Differential gene expression during desiccation stress in the insect-killing nematode Steinernema feltiae IS-6

The developmentally arrested life stage of the entomopathogenic nematode Steinernema feltiae is exposed to threats of survival, including desiccation. We adopted a comprehensive approach to the study of the molecular mechanisms of desiccation stress tolerance in S. feltiae IS-6. We identified, expressed sequence tags (ESTs) that are differentially expressed during desiccation stress in S. feltiae IS-6 infective juveniles using DNA subtractive hybridization. These ESTs included genes that are known to be stress related, genes that are homologous to hypothetical Caenorhabditis elegans proteins, and novel genes that may be involved in traits specific to S. feltiae. Expression pattern characterization revealed that all analyzed ESTs were induced during 8 and 24 hr of dehydration of S. feltiae IS-6. Our results unveiled some of the components of the genetic networks that are activated in S. feltiae IS-6 during dehydration and suggested a differing pattern of temporal regulation during nematode dehydration.     

Members of the NAP/SET family of proteins interact specifically with B- type cyclins

Cyclin-dependent kinase complexes that contain the same catalytic subunit are able to induce different events at different times during the cell cycle, but the mechanisms by which they do so remain largely unknown. To address this problem, we have used affinity chromatography to identify proteins that bind specifically to mitotic cyclins, with the goal of finding proteins that interact with mitotic cyclins to carry out the events of mitosis. This approach has led to the identification of a 60-kD protein called NAP1 that interacts specifically with members of the cyclin B family. This interaction has been highly conserved during evolution: NAP1 in the Xenopus embryo interacts with cyclins B1 and B2, but not with cyclin A, and the S. cerevisiae homolog of NAP1 interacts with Clb2 but not with Clb3. Genetic experiments in budding yeast indicate that NAP1 plays an important role in the function of Clb2, while biochemical experiments demonstrate that purified NAP1 can be phosphorylated by cyclin B/p34cdc2 kinase complexes, but not by cyclin A/p34cdc2 kinase complexes. These results suggest that NAP1 is a protein involved in the specific functions of cyclin B/p34cdc2 kinase complexes. In addition to NAP1, we found a 43-kD protein in Xenopus that is homologous to NAP1 and also interacts specifically with B-type cyclins. This protein is the Xenopus homolog of the human SET protein, which was previously identified as part of a putative oncogenic fusion protein (Von Lindern et al., 1992).     

Effectiveness of Entomopathogenic Nematodes in an Alginate Gel Formulation Against Lepidopterous Pests

An improved calcium alginate gel formulation was developed and tested as a carrier for entomopathogenic nematodes against Spodoptera littoralis and Helicoverpa armigera larvae. Mortality of 100% was caused in 4th instar larvae of the two insects by feeding them on 1000 infective juveniles (IJ) g -1 of Steinernema carpocapsae (ALL strain) in the gel for 24 h. Exposing 2nd to 5th instars of H. armigera and 3rd to 6th of S. littoralis to 500 IJ g -1 of S. carpocapsae (ALL strain) resulted in 70-100% larval mortality. Mature larvae were less susceptible to the nematodes. Mortality of larvae exposed to 500 IJg -1 of S. carpocapsae (ALL strain) ranged from about 45-55% at 4 h to 90-95% at 48 h. Fourth instar larvae fed for 24 h with 250 IJ g -1 of nematode strains in gel showed in S. littoralis ranges of susceptibility in the following descending order: S. feltiae (IS -7 strain) = S. carpocapsae (DT strain) = S. feltiae (IS-6 strain) > S. carpocapsae (Mexican strain) = S. carpocapsae (ALL strain) = Heterorhabditis bacteriophora (HP-88 strain) = H sp. (IS-5 strain) > S. riobravae (Texas strain); in H. armigera the rating was: S. feltiae (IS-7 strain) = H. bacteriophora (HP88 strain) > S. carpocapsae (ALL strain) = S. feltiae (IS-6 strain ) = Heterorhabditis sp. (IS5 strain) > S. carpocapsae (Mexican strain) > S. riobravae (Texas strain) . The number of nematodes per larval cadaver increased with mortality rates. In greenhouse tests at 28 &#45 2°C and 90% relative humidity, gel discs containing 500 IJ g -1 of nematodes were pinned to leaves of potted plants of cotton ( Gossypium hirsutum ) (Acala SJ2) and the plants were offered to S. littoralis larvae. Larval mortality of 89 &#45 12.7% was caused by S. feltiae (IS-7 strain) and most of the plant leaves were protected against the larvae by the nematodes. In the control, larval mortality was 3.3 &#45 0.05% and the plants were almost completely defoliated. Possibilities of using the gel-nematode formulation to protect sheltered crops against insect pests are discussed     

Casein kinase 2 binds and phosphorylates the nucleosome assembly protein-1 (NAP1) in Drosophila melanogaster.

The nucleosome assembly protein-1 (NAP1) was originally identified in HeLa cells as a factor facilitating the in vitro assembly of nucleosomes. However, in yeast cells NAP1 is required in the control of mitotic events induced by the Clb2/p34(CDC28). Here, we show that Drosophila NAP1 is a phosphoprotein that is associated with a kinase able to phosphorylate NAP1. By using an in-gel kinase assay we found that this kinase displays a molecular mass of 38 kDa. Following purification and peptide microsequencing, we identified the kinase phosphorylating NAP1 as the alpha subunit of casein kinase 2 (CK2). With the help of a series of NAP1 segments and synthetic peptides, we assigned the CK2 phosphorylation sites to residues Ser118, Thr120, and Ser284. Interestingly, Ser118 and Thr120 are located within a PEST domain, while Ser284 is adjacent to the nuclear localization signal. Substitution of the identified phosphoresidues by alanine was found to reduce considerably the ability of CK2 to phosphorylate NAP1. The enhanced ability of CK2 to phosphorylate phosphatase-treated NAP1 extracted from Drosophila embryos and the similar tryptic phospho-peptide pattern of in vivo labelled NAP1 and in vitro labelled NAP1 with CK2 indicate that NAP1 is a natural substrate of CK2. Further analysis revealed that both CK2alpha and beta subunits are associated with NAP1 but we found that only the catalytic alpha subunit establishes direct contact with NAP1 on two distinct domains of this protein. The location of CK2 phosphorylation sites in NAP1 suggests that their phosphorylation can contribute to a PEST-mediated protein degradation of NAP1 and the translocation of NAP1 between cytoplasm and nucleus.     

Nuclear export of S6K1 II is regulated by protein kinase CK2 phosphorylation at Ser-17

Ribosomal S6 kinases (S6Ks) are principal players in the regulation of cell growth and energy metabolism. Signaling via phosphatidylinositol 3-kinase and mammalian target of rapamycin pathways mediates the activation of S6K in response to various mitogenic stimuli. The family of S6Ks consists of two forms, S6K1 and -2, that have cytoplasmic and nuclear splicing variants, S6K1 II and S6K1 I, respectively. Nuclear-cytoplasmic shuttling of both isoforms induced by mitogenic stimuli has been reported recently. Here we present the identification of protein kinase CK2 (CK2) as a novel binding and regulatory partner for S6K1 II. The interaction between S6K1 II and CK2beta regulatory subunit was initially identified in a yeast two-hybrid screen and further confirmed by co-immunoprecipitation of transiently expressed and endogenous proteins. The interaction between S6K1 II and CK2 was found to occur in serum-starved and serum-stimulated cells. In addition, we found that S6K1 II is a substrate for CK2. The localization of the CK2 phosphorylation site was narrowed down to Ser-17 in S6K1 II. Mutational analysis and the use of phosphospecific antibody indicate that Ser-17 is a major in vitro and in vivo phosphorylation site for CK2. Functional studies reveal that, in contrast to the wild type kinase, the phosphorylation-mimicking mutant of S6K1 II (S17E) retains its cytoplasmic localization in serum-stimulated cells. Treatment of cells with the nuclear export inhibitor leptomycin B revealed that the S17E mutant accumulates in the nucleus to the same extent as S6K1 II wild type. These results indicate that nuclear import of the S17E mutant is not affected, although the export is significantly enhanced. We also provide evidence that nuclear export of S6K1 is mediated by a CRM1-dependent mechanism. Taken together, this study establishes a functional link between S6K1 II and CK2 signaling, which involves the regulation of S6K1 II nuclear export by CK2-mediated phosphorylation of Ser-17.     

NAP57, a mammalian nucleolar protein with a putative homolog in yeast and bacteria [published erratum appears in J Cell Biol 1998 Jan 26;140(2):447]

We report the identification and molecular characterization of a novel nucleolar protein of rat liver. As shown by coimmunoprecipitation this protein is associated with a previously identified nucleolar protein, Nopp140, in an apparently stoichiometric complex and has therefore been termed NAP57 (Nopp140-associated protein of 57 kD). Immunofluorescence and immunogold electron microscopy with NAP57 specific antibodies show colocalization with Nopp140 to the dense fibrillar component of the nucleolus, to coiled bodies, and to the nucleoplasm. Immunogold staining in the nucleoplasm is occasionally seen in the form of curvilinear tracks between the nucleolus and the nuclear envelope, similar to those previously reported for Nopp140. These data suggest that Nopp140 and NAP57 are indeed associated with each other in these nuclear structures. The cDNA deduced primary structure of NAP57 shows a protein of a calculated molecular mass of 52,070 that contains a putative nuclear localization signal near its amino and carboxy terminus and a hydrophobic amino acid repeat motif extending across 84 residues. Like Nopp140, NAP57 lacks any of the known consensus sequences for RNA binding which are characteristic for many nucleolar proteins. Data bank searches revealed that NAP57 is a highly conserved protein. A putative yeast (S. cerevisiae) homolog is 71% identical. Most strikingly, there also appears to be a smaller prokaryotic (E. coli and B. subtilis) homolog that is nearly 50% identical to NAP57. This indicates that NAP57 and its putative homologs might serve a highly conserved function in both pro- and eukaryotes such as chaperoning of ribosomal proteins and/or of preribosome assembly.     

Enhanced Trehalose Accumulation and Desiccation Survival of Entomopathogenic Nematodes Through Cold Preacclimation

Limited storage stability is a major obstacle to further expansion of the use of entomopathogenic nematodes for pest control. Progress has been made that Steinernema carpocapsae can now be stored under partial anhydrobiosis for up to 6 months at 25°C and 10 months at 5°C in a water-dispersible granular (WG) formulation. However, other species have been more difficult to store in the WG formulation due to migration of nematodes out of the granules and sensitivity of some species to desiccation directly at cold temperatures. As acclimation to cold induces trehalose accumulation (a major cryo- and desiccation protectant) in many invertebrates, it was hypothesized that cold preacclimation of entomopathogenic nematodes will enhance their survival in the WG formulation at cold temperatures. This hypothesis was tested using a temperate species Steinernema feltiae , a subtropical species S. carpocapsae , and a tropical species Steinernema riobrave possessing different thermal niche breadths and reproduction temperature optima. Cold acclimation of infective juveniles increased trehalose accumulation in all three species and the amount of trehalose accumulated was both temperature and species dependent. Trehalose content reached at its peak after 6 days at 5°C in S. feltiae (82.28 μg/mg dry weight), after 10 days at 10°C in S. carpocapsae (94.16 μg/mg dry weight) and after 6 days at 15°C in S. riobrave (47.58 μg/mg dry weight). Cold preacclimation at 5°C for 2 days enhanced desiccation survival of S. feltiae in 25% glycerol (osmotic desiccation) at both 5 and 25° and of S. carpocapsae and S. riobrave only at 5°C. Non-cold acclimated S. carpocapsae and S. riobrave were extremely sensitive to desiccation directly at 5°C in 25% glycerol, resulting in over 98% mortality within 6 days, but S. feltiae was more sensitive to desiccation at 25°C than at 5°C. Cold preacclimation increased survival of all the three species in the WG formulation at both 5 and 25°C. The survival of S. riobrave at 5°C in the WG formulation was positively correlated with the length of preacclimation period at 5°C (R 2 = 0.99) and with the amount of trehalose accumulated during cold preacclimation (R 2 = 0.81). These results support the hypothesis that cold preacclimation enhances desiccation survival of entomopathogenic nematodes at cold temperatures and the increased survival correlates well with the increased trehalose accumulation. Results also demonstrate that cold preacclimation can be used as a tool to enhance survival of nematodes in the formulations with reduced water activity.     

Protein kinase CK2 activates the atypical Rio1p kinase and promotes its cell-cycle phase-dependent degradation in yeast

Using co-immunoprecipitation combined with MS analysis, we identified the alpha' subunit of casein kinase 2 (CK2) as an interaction partner of the atypical Rio1 protein kinase in yeast. Co-purification of Rio1p with CK2 from Deltacka1 or Deltacka2 mutant extracts shows that Rio1p preferentially interacts with Cka2p in vitro. The C-terminal domain of Rio1p is essential and sufficient for this interaction. Six C-terminally located clustered serines were identified as the only CK2 sites present in Rio1p. Replacement of all six serine residues by aspartate, mimicking constitutive phosphorylation, stimulates Rio1p kinase activity about twofold in vitro compared with wild-type or the corresponding (S > A)(6) mutant proteins. Both mutant alleles (S > A)(6) or (S > D)(6) complement in vivo, however, growth of the RIO1 (S > A)(6) mutant is greatly retarded and shows a cell-cycle phenotype, whereas the behaviour of the RIO1 (S > D)(6) mutant is indistinguishable from wild-type. This suggests that phosphorylation by protein kinase CK2 leads to moderate activation of Rio1p in vivo and promotes cell proliferation. Physiological studies indicate that phosphorylation by CK2 renders the Rio1 protein kinase susceptible to proteolytic degradation at the G(1)/S transition in the cell-division cycle, whereas the non-phosphorylated version is resistant.     

Anhydrobiotic potential and long-term storage of entomopathogenic nematodes (Rhabditida: Steinernematidae)

Anhydrobiosis is considered to be an important means of achieving storage stability of entomopathogenic nematodes that are used in biological control. This study explored the effects of anhydrobiosis on longevity and infectivity of infective juveniles (IJs) of three species of entomopathogenic nematodes Steinernema carpocapsae, Steinernema feltiae, and Steinernema riobrave at 5 and 25 degrees C. Anhydrobiosis was induced in water-dispersible granules (WG) at 0.966-0.971 water activity and 25 degrees C following a 7-day preconditioning of IJs at 5 degrees C in tap water. Survival and infectivity of the desiccated (anhydrobiotic) IJs was compared with non-desiccated IJs stored in water for different periods. Anhydrobiosis increased longevity of S. carpocapsae IJs by 3 months and of S. riobrave by 1 month in WG at 25 degrees C as compared with IJs stored in water. However, desiccation decreased S. feltiae longevity at 25 degrees C and of all three species at 5 degrees C. These results demonstrate a shelf-life of 5 months for S. carpocapsae at 25 degrees C and 9 months at 5 degrees C in WG with over 90% IJ survival. For S. feltiae, over 90% survival occurred only for 2 months at 25 degrees C and 5 months at 5 degrees C in WG. Steinernema riobrave had over 90% survival only for 1 month at 25 degrees C and the survival dropped below 85% within 1 month at 5 degrees C. Induction of anhydrobiosis in WG resulted in 85, 79 and 76% reduction in oxygen consumption by S. carpocapsae, S. feltiae, and S. riobrave IJs, respectively. Differences in IJ longevity among three species in water at 25 degrees C were related both to the initial lipid content and the rate of lipid utilisation, but not at 5 degrees C. The one-on-one infection bioassays indicated that desiccation had no negative effect on the infectivity of any of the nematode species suggesting no harmful effect on the IJs and/or their symbiotic bacteria. The species differences in IJ longevity and desiccation survival at different temperatures are discussed in relation to their foraging strategy and temperature adaptation.     

Molecular cloning of cDNA encoding mouse Cdc21 and CDC46 homologs and characterization of the products: physical interaction between P1(MCM3) and CDC46 proteins.

Two new mouse genes encoding proteins that belong to the yeast minichromosome maintenance (MCM) protein family, which is involved in the initiation of DNA replication, were isolated and their nucleotide sequence was determined. They were a putative CDC46/MCM5 homolog and a putative cdc21 homolog. About 30% amino acid identity was obtained between members in the family, and > 40% between the putative mouse and yeast homologs. The expression of these genes was cell-cycle specific at the late G1 to S phase. Immunochemical analyses showed the physical interaction between mouse P1MCM3 and CDC46 protein. These results suggest that MCM proteins function in co-ordination for DNA replication.     

Drosophila Casein Kinase I Alpha Regulates Homolog Pairing and Genome Organization by Modulating Condensin II Subunit Cap-H2 Levels

The spatial organization of chromosomes within interphase nuclei is important for gene expression and epigenetic inheritance. Although the extent of physical interaction between chromosomes and their degree of compaction varies during development and between different cell-types, it is unclear how regulation of chromosome interactions and compaction relate to spatial organization of genomes. Drosophila is an excellent model system for studying chromosomal interactions including homolog pairing. Recent work has shown that condensin II governs both interphase chromosome compaction and homolog pairing and condensin II activity is controlled by the turnover of its regulatory subunit Cap-H2. Specifically, Cap-H2 is a target of the SCF^Slimb E3 ubiquitin-ligase which down-regulates Cap-H2 in order to maintain homologous chromosome pairing, chromosome length and proper nuclear organization. Here, we identify Casein Kinase I alpha (CK1α) as an additional negative-regulator of Cap-H2. CK1α-depletion stabilizes Cap-H2 protein and results in an accumulation of Cap-H2 on chromosomes. Similar to Slimb mutation, CK1α depletion in cultured cells, larval salivary gland, and nurse cells results in several condensin II-dependent phenotypes including dispersal of centromeres, interphase chromosome compaction, and chromosome unpairing. Moreover, CK1α loss-of-function mutations dominantly suppress condensin II mutant phenotypes in vivo. Thus, CK1α facilitates Cap-H2 destruction and modulates nuclear organization by attenuating chromatin localized Cap-H2 protein.     

Protein kinase CK2 is inhibited by human nucleolar phosphoprotein p140 in an inositol hexakisphosphate-dependent manner

Protein kinase CK2 is a ubiquitous protein kinase that can phosphorylate various proteins involved in central cellular processes, such as signal transduction, cell division, and proliferation. We have shown that the human nucleolar phosphoprotein p140 (hNopp140) is able to regulate the catalytic activity of CK2. Unphosphorylated hNopp140 and phospho-hNopp140 bind to the regulatory and catalytic subunits of CK2, respectively, and the interaction between hNopp140 and CK2 was prevented by inositol hexakisphosphate (InsP(6)). Phosphorylation of alpha-casein, genimin, or human phosphatidylcholine transfer protein-like protein by CK2 was inhibited by hNopp140, and InsP(6) recovered the suppressed activity of CK2 by hNopp140. These observations indicated that hNopp140 serves as a negative regulator of CK2 and that InsP(6) stimulates the activity of CK2 by blocking the interaction between hNopp140 and CK2.     

Identification of a Novel Protein Interaction Motif in the Regulatory Subunit of Casein Kinase 2

Casein kinase 2 (CK2) regulates multiple cellular processes and can promote oncogenesis. Interactions with the CK2β regulatory subunit of the enzyme target its catalytic subunit (CK2α or CK2α′) to specific substrates; however, little is known about the mechanisms by which these interactions occur. We previously showed that by binding CK2β, the Epstein-Barr virus (EBV) EBNA1 protein recruits CK2 to promyelocytic leukemia (PML) nuclear bodies, where increased CK2-mediated phosphorylation of PML proteins triggers their degradation. Here we have identified a KSSR motif near the dimerization interface of CK2β as forming part of a protein interaction pocket that mediates interaction with EBNA1. We show that the EBNA1-CK2β interaction is primed by phosphorylation of EBNA1 on S393 (within a polyserine region). This phosphoserine is critical for EBNA1-induced PML degradation but does not affect EBNA1 functions in EBV replication or segregation. Using comparative proteomics of wild-type (WT) and KSSR mutant CK2β, we identified an uncharacterized cellular protein, C18orf25/ARKL1, that also binds CK2β through the KSSR motif and show that this involves a polyserine sequence resembling the CK2β binding sequence in EBNA1. Therefore, we have identified a new mechanism of CK2 interaction used by viral and cellular proteins.     

Cellular expression and localization of DGKζ-interacting NAP1-like proteins in the brain and functional implications under hypoxic stress

Diacylglycerol kinase (DGK) catalyzes conversion of a lipid second messenger diacylglycerol to another messenger molecule phosphatidic acid. Consequently, DGK plays a pivotal role in cellular pathophysiology by regulating the levels of these two messengers. We reported previously that DGKζ translocates from the nucleus to cytoplasm in hippocampal neurons under ischemic/hypoxic stress. In addition, we also identified nucleosome assembly protein 1 (NAP1)-like proteins NAP1L1 and NAP1L4 as novel DGKζ-interacting partners using a proteomic approach and revealed that these NAP1-like proteins induce cytoplasmic translocation of DGKζ in overexpressed cells because NAP1-like proteins associate with the nuclear localization signal of DGKζ and block its nuclear import via importin α. In the present study, we examined whether NAP1-like proteins are expressed in the brain and whether the molecular interaction of DGKζ and NAP1-like proteins would be changed in the brain after hypoxic stress. Immunohistochemistry revealed that NAP1L1 and NAP1L4 are widely expressed in neurons and glial cells in the brain with some differences. After 3 days of transient whole-body hypoxic stress, DGKζ translocated from the nucleus to cytoplasm in hippocampal pyramidal neurons, whereas NAP1-like proteins remained in the cytoplasm. Contrary to our expectations, NAP1-like proteins showed no change in their expression levels. The molecular interaction between DGKζ and NAP1-like proteins was attenuated after hypoxic stress. These results suggest that DGKζ cytoplasmic translocation in neurons under hypoxic stress is regulated by some mechanism which differs from that mediated by NAP1-like proteins.     

Binding of FGF-1 variants to protein kinase CK2 correlates with mitogenicity

Fibroblast growth factor-1 (FGF-1) has both extra- and intracellular functions. To identify intracellular binding partners for FGF-1, we isolated proteins from U2OS human osteosarcoma cells interacting specifically with FGF-1. One of the isolated proteins was identified as protein kinase CK2 (CK2). We here provide evidence that FGF-1 binds to both the catalytic alpha-subunit and to the regulatory beta-subunit of CK2. The interaction between FGF-1 and CK2 alpha and beta was characterized by surface plasmon resonance, giving K(D) values of 0.4 +/- 0.3 and 1.2 +/- 0.2 microM, respectively. By using a novel assay for intracellular protein interaction, FGF-1 and CK2 alpha are shown to interact in vivo. In vitro, FGF-1 and FGF-2 are phosphorylated by CK2, and the presence of FGF-1 or FGF-2 was found to enhance the autophosphorylation of CK2 beta. A correlation between the mitogenic potential of FGF-1 mutants and their ability to bind to CK2 alpha was observed. The possible involvement of CK2 in the FGF-induced stimulation of DNA synthesis is discussed.     

Interacting proteins and differences in nuclear transport reveal specific functions for the NAP1 family proteins in plants

Nucleosome assembly protein 1 (NAP1) is conserved from yeast to human and facilitates the in vitro assembly of nucleosomes as a histone chaperone. Inconsistent with their proposed function in the nucleus, however, many NAP1 proteins had been reported to localize in the cytoplasm. We investigated the subcellular localization of tobacco (Nicotiana tabacum) and rice (Oryza sativa) NAP1 family proteins first by identification of interacting partners and by direct examination of the localization of green fluorescent protein-tagged proteins. Through treatment of tobacco cells with leptomycin B and mutagenesis of nuclear export signal, we demonstrated that Nicta;NAP1;1 and Orysa;NAP1;1 shuttle between the cytoplasm and the nucleus. Together with the demonstration that tobacco NAP1 proteins bind histone H2A and H2B, our results support the current model and provide additional evidence that function of NAP1 as histone chaperones appears to be conserved in plants. In addition, we show that tobacco NAP1 proteins interact with tubulin and the mitotic cyclin Nicta;CYCB1;1, suggesting a role for NAP1 in microtubule dynamics. Interestingly, in spite of their high homology with the above NAP1 proteins, the other three tobacco proteins and Orysa;NAP1;2 did not show nucleocytoplasmic shuttling and were localized only in the cytoplasm. Moreover, Orysa;NAP1;3 that lacks a typical nuclear localization signal sequence was localized in both the cytoplasm and the nucleus. Finally, we show that only Orysa;NAP1;3 could be phosphorylated by casein kinase 2alpha in vitro. However, this phosphorylation was not responsible for nuclear import of Orysa;NAP1;3 as being demonstrated through mutagenesis studies. Together, our results provide an important step toward elucidating the molecular mechanism of function of the NAP1 family proteins in plants.     

Calcium-regulated interaction of Sgt1 with S100A6 (calcyclin) and other S100 proteins

S100A6 (calcyclin), a small calcium-binding protein from the S100 family, interacts with several target proteins in a calcium-regulated manner. One target is Calcyclin-Binding Protein/Siah-1-Interacting Protein (CacyBP/SIP), a component of a novel pathway of beta-catenin ubiquitination. A recently discovered yeast homolog of CacyBP/SIP, Sgt1, associates with Skp1 and regulates its function in the Skp1/Cullin1/F-box complex ubiquitin ligase and in kinetochore complexes. S100A6-binding domain of CacyBP/SIP is in its C-terminal region, where the homology between CacyBP/SIP and Sgt1 is the greatest. Therefore, we hypothesized that Sgt1, through its C-terminal region, interacts with S100A6. We tested this hypothesis by performing affinity chromatography and chemical cross-linking experiments. Our results showed that Sgt1 binds to S100A6 in a calcium-regulated manner and that the S100A6-binding domain in Sgt1 is comprised of 71 C-terminal residues. Moreover, S100A6 does not influence Skp1-Sgt1 binding, a result suggesting that separate Sgt1 domains are responsible for interactions with S100A6 and Skp1. Sgt1 binds not only to S100A6 but also to S100B and S100P, other members of the S100 family. The interaction between S100A6 and Sgt1 is likely to be physiologically relevant because both proteins were co-immunoprecipitated from HEp-2 cell line extract using monoclonal anti-S100A6 antibody. Phosphorylation of the S100A6-binding domain of Sgt1 by casein kinase II was inhibited by S100A6, a result suggesting that the role of S100A6 binding is to regulate the phosphorylation of Sgt1. These findings suggest that protein ubiquitination via Sgt1-dependent pathway can be regulated by S100 proteins.     

Active and exo-site inhibition of human factor Xa: structure of des-Gla factor Xa inhibited by NAP5, a potent nematode anticoagulant protein from Ancylostoma caninum

Hookworms are hematophagous nematodes capable of growth, development and subsistence in living host systems such as humans and other mammals. Approximately one billion, or one in six, people worldwide are infected by hookworms causing gastrointestinal blood loss and iron deficiency anemia. The hematophagous hookworm Ancylostoma caninum produces a family of small, disulfide-linked protein anticoagulants (75-84 amino acid residues). One of these nematode anticoagulant proteins, NAP5, inhibits the amidolytic activity of factor Xa (fXa) with K(i)=43 pM, and is the most potent natural fXa inhibitor identified thus far. The crystal structure of NAP5 bound at the active site of gamma-carboxyglutamic acid domainless factor Xa (des-fXa) has been determined at 3.1 A resolution, which indicates that Asp189 (fXa, S1 subsite) binds to Arg40 (NAP5, P1 site) in a mode similar to that of the BPTI/trypsin interaction. However, the hydroxyl group of Ser39 of NAP5 additionally forms a hydrogen bond (2.5 A) with His57 NE2 of the catalytic triad, replacing the hydrogen bond of Ser195 OG to the latter in the native structure, resulting in an interaction that has not been observed before. Furthermore, the C-terminal extension of NAP5 surprisingly interacts with the fXa exosite of a symmetry-equivalent molecule forming a short intermolecular beta-strand as observed in the structure of the NAPc2/fXa complex. This indicates that NAP5 can bind to fXa at the active site, or the exosite, and to fX at the exosite. However, unlike NAPc2, NAP5 does not inhibit fVIIa of the fVIIa/TF complex.