Biomedical subjects

Paul Rava

Publications and source records attributed to Paul Rava.

4 recordsLinked to original sources

PubMed2006

Phospholipid transfer activity of microsomal triacylglycerol transfer protein is sufficient for the assembly and secretion of apolipoprotein B lipoproteins.

Human microsomal triacylglycerol transfer protein (hMTP) is essential for apolipoprotein B (apoB)-lipoprotein assembly and secretion and is known to transfer triacylglycerols, cholesterol esters, and phospholipids. To understand the relative importance of each lipid transfer activity, we compared the ability of hMTP and its Drosophila ortholog (dMTP) to assemble apoB lipoproteins and to transfer various lipids. apoB48 secretion was induced when co-expressed with either hMTP or dMTP in COS cells, and oleic acid supplementation further augmented secretion without altering particle density. C-terminal epitope-tagged dMTP (dMTP-FLAG) facilitated the secretion of apoB polypeptides in the range of apoB48 to apoB72 but was approximately 50% as efficient as hMTP-FLAG. Comparison of lipid transfer activities revealed that although phospholipid transfer was similar in both orthologs, dMTP was unable to transfer neutral lipids. We conclude that the phospholipid transfer activity of MTP is sufficient for the assembly and secretion of primordial apoB lipoproteins and may represent its earliest function evolved for the mobilization of lipid in invertebrates. Identification of MTP inhibitors, which selectively affect transfer of a specific lipid class, may have therapeutic potential.

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PubMed2005

Microsomal triglyceride transfer protein lipidation and control of CD1d on antigen-presenting cells.

Microsomal triglyceride transfer protein (MTP), an endoplasmic reticulum (ER) chaperone that loads lipids onto apolipoprotein B, also regulates CD1d presentation of glycolipid antigens in the liver and intestine. We show MTP RNA and protein in antigen-presenting cells (APCs) by reverse transcription-polymerase chain reaction and by immunoblotting of mouse liver mononuclear cells and mouse and human B cell lines. Functional MTP, demonstrated by specific triglyceride transfer activity, is present in both mouse splenocytes and a CD1d-positive mouse NKT hybridoma. In a novel in vitro transfer assay, purified MTP directly transfers phospholipids, but not triglycerides, to recombinant CD1d. Chemical inhibition of MTP lipid transfer does not affect major histocompatibility complex class II presentation of ovalbumin, but considerably reduces CD1d-mediated presentation of alpha-galactosylceramide (alpha-galcer) and endogenous antigens in mouse splenic and bone marrow-derived dendritic cells (DCs), as well as in human APC lines and monocyte-derived DCs. Silencing MTP expression in the human monocyte line U937 affects CD1d function, as shown by diminished presentation of alpha-galcer. We propose that MTP acts upstream of the saposins and functions as an ER chaperone by loading endogenous lipids onto nascent CD1d. Furthermore, our studies suggest that a small molecule inhibitor could be used to modulate the activity of NKT cells.

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PubMed2005

Transfer of cholesteryl esters and phospholipids as well as net deposition by microsomal triglyceride transfer protein.

Microsomal triglyceride transfer protein (MTP) activity is classically measured using radioactive lipids. We described a simple fluorescence assay to measure its triacylglycerol (TAG) transfer activity. Here, we describe fluorescence-based methods to measure the transfer of phospholipids (PLs) and cholesteryl esters (CEs) by MTP. Both transfer activities increased with time and MTP amounts and were inhibited to different extents by an MTP antagonist, BMS197636. We also describe a method to measure the net deposition of fluorescent lipids in acceptor vesicles. In this procedure, negatively charged donor vesicles are incubated with MTP and acceptor vesicles, and lipids transferred to acceptors are quantified after the removal of donor vesicles and MTP by the addition of DE52. Lipid deposition in acceptor vesicles was dependent on time and MTP. Using these methods, TAG transfer activity was the most robust activity present in purified MTP; CE and PL transfer activities were 60-71% and 5-13% of the TAG transfer activity, respectively. The method to determine lipid transfer is recommended for routine MTP activity measurements for its simplicity. These methods may help identify specific inhibitors for individual lipid transfer activities, in characterizing different domains involved in transfer, and in the isolation of mutants that bind but cannot transfer lipids.

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PubMed2003

Microsomal triglyceride transfer protein: a multifunctional protein.

Microsomal triglyceride transfer protein (MTP) is a heterodimeric protein that transfers neutral lipids between membranes in vitro. Absence of this lipid transfer activity in the microsomes of abetalipoproteinemia patients established its pivotal function in lipoprotein assembly. Recent studies indicate that the lipid transfer activity is involved in importing triglycerides into the lumen of the endoplasmic reticulum. In addition to its lipid transfer activity, MTP physically interacts with apoB. This led to speculation that MTP may act as a chaperone. It remains to be determined whether the binding of MTP to apoB plays a role in either proper folding or net lipidation of nascent apoB. Both functions, lipid transfer and apoB binding, may be involved in the initial step of lipidation of nascent apoB resulting in the synthesis of primordial lipoprotein particles. Furthermore, it has been shown that MTP stably associates with lipid vesicles. The lipid-associated MTP may be important in core expansion of primordial lipoproteins. In summary, three independent functions (lipid transfer, apoB binding and membrane association) of MTP have been identified. Here, we propose these functions are carried out by a combination of different structural motifs. Based on sequence homology with lipovitellin, the M subunit of MTP is predicted to contain three beta-sheets (A, C, and N) and one alpha-helical domain. The A- and C-sheets may be involved in lipid transfer, the N-sheet and the helical domain in apoB binding, and the N- and A-sheets in membrane association. It is also speculated that MTP may function in physiologic processes beyond lipoprotein assembly.

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