Protein disulfide isomerase is a component of the microsomal triglyceride transfer protein complex

A bovine liver protein which catalyzes the transfer of triglyceride between membranes has previously been isolated from the lumen of the microsomal fraction. When further purified about 100-fold, two polypeptides of molecular mass 58,000 and 88,000 were identified (Wetterau, J. R., and Zilversmit, D. B. (1985) Chem. Phys. Lipids 38, 205-222). We demonstrate here that the two polypeptides (referred to as 58-kDa and 88-kDa, respectively) are associated in a protein-protein complex, and that the triglyceride transfer activity is associated with this complex. Antibodies specific for either polypeptide immunoprecipitated both the 58-kDa and 88-kDa polypeptides as well as the lipid transfer activity. The 58-kDa subunit of the microsomal transfer protein complex was identified as protein disulfide-isomerase (PDI) (EC 5.3.4.1) by 1) a comparison of the amino-terminal sequence of PDI and the 58-kDa subunit of the transfer protein, 2) a comparison of the reverse phase high performance liquid chromatography peptide maps of CNBr digests of PDI and the lipid transfer protein, 3) immunoprecipitation competition experiments in which PDI was found to compete with the lipid transfer protein for immunoprecipitation by the anti-58-kDa polyclonal antibodies, 4) immunological cross-reactivity of the microsomal triglyceride transfer protein complex with polyclonal antibodies raised against PDI, and 5) the appearance of protein disulfide isomerase activity following the dissociation of purified microsomal transfer protein complex with guanidine HCl. In conclusion, the microsomal triglyceride transfer protein has a multi-subunit structure which is unique compared to other intracellular lipid transfer proteins which have been described to be single polypeptides. The unexpected finding that PDI is a component of the microsomal triglyceride transfer protein complex suggests a new previously undescribed role for protein disulfide isomerase.     

Protein disulfide isomerase serves as a molecular chaperone to maintain estrogen receptor alpha structure and function

The effects of the steroid hormone 17beta-estradiol are mediated through its interaction with the nuclear estrogen receptor (ER). Upon binding 17beta-estradiol, the ER initiates changes in gene expression through its interaction with specific DNA sequences, estrogen response elements (EREs), and recruits coregulatory proteins that influence gene expression. To better understand how estrogen-responsive genes are regulated, we have isolated and identified proteins associated with ERalpha when it is bound to the consensus ERE. One of these proteins, protein disulfide isomerase (PDI), has two distinct functions: acting as a molecular chaperone to maintain properly folded proteins and regulating the redox state of proteins by catalyzing the thiol-disulfide exchange reaction through two thioredoxin-like domains. Using a battery of biochemical and molecular techniques, we have demonstrated that PDI colocalizes with ERalpha in MCF-7 nuclei, alters ERalpha conformation, enhances the ERalpha-ERE interaction in the absence and presence of an oxidizing agent, influences the ability of ERalpha to mediate changes in gene expression, and associates with promoter regions of two endogenous estrogen-responsive genes. Our studies suggest that PDI plays a critical role in estrogen responsiveness by functioning as a molecular chaperone and assisting the receptor in differentially regulating target gene expression.     

Subcellular localization of microsomal triglyceride transfer protein

Microsomal triglyceride transfer protein (MTP) is essential for the assembly of apolipoprotein B-containing lipoproteins. Within the endoplasmic reticulum, it transfers lipid from the membrane to the forming lipoprotein. Recent evidence suggests that it may also function within the Golgi apparatus. To address this hypothesis, we developed a polyclonal antibody to MTP and used it in a series of studies on mouse liver and McArdle-RH7777 (McA) cells. Western blot analysis demonstrated the presence of MTP within mouse hepatic-Golgi apparatus-rich fractions. In addition, in vitro lipid transfer assays demonstrated the presence of triglyceride transfer activity within the Golgi fractions. Immunohistochemical studies with mouse liver demonstrated the presence of MTP within all hepatocytes, but not in nonparenchymal cells. The subcellular location of MTP in McA cells was investigated using confocal microscopy. MTP colocalized with the trans-Golgi network (TGN) 38 and Golgi SNARE (soluble N-ethylmalemide-sensitive factor attachment protein receptor) of 28 kDa (GS28), markers for the trans- and cis-Golgi apparatus, respectively. Morphometric analyses indicated that approximately 17% of the MTP signal colocalized with the TGN38, while 33% of the trans-Golgi marker colocalized with the MTP. Approximately 17% of the MTP signal colocalized with the GS28, whereas 53% of the cis-Golgi marker colocalized with the MTP. The results provide unequivocal evidence for the location of MTP within the Golgi apparatus, and further highlight the importance of this organelle in the assembly of lipoproteins.     

微粒体甘油三脂转运蛋白MTP的结构和功能研究概况

微粒体甘油三酯转运蛋白MTP(microsomal triglyceride transfer protein,MTP)首先是从牛的肝细胞微粒体碎片中分离获得的,其作用是加速甘油三脂(triglyceride,TG)、胆固醇(cholesteryl ester,CE)和磷脂酰胆碱(phosphatidylcholine,PC)的转运和细胞或亚细胞膜的生物合成。它后来在肝细胞和小肠的微粒体膜中发现[1],由于它的位置及其转运TG可以推测与血浆脂蛋白中极低密度脂蛋白(very low density lipoprotein,VLDL)和乳糜微粒(chylomicrons,CM)的组装过程有关。     

Protein disulfide isomerase: the structure of oxidative folding

Cellular functions hinge on the ability of proteins to adopt their correct folds, and misfolded proteins can lead to disease. Here, we focus on the proteins that catalyze disulfide bond formation, a step in the oxidative folding pathway that takes place in specialized cellular compartments. In the endoplasmic reticulum of eukaryotes, disulfide formation is catalyzed by protein disulfide isomerase (PDI); by contrast, prokaryotes produce a family of disulfide bond (Dsb) proteins, which together achieve an equivalent outcome in the bacterial periplasm. The recent crystal structure of yeast PDI has increased our understanding of the function and mechanism of PDI. Comparison of the structure of yeast PDI with those of bacterial DsbC and DsbG reveals some similarities but also striking differences that suggest directions for future research aimed at unraveling the catalytic mechanism of disulfide bond formation in the cell.     

The crystal structure of yeast protein disulfide isomerase suggests cooperativity between its active sites

Protein disulfide isomerase plays a key role in catalyzing the folding of secretory proteins. It features two catalytically inactive thioredoxin domains inserted between two catalytically active thioredoxin domains and an acidic C-terminal tail. The crystal structure of yeast PDI reveals that the four thioredoxin domains are arranged in the shape of a twisted "U" with the active sites facing each other across the long sides of the "U." The inside surface of the "U" is enriched in hydrophobic residues, thereby facilitating interactions with misfolded proteins. The domain arrangement, active site location, and surface features strikingly resemble the Escherichia coli DsbC and DsbG protein disulfide isomerases. Biochemical studies demonstrate that all domains of PDI, including the C-terminal tail, are required for full catalytic activity. The structure defines a framework for rationalizing the differences between the two active sites and their respective roles in catalyzing the formation and rearrangement of disulfide bonds.     

Protein disulfide isomerase assisted protein folding in malaria parasites

In eukaryotes, the formation of protein disulfide bonds among cysteine residues is mediated by protein disulfide isomerases and occurs in the highly oxidised environment of the endoplasmic reticulum. This process is poorly understood in malaria parasites. In this paper, we report the gene isolation, sequence and phylogenetic comparisons, protein structure and thioredoxin-domain analyses of nine protein disulfide isomerases-like molecules from five species of malaria parasites including Plasmodium falciparum and Plasmodium vivax (human), Plasmodium knowlesi (simian) and Plasmodium berghei and Plasmodium yoelii (murine). Four of the studied protein disulfide isomerases belong to P. falciparum malaria and have been named PfPDI-8, PfPDI-9, PfPDI-11 and PfPDI-14, based on their chromosomal location. Among these, PfPDI-8 bears the closest similarity to a prototype PDI molecule with two thioredoxin domains (containing CGHC active sites) and a C-terminal Endoplasmic reticulum retrieval signal, SEEL. PfPDI-8 is expressed during all stages of parasite life cycle and is highly conserved (82-96% identity at amino acid level) in the other four Plasmodium species studied. Detailed biochemical analysis of PfPDI-8 revealed that this molecule is a potent oxido-reductase enzyme that facilitated the disulfide-dependent conformational folding of EBA-175, a leading malaria vaccine candidate. These studies open the avenues to understand the process of protein folding and secretory pathway in malaria parasites that in turn might aid in the production of superior recombinant vaccines and provide novel drug targets.     

Identification of a novel isoform of microsomal triglyceride transfer protein

Microsomal triglyceride transfer protein (MTP) has been studied extensively, primarily because of its role in the assembly of very low density lipoproteins by the liver and chylomicrons by the intestine. Recent studies have suggested that MTP may also play key roles in other cellular processes. In this paper we report the identification of a novel splice variant of MTP in mice. This isoform, MTP-B, has a unique first exon located approximately 2.7 kilobases upstream of canonical MTP (MTP-A) exon 1. The alternative exon encodes 35 amino acids compared with 20 amino acids encoded by exon 1 of MTP-A. MTP-B represents approximately 90% of total MTP mRNA in mouse adipocytes and 3T3-L1 cells and <5% in mouse liver and intestine. Expression of the alternate isoform in mouse liver was confirmed by mass spectrometry. Co-transfection of COS cells with truncated forms of apoB and either MTP-A or MTP-B demonstrated that both isoforms are effective in the assembly and secretion of nascent apoB-containing lipoproteins. Confocal microscopy of 3T3-L1 cells transfected with enhanced green fluorescent protein or DsRed fusions of the two proteins revealed that MTP-A is localized to the endoplasmic reticulum, whereas MTP-B localizes primarily to the Golgi complex in these cells. We conclude that MTP-B functions similarly to MTP-A in lipoprotein assembly. However, in nonlipoprotein-secreting cells, such as the adipocyte, MTP-B may have different localization properties, perhaps reflecting a distinct role in lipid storage and mobilization.     

Identification of microsomal triglyceride transfer protein in intestinal brush-border membrane

Microsomal triglyceride transfer protein (MTP) is a heterodimeric complex consisting of a unique large 97-kDa protein and the multifunctional 58-kDa protein disulfide isomerase (PDI). It plays an essential role in the assembly of lipoproteins by shuttling lipids between phospholipid membranes. Based on cell fractionation, early studies have suggested the endoplasmic reticulum (ER) as the exclusive site of MTP. Focusing on the plasma membrane in this study, our attempts with immunoelectron microscopy and specific antibodies surprisingly revealed that labeling was not exclusively confined to the microsomes of rat absorptive cells. Immunogold labeling was also detected over the microvillus membrane of enterocytes. Western blot analysis and biochemical activity measurement confirmed MTP protein expression in brush-border membrane vesicles (BBMV) isolated from the intestinal epithelial cells of various species. Furthermore, MTP was coexpressed in microvilli membrane with PDI that is crucial to maintain the structure and activity of the MTP complex. The treatment of Caco-2 cells with nocodazole and colchicine blocked the appearance of MTP in the apical membrane. Similarly, the addition of BMS-197636, a known inhibitor of MTP transfer activity, suppressed the latter. In conclusion, the present studies suggest that MTP is present in the brush-border membrane of the enterocyte. Understanding the possible physiological role of MTP in this location may reveal additional functions.     

Acquisition of triacylglycerol transfer activity by microsomal triglyceride transfer protein during evolution

Microsomal triglyceride transfer protein (MTP) is essential for the assembly of neutral-lipid-rich apolipoprotein B (apoB) lipoproteins. Previously we reported that the Drosophila MTP transfers phospholipids but does not transfer triglycerides. In contrast, human MTP transfers both lipids. To explore the acquisition of triglyceride transfer activity by MTP, we evaluated amino acid sequences, protein structures, and the biochemical and cellular properties of various MTP orthologues obtained from species that diverged during evolution. All MTP orthologues shared similar secondary and tertiary structures, associated with protein disulfide isomerase, localized to the endoplasmic reticulum, and supported apoB secretion. While vertebrate MTPs transferred triglyceride, invertebrate MTPs lacked this activity. Thus, triglyceride transfer activity was acquired during the transition from invertebrates to vertebrates. Within vertebrates, fish, amphibians, and birds displayed 27%, 40%, and 100% triglyceride transfer activity compared to mammals. We conclude that MTP triglyceride transfer activity first appeared in fish and speculate that the acquisition of triglyceride transfer activity by MTP provided for a significant advantage in the evolution of larger and more complex organisms.     

Novel mutations in the microsomal triglyceride transfer protein gene causing abetalipoproteinemia

Abetalipoproteinemia (ABL) is an inherited disease characterized by the virtual absence of apolipoprotein B (apoB)-containing lipoproteins from plasma. Only limited numbers of families have been screened for mutations in the microsomal triglyceride transfer protein (MTP) gene. To clarify the genetic basis of clinical diversity of ABL, mutations of the MTP gene have been screened in 4 unrelated patients with ABL. Three novel mutations have been identified: a frameshift mutation caused by a single adenine deletion at position 1389 of the cDNA, and a missense mutation, Asn780Tyr, each in homozygous forms; and a splice site mutation, 2218-2A-->G, in a compound heterozygous form. The frameshift and splice site mutations are predicted to encode truncated forms of MTP. When transiently expressed in Cos-1 cells, the Asn780Tyr mutant MTP bound protein disulfide isomerase (PDI) but displayed negligible MTP activity. It is of interest that the patient having the Asn780Tyr mutation, a 27-year-old male, has none of the manifestations characteristic of classic ABL even though his plasma apoB and vitamin E were virtually undetectable. These results indicated that defects of the MTP gene are the proximal cause of ABL.     

Protein disulfide isomerase family proteins involved in soybean protein biogenesis

Protein disulfide isomerase family proteins are known to play important roles in the folding of nascent polypeptides and the formation of disulfide bonds in the endoplasmic reticulum. In this study, we cloned two similar protein disulfide isomerase family genes from soybean leaf (Glycine max L. Merrill cv. Jack) mRNA by RT-PCR using forward and reverse primers designed from the expressed sequence tag clone sequences. The cDNA encodes a protein of either 364 or 362 amino acids, named GmPDIS-1 or GmPDIS-2, respectively. The nucleotide and amino acid sequence identities of GmPDIS-1 and GmPDIS-2 were 68% and 74%, respectively. Both proteins lack the C-terminal, endoplasmic reticulum-retrieval signal, KDEL. Recombinant proteins of both GmPDIS-1 and GmPDIS-2 were expressed in Escherichia coli as soluble folded proteins that showed both an oxidative refolding activity of denatured ribonuclease A and a chaperone activity. Their domain structures were identified as containing two thioredoxin-like domains, a and a', and an ERp29c domain by peptide mapping with either trypsin or V8 protease. In cotyledon cells, both proteins were shown to distribute to the endoplasmic reticulum and protein storage vacuoles by confocal microscopy. Data from coimmunoprecipitation and crosslinking experiments suggested that GmPDIS-1 associates with proglycinin, a precursor of the seed storage protein glycinin, in the cotyledon. Levels of GmPDIS-1, but not of GmPDIS-2, were increased in cotyledons, where glycinin accumulates during seed development. GmPDIS-1, but not GmPDIS-2, was induced under endoplasmic reticulum-stress conditions.     

Autodegradation of protein disulfide isomerase

Protein disulfide isomerase (PDI) and its degradation products were found in HepG2, COS-1, and CHO-K1 cells. Whether or not the products were formed through autodegradation of PDI was examined, since PDI contains the CGHC motif, which is the active center of proteolytic activity in ER-60 protease. Commercial bovine PDI was autodegraded to produce a trimmed PDI. In addition, human recombinant PDI also had autodegradation activity. Mutant recombinant PDIs with CGHC motifs of which cysteine residues were replaced with serine or alanine residues were prepared. However, they were not autodegraded, suggesting the cysteine residues of motifs are necessary for autodegradation.     

A deficiency of microsomal triglyceride transfer protein reduces apolipoprotein B secretion

Microsomal triglyceride transfer protein (MTP) transfers lipids to apolipoprotein B (apoB) within the endoplasmic reticulum, a process that involves direct interactions between apoB and the large subunit of MTP. Recent studies with heterozygous MTP knockout mice have suggested that half-normal levels of MTP in the liver reduce apoB secretion. We hypothesized that reduced apoB secretion in the setting of half-normal MTP levels might be caused by a reduced MTP:apoB ratio in the endoplasmic reticulum, which would reduce the number of apoB-MTP interactions. If this hypothesis were true, half-normal levels of MTP might have little impact on lipoprotein secretion in the setting of half-normal levels of apoB synthesis (since the ratio of MTP to apoB would not be abnormally low) and might cause an exaggerated reduction in lipoprotein secretion in the setting of apoB overexpression (since the MTP:apoB ratio would be even lower). To test this hypothesis, we examined the effects of heterozygous MTP deficiency on apoB metabolism in the setting of normal levels of apoB synthesis, half-normal levels of apoB synthesis (heterozygous Apob deficiency), and increased levels of apoB synthesis (transgenic overexpression of human apoB). Contrary to our expectations, half-normal levels of MTP reduced the plasma apoB100 levels to the same extent ( approximately 25-35%) at each level of apoB synthesis. In addition, apoB secretion from primary hepatocytes was reduced to a comparable extent at each level of apoB synthesis. Thus, these results indicate that the concentration of MTP within the endoplasmic reticulum rather than the MTP:apoB ratio is the critical determinant of lipoprotein secretion. Finally, we found that heterozygosity for an apoB knockout mutation lowered plasma apoB100 levels more than heterozygosity for an MTP knockout allele. Consistent with that result, hepatic triglyceride accumulation was greater in heterozygous apoB knockout mice than in heterozygous MTP knockout mice.     

Generation of hepatoma cell lines deficient in microsomal triglyceride transfer protein

The microsomal triglyceride transfer protein (MTP) is essential for the secretion of apolipoprotein B (apoB)48- and apoB100-containing lipoproteins in the intestine and liver, respectively. Loss of function mutations in MTP cause abetalipoproteinemia. Heterologous cells are used to evaluate the function of MTP in apoB secretion to avoid background MTP activity in liver and intestine-derived cells. However, these systems are not suitable to study the role of MTP in the secretion of apoB100-containing lipoproteins, as expression of a large apoB100 peptide using plasmids is difficult. Here, we report a new cell culture model amenable for studying the role of different MTP mutations on apoB100 secretion. The endogenous MTTP gene was ablated in human hepatoma Huh-7 cells using single guide RNA and RNA-guided clustered regularly interspaced short palindromic repeats-associated sequence 9 ribonucleoprotein complexes. We successfully established three different clones that did not express any detectable MTTP mRNA or MTP protein or activity. These cells were defective in secreting apoB-containing lipoproteins and accumulated lipids. Furthermore, we show that transfection of these cells with plasmids expressing human MTTP cDNA resulted in the expression of MTP protein, restoration of triglyceride transfer activity, and secretion of apoB100. Thus, these new cells can be valuable tools for studying structure-function of MTP, roles of different missense mutations in various lipid transfer activities of MTP, and their ability to support apoB100 secretion, compensatory changes associated with loss of MTP, and in the identification of novel proteins that may require MTP for their synthesis and secretion.     

Discovery of microsomal triglyceride transfer protein (MTP) inhibitors with potential for decreased active metabolite load compared to dirlotapide

Analogues related to dirlotapide (1), a gut-selective inhibitor of microsomal triglyceride transfer protein (MTP) were prepared with the goal of further reducing the potential for unwanted liver MTP inhibition and associated side-effects. Compounds were designed to decrease active metabolite load: reducing MTP activity of likely human metabolites and increasing metabolite clearance to reduce exposure. Introduction of 4′-alkyl and 4′-alkoxy substituents afforded compounds exhibiting improved therapeutic index in rats with respect to liver triglyceride accumulation and enzyme elevation. Likely human metabolites of select compounds were prepared and characterized for their potential to inhibit MTP in vivo. Based on preclinical efficacy and safety data and its potential for producing short-lived, weakly active metabolites, compound 13 (PF-02575799) advanced into phase 1 clinical studies.