scholarly journals Sir3p Domains Involved in the Initiation of Telomeric Silencing in Saccharomyces cerevisiae

Genetics ◽  
1998 ◽  
Vol 150 (3) ◽  
pp. 977-986 ◽  
Author(s):  
Yangsuk Park ◽  
John Hanish ◽  
Arthur J Lustig
Keyword(s):  
Amino Acids ◽  
Active Site ◽  
Fusion Proteins ◽  
Initiation Site ◽  
Negative Control ◽  
Model System ◽  
Mutant Protein ◽  
Regulatory Domain ◽  
C Terminus ◽  
Necessary And Sufficient

Abstract Previous studies from our laboratory have demonstrated that tethering of Sir3p at the subtelomeric/telomeric junction restores silencing in strains containing Rap1-17p, a mutant protein unable to recruit Sir3p. This tethered silencing assay serves as a model system for the early events that follow recruitment of silencing factors, a process we term initiation. A series of LexA fusion proteins in-frame with various Sir3p fragments were constructed and tested for their ability to support tethered silencing. Interestingly, a region comprising only the C-terminal 144 amino acids, termed the C-terminal domain (CTD), is both necessary and sufficient for restoration of silencing. Curiously, the LexA-Sir3N205 mutant protein overcomes the requirement for the CTD, possibly by unmasking a cryptic initiation site. A second domain spanning amino acids 481-835, termed the nonessential for initiation domain (NID), is dispensable for the Sir3p function in initiation, but is required for the recruitment of the Sir4p C terminus. In addition, in the absence of the N-terminal 481 amino acids, the NID negatively influences CTD activity. This suggests the presence of a third region, consisting of the N-terminal half (1-481) of Sir3p, termed the positive regulatory domain (PRD), which is required to initiate silencing in the presence of the NID. These data suggest that the CTD “active” site is under both positive and negative control mediated by multiple Sir3p domains.

Dissecting interactions between EB1, microtubules and APC in cortical clusters at the plasma membrane

2002 ◽  
Vol 115 (8) ◽  
pp. 1583-1590 ◽  
Author(s):  
Angela I. M. Barth ◽  
Kathleen A. Siemers ◽  
W. James Nelson
Keyword(s):  
Amino Acids ◽  
Cluster Formation ◽  
Adenomatous Polyposis ◽  
Polyposis Coli ◽  
Binding Domains ◽  
C Terminus ◽  
Endogenous Proteins ◽  
Apc Protein ◽  
Necessary And Sufficient ◽  
Armadillo Repeats

End-binding protein (EB) 1 binds to the C-terminus of adenomatous polyposis coli (APC) protein and to the plus ends of microtubules (MT) and has been implicated in the regulation of APC accumulation in cortical clusters at the tip of extending membranes. We investigated which APC domains are involved in cluster localization and whether binding to EB1 or MTs is essential for APC cluster localization. Armadillo repeats of APC that lack EB1- and MT-binding domains are necessary and sufficient for APC localization in cortical clusters; an APC fragment lacking the armadillo repeats, but containing MT-and EB1-binding domains, does not localize to the cortical clusters but instead co-aligns with MTs throughout the cell. Significantly, analysis of endogenous proteins reveals that EB1 does not accumulate in the APC clusters. However, overexpressed EB1 does accumulate in APC clusters; the APC-binding domain in EB1 is located in the C-terminal region of EB1 between amino acids 134 and 268. Overexpressed APC- or MT-binding domains of EB1 localize to APC cortical clusters and MT, respectively, without affecting APC cluster formation itself. These results show that localization of APC in cortical clusters is different from that of EB1 at MT plus ends and appears to be independent of EB1.

scholarly journals Regulation of Bestrophin Cl Channels by Calcium: Role of the C Terminus

2008 ◽  
Vol 132 (6) ◽  
pp. 681-692 ◽  
Author(s):  
Qinghuan Xiao ◽  
Andrew Prussia ◽  
Kuai Yu ◽  
Yuan-yuan Cui ◽  
H. Criss Hartzell
Keyword(s):  
Amino Acids ◽  
Density Functional ◽  
Transmembrane Domain ◽  
Channel Gating ◽  
Regulatory Domain ◽  
Acidic Amino Acids ◽  
C Terminus ◽  
Ef Hand ◽  
Cl Channels

Human bestrophin-1 (hBest1), which is genetically linked to several kinds of retinopathy and macular degeneration in both humans and dogs, is the founding member of a family of Cl− ion channels that are activated by intracellular Ca2+. At present, the structures and mechanisms responsible for Ca2+ sensing remain unknown. Here, we have used a combination of molecular modeling, density functional–binding energy calculations, mutagenesis, and patch clamp to identify the regions of hBest1 involved in Ca2+ sensing. We identified a cluster of a five contiguous acidic amino acids in the C terminus immediately after the last transmembrane domain, followed by an EF hand and another regulatory domain that are essential for Ca2+ sensing by hBest1. The cluster of five amino acids (293–308) is crucial for normal channel gating by Ca2+ because all but two of the 35 mutations we made in this region rendered the channel incapable of being activated by Ca2+. Using homology models built on the crystal structure of calmodulin (CaM), an EF hand (EF1) was identified in hBest1. EF1 was predicted to bind Ca2+ with a slightly higher affinity than the third EF hand of CaM and lower affinity than the second EF hand of troponin C. As predicted by the model, the D312G mutation in the putative Ca2+-binding loop (312–323) reduced the apparent Ca2+ affinity by 20-fold. In addition, the D312G and D323N mutations abolished Ca2+-dependent rundown of the current. Furthermore, analysis of truncation mutants of hBest1 identified a domain adjacent to EF1 that is rich in acidic amino acids (350–390) that is required for Ca2+ activation and plays a role in current rundown. These experiments identify a region of hBest1 (312–323) that is involved in the gating of hBest1 by Ca2+ and suggest a model in which Ca2+ binding to EF1 activates the channel in a process that requires the acidic domain (293–308) and another regulatory domain (350–390). Many of the ∼100 disease-causing mutations in hBest1 are located in this region that we have implicated in Ca2+ sensing, suggesting that these mutations disrupt hBest1 channel gating by Ca2+.

scholarly journals Localization of Functional Domains of the Mitogenic Toxin of Pasteurella multocida

2001 ◽  
Vol 69 (12) ◽  
pp. 7839-7850 ◽  
Author(s):  
Gillian D. Pullinger ◽  
R. Sowdhamini ◽  
Alistair J. Lax
Keyword(s):  
Amino Acids ◽  
Fusion Proteins ◽  
Pasteurella Multocida ◽  
Transmembrane Domain ◽  
Polyclonal Antibodies ◽  
Full Length ◽  
Cell Binding ◽  
Terminal Fragment ◽  
C Terminus ◽  
N Terminus

ABSTRACT The locations of the catalytic and receptor-binding domains of thePasteurella multocida toxin (PMT) were investigated. N- and C-terminal fragments of PMT were cloned and expressed as fusion proteins with affinity tags. Purified fusion proteins were assessed in suitable assays for catalytic activity and cell-binding ability. A C-terminal fragment (amino acids 681 to 1285) was catalytically active. When microinjected into quiescent Swiss 3T3 cells, it induced changes in cell morphology typical of toxin-treated cells and stimulated DNA synthesis. An N-terminal fragment with a His tag at the C terminus (amino acids 1 to 506) competed with full-length toxin for binding to surface receptors and therefore contains the cell-binding domain. The inactive mutant containing a mutation near the C terminus (C1165S) also bound to cells in this assay. Polyclonal antibodies raised to the N-terminal PMT region bound efficiently to full-length native toxin, suggesting that the N terminus is surface located. Antibodies to the C terminus of PMT were microinjected into cells and inhibited the activity of toxin added subsequently to the medium, confirming that the C terminus contains the active site. Analysis of the PMT sequence predicted a putative transmembrane domain with predicted hydrophobic and amphipathic helices near the N terminus over the region of homology to the cytotoxic necrotizing factors. The C-terminal end of PMT was predicted to be a mixed α/β domain, a structure commonly found in catalytic domains. Homology to proteins of known structure and threading calculations supported these assignments.

scholarly journals The C Terminus of YopT Is Crucial for Activity and the N Terminus Is Crucial for Substrate Binding

2003 ◽  
Vol 71 (8) ◽  
pp. 4623-4632 ◽  
Author(s):  
Isabel Sorg ◽  
Claudia Hoffmann ◽  
Juergen Dumbach ◽  
Klaus Aktories ◽  
Gudula Schmidt
Keyword(s):  
Amino Acids ◽  
Fusion Proteins ◽  
Cysteine Proteases ◽  
Guanine Nucleotide ◽  
New Family ◽  
C Terminus ◽  
Guanine Nucleotide Dissociation Inhibitor ◽  
Release Assay ◽  
Terminal Amino ◽  
Inactive Protein

ABSTRACT Recently, it was shown that Yersinia outer protein T (YopT) belongs to a new family of cysteine proteases containing invariant C, H, and D residues that are crucial for its activity. YopT cleaves RhoA, Rac, and Cdc42 at their C termini, thereby releasing them from the membrane. Moreover, YopT inhibits the Rho-rhotekin and Rho-guanine nucleotide dissociation inhibitor interactions. To characterize the active domain of YopT, we constructed N- and C-terminal truncations and expressed them as glutathione S-transferase fusion proteins in Escherichia coli. The toxin fragments were tested for stability by trypsin digestion. The activity of the proteins was studied by membrane release assay, rhotekin pulldown experiments, and microinjection. Whereas deletion of the first 74 N-terminal amino acids did not influence the activity of YopT, deletion of 8 amino acids from the C terminus led to complete loss of activity. N-terminal deletion of 100 amino acids led to an inactive protein, although it still contained the amino acids C139, H258, and D274, which are essential for catalysis. Loss of activity of the N-terminal deletions corresponded to the block of interaction with RhoA, indicating that residues 75 to 100 of YopT are essential for binding to the GTPase. By contrast, when up to 15 amino acids of the C terminus were deleted, the protein had no activity but was still able to interact with RhoA, suggesting a role for the C terminus in the enzyme activity of YopT.

scholarly journals The mouse c-rel protein has an N-terminal regulatory domain and a C-terminal transcriptional transactivation domain.

1990 ◽  
Vol 10 (10) ◽  
pp. 5473-5485 ◽  
Author(s):  
P Bull ◽  
K L Morley ◽  
M F Hoekstra ◽  
T Hunter ◽  
I M Verma
Keyword(s):  
Amino Acids ◽  
Fusion Proteins ◽  
Mammalian Cells ◽  
Dna Binding Domain ◽  
Transactivation Domain ◽  
Regulatory Domain ◽  
Amino Acid Residues ◽  
Terminal Portion ◽  
In Cis ◽  
Transcriptional Transactivator

We have shown that the murine c-rel protein can act as a transcriptional transactivator in both yeast and mammalian cells. Fusion proteins generated by linking rel sequences to the DNA-binding domain of the yeast transcriptional activator GAL4 activate transcription from a reporter gene linked in cis to a GAL4 binding site. The full-length mouse c-rel protein (588 amino acids long) is a poor transactivator; however, the C-terminal portion of the protein between amino acid residues 403 to 568 is a potent transcriptional transactivator. Deletion of the N-terminal half of the c-rel protein augments its transactivation function. We propose that c-rel protein has an N-terminal regulatory domain and a C-terminal transactivation domain which together modulate its function as a transcriptional transactivator.

scholarly journals Probing the Structure of Rotavirus NSP4: a Short Sequence at the Extreme C Terminus Mediates Binding to the Inner Capsid Particle

2000 ◽  
Vol 74 (11) ◽  
pp. 5388-5394 ◽  
Author(s):  
Judith A. O'Brien ◽  
John A. Taylor ◽  
A. R. Bellamy
Keyword(s):  
Amino Acids ◽  
Binding Sites ◽  
Limited Proteolysis ◽  
Coiled Coil ◽  
Surface Binding ◽  
Virus Maturation ◽  
C Terminus ◽  
Tetramerization Domain ◽  
Necessary And Sufficient ◽  
Sequence Requirements

ABSTRACT The rotavirus nonstructural glycoprotein NSP4 functions as the receptor for the inner capsid particle (ICP) which buds into the lumen of the endoplasmic reticulum during virus maturation. The structure of the cytoplasmic domain of NSP4 from rotavirus strain SA11 has been investigated by using limited proteolysis and mass spectrometry. Digestion with trypsin and V8 protease reveals a C-terminal protease-sensitive region that is 28 amino acids long. The minimal sequence requirements for receptor function have been defined by constructing fusions with glutathione S-transferase and assessing their ability to bind ICPs. These experiments demonstrate that 17 to 20 amino acids from the extreme C terminus are necessary and sufficient for ICP binding and that this binding is cooperative. These observations are consistent with a model for the structure of the NSP4 cytoplasmic region in which four flexible regions of 28 amino acids are presented by a protease-resistant coiled-coil tetramerization domain, with only the last ∼20 amino acids of each peptide interacting with the surface binding sites on the ICP.

scholarly journals Solid-phase synthesis of imprinted nanoparticles as artificial antibodies against the C-terminus of the cannabinoid CB1 receptor: exploring a viable alternative for bioanalysis

2021 ◽  
Vol 188 (11) ◽  
Author(s):  
Alberto Gómez-Caballero ◽  
Ainhoa Elejaga-Jimeno ◽  
Gontzal García del Caño ◽  
Nora Unceta ◽  
Antonio Guerreiro ◽  
...  
Keyword(s):  
Amino Acids ◽  
Solid Phase ◽  
Spherical Shape ◽  
Cb1 Receptor ◽  
Negative Control ◽  
Solution Temperature ◽  
Dot Blot ◽  
Recombinant Fusion Proteins ◽  
C Terminus ◽  
Target Epitope

AbstractThe production of artificial anti-CB1 antibodies in nanoparticle format is described using the solid-phase imprinting approach. Instead of whole protein imprinting, a linear C-terminus sequence of the receptor comprising 15 amino acids (458-KVTMSVSTDTSAEAL-472) has been used as template, in accordance with the epitope imprinting approach. This sequence is located intracellularly, and it is involved in coupling to Gi/o proteins, being responsible for CB1 receptor desensitisation and internalisation. Developed molecularly imprinted materials were found to be in the nanometre scale, with a particle size of 126.4 ± 10.5 nm at pH 3 (25 ºC) and spherical shape. It was also observed that the size was sensible to temperature changes being reduced to 106.3 ± 15.2 nm at 35 °C. Lower critical solution temperature of this polymer was found to be ≈ 33.4 °C. The affinity and selectivity of the artificial antibody were assessed through dot blot and Western blot experiments. For the latter, recombinant fusion proteins GST-CB1414-472 and GST-CB1414-442 were produced to work respectively as target and negative control proteins. The control protein did not carry the target epitope for being devoid of last 30 amino acids at the C-terminus. The results demonstrated that the anti-CB1 material recognised selectively the target protein, thanks to the presence of the 15-amino acid sequence selected as epitope, which revealed that binding occurred at the C-terminus of the receptor itself. The methodology presented may pave the way for the development of novel imprinted nanomaterials for other proteins included in the superfamily of the G-protein-coupled receptors (GPCR). Graphical abstract

Abstract 237: Structural Analysis of Ang Peptide Specificity in Binding to ACE, ACE2, Neprilysin, and PRCP

Hypertension ◽  
2012 ◽  
Vol 60 (suppl_1) ◽  
Author(s):  
Jeremy W Prokop ◽  
Fabiano C Araujo ◽  
Ingrid K Watanabie ◽  
Dulce E Caserini ◽  
Amy Milsted
Keyword(s):  
Amino Acids ◽  
Active Site ◽  
Active Sites ◽  
Aromatic Amino Acids ◽  
Structural Data ◽  
Angiotensin Peptides ◽  
C Terminus ◽  
N Terminus ◽  
Natural Variants ◽  
Multiple Species

A combination of known biochemistry and physiology data on angiotensin converting enzyme (ACE), ACE2, neprilysin and prolylcarboxypeptidase (PRCP) with structural biochemistry and bioinformatics reveals several key amino acids contributing to the orientation of Ang peptides in the active site of the various enzymes. A total of four structures are known for the ACE N-terminus, twelve for the ACE C-terminus, eleven for ACE2, seven for neprilysin, and one for PRCP. Of all the known structures, none include angiotensin peptides bound into an active site. Autodock experiments reveal possible binding conformations of Ang I, Ang II, Ang-(1-9), Ang-(1-7), or Ang III into the active site of each enzyme. These autodock experiments were validated using combinations of disease associated natural variants, known mutagenesis data, conservation of amino acids in multiple species and inhibitor based structural data, all revealing amino acids of high functionality that interact with Ang peptides. Aromatic amino acids (Phe, Tyr, and Trp) tend to dictate the cleavage site of the Ang peptides through interactions with amino acid 4 (Tyr), 6 (His) or 8 (Phe) of angiotensin. The ACE C-terminus and N-terminus have three conserved aromatic amino acids 10 Å or less from the catalytic Zn, ACE2 has two 11 Å or less from the catalytic Zn, neprilysin has a Phe 8.4Å from the catalytic Zn, and PRCP has five aromatic amino acids 12Å or less from the active site. A high density of serine and threonine amino acids is found in the active sites of some enzymes when compared with total serines and threonines (ACE N-term=39%, ACE C-term=46%, ACE2=32%, neprilysin=15%, PRCP=10%), suggesting a strong likelihood of a hydrogen bonding network with angiotensin peptides in the active site, aiding in proper orientation for cleavage. Positions of serines and threonines (along with Lys, Arg, Val, Tyr, Phe, and Met) differ in the two active sites of ACE and are in contact with docked Ang I. This study shows enzyme differences and similarities in amino acids contacting the Ang peptides. Further, our results suggest that it may be possible to target multiple enzymes with the same (or a single) treatment in hypertensive patients, minimizing additive side effects of multidrug treatments.

The mouse c-rel protein has an N-terminal regulatory domain and a C-terminal transcriptional transactivation domain

1990 ◽  
Vol 10 (10) ◽  
pp. 5473-5485
Author(s):  
P Bull ◽  
K L Morley ◽  
M F Hoekstra ◽  
T Hunter ◽  
I M Verma
Keyword(s):  
Amino Acids ◽  
Fusion Proteins ◽  
Mammalian Cells ◽  
Dna Binding Domain ◽  
Transactivation Domain ◽  
Regulatory Domain ◽  
Amino Acid Residues ◽  
Terminal Portion ◽  
In Cis ◽  
Transcriptional Transactivator

We have shown that the murine c-rel protein can act as a transcriptional transactivator in both yeast and mammalian cells. Fusion proteins generated by linking rel sequences to the DNA-binding domain of the yeast transcriptional activator GAL4 activate transcription from a reporter gene linked in cis to a GAL4 binding site. The full-length mouse c-rel protein (588 amino acids long) is a poor transactivator; however, the C-terminal portion of the protein between amino acid residues 403 to 568 is a potent transcriptional transactivator. Deletion of the N-terminal half of the c-rel protein augments its transactivation function. We propose that c-rel protein has an N-terminal regulatory domain and a C-terminal transactivation domain which together modulate its function as a transcriptional transactivator.

scholarly journals Prolonged Half-Life and Improved Recovery of Recombinant Factor IX-XTEN Fusion Proteins in Hemophilia B Mouse Model

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2271-2271
Author(s):  
Arjan van der Flier ◽  
Zhiqian Lucy Liu ◽  
Zhan Liu ◽  
Oblaise Mercury ◽  
Ayman Ismail ◽  
...  
Keyword(s):  
Amino Acids ◽  
Half Life ◽  
Fusion Proteins ◽  
Research Funding ◽  
Acute Treatment ◽  
Hemophilia B ◽  
Hek293 Cells ◽  
Employment Equity ◽  
C Terminus ◽  
Equity Ownership

Abstract INTRODUCTION Prophylactic treatment for hemophilia B patients is the therapy of choice to improve quality of life and minimize annual bleeding rates and damage to joints. A new generation of extended half-life (EHL) FIX replacement products has been generated to improve patient care by reducing treatment burden. The factors include the currently marketed rFIXFc, as well as rFIX attached to PEG or albumin. The latter two are in clinical trials. All FIX preparations are administered by intravenous dosing, which can be particularly challenging for young patients and patients with limited venous access; these difficulties are reduced, but not eliminated, by the less frequent dosing achieved with EHL rFIX therapies. In the current study, we evaluate an XTEN-recombinant protein technology in order to develop EHL rFIX-XTEN molecules that are suitable for both acute treatment, as well as prophylactic subcutaneous dosing in hemophilia B, and could potentially further reduce the burden of treatment. XTEN are unstructured polypeptide sequences that consist of a limited set of natural amino acids (Pro, Ala, Gly, Glu, Ser, Thr). Fusion of XTEN to proteins alters its hydrodynamic properties and reduces the rate of clearance and degradation of the fusion protein. These XTEN fusion proteins are produced using recombinant technology, without the need for chemical modifications, and degraded by natural pathways. MATERIALS AND METHODS Recombinant FIX and FIXFc molecules were expressed as the natural Arg338Leu (R388L, Padua) variant with improved activity. XTEN polypeptides are fused to either the C-terminus of rFIX or inserted into the EGF2 domain or activation peptide (AP) domain of rFIX or rFIXFc. The fusion proteins were prepared by transient expression in human HEK293 cells followed by affinity purification. Hemophilia B (HemB) mice were dosed by either intravenous or subcutaneous injection with a single bolus of 50 or 200 IU/kg of the rFIX- or rFIXFc-fusion proteins. Plasma activity levels were determined over time using a FIX activated partial thromboplastin time assay (aPTT). PK parameters were determined using non-compartmental modeling with Phoenix WinNonlin 6.2.1 (Pharsight). RESULTS Insertion of XTEN sequences with increasing length (42, 72, 144 or 288 amino acids long) at either C-terminus of rFIX-R338L or in the AP domain showed a size dependent increase in plasma recovery up to 60% following intravenous bolus dosing. Combinations of XTEN insertions in the EGF2 or AP domain with Fc-mediated half-life extension in rFIXFc-R338L, extended the half-life as well as increased the plasma recovery. The AUC/D for rFIX-CT-XTEN.288 and rFIXFc-AP-XTEN.72 were 8.5 and 14.5 fold improved when compared to intravenously dosed rFIX, respectively. Following a subcutaneous dose of either rFIXFc-AP-XTEN.72 or rFIX-CT-XTEN.288, we observed 28 and 40-fold improved AUC/D; 15- and 30-fold improved Cmax/D and 3-fold increased bioavailability. When compared to rFIXFc the improvement in pharmacokinetic parameters was 6- and 9-fold improved AUC/D; 3- and 10-fold Cmax/D and 1.5- and 2-fold improved bioavailability for FIXFc-AP-XTEN.72 and rFIX-CT-XTEN.288, respectively. Taken together, subcutaneous dosing of rFIX-CT-XTEN.288 and rFIXFc-AP-XTEN.72 in HemB mice showed improved AUC/D when compared to intravenous dosing of rFIXFc. CONCLUSIONS rFIXFc-AP-XTEN.72 and rFIX-CT-XTEN.288 show greatly improved subcutaneous pharmacokinetics in HemB mice compared to both rFIX and rFIXFc. These promising preclinical subcutaneous dosing data in HemB mice suggests the potential of once weekly or every two weeks prophylactic subcutaneous dosing of FIX-XTEN molecules in patients. In addition, the molecules have potential for acute treatment by intravenous dosing. Further studies are ongoing to address the efficacy and allometric scaling in preclinical animal models. Disclosures van der Flier: Biogen: Employment. Liu:Biogen: Employment. Liu:Biogen: Employment, Equity Ownership, Honoraria, Research Funding. Mercury:Biogen: Employment. Ismail:Biogen: Employment. Seth-Chhabra:Biogen: Employment, Equity Ownership, Honoraria, Patents & Royalties, Research Funding. Kulman:Biogen: Employment. Schellenberger:Amunix Operating Inc: Employment. Light:Biogen: Employment, Equity Ownership. Peters:Biogen: Employment.

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