Abstract 192: Characterization of Microsomal Triglyceride Transfer Protein Missense Mutations Found in Abetalipoproteinemia and Hybobetalipoproteinemia Subjects

2016 ◽  
Vol 36 (suppl_1) ◽  
Author(s):  
Meghan T Walsh ◽  
Enza Di Leo ◽  
Patrizia Tarugi ◽  
M. Mahmood Hussain
Keyword(s):  
Plasma Lipid ◽  
Microsomal Triglyceride Transfer Protein ◽  
The Other ◽  
Plasma Lipoproteins ◽  
Missense Mutations ◽  
Lipid Transfer ◽  
Novel Proteins ◽  
Transfer Protein ◽  
Apob Secretion ◽  
Familial Hypobetalipoproteinemia

We describe two new hypolipidemic patients with very low plasma triglyceride and apolipoprotein B (apoB) levels and lipid malabsorption with plasma lipid profiles similar to abetalipoproteinemia (ABL) patients. In these patients, we identified two previously uncharacterized missense mutations in the microsomal triglyceride transfer protein (MTP) gene, R46G and D361Y, and studied their effects on function. We also characterized three missense mutations (H297Q, D384A, and G661A) reported earlier in a familial hypobetalipoproteinemia patient. R46G had no effect on MTP expression or function and supported apoB secretion. Similarly, H297Q, D384A, and G661A mutants supported apoB secretion similarly to WT MTP. Contrary to these four missense mutations, D361Y was unable to support apoB secretion. Functional analysis revealed that this mutant was unable to bind protein disulfide isomerase (PDI) or transfer lipids. The negative charge at residue 361 was critical for MTP function as D361E was able to support apoB secretion and transfer lipids. D361Y most likely disrupts the tightly packed middle α-helical region of MTP, mitigates PDI binding, abolishes lipid transfer activity, and causes ABL. On the other hand, the hypolipidemia in the other two patients was not due to MTP dysfunction. Thus, in this study of five missense mutations spread throughout MTP’s three structural domains found in three hypolipidemic patients, we found that four of the mutations did not affect MTP function. Thus, there probably exist novel mutations in other genes that cause severe hypolipidemia and their recognition may identify novel proteins involved in the synthesis and/or catabolism of plasma lipoproteins.

Abstract 232: A Novel Abetalipoproteinemia Missense Mutation Highlights the Importance of the N-Terminal ß-Sheet in the Lipid Transfer and ApoB Secretion Activities of Microsomal Triglyceride Transfer Protein

2014 ◽  
Vol 34 (suppl_1) ◽  
Author(s):  
Meghan T Walsh ◽  
Enza Di Leo ◽  
Eda Ozaydin ◽  
Patrizia Tarugi ◽  
Mahmood Hussain
Keyword(s):  
Missense Mutation ◽  
Structural Integrity ◽  
Microsomal Triglyceride Transfer Protein ◽  
Salt Bridge ◽  
Missense Mutations ◽  
Lipid Transfer ◽  
Transfer Protein ◽  
Transfer Activity ◽  
Apob Secretion ◽  
Β Sheet

Microsomal triglyceride transfer protein (MTP) is critical for the assembly and secretion of apolipoprotein B (apoB)-containing lipoproteins. Mutations in the MTTP gene cause abetalipoproteinemia (ABL). Missense mutations in ABL have revealed that the central α-helical and C-terminal β-sheet domains are important for the lipid transfer activity of MTP and for the assembly and secretion of apoB-containing lipoproteins. The N-terminal domain, on the other hand, has mainly been implicated in apoB- and membrane-binding. Here, we describe a novel ABL missense mutation (D169V) in the N-terminal β-sheet of MTP. Although this mutant MTP (MTPD169V) is expressed and localized to the endoplasmic reticulum, it is unable to transfer triglycerides and phospholipids. Further, MTPD169V does not support the assembly and secretion of apoB-containing lipoproteins. Computational molecular modeling suggests that D169 could form an internal salt bridge with K187 and K189. Indeed, mutagenesis of these lysine residues to leucine abolishes triglyceride transfer and apoB secretion activities of MTP. Furthermore, conservative mutagenesis that preserves charges on these residues partially restores triglyceride transfer and apoB secretion activities of MTP. Therefore, D169 is probably involved in an internal salt bridge with K187 and K189. Disruption of this internal salt bridge in the N-terminal region affects the lipid transfer activity present in the C-terminal end of the MTP molecule. We speculate that this salt bridge, although away from the speculated lipid transfer site, might be important in providing structural integrity necessary for the lipid transfer activity of MTP.

scholarly journals MTP regulated by an alternate promoter is essential for NKT cell development

2007 ◽  
Vol 204 (3) ◽  
pp. 533-545 ◽  
Author(s):  
Stephanie K. Dougan ◽  
Paul Rava ◽  
M. Mahmood Hussain ◽  
Richard S. Blumberg
Keyword(s):  
Lipid Transfer Protein ◽  
Microsomal Triglyceride Transfer Protein ◽  
Cell Development ◽  
Northern Analysis ◽  
Lipid Transfer ◽  
Nkt Cell ◽  
Transfer Protein ◽  
Phospholipid Transfer ◽  
Apob Secretion

Microsomal triglyceride transfer protein (MTP), an endoplasmic reticulum lipid transfer protein critical for apolipoprotein B (apoB) secretion, regulates CD1d antigen presentation. We identified MTP variant 1 (MTPv1), a novel splice variant of mouse MTP, by polymerase chain reaction and Northern analysis in non–apoB-secreting tissues, including thymocytes and antigen-presenting cells (APCs). Edman degradation of MTPv1 isolated from transfected cells revealed three unique residues; however, recombinant MTP and MTPv1 had an equivalent protein disulfide isomerase association, subcellular localization, triglyceride transfer, phospholipid transfer, response to inhibitors, and ability to support apoB secretion. MTP and MTPv1 efficiently transferred phosphatidylethanolamine to CD1d in vitro. NKT cells fail to develop in fetal thymic organ culture (FTOC) treated with MTP antagonists. MTP-inhibited FTOCs produced negligible numbers of CD1d tetramer–positive cells and exhibited marked defects in IL-4 production upon stimulation with anti-CD3 or α-galactosylceramide–pulsed APCs. CD1d expression on CD4+CD8+ FTOC cells was unaffected by MTP inhibition. Thus, our results demonstrate that MTPv1 in thymocytes is critical to NKT cell development. We hypothesize that, when MTP is inactive, CD1d traffics to the cell surface and presents no lipid or a lipid that is incapable of mediating NKT cell selection and/or is refractory to lysosomal editing.

scholarly journals Structure of the lutein-binding domain of human StARD3 at 1.74 Å resolution and model of a complex with lutein

2016 ◽  
Vol 72 (8) ◽  
pp. 609-618 ◽  
Author(s):  
Martin P. Horvath ◽  
Evan W. George ◽  
Quang T. Tran ◽  
Kody Baumgardner ◽  
Gabe Zharov ◽  
...  
Keyword(s):  
Lipid Transfer Protein ◽  
Transport Proteins ◽  
Binding Domain ◽  
Regions Of Interest ◽  
The Other ◽  
Lipid Transfer ◽  
Transfer Protein ◽  
Biochemical Function ◽  
Rigid Body Docking ◽  
Resolution Structure

A crystal structure of the lutein-binding domain of human StARD3 (StAR-related lipid-transfer protein 3; also known as MLN64) has been refined to 1.74 Å resolution. A previous structure of the same protein determined to 2.2 Å resolution highlighted homology with StARD1 and shared cholesterol-binding character. StARD3 has since been recognized as a carotenoid-binding protein in the primate retina, where its biochemical function of binding lutein with specificity appears to be well suited to recruit this photoprotective molecule. The current and previous structures correspond closely to each other (r.m.s.d. of 0.25 Å), especially in terms of the helix-grip fold constructed around a solvent-filled cavity. Regions of interest were defined with alternate conformations in the current higher-resolution structure, including Arg351 found within the cavity and Ω1, a loop of four residues found just outside the cavity entrance. Models of the complex with lutein generated by rigid-body docking indicate that one of the ionone rings must protrude outside the cavity, and this insight has implications for molecular interactions with transport proteins and enzymes that act on lutein. Interestingly, models with the ∊-ionone ring characteristic of lutein pointing towards the bottom of the cavity were associated with fewer steric clashes, suggesting that steric complementarity and ligand asymmetry may play a role in discriminating lutein from the other ocular carotenoids zeaxanthin andmeso-zeaxanthin, which only have β-ionone rings.

Purification, characterization, and conformational analysis of rabbit plasma lipid transfer protein

Biochemistry ◽  
1993 ◽  
Vol 32 (26) ◽  
pp. 6729-6736 ◽  
Author(s):  
Kerry W. S. Ko ◽  
Kim Oikawa ◽  
Taira Ohnishi ◽  
Cyril M. Kay ◽  
Shinji Yokoyama
Keyword(s):  
Conformational Analysis ◽  
Lipid Transfer Protein ◽  
Plasma Lipid ◽  
Lipid Transfer ◽  
Rabbit Plasma ◽  
Transfer Protein

scholarly journals The lipid transfer properties of CETP define the concentration and composition of plasma lipoproteins

2020 ◽  
Vol 61 (8) ◽  
pp. 1168-1179 ◽  
Author(s):  
Richard E. Morton ◽  
Yan Liu
Keyword(s):  
Substrate Preference ◽  
Plasma Lipoproteins ◽  
Lipid Transfer ◽  
Hepatic Expression ◽  
Transfer Protein ◽  
Novel Approach ◽  
Human Ortholog ◽  
Low Levels ◽  
Transfer Properties

Cholesteryl ester transfer protein (CETP) facilitates the net transfer of cholesteryl esters (CEs) and TGs between lipoproteins, impacting the metabolic fate of these lipoproteins. Previous studies have shown that a CETP antibody can alter CETP’s preference for CE versus TG as transfer substrate, suggesting that CETP substrate preference can be manipulated in vivo. Hamster and human CETPs have very different preferences for CE and TG. To assess the effect of altering CETP’s substrate preference on lipoproteins in vivo, here, we expressed human CETP in hamsters. Chow-fed hamsters received adenoviruses expressing no CETP, hamster CETP, or human CETP. Plasma CETP mass increased 2-fold in both the hamster and human CETP groups. Although the animals expressing human CETP still had low levels of hamster CETP, the CE versus TG preference of their plasma CETP was similar to that of the human ortholog. Hamster CETP overexpression had little impact on lipoproteins. However, expression of human CETP reduced HDL up to 50% and increased VLDL cholesterol 2.5-fold. LDL contained 20% more CE, whereas HDL CE was reduced 40%, and TG increased 6-fold. The HDL3:HDL2 ratio increased from 0.32 to 0.60. Hepatic expression of three cholesterol-related genes (LDLR, SCARB1, and CYP7A1) was reduced up to 40%. However, HDL-associated CE excretion into feces was unchanged. We conclude that expression of human CETP in hamsters humanizes their lipoprotein profile with respect to the relative concentrations of VLDL, LDL, HDL, and the HDL3:HDL2 ratio. Altering the lipid substrate preference of CETP provides a novel approach for modifying plasma lipoproteins.

scholarly journals Human CETP lacks lipopolysaccharide transfer activity, but worsens inflammation and sepsis outcomes in mice

2020 ◽  
pp. jlr.RA120000704
Author(s):  
Aloïs Dusuel ◽  
Valérie Deckert ◽  
Jean-Paul PAIS DE BARROS ◽  
Kevin Van Dongen ◽  
Hélène Choubley ◽  
...  
Keyword(s):  
Cecal Ligation ◽  
Plasma Lipid ◽  
Plasma Lipoproteins ◽  
Protective Effects ◽  
Lipid Transfer ◽  
Transfer Property ◽  
Transfer Activity ◽  
Lps Binding

Bacterial lipopolysaccharides (LPSs or endotoxins) can bind most proteins of the lipid transfer/LPS-binding protein (LT/LBP) family in host organisms. The LPS-bound LT/LBP proteins then trigger either an LPS-induced proinflammatory cascade or LPS binding to lipoproteins that are involved in endotoxin inactivation and detoxification. Cholesteryl ester transfer protein (CETP) is an LT/LBP member, but its impact on LPS metabolism and sepsis outcome is unclear. Here, we performed fluorescent LPS transfer assays to assess the ability of CETP to bind and transfer LPS. The effects of intravenous (iv) infusion of purified LPS or polymicrobial infection (cecal ligation and puncture [CLP]) were compared in transgenic mice expressing human CETP and wild-type mice naturally having no CETP activity. CETP displayed no LPS transfer activity in vitro, but it tended to reduce biliary excretion of LPS in vivo. The CETP expression in mice was associated with significantly lower basal plasma lipid levels and with higher mortality rates in both models of endotoxemia and sepsis. Furthermore, CETPTg plasma modified cytokine production of macrophages in vitro. In conclusion, despite having no direct LPS binding and transfer property, human CETP worsens sepsis outcomes in mice by altering the protective effects of plasma lipoproteins against endotoxemia, inflammation, and infection.

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