scholarly journals Modular structure of chromosomal proteins HMG-14 and HMG-17: definition of a transcriptional enhancement domain distinct from the nucleosomal binding domain.

1995 ◽  
Vol 15 (12) ◽  
pp. 6663-6669 ◽  
Author(s):  
L Trieschmann ◽  
Y V Postnikov ◽  
A Rickers ◽  
M Bustin
Keyword(s):  
Amino Acids ◽  
Structural Features ◽  
Binding Domain ◽  
Full Length ◽  
Modular Structure ◽  
Chromosomal Proteins ◽  
Nucleosome Core ◽  
Terminal Amino

Chromosomal proteins HMG-14 and HMG-17 are the only known nuclear proteins which specifically bind to the nucleosome core particle and are implicated in the generation and/or maintenance of structural features specific to active chromatin. The two proteins facilitate polymerase II and III transcription from in vitro- and in vivo-assembled circular chromatin templates. Here we used deletion mutants and specific peptides to identify the transcriptional enhancement domain and delineate the nucleosomal binding domain of the HMG-14 and -17 proteins. Deletion of the 22 C-terminal amino acids of HMG-17 or 26 C-terminal amino acids of HMG-14 reduces significantly the ability of the proteins to enhance transcription from chromatin templates. In contrast, N-terminal truncation mutants had the same transcriptional enhancement activity as the full-length proteins. We conclude that the negatively charged C-terminal region of the proteins is required for transcriptional enhancement. Chromatin transcription enhancement assays, which involve binding competition between the full-length proteins and peptides derived from their nucleosomal binding regions, indicate that the minimal nucleosomal binding domain of human HMG-17 is 24 amino acids long and spans residues 17 to 40. The results suggest that HMG-14 and -17 proteins have a modular structure and contain distinct functional domains.

scholarly journals Microtubule Actin Cross-Linking Factor (Macf)

1999 ◽  
Vol 147 (6) ◽  
pp. 1275-1286 ◽  
Author(s):  
Conrad L. Leung ◽  
Dongming Sun ◽  
Min Zheng ◽  
David R. Knowles ◽  
Ronald K.H. Liem
Keyword(s):  
Calcium Binding ◽  
Coiled Coil ◽  
Specific Protein ◽  
Binding Domain ◽  
Full Length ◽  
Actin Binding ◽  
Cross Linking ◽  
Actin Binding Domain

We cloned and characterized a full-length cDNA of mouse actin cross-linking family 7 (mACF7) by sequential rapid amplification of cDNA ends–PCR. The completed mACF7 cDNA is 17 kb and codes for a 608-kD protein. The closest relative of mACF7 is the Drosophila protein Kakapo, which shares similar architecture with mACF7. mACF7 contains a putative actin-binding domain and a plakin-like domain that are highly homologous to dystonin (BPAG1-n) at its NH2 terminus. However, unlike dystonin, mACF7 does not contain a coiled–coil rod domain; instead, the rod domain of mACF7 is made up of 23 dystrophin-like spectrin repeats. At its COOH terminus, mACF7 contains two putative EF-hand calcium-binding motifs and a segment homologous to the growth arrest–specific protein, Gas2. In this paper, we demonstrate that the NH2-terminal actin-binding domain of mACF7 is functional both in vivo and in vitro. More importantly, we found that the COOH-terminal domain of mACF7 interacts with and stabilizes microtubules. In transfected cells full-length mACF7 can associate not only with actin but also with microtubules. Hence, we suggest a modified name: MACF (microtubule actin cross-linking factor). The properties of MACF are consistent with the observation that mutations in kakapo cause disorganization of microtubules in epidermal muscle attachment cells and some sensory neurons.

Structure–function analysis of full-length midkine reveals novel residues important for heparin binding and zebrafish embryogenesis

2013 ◽  
Vol 451 (3) ◽  
pp. 407-415 ◽  
Author(s):  
Jackwee Lim ◽  
Sheng Yao ◽  
Martin Graf ◽  
Christoph Winkler ◽  
Daiwen Yang
Keyword(s):  
Function Analysis ◽  
Structural Features ◽  
Full Length ◽  
Domain Growth ◽  
Heparin Binding ◽  
Cellular Mechanisms ◽  
The Individual ◽  
Zebrafish Embryogenesis

Midkine is a heparin-binding di-domain growth factor, implicated in many biological processes as diverse as angiogenesis, neurogenesis and tumorigenesis. Elevated midkine levels reflect poor prognosis for many carcinomas, yet the molecular and cellular mechanisms orchestrating its activity remain unclear. At the present time, the individual structures of isolated half domains of human midkine are known and its functionally active C-terminal half domain remains a popular therapeutic target. In the present study, we determined the structure of full-length zebrafish midkine and show that it interacts with fondaparinux (a synthetic highly sulfated pentasaccharide) and natural heparin through a previously uncharacterized, but highly conserved, hinge region. Mutating six consecutive residues in the conserved hinge to glycine strongly abates heparin binding and midkine embryogenic activity. In contrast with previous in vitro studies, we found that the isolated C-terminal half domain is not active in vivo in embryos. Instead, we have demonstrated that the N-terminal half domain is needed to enhance heparin binding and mediate midkine embryogenic activity surprisingly in both heparin-dependent and -independent manners. Our findings provide new insights into the structural features of full-length midkine relevant for embryogenesis, and unravel additional therapeutic routes targeting the N-terminal half domain and conserved hinge.

scholarly journals Hepatitis C Virus Nonstructural 5A Protein Induces Interleukin-8, Leading to Partial Inhibition of the Interferon-Induced Antiviral Response

2001 ◽  
Vol 75 (13) ◽  
pp. 6095-6106 ◽  
Author(s):  
Stephen J. Polyak ◽  
Khalid S. A. Khabar ◽  
Denise M. Paschal ◽  
Heather J. Ezelle ◽  
Gilles Duverlie ◽  
...  
Keyword(s):  
Amino Acids ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Antiviral Response ◽  
Full Length ◽  
Interleukin 8 ◽  
Antiviral Resistance ◽  
Ns5a Protein ◽  
Terminal Amino

ABSTRACT Hepatitis C virus (HCV), a major cause of liver disease worldwide, is frequently resistant to the antiviral alpha interferon (IFN). The HCV nonstructural 5A (NS5A) protein has been implicated in HCV antiviral resistance in many studies. NS5A antagonizes the IFN antiviral response in vitro, and one mechanism is via inhibition of a key IFN-induced enzyme, the double-stranded-RNA-activated protein kinase (PKR). In the present study we determined if NS5A uses other strategies to subvert the IFN system. Expression of full-length NS5A proteins from patients who exhibited a complete response (FL-NS5A-CR) or were nonresponsive (FL-NS5A-NR) to IFN therapy in HeLa cells had no effect on IFN induction of IFN-stimulated gene factor 3 (ISGF-3). Expression of mutant NS5A proteins lacking 110 (NS5A-ΔN110), 222 (NS5A-ΔN222), and 334 amino-terminal amino acids and mutants lacking 117 and 230 carboxy-terminal amino acids also had no effect on ISGF-3 induction by IFN. Expression of FL-NS5A-CR and FL-NS5A-NR did not affect IFN-induced STAT-1 tyrosine phosphorylation or upregulation of PKR and major histocompatibility complex class I antigens. However, NS5A expression in human cells induced interleukin 8 (IL-8) mRNA and protein, and this effect correlated with inhibition of the antiviral effects of IFN in an in vitro bioassay. NS5A induced transcription of a reporter gene driven by the IL-8 promoter, and the first 133 bp of the IL-8 promoter made up the minimal domain required for NS5A transactivation. NS5A-ΔN110 and NS5A-ΔN222 stimulated the IL-8 promoter to higher levels than did the full-length NS5A protein, and this correlated with increased nuclear localization of the proteins. Additional mutagenesis of the IL-8 promoter suggested that NF-κB and AP-1 were important in NS5A-ΔN222 transactivation in the presence of tumor necrosis factor alpha and that NF–IL-6 was inhibitory to this process. This study suggests that NS5A inhibits the antiviral actions of IFN by at least two mechanisms and provides the first evidence for a biological effect of the transcriptional activity of the NS5A protein. During HCV infection, viral proteins may induce chemokines that contribute to HCV antiviral resistance and pathogenesis.

scholarly journals Regulation of E2F1-Dependent Gene Transcription and Apoptosis by the ETS-Related Transcription Factor GABPγ1

2002 ◽  
Vol 22 (7) ◽  
pp. 2147-2158 ◽  
Author(s):  
Ludger Hauck ◽  
Rudolf G. Kaba ◽  
Martin Lipp ◽  
Rainer Dietz ◽  
Rüdiger von Harsdorf
Keyword(s):  
Transcription Factor ◽  
Transcription Factors ◽  
Cell Fate ◽  
Binding Domain ◽  
Pocket Proteins ◽  
Related Transcription Factor ◽  
Terminal Amino ◽  
Fate Decision

ABSTRACT The E2F family of transcription factors comprises six related members which are involved in the control of the coordinated progression through the G1/S-phase transition of cell cycle or in cell fate decision. Their activity is regulated by pocket proteins, including pRb, p107, and p130. Here we show that E2F1 directly interacts with the ETS-related transcription factor GABPγ1 in vitro and in vivo. The binding domain interacting with GABPγ1 was mapped to the C-terminal amino acids 310 to 437 of E2F1, which include its transactivation and pRb binding domain. Among the E2F family of transcription factors, the interaction with GABPγ1 is restricted to E2F1. DNA-binding E2F1 complexes containing GABPγ1 are characterized by enhanced E2F1-dependent transcriptional activity. Moreover, GABPγ1 suppresses E2F1-dependent apoptosis by mechanisms other than the inhibition of the transactivation capacity of E2F1. In summary, our results provide evidence for a novel pRb-independent mechanism regulating E2F1-dependent transcription and apoptosis.

scholarly journals The C-Terminal 88 Amino Acids of the Sendai Virus P Protein Have Multiple Functions Separable by Mutation

2002 ◽  
Vol 76 (1) ◽  
pp. 68-77 ◽  
Author(s):  
Jeffery Tuckis ◽  
Sherin Smallwood ◽  
Joyce A. Feller ◽  
Sue A. Moyer
Keyword(s):  
Amino Acids ◽  
Protein Interactions ◽  
Sendai Virus ◽  
Intact Cell ◽  
Binding Domain ◽  
Site Directed Mutagenesis ◽  
Genome Replication ◽  
Multiple Functions

ABSTRACT The Sendai virus P-L polymerase complex binds the NP-encapsidated nucleocapsid (NC) template through a P-NP interaction. To identify P amino acids responsible for binding we performed site-directed mutagenesis on the C-terminal 88 amino acids in the NC binding domain. The mutant P proteins expressed from plasmids were assayed for viral RNA synthesis and for various protein-protein interactions. All the mutants formed P oligomers and bound to L protein. While two mutants, JT3 and JT8, retained all P functions at or near the levels of wild-type (wt) P, three others—JT4, JT6, and JT9—were completely defective for both transcription and genome replication in vitro. Each of the inactive mutants retained significant NC binding but had a different spectrum of other binding interactions and activities, suggesting that the NC binding domain also affects the catalytic function of the polymerase. NC binding was inhibited by combinations of the inactive mutations. The remaining P mutants were active in transcription but defective in various aspects of genome replication. Some P mutants were defective in NP0 binding and abolished the reconstitution of replication from separate P-L and NP0-P complexes. In some of these cases the coexpression of the wt polymerase with the mutant NP0-P complex could rescue the defect in replication, suggesting an interaction between these complexes. For some P mutants replication occurred in vivo, but not in vitro, suggesting that the intact cell is providing an unknown function that cannot be reproduced in extracts of cells. Thus, the C-terminal region of P is complex and possesses multiple functions besides NC binding that can be separated by mutation.

scholarly journals The Gal3p-Gal80p-Gal4p Transcription Switch of Yeast: Gal3p Destabilizes the Gal80p-Gal4p Complex in Response to Galactose and ATP

1999 ◽  
Vol 19 (11) ◽  
pp. 7828-7840 ◽  
Author(s):  
Alok Kumar Sil ◽  
Samina Alam ◽  
Ping Xin ◽  
Ly Ma ◽  
Melissa Morgan ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acids ◽  
Genetic Interaction ◽  
Transcription Activation ◽  
Full Length ◽  
Signal Transducer ◽  
Binding Peptide ◽  
Two Hybrid

ABSTRACT The Gal3, Gal80, and Gal4 proteins of Saccharomyces cerevisiae comprise a signal transducer that governs the galactose-inducible Gal4p-mediated transcription activation ofGAL regulon genes. In the absence of galactose, Gal80p binds to Gal4p and prohibits Gal4p from activating transcription, whereas in the presence of galactose, Gal3p binds to Gal80p and relieves its inhibition of Gal4p. We have found that immunoprecipitation of full-length Gal4p from yeast extracts coprecipitates less Gal80p in the presence than in the absence of Gal3p, galactose, and ATP. We have also found that retention of Gal80p by GSTG4AD (amino acids [aa] 768 to 881) is markedly reduced in the presence compared to the absence of Gal3p, galactose, and ATP. Consistent with these in vitro results, an in vivo two-hybrid genetic interaction between Gal80p and Gal4p (aa 768 to 881) was shown to be weaker in the presence than in the absence of Gal3p and galactose. These compiled results indicate that the binding of Gal3p to Gal80p results in destabilization of a Gal80p-Gal4p complex. The destabilization was markedly higher for complexes consisting of G4AD (aa 768 to 881) than for full-length Gal4p, suggesting that Gal80p relocated to a second site on full-length Gal4p. Congruent with the idea of a second site, we discovered a two-hybrid genetic interaction involving Gal80p and the region of Gal4p encompassing aa 225 to 797, a region of Gal4p linearly remote from the previously recognized Gal80p binding peptide within Gal4p aa 768 to 881.

Calreticulin and Calreticulin Fragments Are Endothelial Cell Inhibitors That Suppress Tumor Growth

Blood ◽  
1999 ◽  
Vol 94 (7) ◽  
pp. 2461-2468 ◽  
Author(s):  
Sandra E. Pike ◽  
Lei Yao ◽  
Joyce Setsuda ◽  
Karen D. Jones ◽  
Barry Cherney ◽  
...  
Keyword(s):  
Amino Acids ◽  
Cell Proliferation ◽  
Endothelial Cell ◽  
Tumor Growth ◽  
Angiogenesis Inhibitors ◽  
Full Length ◽  
Endothelial Cell Proliferation ◽  
Proliferation In Vitro

Several angiogenesis inhibitors are fragments of larger proteins that are themselves not active as angiogenesis inhibitors. Vasostatin, the N-terminal domain of calreticulin inclusive of amino acids 1-180, is an angiogenesis inhibitor that exerts antitumor effects in vivo. In the present study, we examined whether the full-length calreticulin molecule shares the antiangiogenic and antitumor activities of vasostatin. Similar to vasostatin, calreticulin selectively inhibited endothelial cell proliferation in vitro, but not cells of other lineages, and suppressed angiogenesis in vivo. When inoculated into athymic mice, calreticulin inhibited Burkitt tumor growth comparably with vasostatin. Calreticulin lacking the N-terminal 1-120 amino acids inhibited endothelial cell proliferation in vitro and Burkitt tumor growth in vivo comparably with vasostatin. An internal calreticulin fragment encompassing amino acids 120-180 also inhibited endothelial cell proliferation in vitro and angiogenesis in vivo comparably with calreticulin and vasostatin. These results suggest that the antiangiogenic activities of vasostatin reside in a domain that is accessible from the full-length calreticulin molecule and localize to calreticulin N-terminal amino acids 120-180. Thus, calreticulin and calreticulin fragments are inhibitors of angiogenesis that directly target endothelial cells, inhibit angiogenesis, and suppress tumor growth. This information may be critical in designing targeted inhibitors of pathological angiogenesis that underlies cancer and other diseases.

scholarly journals Interactions between Papillomavirus L1 and L2 Capsid Proteins

2003 ◽  
Vol 77 (8) ◽  
pp. 4818-4826 ◽  
Author(s):  
Renée L. Finnen ◽  
Kimberly D. Erickson ◽  
Xiaojiang S. Chen ◽  
Robert L. Garcea
Keyword(s):  
Amino Acids ◽  
Capsid Protein ◽  
Single Molecule ◽  
Hydrophobic Interactions ◽  
Protein Complexes ◽  
Binding Domain ◽  
Site Directed Mutagenesis ◽  
Hydrophobic Region

ABSTRACT The human papillomavirus (HPV) capsid consists of 360 copies of the major capsid protein, L1, arranged as 72 pentamers on a T=7 icosahedral lattice, with substoichiometric amounts of the minor capsid protein, L2. In order to understand the arrangement of L2 within the HPV virion, we have defined and biochemically characterized a domain of L2 that interacts with L1 pentamers. We utilized an in vivo binding assay involving the coexpression of recombinant HPV type 11 (HPV11) L1 and HPV11 glutathione S-transferase (GST) L2 fusion proteins in Escherichia coli. In this system, L1 forms pentamers, GST=L2 associates with these pentamers, and L1+L2 complexes are subsequently isolated by using the GST tag on L2. The stoichiometry of L1:L2 in purified L1+L2 complexes was 5:1, indicating that a single molecule of L2 interacts with an L1 pentamer. Coexpression of HPV11 L1 with deletion mutants of HPV11 L2 defined an L1-binding domain contained within amino acids 396 to 439 near the carboxy terminus of L2. L2 proteins from eight different human and animal papillomavirus serotypes were tested for their ability to interact with HPV11 L1. This analysis targeted a hydrophobic region within the L1-binding domain of L2 as critical for L1 binding. Introduction of negative charges into this hydrophobic region by site-directed mutagenesis disrupted L1 binding. L1-L2 interactions were not significantly disrupted by treatment with high salt concentrations (2 M NaCl), weak detergents, and urea concentrations of up to 2 M, further indicating that L1 binding by this domain is mediated by strong hydrophobic interactions. L1+L2 protein complexes were able to form virus-like particles in vitro at pH 5.2 and also at pH 6.8, a pH that is nonpermissive for assembly of L1 protein alone. Thus, L1/L2 interactions are primarily hydrophobic, encompass a relatively short stretch of amino acids, and have significant effects upon in vitro assembly.

scholarly journals Murine Coronavirus Evolution In Vivo: Functional Compensation of a Detrimental Amino Acid Substitution in the Receptor Binding Domain of the Spike Glycoprotein

2005 ◽  
Vol 79 (12) ◽  
pp. 7629-7640 ◽  
Author(s):  
Sonia Navas-Martin ◽  
Susan T. Hingley ◽  
Susan R. Weiss
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Receptor Binding ◽  
Amino Acid Substitution ◽  
Binding Domain ◽  
Receptor Binding Domain ◽  
Spike Glycoprotein ◽  
Functional Compensation

ABSTRACT Murine coronavirus A59 strain causes mild to moderate hepatitis in mice. We have previously shown that mutants of A59, unable to induce hepatitis, may be selected by persistent infection of primary glial cells in vitro. These in vitro isolated mutants encoded two amino acids substitutions in the spike (S) gene: Q159L lies in the putative receptor binding domain of S, and H716D, within the cleavage signal of S. Here, we show that hepatotropic revertant variants may be selected from these in vitro isolated mutants (Q159L-H716D) by multiple passages in the mouse liver. One of these mutants, hr2, was chosen for more in-depth study based on a more hepatovirulent phenotype. The S gene of hr2 (Q159L- R654H -H716D- E1035D ) differed from the in vitro isolates (Q159L-H716D) in only 2 amino acids (R654H and E1035D). Using targeted RNA recombination, we have constructed isogenic recombinant MHV-A59 viruses differing only in these specific amino acids in S (Q159L-R654H-H716D-E1035D). We demonstrate that specific amino acid substitutions within the spike gene of the hr2 isolate determine the ability of the virus to cause lethal hepatitis and replicate to significantly higher titers in the liver compared to wild-type A59. Our results provide compelling evidence of the ability of coronaviruses to rapidly evolve in vivo to highly virulent phenotypes by functional compensation of a detrimental amino acid substitution in the receptor binding domain of the spike glycoprotein.

The N-Terminus of Hepcidin Is Essential for Its Interaction with Ferroportin: Structure-Function Study.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3588-3588
Author(s):  
Elizabeta Nemeth ◽  
Gloria C. Preza ◽  
Alan J. Waring ◽  
Tomas Ganz
Keyword(s):  
Amino Acids ◽  
Disulfide Bonds ◽  
Human Subjects ◽  
Amino Acid Peptide ◽  
Cellular Iron ◽  
N Terminus ◽  
A Cell ◽  
Terminal Amino

Abstract Hepcidin, a small peptide produced by the liver, is the principal iron-regulatory hormone. It acts by binding to the iron exporter ferroportin, inducing its internalization and degradation, thereby blocking cellular iron efflux. The bioactive 25 amino acid peptide has a hairpin structure stabilized by 4 disulfide bonds. We synthesized a series of hepcidin derivatives and determined their bioactivity in a cell line expressing ferroportin-GFP fusion protein, by measuring the degradation of ferroportin-GFP and the accumulation of ferritin after peptide treatment. Bioactivity was also assayed in vivo, by measuring hypoferremia in mice injected with hepcidin derivatives. Serial deletion of N-terminal amino acids caused progressive decrease in activity which was completely lost when five N-terminal aa were deleted. Synthetic 3-aa and 6-aa N-terminal peptides alone, however, did not internalize ferroportin, and did not interfere with ferroportin internalization by native hepcidin. Deletion of two C-terminal amino acids did not affect peptide activity. Removal of individual disulfide bonds by pairwise substitution of cysteines with alanines also did not impact peptide activity in vitro. However, these peptides were significantly less active in vivo, likely due to their decreased stability in circulation. Peptides with a substitution G71D or K83R, previously described in human subjects, were fully active in vitro and in vivo. Zebrafish hepcidin, which is only 60% similar to human hepcidin, but with conservative substitutions at the N-terminus, was as active as its human counterpart. Apart from the essential nature of the N-terminus, hepcidin structure appears permissive for mutations. Further studies of hepcidin structure in relation to its function are essential for the design of hepcidin antagonists and agonists which could be used for treatment of iron disorders.

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