scholarly journals First eight residues of apolipoprotein A-I mediate the C-terminus control of helical bundle unfolding and its lipidation

2019 ◽  
Author(s):  
Gregory Brubaker ◽  
Shuhui W. Lorkowski ◽  
Kailash Gulshan ◽  
Stanley L. Hazen ◽  
Valentin Gogonea ◽  
...  
Keyword(s):  
Cholesterol Efflux ◽  
Thermal Denaturation ◽  
Point Mutations ◽  
Lipid Binding ◽  
Site Directed Mutagenesis ◽  
Amino Terminus ◽  
Helical Bundle ◽  
Apolipoprotein A ◽  
C Terminus ◽  
Bundle Structure

AbstractThe crystal structure of a C-terminal deletion of apolipoprotein A-I (apoA1) shows a large helical bundle structure in the amino half of the protein, from residues 8 to 115. Using site directed mutagenesis, guanidine or thermal denaturation, cell free liposome clearance, and cellular ABCA1-mediated cholesterol efflux assays, we demonstrate that apoA1 lipidation can occur when the barrier to this bundle unfolding is lowered. The absence of the C-terminus renders the bundle harder to unfold resulting in loss of apoA1 lipidation that can be reversed by point mutations, such as Trp8Ala, and by truncations as short as 8 residues in the amino terminus, both of which lower the barrier to helical bundle unfolding. Locking the bundle via a disulfide bond leads to loss of apoA1 lipidation. We propose a model in which the C-terminus acts on the N-terminus to destabilize helical bundle. Upon lipid binding to the C-terminus, Trp8 is displaced from its interaction with Phe57, Arg61, Leu64, Val67, Phe71, and Trp72 to destabilize the bundle. However, when the C-terminus is deleted, Trp8 cannot be displaced, the bundle cannot unfold, and apoA1 cannot be lipidated.

N-homocysteinylation of apolipoprotein A-I impairs the protein’s antioxidant ability but not its cholesterol efflux capacity

2014 ◽  
Vol 395 (6) ◽  
pp. 641-648 ◽  
Author(s):  
Akari Miyazaki ◽  
Nozomi Sagae ◽  
Yoko Usami ◽  
Megumi Sato ◽  
Takahiro Kameda ◽  
...  
Keyword(s):  
Cholesterol Efflux ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Lipid Binding ◽  
Binding Activity ◽  
Antioxidant Ability ◽  
Wild Type ◽  
Cholesterol Efflux Capacity ◽  
Apolipoprotein A ◽  
Significant Difference

Abstract A high homocysteine (Hcy) level is a risk factor for atherosclerosis. Hcy can be added to proteins through a process known as N-homocysteinylation. This is thought to be a potential cause of atherosclerosis induction. We previously reported that N-homocysteinylated apolipoprotein A-I (N-Hcy-apoA-I) was identified in normal human plasma. In this study, the effect of N-homocysteinylation on the functions of apoA-I was examined. A kinetic study using dimyristoyl phosphatidylcholine (DMPC) liposomes indicated that N-Hcy-apoA-I showed increased lipid-binding activity compared to wild-type apoA-I. Two reconstituted high-density lipoprotein (rHDL) particles of different sizes (approximately 8.2 nm and 7.6 nm in diameter) were produced by mixing apoA-I and 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC). However, an increased ratio of large to small particles was found in rHDL prepared with N-Hcy-apoA-I. The normal apoA-I antioxidant ability, estimated by the suppression of conjugated diene formation in low-density lipoprotein (LDL) induced by copper sulfate oxidation, was considerably impaired when using N-Hcy-apoA-I. Although N-Hcy-apoA-I functioned as an oxidant, no significant difference was observed in the cholesterol efflux capacity from THP-1 macrophages between wild-type apoA-I and N-Hcy-apoA-I. These results suggest that N-Hcy-apoA-I might be proatherogenic due to its oxidative behavior but not an attenuation of cholesterol efflux capacity.

Transthyretin is a metallopeptidase with an inducible active site

2012 ◽  
Vol 443 (3) ◽  
pp. 769-778 ◽  
Author(s):  
Márcia A. Liz ◽  
Sérgio C. Leite ◽  
Luiz Juliano ◽  
Maria J. Saraiva ◽  
Ana M. Damas ◽  
...  
Keyword(s):  
Active Site ◽  
Three Dimensional ◽  
Point Mutations ◽  
Site Directed Mutagenesis ◽  
Side Chain ◽  
Metal Chelators ◽  
Serine Residue ◽  
General Base ◽  
Apolipoprotein A ◽  
Β Amyloid

TTR (transthyretin) was found recently to possess proteolytic competency besides its well-known transport capabilities. It was described as a cryptic serine peptidase cleaving multiple natural substrates (including β-amyloid and apolipoprotein A-I) involved in diseases such as Alzheimer's disease and atherosclerosis. In the present study, we aimed to elucidate the catalytic machinery of TTR. All attempts to identify a catalytic serine residue were unsuccessful. However, metal chelators abolished TTR activity. Proteolytic inhibition by EDTA or 1,10-phenanthroline could be reversed with Zn2+ and Mn2+. These observations, supported by analysis of three-dimensional structures of TTR complexed with Zn2+, led to the hypothesis that TTR is a metallopeptidase. Site-directed mutagenesis of selected amino acids unambiguously confirmed this hypothesis. The TTR active site is inducible and constituted via a protein rearrangement resulting in ~7% of proteolytically active TTR at pH 7.4. The side chain of His88 is shifted near His90 and Glu92 establishing a Zn2+-chelating pattern HXHXE not found previously in any metallopeptidase and only conserved in TTR of humans and some other primates. Point mutations of these three residues yielded proteins devoid of proteolytic activity. Glu72 was identified as the general base involved in activation of the catalytic water. Our results unveil TTR as a metallopeptidase and define its catalytic machinery.

scholarly journals First eight residues of apolipoprotein A-I mediate the C-terminus control of helical bundle unfolding and its lipidation

PLoS ONE ◽  
2020 ◽  
Vol 15 (1) ◽  
pp. e0221915
Author(s):  
Gregory Brubaker ◽  
Shuhui W. Lorkowski ◽  
Kailash Gulshan ◽  
Stanley L. Hazen ◽  
Valentin Gogonea ◽  
...  
Keyword(s):  
Helical Bundle ◽  
Apolipoprotein A ◽  
C Terminus

scholarly journals The C-Terminal Region of Bacteroides fragilis Toxin Is Essential to Its Biological Activity

2006 ◽  
Vol 74 (10) ◽  
pp. 5595-5601 ◽  
Author(s):  
Cynthia L. Sears ◽  
Simy L. Buckwold ◽  
Jai W. Shin ◽  
Augusto A. Franco
Keyword(s):  
Biological Activity ◽  
Amino Acid ◽  
Point Mutations ◽  
Bacteroides Fragilis ◽  
Terminal Region ◽  
Site Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Wild Type ◽  
Reverse Transcription Pcr ◽  
C Terminus

ABSTRACT To evaluate the role of the C-terminal region in Bacteroides fragilis toxin (BFT) activity, processing, and secretion, sequential C-terminal truncation and point mutations were created by site-directed mutagenesis. Determination of BFT activity on HT29/C1 cells, cleavage of E-cadherin, and the capacity to induce interleukin-8 secretion by wild-type BFT and C-terminal deletion mutants showed that deletion of only 2 amino acid residues at the C terminus significantly reduced BFT biological activity and deletion of eight or more amino acid residues obliterated BFT biologic activity. Western blot and reverse transcription-PCR analyses indicated that BFT mutants lacking seven or fewer amino acid residues in the C-terminal region are processed and expressed similar to wild-type BFT. However, BFT mutants lacking eight or more amino acids at the C terminus are expressed similar to wild-type BFT but are unstable. We concluded that the C terminus of BFT is not tolerant of modest amino acid deletions, suggesting that it is biologically important for BFT activity.

scholarly journals Structure-function studies of apoA-I variants

2002 ◽  
Vol 43 (8) ◽  
pp. 1283-1292 ◽  
Author(s):  
Dmitri Sviridov ◽  
Anh Hoang ◽  
Wei Huang ◽  
Jun Sasaki
Keyword(s):  
Central Region ◽  
Binding Sites ◽  
Cholesterol Efflux ◽  
Lipid Binding ◽  
Binding Properties ◽  
Apolipoprotein A ◽  
Self Association ◽  
Rate Limiting ◽  
Lower Capacity ◽  
Combined Data

Five mutants of apolipoprotein A-I (apoA-I), apoA-I(Δ63–73), apoA-I(Δ140–150), apoA-I(63–73@140–150), apoA-I(R149V), and apoA-I(P143A) were compared with human plasma apoA-I for their ability to promote cholesterol and phospholipid efflux from HepG2 cells. A significantly lower capacity to promote cholesterol and phospholipid efflux was observed with lipid-free apoA-I(Δ63–73), while mutations apoA-I(Δ140–150) and apoA-I(P143A) affected phospholipid efflux only. When added as apoA-I/palmitoyloleoyl phosphatidylcholine (POPC) complex, mutations apoA-I(63–73@140–150) and apoA-I(Δ140–150) affected cholesterol efflux. None of the mutations affected α-helicity of the lipid-free mutants or their self-association. Five natural mutations of apoA-I, apoA-I(A95D), apoA-I (Y100H), apoA-I(E110K), apoA-I(V156E), and apoA-I (H162Q) were studied for their ability to bind lipids and promote cholesterol efflux. None of the mutations affected lipid-binding properties, cholesterol efflux, or α-helicity of lipid-free mutants. Two mutations affected self-association of apoA-I: apoA-I(A95D) was more prone to self-association, while apoA-I(E100H) did not self-associate.The following conclusions could be made from the combined data: i) regions 210–243 and 63–100 are the lipid-binding sites of apoA-I and are also required for the efflux of lipids to lipid-free apoA-I, suggesting that initial lipidation of apoA-I is rate limiting in efflux; ii) in addition to the lipid-binding regions, the central region is important for cholesterol efflux to lipidated apoA-I, suggesting its possible involvement in interaction with cells.

scholarly journals Point mutations that inactivate MGAT4D-L, an inhibitor of MGAT1 and complex N-glycan synthesis

2020 ◽  
Vol 295 (41) ◽  
pp. 14053-14064
Author(s):  
Ayodele Akintayo ◽  
Joshua Mayoral ◽  
Masahiro Asada ◽  
Jian Tang ◽  
Subha Sundaram ◽  
...  
Keyword(s):  
Amino Acids ◽  
Inhibitory Activity ◽  
Mammalian Cells ◽  
Point Mutations ◽  
Site Directed Mutagenesis ◽  
Mechanism Of Inhibition ◽  
C Terminus ◽  
Membrane Bound ◽  
Galanthus Nivalis ◽  
Glcnac Transferase

The membrane-bound, long form of MGAT4D, termed MGAT4D-L, inhibits MGAT1 activity in transfected cells and reduces the generation of complex N-glycans. MGAT1 is the GlcNAc-transferase that initiates complex and hybrid N-glycan synthesis. We show here that Drosophila MGAT1 was also inhibited by MGAT4D-L in S2 cells. In mammalian cells, expression of MGAT4D-L causes the substrate of MGAT1 (Man5GlcNAc2Asn) to accumulate on glycoproteins, a change that is detected by the lectin Galanthus nivalis agglutinin (GNA). Using GNA binding as an assay for the inhibition of MGAT1 in MGAT4D-L transfectants, we performed site-directed mutagenesis to determine requirements for MGAT1 inhibition. Deletion of 25 amino acids (aa) from the C terminus inactivated MGAT4D-L, but deletion of 20 aa did not. Conversion of the five key amino acids (PSLFQ) to Ala, or deletion of PSLFQ in the context of full-length MGAT4D-L, also inactivated MGAT1 inhibitory activity. Nevertheless, mutant, inactive MGAT4D-L interacted with MGAT1 in co-immuno-precipitation experiments. The PSLFQ sequence also occurs in MGAT4A and MGAT4B GlcNAc-transferases. However, neither inhibited MGAT1 in transfected CHO cells. MGAT4D-L inhibitory activity could be partially transferred by attaching PSLFQ or the 25-aa C terminus of MGAT4D-L to the C terminus of MGAT1. Mutation of each amino acid in PSLFQ to Ala identified both Leu and Phe as independently essential for MGAT4D-L activity. Thus, replacement of either Leu-395 or Phe-396 with Ala led to inactivation of MGAT4D-L inhibitory activity. These findings provide new insights into the mechanism of inhibition of MGAT1 by MGAT4D-L, and for the development of small molecule inhibitors of MGAT1.

scholarly journals The C Terminus of Apolipoprotein A-V Modulates Lipid-binding Activity

2007 ◽  
Vol 282 (21) ◽  
pp. 15484-15489 ◽  
Author(s):  
Jennifer A. Beckstead ◽  
Kasuen Wong ◽  
Vinita Gupta ◽  
Chung-Ping L. Wan ◽  
Victoria R. Cook ◽  
...  
Keyword(s):  
Lipid Binding ◽  
Binding Activity ◽  
Apolipoprotein A ◽  
C Terminus
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