The plant CDF family member TgMTP1 from the Ni/Zn hyperaccumulator Thlaspi goesingense acts to enhance efflux of Zn at the plasma membrane when expressed in Saccharomyces cerevisiae

To avoid metal toxicity, organisms have evolved mechanisms including efflux of metal ions from cells and sequestration into internal cellular compartments. Members of the ubiquitous cation diffusion facilitator (CDF) family are known to play an important role in these processes. Overexpression of the plant CDF family member metal tolerance protein 1 (MTP1) from the Ni/Zn hyperaccumulator Thlaspi goesingense (TgMTP1), in the Saccharomyces cerevisiaeDelta zinc resistance conferring (zrc)1Delta cobalt transporter (cot)1 double mutant, suppressed the Zn sensitivity of this strain. T. goesingense was found to contain several allelic variants of TgMTP1, all of which confer similar resistance to Zn in Deltazrc1Deltacot1. Similarly, MTP1 from various hyperaccumulator and non-accumulator species also confer similar resistance to Zn. Deltazrc1Deltacot1 lacks the ability to accumulate Zn in the vacuole and has lower accumulation of Zn after either long- or short-term Zn exposure. Expression of TgMTP1 in Deltazrc1Deltacot1 leads to further lowering of Zn accumulation and an increase in Zn efflux from the cells. Expression of TgMTP1 in a V-type ATPase-deficient S. cerevisiae strain also confers increased Zn resistance. In vivo and in vitro immunological staining of hemagglutinin (HA)-tagged TgMTP1::HA reveals the protein to be localized in both the S. cerevisiae vacuolar and plasma membranes. Taken together, these data are consistent with MTP1 functioning to enhance plasma membrane Zn efflux, acting to confer Zn resistance independent of the vacuole in S. cerevisiae. Transient expression in Arabidopsis thaliana protoplasts also reveals that TgMTP1::green fluorescent protein (GFP) is localized at the plasma membrane, suggesting that TgMTP1 may also enhance Zn efflux in plants.     

Reduction of VLDL Secretion Decreases Cholesterol Excretion in Niemann-Pick C1-Like 1 Hepatic Transgenic Mice

An effective way to reduce LDL cholesterol, the primary risk factor of atherosclerotic cardiovascular disease, is to increase cholesterol excretion from the body. Our group and others have recently found that cholesterol excretion can be facilitated by both hepatobiliary and transintestinal pathways. However, the lipoprotein that moves cholesterol through the plasma to the small intestine for transintestinal cholesterol efflux (TICE) is unknown. To test the hypothesis that hepatic very low-density lipoproteins (VLDL) support TICE, antisense oligonucleotides (ASO) were used to knockdown hepatic expression of microsomal triglyceride transfer protein (MTP), which is necessary for VLDL assembly. While maintained on a high cholesterol diet, Niemann-Pick C1-like 1 hepatic transgenic (L1Tg) mice, which predominantly excrete cholesterol via TICE, and wild type (WT) littermates were treated with control ASO or MTP ASO. In both WT and L1Tg mice, MTP ASO decreased VLDL triglyceride (TG) and cholesterol secretion. Regardless of treatment, L1Tg mice had reduced biliary cholesterol compared to WT mice. However, only L1Tg mice treated with MTP ASO had reduced fecal cholesterol excretion. Based upon these findings, we conclude that VLDL or a byproduct such as LDL can move cholesterol from the liver to the small intestine for TICE.     

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.     

Endogenous expression of matriptase in neural progenitor cells promotes cell migration and neuron differentiation

Recent studies show that type II transmembrane serine proteases play important roles in diverse cellular activities and pathological processes. Their expression and functions in the central nervous system, however, are largely unexplored. In this study, we show that the expression of one such member, matriptase (MTP), was cell type-restricted and primarily expressed in neural progenitor (NP) cells and neurons. Blocking MTP expression or MTP activity prevented NP cell traverse of reconstituted basement membrane, whereas overexpression of MTP promoted it. The NP cell mobilization induced by either vascular endothelial growth factor or hepatocyte growth factor was also impaired by knocking down MTP expression. MTP acts upstream of matrix metalloproteinase 2 in promoting NP cell mobility. In embryonic stem cell differentiation to neural cells, MTP knockdown had no effect on entry of embryonic stem cells into the neural lineage. High MTP expression or activity, however, shifts the population dynamics from NP cells toward neurons to favor neuronal differentiation. This is the first report to demonstrate the direct involvement of type II transmembrane serine protease in NP cell function.     

Binding of microsomal triglyceride transfer protein to lipids results in increased affinity for apolipoprotein B: evidence for stable microsomal MTP-lipid complexes.

Apolipoprotein B (apoB) and microsomal triglyceride transfer protein (MTP) are known to interact with each other. We evaluated the effect of different lipids on the protein-protein interactions between MTP and apoB100 or its C-terminally truncated forms. Negatively charged lipids decreased protein-protein interactions between apoB and MTP. In contrast, zwitterionic phospholipids enhanced (2-4-fold) the binding of apoB100 to MTP by increasing affinity (1.5-3-fold) between these proteins without affecting the number of binding sites. Similarly, phospholipids augmented (1.5-4-fold) the binding of various C-terminally truncated apoB peptides to MTP. The increased binding was greater for apoB peptides containing lipid-binding domains, such as apoB28 and apoB42. Surprisingly, preincubation of apoB28 with lipid vesicles had no effect on MTP binding. In contrast, incubation of MTP with lipid vesicles resulted in a stable association of MTP with vesicles, and MTP-lipid vesicles bound better (5-fold increase) to LDL than did lipid-free MTP. To determine whether MTP exists stably associated with lipids in cells, microsomal contents from COS cells expressing MTP, HepG2 cells, and mouse liver were ultracentrifuged, and MTP was visualized in different density fractions. MTP was found associated and unassociated with lipids. In contrast, apoB17 and apoB:270-570 were present unassociated with lipids in COS cells. These studies show that the binding of MTP to lipids results in increased affinity for apoB and that stable MTP-lipid complexes exist in the lumen of the endoplasmic reticulum. Protein-protein interactions between apoB and MTP may juxtapose lipids associated with MTP to lipid-binding domains of apoB and facilitate hydrophobic interactions leading to enhance affinity. We speculate that MTP-lipid complexes may serve as nuclei to form "primordial lipoproteins" and may also play a role in the bulk addition of lipids during the "core expansion" of these lipoproteins.     

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.     

Discovery of potential novel microsomal triglyceride transfer protein inhibitors via virtual screening of pharmacophore modelling and molecular docking

In order to identify potential natural inhibitors against the microsomal triglyceride transfer protein (MTP), HipHop models were generated using 20 known inhibitors from the Binding Database. Using evaluation indicators, the best hypothesis model, Hypo1, was selected and utilised to screen the Traditional Chinese Medicine Database, which resulted in a hit list of 58 drug-like compounds. A homology model of MTP was built by MODELLER and was minimised by CHARMm force field. It was then validated by Ramachandran plot and Verify-3D so as to obtain a stable structure, which was further used to refine the 58 hits using molecular docking studies. Then, five compounds with higher docking scores which satisfied the docking requirements were discovered. Among them, Ginkgetin and Dauricine were most likely to be candidates that exhibition inhibiting effect on MTP. The screening strategy in this study is relatively new to the discovery of MTP inhibitors in medicinal chemistry. Moreover, it is important to note that, lomitapide, an approved MTP inhibitor, fits well with Hypo1 as well as our homology model of MTP, which confirmed the rationality of our studies. The results indicated the applicability of molecular modeling for the discovery of potential natural MTP inhibitors from traditional Chinese herbs.     

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.     

Molecular cloning,expression, and hormonal regulation of the chicken microsomal triglyceride transfer protein

During an egg-laying cycle, oviparous animals transfer massive amounts of triglycerides, the major lipid component of very low density lipoprotein (VLDL), from the liver to the developing oocytes. A major stimulus for this process is the rise in estrogen associated with the onset of an egg-laying cycle. In mammals, the microsomal triglyceride transfer protein (MTP) is required for VLDL assembly and secretion. To enable studies to determine if MTP plays a role in basal and estrogen-stimulated VLDL assembly and secretion in an oviparous vertebrate, we have cloned and sequenced the chicken MTP cDNA. This cDNA encodes a protein of 893 amino acids with an N-terminal signal sequence. The primary sequence of chicken MTP is, on average, 65% identical to that of mammalian homologs, and 23% identical to the Drosophila melanogaster protein. We have obtained a clone of chicken embryo fibroblast cells that stably express the avian MTP cDNA and show that these cells display MTP activity as measured by the transfer of a fluorescently labeled neutral lipid. As in mammals, chicken MTP is localized to the endoplasmic reticulum as revealed by indirect immunofluorescence and by the fact that its N-linked oligosaccharide moiety remains sensitive to endoglycosidase H. Endogenous, enzymatically active MTP is also expressed in an estrogen receptor-expressing chicken hepatoma cell line that secretes apolipoprotein B-containing lipoproteins. In this cell line and in vivo, the expression and activity of MTP are not influenced by estrogen. Therefore, up-regulation of MTP in the liver is not required for the increased VLDL assembly during egg production in the chicken. This indicates that MTP is not rate-limiting, even for the massive estrogen-induced secretion of VLDL accompanying an egg-laying cycle.     

Inhibiting proteasomal degradation of microsomal triglyceride transfer protein prevents CCl4-induced steatosis

Carbon tetrachloride (CCl(4)) interferes with triglyceride secretion and causes steatosis, fibrosis, and necrosis. In mice, CCl(4) decreased plasma triglyceride-rich lipoproteins, increased cellular lipids, and reduced microsomal triglyceride transfer protein (MTP) without diminishing mRNA levels. Similarly, CCl(4) decreased apoB-lipoprotein production and MTP activity but had no effect on mRNA levels in primary enterocytes and colon carcinoma and hepatoma cells. CCl(4) did not affect MTP synthesis but induced post-translational degradation involving ubiquitinylation and proteasomes in McA-RH7777 cells. By contrast, MTP inhibitor increased cellular lipids without affecting MTP protein. MTP was covalently modified when cells were incubated with (14)CCl(4). This modification was prevented by the inhibition of P450 oxygenases, indicating that CCl(3)(.) generated by these enzymes targets MTP for degradation. To determine whether inhibition of proteolysis could prevent CCl(4) toxicity, mice were fed with CCl(4) with or without lactacystin. Lactacystin increased ubiquitinylated MTP and prevented lipid accumulation in tissues. Thus, CCl(4) induces post-translational degradation without affecting lipid transfer activity, whereas MTP antagonist inhibits lipid transfer activity without causing its destruction. These studies identify MTP as a major target of CCl(4) and its degradation as a novel mechanism involved in the onset of steatosis, suggesting that inhibition of proteolysis may prevent some forms of steatosis.     

Biosynthesis of phytohormones from novel rhizobacterial isolates and their in vitro plant growth-promoting efficacy

The health of the plant and soil fertility is dependent on the plant–microbes interaction in the rhizosphere. Microbial life tends to endure various rhizosphere plant–microbe interactions. Phytohormones such as auxins, cytokinins, gibberellic acid, ethylene and abscisic acid are termed as the classical group of hormones. Out of the 70 rhizobacterial strains isolated from the Coleus rhizosphere, three different rhizobacterial strains Pseudomonas stutzeri MTP40, Stenotrophomonas maltophilia MTP42 and Pseudomonas putida MTP50 having plant growth-promoting attributes were isolated and characterized for its phytohormone-producing ability. The phytohormones such as indole 3-acetic acid (IAA), gibberellic acid and cytokinin (kinetin and 6-benzyladenosine) were affirmed in culture supernatant of the above isolates. IAA was detected in all the three isolates, where in highest production was found in S. maltophilia MTP42 (240?µg/mL) followed by P. stutzeri MTP40 (250?µg/mL) and P. putida MTP50 (233?µg/mL). Gibberellic acid production was found maximum in MTP40 (34?µg/mL), followed by MTP42 (31?µg/mL) and MTP50 (27?µg/mL). The cytokinin production from the isolates, namely, MTP40, MTP42 and MTP50 were 13, 11 and 7.5?µg/mL, respectively. The isolates showing the production of plant growth enhancing phytohormones can be commercialized as potent bioformulations.     

Acute suppression of apo B secretion by insulin occurs independently of MTP

Secretion of apolipoprotein (apo) B-containing lipoproteins by the liver depends mainly upon apo B availability and microsomal triglyceride transfer protein (MTP) activity and is subject to insulin regulation. Hepatic MTP mRNA expression is negatively regulated by insulin which correlates with inhibition of apo B secretion suggesting that insulin might suppress apo B secretion through an MTP-dependent mechanism. To investigate this possibility, we examined the acute effect of insulin on hepatic MTP expression and activity levels in vivo utilizing apobec-1^−/− mice. Insulin did not significantly alter hepatic MTP mRNA levels or lipid transfer activity 2 h following injection, but suppressed expression of genes important in gluconeogenesis. To study the specific role of MTP, we expressed human MTP (hMTP) in primary rat hepatocytes using adenoviral gene transfer. Increased expression of hMTP resulted in a 47.6 ± 17.9% increase in total apo B secreted. Incubation of hepatocytes with insulin suppressed apo B secretion by 50.1 ± 10.8% in cells over-expressing hMTP and by 53.0 ± 12.4% in control transfected hepatocytes. Results indicate that even under conditions of increased hepatic apo B secretion mediated by MTP, responsiveness of hepatocytes to insulin to suppress apo B secretion is maintained.     

Immunolocalization, ontogeny, and regulation of microsomal triglyceride transfer protein in human fetal intestine

To examine the multiple stages of lipoprotein packaging during development, we studied localization, ontogeny, and regulation of microsomal transfer protein (MTP), a crucial protein for lipid transport. With the use of immunofluorescence, MTP was identified in villus and crypt epithelial cells in different regions of human fetal intestine, including colon. Staining was detected as early as the 13th wk of gestation in all gut segments and was almost entirely confined to the columnar epithelial cells of the jejunum and colon. Unlike immunofluorescence, which provides qualitative but not quantitative information on MTP signal, enzymatic assays revealed a decreasing gradient from proximal small intestine to distal, as confirmed by immunoblot. Activity of MTP in small intestinal explants cultured for different incubation periods (0, 4, 8, and 24 h) peaked at 4 h but remained insensitive to different concentrations of oleic acid. Also, a trend toward increasing MTP activity was observed at 20-22 wk of gestation. Finally, in strong contrast to jejunal efficiency, colonic explants displayed impaired lipid production, apolipoprotein biogenesis, and lipoprotein assembly, in association with poor expression of MTP. These findings provide the first evidence that human fetal gut is able to express MTP and emphasize the distinct regional distribution, regulation by oleic acid, and ontogeny of MTP.     

Intestine-specific MTP and global ACAT2 deficiency lowers acute cholesterol absorption with chylomicrons and HDLs

Intestinal cholesterol absorption involves the chylomicron and HDL pathways and is dependent on microsomal triglyceride transfer protein (MTP) and ABCA1, respectively. Chylomicrons transport free and esterified cholesterol, whereas HDLs transport free cholesterol. ACAT2 esterifies cholesterol for secretion with chylomicrons. We hypothesized that free cholesterol accumulated during ACAT2 deficiency may be secreted with HDLs when chylomicron assembly is blocked. To test this, we studied cholesterol absorption in mice deficient in intestinal MTP, global ACAT2, and both intestinal MTP and global ACAT2. Intestinal MTP ablation significantly increased intestinal triglyceride and cholesterol levels and reduced their transport with chylomicrons. In contrast, global ACAT2 deficiency had no effect on triglyceride absorption but significantly reduced cholesterol absorption with chylomicrons and increased cellular free cholesterol. Their combined deficiency reduced cholesterol secretion with both chylomicrons and HDLs. Thus, contrary to our hypothesis, free cholesterol accumulated in the absence of MTP and ACAT2 is unavailable for secretion with HDLs. Global ACAT2 deficiency causes mild hypertriglyceridemia and reduces hepatosteatosis in mice fed high cholesterol diets by increasing hepatic lipoprotein production by unknown mechanisms. We show that this phenotype is preserved in the absence of intestinal MTP in global ACAT2-deficient mice fed a Western diet. Further, we observed increases in hepatic MTP activity in these mice. Thus, ACAT2 deficiency might increase MTP expression to avoid hepatosteatosis in cholesterol-fed animals. Therefore, ACAT2 inhibition might avert hepatosteatosis associated with high cholesterol diets by increasing hepatic MTP expression and lipoprotein production.     

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.