scholarly journals Surface Signaling in Ferric Citrate Transport Gene Induction: Interaction of the FecA, FecR, and FecI Regulatory Proteins

2000 ◽  
Vol 182 (3) ◽  
pp. 637-646 ◽  
Author(s):  
Sabine Enz ◽  
Susanne Mahren ◽  
Uwe H. Stroeher ◽  
Volkmar Braun
Keyword(s):  
Nitrilotriacetic Acid ◽  
Ferric Citrate ◽  
Binding Assays ◽  
Surface Binding ◽  
Citrate Transport ◽  
C Terminus ◽  
N Terminus ◽  
Surface Signaling

ABSTRACT In Escherichia coli, transcription of the ferric citrate transport genes fecABCDE is controlled by a novel signal transduction mechanism that starts at the cell surface. Binding of ferric citrate to the outer membrane protein FecA initiates a signal that is transmitted by FecR across the cytoplasmic membrane into the cytoplasm where FecI, the sigma factor, is activated. Interaction between the signaling proteins was demonstrated by utilizing two methods. In in vitro binding assays, FecR that was His tagged at the N terminus [(His)10-FecR] and bound to a Ni-nitrilotriacetic acid agarose column was able to retain FecA, and FecR that was His tagged at the C terminus [FecR-(His)6] retained FecI on the column. An N-terminally truncated, induction-negative but transport-active FecA protein did not bind to (His)10-FecR. The in vivo assay involved the determination of the FecA, FecR, and FecI interacting domains with the bacterial two-hybrid Lex-based system. FecA1–79 interacts with FecR101–317 and FecR1–85 interacts with FecI1–173. These data clearly support a model that proposes interaction of the periplasmic N terminus of FecA with the periplasmic C-terminal portion of FecR and interaction of the cytoplasmic N terminus of FecR with FecI, which results in FecI activation.

scholarly journals Functional Characterization of the Interaction of Ste50p with Ste11p MAPKKK inSaccharomyces cerevisiae

1999 ◽  
Vol 10 (7) ◽  
pp. 2425-2440 ◽  
Author(s):  
Cunle Wu ◽  
Ekkehard Leberer ◽  
David Y. Thomas ◽  
Malcolm Whiteway
Keyword(s):  
Protein Kinase ◽  
Functional Characterization ◽  
Hog Pathway ◽  
C Terminus ◽  
Extracellular Signal ◽  
Osmotic Stress Response ◽  
N Terminus

The Saccharomyces cerevisiae Ste11p protein kinase is a homologue of mammalian MAPK/extracellular signal-regulated protein kinase kinase kinases (MAPKKKs or MEKKs) as well as theSchizosaccharomyces pombe Byr2p kinase. Ste11p functions in several signaling pathways, including those for mating pheromone response and osmotic stress response. The Ste11p kinase has an N-terminal domain that interacts with other signaling molecules to regulate Ste11p function and direct its activity in these pathways. One of the Ste11p regulators is Ste50p, and Ste11p and Ste50p associate through their respective N-terminal domains. This interaction relieves a negative activity of the Ste11p N terminus, and removal of this negative function is required for Ste11p function in the high-osmolarity glycerol (HOG) pathway. The Ste50p/Ste11p interaction is also important (but not essential) for Ste11p function in the mating pathway; in this pathway binding of the Ste11p N terminus with both Ste50p and Ste5p is required, with the Ste5p association playing the major role in Ste11p function. In vitro, Ste50p disrupts an association between the catalytic C terminus and the regulatory N terminus of Ste11p. In addition, Ste50p appears to modulate Ste11p autophosphorylation and is itself a substrate of the Ste11p kinase. Therefore, both in vivo and in vitro data support a role for Ste50p in the regulation of Ste11p activity.

scholarly journals The C-terminal Domain of p21 Inhibits Nucleotide Excision Repair In Vitro and In Vivo

1999 ◽  
Vol 10 (7) ◽  
pp. 2119-2129 ◽  
Author(s):  
Marcus P. Cooper ◽  
Adayabalam S. Balajee ◽  
Vilhelm A. Bohr
Keyword(s):  
Dna Repair ◽  
Nucleotide Excision Repair ◽  
Excision Repair ◽  
Binding Domain ◽  
In Vivo Studies ◽  
C Terminus ◽  
Nucleotide Excision ◽  
N Terminus

The protein p21Cip1, Waf1, Sdi1 is a potent inhibitor of cyclin-dependent kinases (CDKs). p21 can also block DNA replication through its interaction with the proliferating cell nuclear antigen (PCNA), which is an auxiliary factor for polymerase δ. PCNA is also implicated in the repair resynthesis step of nucleotide excision repair (NER). Previous studies have yielded contradictory results on whether p21 regulates NER through its interaction with PCNA. Resolution of this controversy is of interest because it would help understand how DNA repair and replication are regulated. Hence, we have investigated the effect of p21 on NER both in vitro and in vivo using purified fragments of p21 containing either the CDK-binding domain (N terminus) or the PCNA binding domain (C terminus) of the protein. In the in vitro studies, DNA repair synthesis was measured in extracts from normal human fibroblasts using plasmids damaged by UV irradiation. In the in vivo studies, we used intact and permeabilized cells. The results show that the C terminus of the p21 protein inhibits NER both in vitro and in vivo. These are the first in vivo studies in which this question has been examined, and we demonstrate that inhibition of NER by p21 is not merely an artificial in vitro effect. A 50% inhibition of in vitro NER occurred at a 50:1 molar ratio of p21 C-terminus fragment to PCNA monomer. p21 differentially regulates DNA repair and replication, with repair being much less sensitive to inhibition than replication. Our in vivo results suggest that the inhibition occurs at the resynthesis step of the repair process. It also appears that preassembly of PCNA at repair sites mitigates the inhibitory effect of p21. We further demonstrate that the inhibition of DNA repair is mediated via binding of p21 to PCNA. The N terminus of p21 had no effect on DNA repair, and the inhibition of DNA repair by the C terminus of p21 was relieved by the addition of purified PCNA protein.

scholarly journals Camelpox virus encodes a schlafen-like protein that affects orthopoxvirus virulence

2007 ◽  
Vol 88 (6) ◽  
pp. 1667-1676 ◽  
Author(s):  
Caroline Gubser ◽  
Rory Goodbody ◽  
Andrea Ecker ◽  
Gareth Brady ◽  
Luke A. J. O'Neill ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Sequence Similarity ◽  
Small Animal ◽  
Wild Type ◽  
Recombinant Viruses ◽  
C Terminus ◽  
N Terminus ◽  
Type V

Camelpox virus (CMLV) gene 176R encodes a protein with sequence similarity to murine schlafen (m-slfn) proteins. In vivo, short and long members of the m-slfn family inhibited T-cell development, whereas in vitro, only short m-slfns caused arrest of fibroblast growth. CMLV 176 protein (v-slfn) is most closely related to short m-slfns; however, when expressed stably in mammalian cells, v-slfn did not inhibit cell growth. v-slfn is a predominantly cytoplasmic 57 kDa protein that is expressed throughout infection. Several other orthopoxviruses encode v-slfn proteins, but the v-slfn gene is fragmented in all sequenced variola virus and vaccinia virus (VACV) strains. Consistent with this, all 16 VACV strains tested do not express a v-slfn detected by polyclonal serum raised against the CMLV protein. In the absence of a small animal model to study CMLV pathogenesis, the contribution of CMLV v-slfn to orthopoxvirus virulence was studied via its expression in an attenuated strain of VACV. Recombinant viruses expressing wild-type v-slfn or v-slfn tagged at its C terminus with a haemagglutinin (HA) epitope were less virulent than control viruses. However, a virus expressing v-slfn tagged with the HA epitope at its N terminus had similar virulence to controls, implying that the N terminus has an important function. A greater recruitment of lymphocytes into infected lung tissue was observed in the presence of wild-type v-slfn but, interestingly, these cells were less activated. Thus, v-slfn is an orthopoxvirus virulence factor that affects the host immune response to infection.

scholarly journals Distinct domains of antizyme required for binding and proteolysis of ornithine decarboxylase.

1994 ◽  
Vol 14 (1) ◽  
pp. 87-92 ◽  
Author(s):  
X Li ◽  
P Coffino
Keyword(s):  
Conformational Change ◽  
Ornithine Decarboxylase ◽  
Polyamine Biosynthesis ◽  
Selective Degradation ◽  
Cell Biol ◽  
C Terminus ◽  
N Terminus ◽  
Inducible Protein

Selective degradation by proteasomes of ornithine decarboxylase, the initial enzyme in polyamine biosynthesis, is mediated by the polyamine-inducible protein antizyme. Antizyme binds to a region near the N terminus of ornithine decarboxylase (X. Li and P. Coffino, Mol. Cell. Biol. 12:3556-3562, 1992). This interaction induces a conformational change in ornithine decarboxylase that exposes its C terminus and inactivates the enzyme (X. Li and P. Coffino, Mol. Cell. Biol. 13:1487-1492, 1993). Here we show that the C-terminal half of antizyme alone can inactivate ornithine decarboxylase and alter its conformation, but it cannot direct degradation of the enzyme, either in vitro or in vivo. A portion of the N-terminal half of antizyme must be present to promote degradation.

scholarly journals Cep57, a multidomain protein with unique microtubule and centrosomal localization domains

2008 ◽  
Vol 412 (2) ◽  
pp. 265-273 ◽  
Author(s):  
Ko Momotani ◽  
Alexander S. Khromov ◽  
Tsuyoshi Miyake ◽  
P. Todd Stukenberg ◽  
Avril V. Somlyo
Keyword(s):  
Ectopic Expression ◽  
Coiled Coil ◽  
Full Length ◽  
Centrosomal Protein ◽  
Multidomain Protein ◽  
Coiled Coil Domain ◽  
C Terminus ◽  
N Terminus

The present study demonstrates different functional domains of a recently described centrosomal protein, Cep57 (centrosomal protein 57). Endogenous Cep57 protein and ectopic expression of full-length protein or the N-terminal coiled-coil domain localize to the centrosome internal to γ-tubulin, suggesting that it is either on both centrioles or on a centromatrix component. The N-terminus can also multimerize with the N-terminus of other Cep57 molecules. The C-terminus contains a second coiled-coil domain that directly binds to MTs (microtubules). This domain both nucleates and bundles MTs in vitro. This activity was also seen in vivo, as overexpression of full-length Cep57 or the C-terminus generates nocodazole-resistant MT cables in cells. Based on the present findings, we propose that Cep57 serves as a link with its N-terminus anchored to the centriole or centromatrix and its C-terminus to MTs.

scholarly journals Cotranslational assembly imposes evolutionary constraints on homomeric proteins

2016 ◽  
Author(s):  
Eviatar Natan ◽  
Tamaki Endoh ◽  
Liora Haim-Vilmovsky ◽  
Guilhem Chalancon ◽  
Tilman Flock ◽  
...  
Keyword(s):  
Protein Function ◽  
Protein Complexes ◽  
Evolutionary Constraints ◽  
E Coli ◽  
Protein Library ◽  
C Terminus ◽  
N Terminus ◽  
Polypeptide Chains

AbstractThere is increasing evidence that some proteins fold during translation, i.e. cotranslationally, which implies that partial protein function, including interactions with other molecules, could potentially be unleashed early on during translation. Although little is known about cotranslational assembly mechanisms, for homomeric protein complexes, translation by the ribosome, folding and assembly, should be well-coordinated to avoid misassembly in the context of polysomes. We analysed 3D structures of homomers and identified a statistically significant trend conserved across evolution that supports this hypothesis: namely that homomeric contacts tend to be localized towards the C-terminus rather than N-terminus of homomeric polypeptide chains. To probe this in more detail, we expressed a GFP-based library of 611 homomeric E. coli genes, and analyzing their folding and assembly in vivo. Consistent with our hypothesis, interface residues tend to be located near the N-terminus in cotranslationally aggregating homomers. In order to dissect the mechanisms of folding and assembly under controlled conditions, we engineered a protein library with three variable components: (i) the position and type homomerization domain, (ii) the reporter domain and (iii) the linker length that connects the two. By analyzing the misassembly rates of these engineered constructs in vivo, in vitro and in silico, we confirmed our hypothesis that C-terminal homomerization is favorable to N-terminal homomerization. More generally, these results provide a set of spatiotemporal constraints within polypeptide chains that favor efficient assembly, with implications for protein evolution and design.

Distinct domains of antizyme required for binding and proteolysis of ornithine decarboxylase

1994 ◽  
Vol 14 (1) ◽  
pp. 87-92
Author(s):  
X Li ◽  
P Coffino
Keyword(s):  
Conformational Change ◽  
Ornithine Decarboxylase ◽  
Polyamine Biosynthesis ◽  
Selective Degradation ◽  
Cell Biol ◽  
C Terminus ◽  
N Terminus ◽  
Inducible Protein

Selective degradation by proteasomes of ornithine decarboxylase, the initial enzyme in polyamine biosynthesis, is mediated by the polyamine-inducible protein antizyme. Antizyme binds to a region near the N terminus of ornithine decarboxylase (X. Li and P. Coffino, Mol. Cell. Biol. 12:3556-3562, 1992). This interaction induces a conformational change in ornithine decarboxylase that exposes its C terminus and inactivates the enzyme (X. Li and P. Coffino, Mol. Cell. Biol. 13:1487-1492, 1993). Here we show that the C-terminal half of antizyme alone can inactivate ornithine decarboxylase and alter its conformation, but it cannot direct degradation of the enzyme, either in vitro or in vivo. A portion of the N-terminal half of antizyme must be present to promote degradation.

scholarly journals The FecI Extracytoplasmic-Function Sigma Factor of Escherichia coli Interacts with the β′ Subunit of RNA Polymerase

2003 ◽  
Vol 185 (6) ◽  
pp. 1796-1802 ◽  
Author(s):  
Susanne Mahren ◽  
Volkmar Braun
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Sigma Factor ◽  
Nitrilotriacetic Acid ◽  
Ferric Citrate ◽  
Β Subunit ◽  
Ecf Sigma Factor ◽  
K 12

ABSTRACT Transcription of the ferric citrate transport system of Escherichia coli K-12 is mediated by the extracytoplasmic-function (ECF) sigma factor FecI, which is activated by ferric citrate in the growth medium. By using a bacterial two-hybrid system, it was shown in vivo that FecI binds to the β′ subunit of RNA polymerase. The inactive mutant protein FecI(K155E) displayed reduced binding to β′, and small deletions along the entire FecI protein led to total impairment of β′ binding. In vitro, FecI was retained on Ni2+-nitrilotriacetic acid agarose loaded with a His-tagged β′1-313 fragment and coeluted with β′1-313. Binding of FecI to β′ and β′1-313 was enhanced by FecR1-85, which represents the cytoplasmic portion of the FecR protein that transmits the inducing signal across the cytoplasmic membrane. Interaction of FecR with FecI was demonstrated by showing that isolated FecR inhibited degradation of FecI by trypsin. This is the first demonstration of binding of an ECF sigma factor of the FecI type to the β′ subunit of RNA polymerase and of binding being enhanced by the protein that activates the ECF sigma factor.

scholarly journals A Peptide Found in Human Serum, Derived from the C-Terminus of Albumin, Shows Antifungal Activity In Vitro and In Vivo

2020 ◽  
Vol 8 (10) ◽  
pp. 1627
Author(s):  
Tecla Ciociola ◽  
Pier Paolo Zanello ◽  
Tiziana D’Adda ◽  
Serena Galati ◽  
Stefania Conti ◽  
...  
Keyword(s):  
Antifungal Activity ◽  
Human Serum ◽  
Fungicidal Activity ◽  
Galleria Mellonella ◽  
Serum Proteins ◽  
Morphological Alterations ◽  
C Terminus ◽  
Different Sources

The growing problem of antimicrobial resistance highlights the need for alternative strategies to combat infections. From this perspective, there is a considerable interest in natural molecules obtained from different sources, which are shown to be active against microorganisms, either alone or in association with conventional drugs. In this paper, peptides with the same sequence of fragments, found in human serum, derived from physiological proteins, were evaluated for their antifungal activity. A 13-residue peptide, representing the 597–609 fragment within the albumin C-terminus, was proved to exert a fungicidal activity in vitro against pathogenic yeasts and a therapeutic effect in vivo in the experimental model of candidal infection in Galleria mellonella. Studies by confocal microscopy and transmission and scanning electron microscopy demonstrated that the peptide penetrates and accumulates in Candida albicans cells, causing gross morphological alterations in cellular structure. These findings add albumin to the group of proteins, which already includes hemoglobin and antibodies, that could give rise to cryptic antimicrobial fragments, and could suggest their role in anti-infective homeostasis. The study of bioactive fragments from serum proteins could open interesting perspectives for the development of new antimicrobial molecules derived by natural sources.

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