The amino-terminal domain of pyrrolysyl-tRNA synthetase is dispensable in vitro but required for in vivo activity

ABSTRACT Most members of the AraC/XylS family contain a conserved carboxy-terminal DNA binding domain and a less conserved amino-terminal domain involved in binding small-molecule effectors and dimerization. However, there is no evidence that Rns, a regulator of enterotoxigenic Escherichia coli virulence genes, responds to an effector ligand, and in this study we found that the amino-terminal domain of Rns does not form homodimers in vivo. Exposure of Rns to the chemical cross-linker glutaraldehyde revealed that the full-length protein is also a monomer in vitro. Nevertheless, deletion analysis of Rns demonstrated that the first 60 amino acids of the protein are essential for the activation and repression of Rns-regulated promoters in vivo. Amino-terminal truncation of Rns abolished DNA binding in vitro, and two randomly generated mutations, I14T and N16D, that independently abolished Rns autoregulation were isolated. Further analysis of these mutations revealed that they have disparate effects at other Rns-regulated promoters and suggest that they may be involved in an interaction with the carboxy-terminal domain of Rns. Thus, evolution may have preserved the amino terminus of Rns because it is essential for the regulator's activity even though it apparently lacks the two functions, dimerization and ligand binding, usually associated with the amino-terminal domains of AraC/XylS family members.

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BBK07, a Dominant In Vivo Antigen of Borrelia burgdorferi, Is a Potential Marker for Serodiagnosis of Lyme Disease

Clinical and Vaccine Immunology ◽

10.1128/cvi.00301-09 ◽

2009 ◽

Vol 16 (11) ◽

pp. 1569-1575 ◽

Cited By ~ 8

Author(s):

Adam S. Coleman ◽

Utpal Pal

Keyword(s):

Lyme Disease ◽

Borrelia Burgdorferi ◽

Amino Acid Identity ◽

Amino Terminal ◽

Terminal Domain ◽

Potential Marker ◽

Mammalian Infection ◽

Amino Terminal Domain

ABSTRACT One of the recently identified Borrelia burgdorferi immunogens, BBK07, is characterized for its expression in the spirochete infection cycle and evaluated for its potential use as a serodiagnostic marker for Lyme disease. We show that the BBK07 gene is expressed at extremely low levels in vitro and in ticks but is dramatically induced by spirochetes once introduced into the host and is highly expressed throughout mammalian infection. In contrast, the expression of BBK12, a paralog of BBK07 with 87% amino acid identity, although expressed in vitro, remained undetectable in vivo throughout murine infection and in ticks. BBK07 is localized in the outer membrane, and the amino-terminal domain of the antigen is exposed on the microbial surface. A truncated BBK07 protein representing the amino-terminal domain is able to effectively detect antibodies to B. burgdorferi, both in experimentally infected mice and in humans. Further characterization of the immunodominant antigens of B. burgdorferi, such as BBK07, could contribute to the development of novel serodiagnostic markers for detection of Lyme disease.

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ADA3, a putative transcriptional adaptor, consists of two separable domains and interacts with ADA2 and GCN5 in a trimeric complex.

Molecular and Cellular Biology ◽

10.1128/mcb.15.3.1203 ◽

1995 ◽

Vol 15 (3) ◽

pp. 1203-1209 ◽

Cited By ~ 104

Author(s):

J Horiuchi ◽

N Silverman ◽

G A Marcus ◽

L Guarente

Keyword(s):

Activation Domains ◽

Amino Terminal ◽

Terminal Domain ◽

Trimeric Complex ◽

Nonoverlapping Domains ◽

Carboxyl Terminal Domain ◽

Carboxyl Terminal ◽

Amino Terminal Domain

Mutations in yeast ADA2, ADA3, and GCN5 weaken the activation potential of a subset of acidic activation domains. In this report, we show that their gene products form a heterotrimeric complex in vitro, with ADA2 as the linchpin holding ADA3 and GCN5 together. Further, activation by LexA-ADA3 fusions in vivo are regulated by the levels of ADA2. Combined with a prior observation that LexA-ADA2 fusions are regulated by the levels of ADA3 (N. Silverman, J. Agapite, and L. Guarente, Proc. Natl. Acad. Sci. USA 91:11665-11668, 1994), this finding suggests that these proteins also form a complex in cells. ADA3 can be separated into two nonoverlapping domains, an amino-terminal domain and a carboxyl-terminal domain, which do not separately complement the slow-growth phenotype or transcriptional defect of a delta ada3 strain but together supply full complementation. The carboxyl-terminal domain of ADA3 alone suffices for heterotrimeric complex formation in vitro and activation of LexA-ADA2 in vivo. We present a model depicting the ADA complex as a coactivator in which the ADA3 amino-terminal domain mediates an interaction between activation domains and the ADA complex.

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Dual Cyclin-Binding Domains Are Required for p107 To Function as a Kinase Inhibitor

Molecular and Cellular Biology ◽

10.1128/mcb.18.9.5380 ◽

1998 ◽

Vol 18 (9) ◽

pp. 5380-5391 ◽

Cited By ~ 39

Author(s):

Enrique Castaño ◽

Yelena Kleyner ◽

Brian David Dynlacht

Keyword(s):

Mammalian Cells ◽

Kinase Inhibitor ◽

Point Mutations ◽

Growth Suppression ◽

Kinase Substrate ◽

Amino Terminal ◽

Terminal Domain ◽

Amino Terminal Domain

ABSTRACT The retinoblastoma (pRB) family of proteins includes three proteins known to suppress growth of mammalian cells. Previously we had found that growth suppression by two of these proteins, p107 and p130, could result from the inhibition of associated cyclin-dependent kinases (cdks). One important unresolved issue, however, is the mechanism through which inhibition occurs. Here we present in vivo and in vitro evidence to suggest that p107 is a bona fide inhibitor of both cyclin A-cdk2 and cyclin E-cdk2 that exhibits an inhibitory constant (Ki ) comparable to that of the cdk inhibitor p21/WAF1. In contrast, pRB is unable to inhibit cdks. Further reminiscent of p21, a second cyclin-binding site was mapped to the amino-terminal portions of p107 and p130. This amino-terminal domain is capable of inhibiting cyclin-cdk2 complexes, although it is not a potent substrate for these kinases. In contrast, a carboxy-terminal fragment of p107 that contains the previously identified cyclin-binding domain serves as an excellent kinase substrate although it is unable to inhibit either kinase. Clustered point mutations suggest that the amino-terminal domain is functionally important for cyclin binding and growth suppression. Moreover, peptides spanning the cyclin-binding region are capable of interfering with p107 binding to cyclin-cdk2 complexes and kinase inhibition. Our ability to distinguish between p107 and p130 as inhibitors rather than simple substrates suggests that these proteins may represent true inhibitors of cdks.

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Amino-Terminal Domain Exchange Redirects Origin-Specific Interactions of Adeno-Associated Virus Rep78 In Vitro

Journal of Virology ◽

10.1128/jvi.75.7.3230-3239.2001 ◽

2001 ◽

Vol 75 (7) ◽

pp. 3230-3239 ◽

Cited By ~ 21

Author(s):

Miran Yoon ◽

Deborah H. Smith ◽

Peter Ward ◽

Francisco J. Medrano ◽

Aneel K. Aggarwal ◽

...

Keyword(s):

Dna Replication ◽

Catalytic Domain ◽

Adeno Associated Virus ◽

Free System ◽

Site Specific ◽

Amino Terminal ◽

Terminal Domain ◽

Site Specific Integration ◽

Amino Terminal Domain

ABSTRACT The unique ability of adeno-associated virus type 2 (AAV) to site-specifically integrate its genome into a defined sequence on human chromosome 19 (AAVS1) makes it of particular interest for use in targeted gene delivery. The objective underlying this study is to provide evidence for the feasibility of retargeting site-specific integration into selected loci within the human genome. Current models postulate that AAV DNA integration is initiated through the interactions of the products of a single viral open reading frame,REP, with sequences present in AAVS1 that resemble the minimal origin for AAV DNA replication. Here, we present a cell-free system designed to dissect the Rep functions required to target site-specific integration using functional chimeric Rep proteins derived from AAV Rep78 and Rep1 of the closely related goose parvovirus. We show that amino-terminal domain exchange efficiently redirects the specificity of Rep to the minimal origin of DNA replication. Furthermore, we establish that the amino-terminal 208 amino acids of Rep78/68 constitute a catalytic domain of Rep sufficient to mediate site-specific endonuclease activity.

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In vitro binding of LexA repressor to DNA: evidence for the involvement of the amino-terminal domain.

The EMBO Journal ◽

10.1002/j.1460-2075.1986.tb04283.x ◽

1986 ◽

Vol 5 (4) ◽

pp. 793-798 ◽

Cited By ~ 31

Author(s):

S. Hurstel ◽

M. Granger-Schnarr ◽

M. Daune ◽

M. Schnarr

Keyword(s):

Amino Terminal ◽

Dna Evidence ◽

In Vitro Binding ◽

Terminal Domain ◽

Lexa Repressor ◽

Vitro Binding ◽

Amino Terminal Domain

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The Amino-Terminal Domain of Chloroplast Hsp93 Is Important for Its Membrane Association and Functions in Vivo

PLANT PHYSIOLOGY ◽

10.1104/pp.112.193300 ◽

2012 ◽

Vol 158 (4) ◽

pp. 1656-1665 ◽

Cited By ~ 13

Author(s):

Chiung-Chih Chu ◽

Hsou-min Li

Keyword(s):

Membrane Association ◽

Amino Terminal ◽

Terminal Domain ◽

Amino Terminal Domain

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Ezrin links syndecan-2 to the cytoskeleton

Journal of Cell Science ◽

10.1242/jcs.113.7.1267 ◽

2000 ◽

Vol 113 (7) ◽

pp. 1267-1276 ◽

Cited By ~ 5

Author(s):

F. Granes ◽

J.M. Urena ◽

N. Rocamora ◽

S. Vilaro

Keyword(s):

Actin Cytoskeleton ◽

Protein Complex ◽

Membrane Receptors ◽

In Vitro Assays ◽

Amino Terminal ◽

Terminal Domain ◽

Rhoa Gtpase ◽

Triton X ◽

Amino Terminal Domain

The syndecan family of heparan sulfate proteoglycans is known to associate with the actin cytoskeleton, possibly transducing signals from the extracellular matrix. In the search for proteins that could mediate the association of syndecan-2 with the actin cytoskeleton we found that ezrin, a protein which links membrane receptors to the cytoskeleton, coimmunoprecipitated with syndecan-2 in COS-1 cells. In vitro assays indicated a direct association between the amino-terminal domain of ezrin and the cytoplasmic domain of syndecan-2. Confocal microscopy showed colocalization of ezrin and syndecan-2 in actin-rich microspikes in COS-1 cells. The syndecan-2/ezrin protein complex was resistant to 0.2% Triton X-100 extraction but the syndecan-2/amino-terminal domain of ezrin complex was not, which indicated that carboxi-terminal domain of ezrin is involved in the cytoskeleton anchorage of this protein complex. Additionally we observed that the activation of rhoA GTPase increased syndecan-2 insolubility in 0.2% Triton X-100 and syndecan-2/ezrin association. Taken together, these results indicate that ezrin connects syndecan-2 to the actin cytoskeleton.

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Recombinant Polypeptides Derived from the Fibrin Binding Domain of Fibronectin Are Potential Agents for the Imaging of Blood Clots

Thrombosis and Haemostasis ◽

10.1055/s-0038-1656052 ◽

1997 ◽

Vol 77 (04) ◽

pp. 796-803 ◽

Cited By ~ 5

Author(s):

N Ezov ◽

A Nimrod ◽

B Parizada ◽

M M Werber ◽

A Goldlust ◽

...

Keyword(s):

Thrombus Formation ◽

Recombinant Polypeptide ◽

Covalent Linkage ◽

Steel Coil ◽

Amino Terminal ◽

Imaging Tool ◽

Recombinant Polypeptides ◽

Amino Terminal Domain

SummaryThrombus formation in the circulation is accompanied by covalent linkage of fibronectin (FN) through transglutamination of glutamine no. 3 in the fibrin binding amino terminal domain (FBD) of FN. We have exploited this phenomenon for thrombus detection by the employment of radioactively-labelled recombinant polypeptide molecules derived from the 5-finger FBD of human FN. Three recombinant FBD polypeptides, 12 kDa (“2 fingers”), 18.5 kDa (“3 fingers”) and 31 kDa FBD (“5 fingers”), were prepared and compared to native FN-derived 31 kDa-FBD with respect to their ability to attach to fibrin clots in vitro and in vivo. The accessibility of Gln-3 in these molecules was demonstrated by the incorporation of stoichiometric amounts of 14C- putrescine in the presence of plasma transglutaminase. Competitive binding experiments to fibrin have indicated that, although the binding affinities of the FBD molecules are lower than that of FN, substantial covalent linkage was obtained in the presence of transglutaminase, and even in the presence of excess FN or heparin. The biological clearance rates of radioactively labelled FBD molecules in rats and rabbits were much higher than those of FN and fibrinogen, thus indicating their potential advantage for use as a diagnostic imaging tool. Of the three molecules, the 12 kDa FBD exhibited the highest rate of clearance. The potential of the 12 kDa and 31 kDa FBDs as imaging agents was examined in a stainless steel coil-induced thrombus model in rats and in a jugular vein thrombus model in rabbits, using either [125I] or [111ln]-labelled materials. At 24 h, clot-to-blood ratios ranged between 10 and 22 for [125I]-12 kDa FBD and 40 and 60 for [luIn]-12 kDa FBD. In the rat model, heparin did not inhibit the uptake of FBD. Taken together, the results indicate that recombinant 12 kDa FBD is a good candidate for the diagnosis of venous thrombosis.

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Agouti-Related Protein Is Posttranslationally Cleaved by Proprotein Convertase 1 to Generate Agouti-Related Protein (AGRP)83–132: Interaction between AGRP83–132 and Melanocortin Receptors Cannot Be Influenced by Syndecan-3

Endocrinology ◽

10.1210/en.2005-1373 ◽

2006 ◽

Vol 147 (4) ◽

pp. 1621-1631 ◽

Cited By ~ 72

Author(s):

John W. M. Creemers ◽

Lynn E. Pritchard ◽

Amy Gyte ◽

Philippe Le Rouzic ◽

Sandra Meulemans ◽

...

Keyword(s):

Food Intake ◽

Core Body Temperature ◽

Full Length ◽

Related Protein ◽

Amino Terminal ◽

Terminal Domain ◽

Carboxyl Terminal ◽

Agouti Related Protein

Agouti-related protein (AGRP) plays a key role in energy homeostasis. The carboxyl-terminal domain of AGRP acts as an endogenous antagonist of the melanocortin-4 receptor (MC4-R). It has been suggested that the amino-terminal domain of AGRP binds to syndecan-3, thereby modulating the effects of carboxyl-terminal AGRP at the MC4-R. This model assumes that AGRP is secreted as a full-length peptide. In this study we found that AGRP is processed intracellularly after Arg79-Glu80-Pro81-Arg82. The processing site suggests cleavage by proprotein convertases (PCs). RNA interference and overexpression experiments showed that PC1/3 is primarily responsible for cleavage in vitro, although both PC2 and PC5/6A can also process AGRP. Dual in situ hybridization demonstrated that PC1/3 is expressed in AGRP neurons in the rat hypothalamus. Moreover, hypothalamic extracts from PC1-null mice contained 3.3-fold more unprocessed full-length AGRP, compared with wild-type mice, based on combined HPLC and RIA analysis, demonstrating that PC1/3 plays a role in AGRP cleavage in vivo. We also found that AGRP83–132 is more potent an antagonist than full-length AGRP, based on cAMP reporter assays, suggesting that posttranslational cleavage is required to potentiate the effect of AGRP at the MC4-R. Because AGRP is cleaved into distinct amino-terminal and carboxyl-terminal peptides, we tested whether amino-terminal peptides modulate food intake. However, intracerebroventricular injection of rat AGRP25–47 and AGRP50–80 had no effect on body weight, food intake, or core body temperature. Because AGRP is cleaved before secretion, syndecan-3 must influence food intake independently of the MC4-R.

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