Regulation of SulA cleavage by Lon protease by the C-terminal amino acid of SulA, histidine

2001 ◽  
Vol 358 (2) ◽  
pp. 473-480 ◽  
Author(s):  
Yoshiyuki ISHII ◽  
Fumio AMANO
Keyword(s):  
Amino Acid ◽  
Protein A ◽  
Lon Protease ◽  
Histidine Residue ◽  
High Accumulation ◽  
C Terminus ◽  
A Cell ◽  
Cell Division Inhibitor

SulA protein, a cell division inhibitor in Escherichia coli, is degraded by Lon protease. The C-terminal eight residues of SulA have been shown to be recognized by Lon; however, it remains to be elucidated which amino acid in the C-terminus of SulA is critical for the recognition of SulA by Lon. To clarify this point, we constructed mutants of SulA with changes in the C-terminal residues, and examined the accumulation and stability of the resulting mutant SulA proteins in vivo. Substitution of the extreme C-terminal histidine residue with another amino acid led to marked accumulation and high stability of SulA in lon+ cells. A SulA mutant in which the C-terminal eight residues were deleted (SulAC161) showed high accumulation and stability, but the addition of histidine to the C-terminus of SulAC161 (SulAC161+H) made it labile. Similarly, SulAC161+H fused to maltose-binding protein (MBP–SulAC161+H) formed a tight complex with and was degraded rapidly by Lon in vitro. Histidine competitively inhibited the degradation of MBP–SulA by Lon, while other amino acids did not. These results suggest that the histidine residue at the extreme C-terminus of SulA is recognized specifically by Lon, leading to a high-affinity interaction between SulA and Lon.

scholarly journals Mutagenesis of the Rns regulator of enterotoxigenic Escherichia coli reveals roles for a linker sequence and two helix–turn–helix motifs

Microbiology ◽  
2010 ◽  
Vol 156 (9) ◽  
pp. 2796-2806 ◽  
Author(s):  
Vivienne Mahon ◽  
Cyril J. Smyth ◽  
Stephen G. J. Smith
Keyword(s):  
Gene Expression ◽  
Escherichia Coli ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Protein A ◽  
Diarrhoeal Disease ◽  
Enterotoxigenic Escherichia Coli ◽  
Insertion Mutagenesis

The pathogenesis of diarrhoeal disease due to human enterotoxigenic Escherichia coli absolutely requires the expression of fimbriae. The expression of CS1 fimbriae is positively regulated by the AraC-like protein Rns. AraC-like proteins are DNA-binding proteins that typically contain two helix–turn–helix (HTH) motifs. A program of pentapeptide insertion mutagenesis of the Rns protein was performed, and this revealed that both HTH motifs are required by Rns to positively regulate CS1 fimbrial gene expression. Intriguingly, a pentapeptide insertion after amino acid C102 reduced the ability of Rns to transactivate CS1 fimbrial expression. The structure of Rns in this vicinity (NACRS) was predicted to be disordered and thus might act as a flexible linker. This hypothesis was confirmed by deletion of this amino acid sequence from the Rns protein; a truncated protein that lacked this sequence was no longer functional. Strikingly, this sequence could be functionally substituted in vivo and in vitro by a flexible seven amino acid sequence from another E. coli AraC-like protein RhaS. Our data indicate that HTH motifs and a flexible sequence are required by Rns for maximal activation of fimbrial gene expression.

scholarly journals The PEST-like sequence of I kappa B alpha is responsible for inhibition of DNA binding but not for cytoplasmic retention of c-Rel or RelA homodimers.

1995 ◽  
Vol 15 (2) ◽  
pp. 872-882 ◽  
Author(s):  
M K Ernst ◽  
L L Dunn ◽  
N R Rice
Keyword(s):  
Amino Acid ◽  
Dna Binding ◽  
Direct Interaction ◽  
Terminal Region ◽  
Nf Kappa B ◽  
I Kappa B Alpha ◽  
Functional Capabilities ◽  
C Terminus

In most cells, proteins belonging to the Rel/NF-kappa B family of transcription factors are held in inactive form in the cytoplasm by an inhibitor protein, I kappa B alpha. Stimulation of the cells leads to degradation of the inhibitor and transit of active DNA-binding Rel/NF-kappa B dimers to the nucleus. I kappa B alpha is also able to inhibit DNA binding by Rel/NF-kappa B dimers in vitro, suggesting that it may perform the same function in cells when the activating signal is no longer present. Structurally, the human I kappa B alpha molecule can be divided into three sections: a 70-amino-acid N terminus with no known function, a 205-residue midsection composed of six ankyrin-like repeats, and a very acidic 42-amino-acid C terminus that resembles a PEST sequence. In this study we examined how the structural elements of the I kappa B alpha protein correlate with its functional capabilities both in vitro and in vivo. Using a battery of I kappa B alpha mutants, we show that (i) a dimer binds a single I kappa B alpha molecule, (ii) the acidic C-terminal region of I kappa B alpha is not required for protein-protein binding and does not mask the nuclear localization signal of the dimer, (iii) the same C-terminal region is required for inhibition of DNA binding, and (iv) this inhibition may be accomplished by direct interaction between the PEST-like region and the DNA-binding region of one of the subunits of the dimer.

A 113-amino-acid truncation at the NS1 C-terminus is a determinant for viral replication of H5N6 avian influenza virus in vitro and in vivo

2018 ◽  
Vol 225 ◽  
pp. 6-16 ◽  
Author(s):  
Xiaole Cui ◽  
Yanhong Ji ◽  
Zhengxiang Wang ◽  
Yingying Du ◽  
Haoran Guo ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Amino Acid ◽  
Avian Influenza ◽  
Viral Replication ◽  
Avian Influenza Virus ◽  
C Terminus

Abstract 5432: Mutation of Three Amino Acids in the Disulfide-Ring of a CNP Based Chimeric Natriuretic Peptide Alters its Vascular Properties

Circulation ◽  
2008 ◽  
Vol 118 (suppl_18) ◽  
Author(s):  
Horng H Chen ◽  
Brenda K Huntley ◽  
Candace Y Lee ◽  
Fernando L Martin ◽  
John A Schirger ◽  
...  
Keyword(s):  
Amino Acid ◽  
Natriuretic Peptide ◽  
Right Atrial ◽  
Bolus Administration ◽  
Amino Acid Peptide ◽  
Arterial Blood ◽  
C Terminus ◽  
Cardiac Unloading

BACKGROUND: C-type natriuretic peptide (CNP) is a 22-amino-acid peptide produced mainly in the endothelium with potent cardiac unloading and modest blood pressure lowering actions, but minimal renal actions. Based on our previous knowledge, we recently fused a 6 aa sequence from BNP to the C-terminus and a 5 aa sequence from ANP to the N-terminus of CNP. This novel hybrid peptide, CBA-NP, has cardiac unloading actions and mild hypotensive effects similar to CNP. Importantly however, the N and C terminus alterations resulted in potent renal excretory actions. here we test the hypothesis that the 3 aa GSM 15–17 in the disulfide-ring mediate the vascular and hypotensive actions. We therefore mutated GSM 15–17 to REA 15–17 , which we named ABC-NP and compared its in vivo and in vitro actions to CBA-NP. METHODS: We determined the cardiorenal and humoral actions of intravenous bolus administration of CBA-NP (n=5) and ABC-NP (n=5) at 25 microgram/Kg in 2 separate group of normal anesthetized dogs. We also assessed the cGMP response of both peptides in human aortic endothelial cells (HAEC), human cardiac fibroblast (HCF) and isolated canine glomeruli. * p<0.05 RESULTS: IV bolus administration of CBA-NP and ABC-NP resulted in diuresis* and natriuresis*. There was a significant decrease in mean arterial blood (MAP) pressure with CBA-NP (126±6 to113±7 mmHg*) but no change with ABC-NP(126±8 to126±8 mmHg) . In addition, the reduction in pulmonary capillary wedge pressure (PCWP) and right atrial pressure (RAP) was significantly greater with CBA-NP as compared to ABC-NP. cGMP generation in HAEC and HCF was minimal with ABC-NP and was significantly higher with CBA-NP*. In contrast, cGMP generation was similar in isolated glomeruli between the two peptides. CONCLUSION: Our studies demonstrates that mutation of three amino acid (aa) residues within the CNP ring of CBA-NP from GSM 15–17 to REA alters the vascular but not the renal excretory properties. Hence by this strategic mutation within the ring of CBA-NP, we have designed a renal specific peptide ABC-NP resulting in new sequence specific functional information which can be used to design organ specific therapeutic peptides with unique properties tailored for a specific disease state.

Glial fibrillary acidic protein - a cell type specific marker for Ito cells in vivo and in vitro

1996 ◽  
Vol 24 (6) ◽  
pp. 719-730 ◽  
Author(s):  
Katrin Neubauer ◽  
Thomas Knittel ◽  
Sabine Aurisch ◽  
Peter Fellmer ◽  
Giuliano Ramadori
Keyword(s):  
Glial Fibrillary Acidic Protein ◽  
Protein A ◽  
Acidic Protein ◽  
Specific Marker ◽  
Cell Type ◽  
Ito Cells ◽  
A Cell ◽  
Cell Type Specific

scholarly journals A novel, resistance-linked ovine PrP variant and its equivalent mouse variant modulate the in vitro cell-free conversion of rPrP to PrPres

2006 ◽  
Vol 87 (12) ◽  
pp. 3747-3751 ◽  
Author(s):  
Louise Kirby ◽  
Wilfred Goldmann ◽  
Fiona Houston ◽  
Andrew C. Gill ◽  
Jean C. Manson
Keyword(s):  
Amino Acid ◽  
Prion Diseases ◽  
Cellular Prion Protein ◽  
Transmission Characteristics ◽  
Different Types ◽  
A Cell ◽  
Conversion Efficiencies ◽  
Equivalent Variant

Prion diseases are associated with the conversion of the normal cellular prion protein, PrPc, to the abnormal, disease-associated form, PrPSc. This conversion can be mimicked in vitro by using a cell-free conversion assay. It has recently been shown that this assay can be modified to use bacterial recombinant PrP as substrate and mimic the in vivo transmission characteristics of rodent scrapie. Here, it is demonstrated that the assay replicates the ovine polymorphism barriers of scrapie transmission. In addition, the recently identified ovine PrP variant ARL168Q, which is associated with resistance of sheep to experimental BSE, modulates the cell-free conversion of ovine recombinant PrP to PrPres by three different types of PrPSc, reducing conversion efficiencies to levels similar to those of the ovine resistance-associated ARR variant. Also, the equivalent variant in mice (L164) is resistant to conversion by 87V scrapie. Together, these results suggest a significant role for this position and/or amino acid in conversion.

scholarly journals Vaccinia Virus G7L Protein Interacts with the A30L Protein and Is Required for Association of Viral Membranes with Dense Viroplasm To Form Immature Virions

2003 ◽  
Vol 77 (6) ◽  
pp. 3418-3429 ◽  
Author(s):  
Patricia Szajner ◽  
Howard Jaffe ◽  
Andrea S. Weisberg ◽  
Bernard Moss
Keyword(s):  
Vaccinia Virus ◽  
Protein A ◽  
Direct Interaction ◽  
Uncharacterized Protein ◽  
Lethal Mutant ◽  
C Terminus ◽  
Infected Cells ◽  
The Stability

ABSTRACT The vaccinia virus A30L protein is required for the association of electron-dense, granular, proteinaceous material with the concave surfaces of crescent membranes, an early step in viral morphogenesis. For the identification of additional proteins involved in this process, we used an antibody to the A30L protein, or to an epitope appended to its C terminus, to capture complexes from infected cells. A prominent 42-kDa protein was resolved and identified by mass spectrometry as the vaccinia virus G7L protein. This previously uncharacterized protein was expressed late in infection and was associated with immature virions and the cores of mature particles. In order to study the role of the G7L protein, a conditional lethal mutant was made by replacing the G7L gene with an inducible copy. Expression of G7L and formation of infectious virus was dependent on the addition of inducer. Under nonpermissive conditions, morphogenesis was blocked and viral crescent membranes and immature virions containing tubular elements were separated from the electron-dense granular viroplasm, which accumulated in large spherical masses. This phenotype was identical to that previously obtained with an inducible, conditional lethal A30L mutant. Additional in vivo and in vitro experiments provided evidence for the direct interaction of the A30L and G7L proteins and demonstrated that the stability of each one was dependent on its association with the other.

scholarly journals Cloning and Molecular Characterization of an Immunogenic LigA Protein of Leptospira interrogans

2002 ◽  
Vol 70 (11) ◽  
pp. 5924-5930 ◽  
Author(s):  
Raghavan U. M. Palaniappan ◽  
Yung-Fu Chang ◽  
S. S. D. Jusuf ◽  
S. Artiushin ◽  
John F. Timoney ◽  
...  
Keyword(s):  
Amino Acid ◽  
Structure Prediction ◽  
Tandem Repeats ◽  
Secondary Structure Prediction ◽  
Protein A ◽  
Immunoblot Analysis ◽  
Leptospira Interrogans ◽  
Reading Frame

ABSTRACT A clone expressing a novel immunoreactive leptospiral immunoglobulin-like protein A of 130 kDa (LigA) from Leptospira interrogans serovar pomona type kennewicki was isolated by screening a genomic DNA library with serum from a mare that had recently aborted due to leptospiral infection. LigA is encoded by an open reading frame of 3,675 bp, and the deduced amino acid sequence consists of a series of 90-amino-acid tandem repeats. A search of the NCBI database found that homology of the LigA repeat region was limited to an immunoglobulin-like domain of the bacterial intimin binding protein of Escherichia coli, the cell adhesion domain of Clostridium acetobutylicum, and the invasin of Yersinia pestis. Secondary structure prediction analysis indicates that LigA consists mostly of beta sheets with a few alpha-helical regions. No LigA was detectable by immunoblot analysis of lysates of the leptospires grown in vitro at 30°C or when cultures were shifted to 37°C. Strikingly, immunohistochemistry on kidney from leptospira-infected hamsters demonstrated LigA expression. These findings suggest that LigA is specifically induced only in vivo. Sera from horses, which aborted as a result of natural Leptospira infection, strongly recognize LigA. LigA is the first leptospiral protein described to have 12 tandem repeats and is also the first to be expressed only during infection. Thus, LigA may have value in serodiagnosis or as a protective immunogen in novel vaccines.

scholarly journals The C Terminus of the Histone Chaperone Asf1 Cross-Links to Histone H3 in Yeast and Promotes Interaction with Histones H3 and H4

2012 ◽  
Vol 33 (3) ◽  
pp. 605-621 ◽  
Author(s):  
Briana K. Dennehey ◽  
Seth Noone ◽  
Wallace H. Liu ◽  
Luke Smith ◽  
Mair E. A. Churchill ◽  
...  
Keyword(s):  
Amino Acid ◽  
Phosphorylation Site ◽  
Histone H3 ◽  
Unnatural Amino Acid ◽  
Full Length ◽  
Content Type ◽  
C Terminus ◽  
Cross Links

ABSTRACTThe central histone H3/H4 chaperone Asf1 comprises a highly conserved globular core and a divergent C-terminal tail. While the function and structure of the Asf1 core are well known, the function of the tail is less well understood. Here, we have explored the role of the yeast (yAsf1) and human (hAsf1a and hAsf1b) Asf1 tails inSaccharomyces cerevisiae. We show, using a photoreactive, unnatural amino acid, that Asf1 tail residue 210 cross-links to histone H3in vivoand, further, that loss of C-terminal tail residues 211 to 279 weakens yAsf1-histone binding affinityin vitronearly 200-fold. Via several yAsf1 C-terminal truncations and yeast-human chimeric proteins, we found that truncations at residue 210 increase transcriptional silencing and that the hAsf1a tail partially substitutes for full-length yAsf1 with respect to silencing but that full-length hAsf1b is a better overall substitute for full-length yAsf1. In addition, we show that the C-terminal tail of Asf1 is phosphorylated at T270 in yeast. Loss of this phosphorylation site does not prevent coimmunoprecipitation of yAsf1 and Rad53 from yeast extracts, whereas amino acid residue substitutions at the Asf1-histone H3/H4 interface do. Finally, we show that residue substitutions in yAsf1 near the CAF-1/HIRA interface also influence yAsf1's function in silencing.

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