scholarly journals NPC2 facilitates bidirectional transfer of cholesterol between NPC1 and lipid bilayers, a step in cholesterol egress from lysosomes

2008 ◽  
Vol 105 (40) ◽  
pp. 15287-15292 ◽  
Author(s):  
Rodney E. Infante ◽  
Michael L. Wang ◽  
Arun Radhakrishnan ◽  
Hyock Joo Kwon ◽  
Michael S. Brown ◽  
...  
Keyword(s):  
Amino Acids ◽  
Lipid Bilayers ◽  
Clinical Phenotype ◽  
Phosphatidylcholine Liposomes ◽  
Terminal Domain ◽  
Naturally Occurring ◽  
Membrane Bound ◽  
Niemann Pick ◽  
Lysosomal Proteins

Egress of lipoprotein-derived cholesterol from lysosomes requires two lysosomal proteins, polytopic membrane-bound Niemann–Pick C1 (NPC1) and soluble Niemann–Pick C2 (NPC2). The reason for this dual requirement is unknown. Previously, we showed that the soluble luminal N-terminal domain (NTD) of NPC1 (amino acids 25–264) binds cholesterol. This NTD is designated NPC1(NTD). We and others showed that soluble NPC2 also binds cholesterol. Here, we establish an in vitro assay to measure transfer of [3H]cholesterol between these two proteins and phosphatidylcholine liposomes. Whereas NPC2 rapidly donates or accepts cholesterol from liposomes, NPC1(NTD) acts much more slowly. Bidirectional transfer of cholesterol between NPC1(NTD) and liposomes is accelerated >100-fold by NPC2. A naturally occurring human mutant of NPC2 (Pro120Ser) fails to bind cholesterol and fails to stimulate cholesterol transfer from NPC1(NTD) to liposomes. NPC2 may be essential to deliver or remove cholesterol from NPC1, an interaction that links both proteins to the cholesterol egress process from lysosomes. These findings may explain how mutations in either protein can produce a similar clinical phenotype.

scholarly journals Role of a tryptophan anchor in human topoisomerase I structure, function and inhibition

2008 ◽  
Vol 411 (3) ◽  
pp. 523-530 ◽  
Author(s):  
Gary S. Laco ◽  
Yves Pommier
Keyword(s):  
Amino Acids ◽  
Topoisomerase I ◽  
Electrostatic Interactions ◽  
Functional Domain ◽  
Site Mutation ◽  
Supercoiled Dna ◽  
Terminal Domain ◽  
Tryptophan Residues ◽  
N Terminus

Human Top1 (topoisomerase I) relaxes supercoiled DNA during cell division and transcription. Top1 is composed of 765 amino acids and contains an unstructured N-terminal domain of 200 amino acids, and a structured functional domain of 565 amino acids that binds and relaxes supercoiled DNA. In the present study we examined the region spanning the junction of the N-terminal domain and functional domain (junction region). Analysis of several published Top1 structures revealed that three tryptophan residues formed a network of aromatic stacking interactions and electrostatic interactions that anchored the N-terminus of the functional domain to sub-domains containing the nose cone and active site. Mutation of the three tryptophan residues (Trp203/Trp205/Trp206) to an alanine residue, either individually or together, in silico revealed that the individual tryptophan residue's contribution to the tryptophan ‘anchor’ was additive. When the three tryptophan residues were mutated to alanine in vitro, the resulting mutant Top1 differed from wild-type Top1 in that it lacked processivity, exhibited resistance to camptothecin and was inactivated by urea. The results indicated that the tryptophan anchor stabilized the N-terminus of the functional domain and prevented the loss of Top1 structure and function.

New Insights into the Function of N-Terminal 11 Amino Acids of Band 3 from Structural and Functional Study of a Naturally Occuring Band 3 Variant.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 577-577 ◽  
Author(s):  
Silverio Perrotta ◽  
Borriello Adriana ◽  
Lucia De Franceschi ◽  
Bruno Nobili ◽  
Achille Iolascon ◽  
...  
Keyword(s):  
Amino Acids ◽  
Band 3 ◽  
Red Cell ◽  
Red Cell Membrane ◽  
Exon 2 ◽  
Protein 4.2 ◽  
Membrane Bound ◽  
Terminal Amino ◽  
Intron 2

Abstract The 911 amino acid human erythroid AE1 (eAE1) Cl-/HCO3- exchanger SLC4A1 (usually called band 3) is the major intrinsic membrane protein of red cells. The N-terminal cytoplasmic domain of AE1 represents the anchoring site for membrane-associated proteins such as ankyrin, protein 4.2, protein 4.1, glycolytic enzymes (including aldolase and glyceraldeyde-3-phosphate dehydrogenase (GAPDH) and hemoglobin. We identified marked band 3 deficiency in the second son of a consanguineous marriage with a life-threatening nonimmune hemolytic anemia. The patient was transfusion-dependent prior to splenectomy. SDS-PAGE and immunoblotting analysis of the proband red cell membrane proteins showed approximately 12±4% of band 3 and protein 4.2 compared to controls. Direct nucleotide sequence of SLC4A1 gene showed a single base substitution (T->C) at position +2 in the donor splice site of intron 2 (Band 3 Neapolis). Functionally, the mutation causes an altered splicing with the consequent formation of two different mature mRNAs, one including intron 2 and one skipping exon 2. While intron 2 retention leads to premature translation termination, exon 2 skipping causes the loss of the normal start site of eAE1 protein translation. The purification of mutant band 3 and its characterization by MALDI mass spectrometry demonstrated the lack of the first 11 amino acids due to the usage of second in frame start site. Real-time RT-PCR analyses of reticulocyte mRNA showed a marked decrement in band 3 transcription accounting for protein deficiency. The lack of the 11 N-terminal amino acids resulted in complete absence of membrane bound aldolase while other glycolitic enzymes (for example GAPDH) were membrane bound. Syk tyrosine kinase recognized the truncated band 3 as a substrate in vitro. In spite of this ability to be phosphorylated by Syk and to recruit Lyn tyrosine kinase in vitro, we were unable to demonstrate Tyr-phosphorylation of mutant band 3 in intact erythrocytes following stimulation by oxidative stress. This finding implies a requirement for the 11 N-terminal amino acids for the sequential Tyr-phosphorylation of band 3 in intact red cell membranes. The mutant band 3 was largely present in the high molecular weight aggregate fraction (about 5.2 fold higher than control), indicating its increased tendency to cluster in the membrane. The spontaneous clustering of truncated band 3 strongly suggests that the negatively charged N-terminal domain may regulate oligomeric state of band 3 in the membrane. Biophysical characterization showed that band 3 deficiency resulted in decreased cohesion between lipid bilyer and spectrin based membrane skeleton accounting for membrane loss. The structural and functional characterization of the naturally occuring mutant band 3 has enabled us to identify a significant role for the 11 N-terminal amino acids in band 3 function and in red cell membrane physiology.

scholarly journals Functions of the C-terminal domain of CTP: phosphocholine cytidylyltransferase. Effects of C-terminal deletions on enzyme activity, intracellular localization and phosphorylation potential

1995 ◽  
Vol 310 (2) ◽  
pp. 699-708 ◽  
Author(s):  
R B Cornell ◽  
G B Kalmar ◽  
R J Kay ◽  
M A Johnson ◽  
J S Sanghera ◽  
...  
Keyword(s):  
Amino Acids ◽  
Lipid Vesicles ◽  
Casein Kinase Ii ◽  
Casein Kinase ◽  
Regulatory Domain ◽  
Cdc2 Kinase ◽  
Phosphocholine Cytidylyltransferase ◽  
Membrane Bound

The role of the C-terminal domain of CTP: phosphocholine cytidylyltransferase (CT) was explored by the creation of a series of deletion mutations in rat liver cDNA, which were expressed in COS cells as a major protein component. Deletion of up to 55 amino acids from the C-terminus had no effect on the activity of the enzyme, its stimulation by lipid vesicles or on its intracellular distribution between soluble and membrane-bound forms. However, deletion of the C-terminal 139 amino acids resulted in a 90% decrease in activity, loss of response to lipid vesicles and a significant decrease in the fraction of membrane-bound enzyme. Identification of the domain that is phosphorylated in vivo was determined by analysis of 32P-labelled CT mutants and by chymotrypsin proteolysis of purified CT that was 32P-labelled in vivo. Phosphorylation was restricted to the C-terminal 52 amino acids (domain P) and occurred on multiple sites. CT phosphorylation in vitro was catalysed by casein kinase II, cell division control 2 kinase (cdc2 kinase), protein kinases C alpha and beta II, and glycogen synthase kinase-3 (GSK-3), but not by mitogen-activated kinase (MAP kinase). Casein kinase II phosphorylation was directed exclusively to Ser-362. The sites phosphorylated by cdc2 kinase and GSK-3 were restricted to several serines within three proline-rich motifs of domain P. Sites phosphorylated in vitro by protein kinase C, on the other hand, were distributed over the N-terminal catalytic as well as the C-terminal regulatory domain. The stoichiometry of phosphorylation catalysed by any of these kinases was less than 0.2 mol P/mol CT, and no effects on enzyme activity were detected. This study supports a tripartite structure for CT with an N-terminal catalytic domain and a C-terminal regulatory domain comprised of a membrane-binding domain (domain M) and a phosphorylation domain (domain P). It also identifies three kinases as potential regulators in vivo of CT, casein kinase II, cyclin-dependent kinase and GSK-3.

scholarly journals Molecular characterization of the TonB2 protein from the fish pathogen Vibrio anguillarum

2009 ◽  
Vol 418 (1) ◽  
pp. 49-59 ◽  
Author(s):  
Claudia S. López ◽  
R. Sean Peacock ◽  
Jorge H. Crosa ◽  
Hans J. Vogel
Keyword(s):  
Amino Acids ◽  
Solution Structure ◽  
Bacterial Species ◽  
Vibrio Anguillarum ◽  
Fish Pathogen ◽  
E Coli ◽  
Terminal Domain ◽  
C Terminus

In the fish pathogen Vibrio anguillarum the TonB2 protein is essential for the uptake of the indigenous siderophore anguibactin. Here we describe deletion mutants and alanine replacements affecting the final six amino acids of TonB2. Deletions of more than two amino acids of the TonB2 C-terminus abolished ferric-anguibactin transport, whereas replacement of the last three residues resulted in a protein with wild-type transport properties. We have solved the high-resolution solution structure of the TonB2 C-terminal domain by NMR spectroscopy. The core of this domain (residues 121–206) has an αββαβ structure, whereas residues 76–120 are flexible and extended. This overall folding topology is similar to the Escherichia coli TonB C-terminal domain, albeit with two differences: the β4 strand found at the C-terminus of TonB is absent in TonB2, and loop 3 is extended by 9 Å (0.9 nm) in TonB2. By examining several mutants, we determined that a complete loop 3 is not essential for TonB2 activity. Our results indicate that the β4 strand of E. coli TonB is not required for activity of the TonB system across Gram-negative bacterial species. We have also determined, through NMR chemical-shift-perturbation experiments, that the E. coli TonB binds in vitro to the TonB box from the TonB2-dependent outer membrane transporter FatA; moreover, it can substitute in vivo for TonB2 during ferric-anguibactin transport in V. anguillarum. Unexpectedly, TonB2 did not bind in vitro to the FatA TonB-box region, suggesting that additional factors may be required to promote this interaction. Overall our results indicate that TonB2 is a representative of a different class of TonB proteins.

scholarly journals Inducible degradation of I kappa B alpha in vitro and in vivo requires the acidic C-terminal domain of the protein.

1995 ◽  
Vol 15 (5) ◽  
pp. 2413-2419 ◽  
Author(s):  
M S Rodriguez ◽  
I Michalopoulos ◽  
F Arenzana-Seisdedos ◽  
R T Hay
Keyword(s):  
Amino Acids ◽  
Proteolytic Activity ◽  
Cell Activation ◽  
Nf Kappa B ◽  
Free System ◽  
In Vitro System ◽  
I Kappa B Alpha ◽  
Terminal Domain

After exposure of cells to tumor necrosis factor (TNF), I kappa B alpha is rapidly degraded by a proteolytic activity that is required for nuclear localization and activation of transcription factor NF-kappa B. To investigate this problem, we have developed a cell-free system to study the degradation of I kappa B alpha initiated in vivo. In this in vitro system, characteristics of endogenous I kappa B alpha degradation were comparable to those observed in vivo. Recombinant I kappa B alpha, when added to lysates from cells exposed to TNF, was specifically degraded by a cellular proteolytic activity; however, it was stable in extracts from unstimulated cells. Inhibition characteristics of the proteolytic activity responsible for I kappa B alpha degradation suggest the involvement of a serine protease. Analysis of mutated forms of I kappa B alpha in the in vitro system demonstrated that an I kappa B alpha species which was unable to interact with NF-kappa B was still efficiently degraded. In contrast, deletion of the C-terminal 61 amino acids from I kappa B alpha rendered the protein resistant to proteolytic degradation. Expression of I kappa B alpha mutated forms in COS-7 cells confirmed the importance of the C-terminal domain for the degradation of the protein in vivo following cell activation. Thus, it is likely that the acidic, negatively charged region represented by the C-terminal 61 amino acids of the protein contains residues critical for TNF-inducible degradation of I kappa B alpha.

scholarly journals A Naturally Occurring Deletion in FliE from Salmonella enterica Serovar Dublin Results in an Aflagellate Phenotype and Defective Proinflammatory Properties

2017 ◽  
Vol 86 (1) ◽  
Author(s):  
Sebastián Sasías ◽  
Adriana Martínez-Sanguiné ◽  
Laura Betancor ◽  
Arací Martínez ◽  
Bruno D'Alessandro ◽  
...  
Keyword(s):  
Amino Acids ◽  
Dna Sequences ◽  
High Frequency ◽  
Salmonella Enterica ◽  
Wild Type ◽  
Content Type ◽  
Naturally Occurring ◽  
Salmonella Enterica Serovar Dublin

ABSTRACTSalmonella entericaserovar Dublin is adapted to cattle but is able to infect humans with high invasiveness. An acute inflammatory response at the intestine helps to preventSalmonelladissemination to systemic sites. Flagella contribute to this response by providing motility and FliC-mediated signaling through pattern recognition receptors. In a previous work, we reported a high frequency (11 out of 25) ofS. Dublin isolates lacking flagella in a collection obtained from humans and cattle. The aflagellate strains were impaired in their proinflammatory propertiesin vitroandin vivo. The aim of this work was to elucidate the underlying cause of the absence of flagella inS. Dublin isolates. We report here that class 3 flagellar genes are repressed in the human aflagellate isolates, due to impaired secretion of FliA anti-sigma factor FlgM. This phenotype is due to an in-frame 42-nucleotide deletion in thefliEgene, which codes for a protein located in the flagellar basal body. The deletion is predicted to produce a protein lacking amino acids 18 to 31. The aflagellate phenotype was highly stable; revertants were obtained only whenfliAwas artificially overexpressed combined with several successive passages in motility agar. DNA sequence analysis revealed that motile revertants resulted from duplications of DNA sequences infliEadjacent to the deleted region. These duplications produced a FliE protein of similar length to the wild type and demonstrate that amino acids 18 to 31 of FliE are not essential. The same deletion was detected inS. Dublin isolates obtained from cattle, indicating that this mutation circulates in nature.

scholarly journals Functional Domains of Yeast Plasmid-Encoded Rep Proteins

2001 ◽  
Vol 183 (7) ◽  
pp. 2306-2315 ◽  
Author(s):  
A. Sengupta ◽  
K. Blomqvist ◽  
A. J. Pickett ◽  
Y. Zhang ◽  
J. S. K. Chew ◽  
...  
Keyword(s):  
Amino Acids ◽  
Protein Interaction ◽  
Yeast Plasmid ◽  
Amino Terminal ◽  
Plasmid Segregation ◽  
Terminal Domain ◽  
Rep Proteins ◽  
Self Association ◽  
Carboxy Terminal

ABSTRACT Both of the Saccharomyces cerevisiae 2μm circle-encoded Rep1 and Rep2 proteins are required for efficient distribution of the plasmid to daughter cells during cellular division. In this study two-hybrid and in vitro protein interaction assays demonstrate that the first 129 amino acids of Rep1 are sufficient for self-association and for interaction with Rep2. Deletion of the first 76 amino acids of Rep1 abolished the Rep1-Rep2 interaction but still allowed some self-association, suggesting that different but overlapping domains specify these interactions. Amino- or carboxy-terminally truncated Rep1 fusion proteins were unable to complement defective segregation of a 2μm-based stability vector withrep1 deleted, supporting the idea of the requirement of Rep protein interaction for plasmid segregation but indicating a separate required function for the carboxy-terminal portion of Rep1. The results of in vitro baiting assays suggest that Rep2 contains two nonoverlapping domains, both of which are capable of mediating Rep2 self-association. The amino-terminal domain interacts with Rep1, while the carboxy-terminal domain was shown by Southwestern analysis to have DNA-binding activity. The overlapping Rep1 and Rep2 interaction domains in Rep1, and the ability of Rep2 to interact with Rep1, Rep2, and DNA, suggest a model in which the Rep proteins polymerize along the 2μm circle plasmid stability locus, forming a structure that mediates plasmid segregation. In this model, competition between Rep1 and Rep2 for association with Rep1 determines the formation or disassembly of the segregation complex.

scholarly journals Two domains of p53 interact with the TATA-binding protein, and the adenovirus 13S E1A protein disrupts the association, relieving p53-mediated transcriptional repression.

1995 ◽  
Vol 15 (1) ◽  
pp. 227-234 ◽  
Author(s):  
N Horikoshi ◽  
A Usheva ◽  
J Chen ◽  
A J Levine ◽  
R Weinmann ◽  
...  
Keyword(s):  
Amino Acids ◽  
Transcriptional Activation ◽  
Transcriptional Repression ◽  
Binding Protein ◽  
Direct Interaction ◽  
Tata Binding Protein ◽  
Amino Terminal ◽  
Terminal Domain ◽  
Carboxy Terminal

The tumor suppressor gene product p53 can activate and repress transcription. Both transcriptional activation and repression are thought to involve the direct interaction of p53 with the basal transcriptional machinery. Previous work has demonstrated an in vitro interaction between p53 and the TATA-binding protein that requires amino acids 20 to 57 of p53 and amino acids 220 to 271 of the TATA-binding protein. The present results show that a 75-amino-acid segment from the carboxy terminus of p53 also can bind to the TATA-binding protein in vitro, and this interaction requires amino acids 217 to 268 of the TATA-binding protein, essentially the same domain that is required for interaction with the amino-terminal domain of p53. A carboxy-terminal segment of p53 can mediate repression when bound to DNA as a GAL4-p53 fusion protein. The amino- and carboxy-terminal p53 interactions occur within the domain on the TATA-binding protein to which the adenovirus 13S E1A oncoprotein has previously been shown to bind. The 13S E1A oncoprotein can dissociate the complex formed between the carboxy-terminal domain of p53 and the TATA-binding protein and relieve p53-mediated transcriptional repression. These results demonstrate that two independent domains of p53 can potentially interact with the TATA-binding protein, and they define a mechanism--relief of repression--by which the 13S E1A oncoprotein can activate transcription through the TATA motif.

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