian tadpole larvae

We have investigated molecular mechanisms of the embryonic development of an ascidian, a primitive chordate which shares features of both invertebrates and vertebrates, with a view to identifying genes involved in development and metamorphosis, We isolated 12 partial cDNA sequences which were expressed in a stage-specific manner using differential display, We report here the isolation of a full-length cDNA sequence for one of these genes which was specifically expressed during the tailbud and larval stages of ascidian development, This cDNA, 1213 bp in length, is predicted to encode a protein of 337 amino acids containing four epidermal growth factor (EGF)-like repeats and three novel cysteine-rich repeats, Characterization of its spatial expression pattern by in situ hybridisation in late tailbud and larval embryos demonstrated strong expression localised throughout the papillae and anteriormost trunk and weaker expression in the epidermis of the remainder of the embryo, As recent evidence indicates that the signal for metamorphosis originates in the anterior trunk region, these results suggest that this gene may have a role in signalling the initiation of metamorphosis. (C) 1997 Wiley-Liss, Inc.

Inactivation of a c-Myb/estrogen receptor fusion protein in transformed primary cells leads to granulocyte/macrophage differentiation and down regulation of c-kit but not c-myc or cdc2

Primary murine fetal hemopoietic cells were transformed with a fusion protein consisting of the ligand-binding domain of the estrogen receptor and a carboxyl-terminally truncated c-Myb protein (ERMYB), The ERMYB-transformed hemopoietic cells exhibit an immature myeloid phenotype when grown in the presence of beta-estradiol. Upon removal of beta-estradiol, the ERMYB cells display increased adherence, decreased clonogenicity and differentiate to cells exhibiting granulocyte or macrophage morphology, The expression of the c-myc, c-kit, cdc2 and bcl-2 genes, which are putatively regulated by Myb, was investigated in ERMYB cells grown in the presence or absence of beta-estradiol. Neither c-myc nor cdc2 expression was down-regulated after removal of beta-estradiol demonstrating that differentiation is not a consequence of decreased transactivation of these genes by ERMYB. While bcl-2 expression was reduced by 50% in ERMYB cells grown in the absence of beta-estradiol, there was no increase in DNA laddering, suggesting that Myb was not protecting ERMYB cells from apoptosis, In contrast, a substantial (200-fold) decrease in c-kit mRNA level was observed following differentiation of ERMYB cells, and c-kit mRNA could be partially re-induced by the re-addition of beta-estradiol. Furthermore, a reporter construct containing the c-kit promoter was activated when cotransfected with a Myb expression vector, providing further evidence of a role for Myb in the regulation of c-kit.

Protein phosphatases 1 and 2A promote Raf-1 activation by regulating 14-3-3 interactions

Raf-1 activation is a complex process which involves plasma membrane recruitment, phosphorylation, protein-protein and lipid-protein interactions, We now show that PP1 and PP2A serine-threonine phosphatases also have a positive role in Ras dependent Raf-1 activation, General serine-threonine phosphatase inhibitors such sodium fluoride, or beta-glycerophosphate and sodium pyrophosphate, or specific PP1 and PP2A inhibitors including microcystin-LR, protein phosphatase 2A inhibitor I-1 or protein phosphatase inhibitor 2 all abrogate H-Ras and K-Ras dependent Raf-1 activation in vitro. A critical Raf-1 target residue for PP1 and PP2A is S259. Serine phosphatase inhibitors block the dephosphorylation of S259, which accompanies Raf-1 activation, and Ras dependent activation of mutant Raf259A is relatively resistant to serine phosphatase inhibitors. Sucrose gradient analysis demonstrates that serine phosphatase inhibition increases the total amount of 14-3-3 and Raf-1 associated with the plasma membrane and significantly alters the distribution of 14-3-3 and Raf-1 across different plasma membrane microdomains, These observations suggest that dephosphorylation of S259 is a critical early step in Ras dependent Raf-1 activation which facilitates 14-3-3 displacement. Inhibition of PP1 and PP2A therefore causes plasma membrane accumulation of Raf-1/14-3-3 complexes which cannot be activated.

The leucine-rich repeat as a protein recognition motif

Leucine-rich repeats (LRRs) are 20-29-residue sequence motifs present in a number of proteins with diverse functions. The primary function of these motifs appears to be to provide a versatile structural framework for the formation of protein-protein interactions. The past two years have seen an explosion of new structural information on proteins with LRRs. The new structures represent different LRR subfamilies and proteins with diverse functions, including GTPase-activating protein rna 1 p from the ribonuclease-inhibitor-like subfamily; spliceosomal protein U2A', Rab geranylgeranyltransferase, internalin B, dynein light chain 1 and nuclear export protein TAP from the SDS22-like subfamily; Skp2 from the cysteine-containing subfamily; and YopM from the bacterial subfamily. The new structural information has increased our understanding of the structural determinants of LRR proteins and our ability to model such proteins with unknown structures, and has shed new light on how these proteins participate in protein-protein interactions.

The solution structure of C1-T1, a two-domain proteinase inhibitor derived from a circular precursor protein from Nicotiana alata

A two-domain portion of the proteinase inhibitor precursor from Nicotiana alata (NaProPI) has been expressed and its structure determined by NMR spectroscopy. NaProPI contains six almost identical 53 amino acid repeats that fold into six highly similar domains; however, the sequence repeats do nut coincide with the structural domains. Five of the structural domains comprise the C-terminal portion of one repeat and the N-terminal portion of the next. The sixth domain contains the C-terminal portion of the sixth repeat and the N-terminal portion of the first repeat. Disulphide bonds link these C and N-terminal fragments to generate the clasped-bracelet fold of NaProPI. The three-dimensional structure of NaProPI is not known, but it is conceivable that adjacent domains in NaProPI interact to generate the circular bracelet with the N and C termini in close enough proximity to facilitate formation of the disulphide bonds that form the clasp The expressed protein, examined in the current study, comprises residues 25-135 of NaProPI and encompasses the first two contiguous structural domains, namely the chymotrypsin inhibitor C1 and the trypsin inhibitor T1, joined by a five-residue linker, and is referred to as C1-T1. The tertiary structure of each domain in C1-T1 is identical to that found in the isolated inhibitors. However, no nuclear Overhauser effect contacts are observed between the two domains and the five-residue linker adopts an extended conformation. The absence of interactions between the domains indicates that adjacent domains do not specifically interact to drive the circularisation of NaProPI. These results are in agreement with recent data which describe similar PI precursors from other members of the Solanaceae having two, three, or four repeats. The lack of strong interdomain association is likely to be important for the function of individual inhibitors by ensuring that there is no masking of reactive sites upon release from the precursor. (C) 2001 Academic Press.

The GRIP domain is a specific targeting sequence for a population of trans-Golgi network derived tubulo-vesicular carriers

Vesicular carriers for intracellular transport associate with unique sets of accessory molecules that dictate budding and docking on specific membrane domains. Although many of these accessory molecules are peripheral membrane proteins, in most cases the targeting sequences responsible for their membrane recruitment have yet to be identified. We have previously defined a novel Golgi targeting domain (GRIP) shared by a family of coiled-coil peripheral membrane Golgi proteins implicated in membrane trafficking. We show here that the docking site for the GRIP motif of p230 is a specific domain of Golgi. membranes. By immunoelectron microscopy of HeLa cells stably expressing a green fluorescent protein (GFP)-p230(GRIP) fusion protein, we show binding specifically to a subset of membranes of the trans-Golgi network (TGN). Real-time imaging of live HeLa cells revealed that the GFP-p230(GRIP) was associated with highly dynamic tubular extensions of the TGN, which have the appearance and behaviour of transport carriers. To further define the nature of the GRIP membrane binding site, in vitro budding assays were performed using purified rat liver Golgi membranes and cytosol from GFP-p230(GRIP) transfected cells. Analysis of Golgi-derived vesicles by sucrose gradient fractionation demonstrated that GFP-p230(GRIP) binds to a specific population of vesicles distinct from those labelled for beta -COP or gamma -adaptin. The GFP-p230(GRIP) fusion protein is recruited to the same vesicle population as full-length p230, demonstrating that the GRIP domain is solely proficient as a targeting signal for membrane binding of the native molecule. Therefore, p230 GRIP is a targeting signal for recruitment to a highly selective membrane attachment site on a specific population of trans-Golgi network tubulovesicular carriers.

A lipid core peptide construct containing a conserved region determinant of the group a streptococcal M protein elicits heterologous opsonic antibodies

The study reported here investigated the immunogenicity and protective potential of a lipid core peptide (LCP) construct containing a conserved region determinant of M protein, defined as peptide J8. Parenteral immunization of mice with LCP-J8 led to the induction of high-titer serum immunoglobulin G J18-specific antibodies when the construct was coadministered with complete Freund's adjuvant (CFA) or administered alone. LCP-J8 in CFA had significantly enhanced immunogenicity compared with the monomeric peptide J8 given in CFA. Moreover, LCP-J8/CFA and LCP-J8 antisera opsonized four different group A streptococcal (GAS) strains, and the antisera did not cross-react with human heart tissue proteins. These data indicate the potential of an LCP-based M protein conserved region GAS vaccine in the induction of broadly protective immune responses in the absence of a conventional adjuvant.

The effects of level of dietary protein on the milk production and rumen physiology of dairy cows fed a diet based on a tropical grass hay

zFour rumen-fistulated, multiparous Holstein-Friesian cows in early lactation were offered mixed diets based on rhodes grass hay (Chloris gayana) cv. Callide containing 13, 14, 15 or 16% crude protein (CP) on a dry matter basis, in a 4 x 4 latin square design. The estimated undegradable protein concentration in these diets was similar with rumen degradable protein concentration varying. Cows fed a diet containing 13% CP had lower (P = 0.07) milk yields than cows in other treatments (20.4 vs 21.9, 22.0 and 22.2 L/d for 13, 14, 15 and 16% CP, respectively). A positive linear relationship was found (P = 0.06) between organic matter intake and dietary CP%. There were negative linear relationships between dietary CP% and digestibilities of dry matter (P = 0.09), organic matter (P = 0.06) and neutral detergent fibre (P = 0.02). Feeding a diet containing 13% CP resulted in significantly lower (P = 0.001) molar proportions (%) of rumen valerate in comparison with other treatments. The molar proportions of isovalerate differed (P = 0.001) between treatments (0.66, 0.78, 0.89 and 1.04%) for 13, 14, 15 and 16% CP, respectively). Dietary protein level had no effect on rates of passage, in situ digestion of rhodes grass hay or ratios of allantoin: creatinine in urine. These data showed that increasing the dietary CP concentration of lactating cows fed diets based on rhodes grass hay increased intakes and not significantly improved at dietary CP concentrations above 14% DM.

Synthesis and structural analysis of the N-terminal domain of the thyroid hormone-binding protein transthyretin

Transthyretin (TTR) is a 55 kDa protein responsible for the transport of thyroid hormones and retinol in human serum. Misfolded forms of the protein are implicated in the amyloid diseases familial amyloidotic polyneuropathy and senile systemic amyloidosis. Its folding properties and stabilization by ligands are of current interest due to their importance in understanding and combating these diseases. To assist in such studies we developed a method for the solid phase synthesis of the monomeric unit of a TTR analogue and its folding to form a functional 55 kDa tetramer. The monomeric unit of the protein was chemically synthesized in three parts, comprising amino acid residues 151, 5499 and 102127, and ligated using chemoselective thioether ligation chemistry. The synthetic protein was folded and assembled to a tetrameric structure in the presence of the TTRs native ligand, thyroxine, as shown by gel filtration chromatography, native gel electrophoresis, TTR antibody recognition and thyroid hormone binding. In the current study the solution structure of the first of these fragment peptides, TTR(151) is examined to determine its intrinsic propensity to form beta-sheet structure, potentially involved in amyloid fibril formation by TTR. Despite the presence of extensive beta-structure in the native form of the protein, the Nterminal fragment adopts an essentially random coil conformation in solution.

The AF-1 Domain of the Orphan Nuclear Receptor NOR-1 Mediates Trans-activation, Coactivator Recruitment, and Activation by the Purine Anti-metabolite 6-Mercaptopurine

NOR-1/NR4A3 is an orphan member of the nuclear hormone receptor superfamily. NOR-1 and its close relatives Nurr1 and Nur77 are members of the NR4A subgroup of nuclear receptors. Members of the NR4A subgroup are induced through multiple signal transduction pathways. They have been implicated in cell proliferation, differentiation, T-cell apoptosis, chondrosarcomas, neurological disorders, inflammation, and atherogenesis. However, the mechanism of transcriptional activation, coactivator recruitment, and agonist-mediated activation remain obscure. Hence, we examined the molecular basis of NOR-1-mediated activation. We observed that NOR-1 trans-activates gene expression in a cell- and target-specific manner; moreover, it operates in an activation function (AF)-1-dependent manner. The N-terminal AF-1 domain delimited to between amino acids 1 and 112, preferentially recruits the steroid receptor coactivator (SRC). Furthermore, SRC-2 modulates the activity of the AF-1 domain but not the C-terminal ligand binding domain (LBD). Homology modeling indicated that the NOR-1 LBD was substantially different from that of hRORbeta, a closely related AF-2-dependent receptor. In particular, the hydrophobic cleft characteristic of nuclear receptors was replaced with a very hydrophilic surface with a distinct topology. This observation may account for the inability of this nuclear receptor LBD to efficiently mediate cofactor recruitment and transcriptional activation. In contrast, the N-terminal AF-1 is necessary for cofactor recruitment and can independently conscript coactivators. Finally, we demonstrate that the purine anti-metabolite 6-mercaptopurine, a widely used antineoplastic and anti-inflammatory drug, activates NOR-1 in an AF-1-dependent manner. Additional 6-mercaptopurine analogs all efficiently activated NOR-1, suggesting that the signaling pathways that modulate proliferation via inhibition of de novo purine and/or nucleic acid biosynthesis are involved in the regulation NR4A activity. We hypothesize that the NR4A subgroup mediates the genotoxic stress response and suggest that this subgroup may function as sensors that respond to genotoxicity.

Functional recycling of C2 domains throughout evolution: A comparative study of synaptotagmin, protein kinase C and phospholipase C by sequence, structural and modelling approaches

The C2 domain is one of the most frequent and widely distributed calcium-binding motifs. Its structure comprises an eight-stranded beta-sandwich with two structural types as if the result of a circular permutation. Combining sequence, structural and modelling information, we have explored, at different levels of granularity, the functional characteristics of several families of C2 domains. At the coarsest level,the similarity correlates with key structural determinants of the C2 domain fold and, at the finest level, with the domain architecture of the proteins containing them, highlighting the functional diversity between the various subfamilies. The functional diversity appears as different conserved surface patches throughout this common fold. In some cases, these patches are related to substrate-binding sites whereas in others they correspond to interfaces of presumably permanent interaction between other domains within the same polypeptide chain. For those related to substrate-binding sites, the predictions overlap with biochemical data in addition to providing some novel observations. For those acting as protein-protein interfaces' our modelling analysis suggests that slight variations between families are a result of not only complementary adaptations in the interfaces involved but also different domain architecture. In the light of the sequence and structural genomic projects, the work presented here shows that modelling approaches along with careful sub-typing of protein families will be a powerful combination for a broader coverage in proteomics. (C) 2003 Elsevier Ltd. All rights reserved.