Bulky hydrophobic side chains in the ß1-sandwich of microsomal triglyceride transfer protein are critical for the transfer of both triglycerides and phospholipids.

Hyperlipidemia predisposes individuals to cardiometabolic diseases, the most common cause of global mortality. Microsomal triglyceride transfer protein (MTP) transfers multiple lipids and is essential for the assembly of apolipoprotein B-containing lipoproteins. MTP inhibition lowers plasma lipids but causes lipid retention in the liver and intestine. Previous studies suggested two lipid transfer domains in MTP and that specific inhibition of triglyceride (TG) and not phospholipid (PL) transfer can lower plasma lipids without significant tissue lipid accumulation. However, how MTP transfers different lipids and the domains involved in these activities are unknown. Here, we tested a hypothesis that two different ß-sandwich domains in MTP transfer TG and PL. Mutagenesis of charged amino acids in ß2-sandwich had no effect on PL transfer activity indicating that they are not critical. In contrast, amino acids with bulky hydrophobic side chains in ß1-sandwich were critical for both TG and PL transfer activities. Substitutions of these residues with smaller hydrophobic side chains or positive charges reduced, whereas negatively charged side chains severely attenuated MTP lipid transfer activities. These studies point to a common lipid transfer domain for TG and PL in MTP that is enriched with bulky hydrophobic amino acids. Furthermore, we observed a strong correlation in different MTP mutants with respect to loss of both the lipid transfer activities, again implicating a common binding site for TG and PL in MTP. We propose that targeting of areas other than the identified common lipid transfer domain might reduce plasma lipids without causing cellular lipid retention.

Intracellular tPA-PAI-1 interaction determines VLDL assembly in hepatocytes.

Apolipoprotein B (apoB)-lipoproteins initiate and promote atherosclerotic cardiovascular disease. Plasma tissue plasminogen activator (tPA) activity is negatively associated with atherogenic apoB-lipoprotein cholesterol levels in humans, but the mechanisms are unknown. We found that tPA, partially through the lysine-binding site on its Kringle 2 domain, binds to the N terminus of apoB, blocking the interaction between apoB and microsomal triglyceride transfer protein (MTP) in hepatocytes, thereby reducing very-low-density lipoprotein (VLDL) assembly and plasma apoB-lipoprotein cholesterol levels. Plasminogen activator inhibitor 1 (PAI-1) sequesters tPA away from apoB and increases VLDL assembly. Humans with PAI-1 deficiency have smaller VLDL particles and lower plasma levels of apoB-lipoprotein cholesterol. These results suggest a mechanism that fine-tunes VLDL assembly by intracellular interactions among tPA, PAI-1, and apoB in hepatocytes.

A coding variant in the microsomal triglyceride transfer protein reduces both hepatic steatosis and plasma lipids.

BACKGROUND: Genetic inactivation and pharmacologic inhibition of the microsomal triglyceride transfer protein (MTP; gene name MTTP) inhibits hepatic secretion of VLDL, thereby reducing serum lipids and apoB at the expense of increasing hepatic steatosis. AIM: To examine the effects of missense variants in MTTP on hepatic and circulating lipids. METHODS: We analysed the association of MTTP missense variants with metabolic, hepatic and clinical phenotypes in the Penn Medicine Biobank (PMBB; n = 37,960) and the UKBiobank (UKB; n = 451,444). RESULTS: We analysed 24 missense variants in MTTP in PMBB for association with biopsy-proven hepatic steatosis and found that an isoleucine 128 to threonine variant (I128T: rs3816873-A, frequency 26%) was associated with reduced steatosis (p < 0.001). PMBB subjects with imaging-proven steatosis also revealed significantly fewer carriers of MTTP I128T compared to controls. Analysis in UKB also showed that MTTP I128T was associated with reduced risk of hepatic steatosis. Unexpectedly, MTTP I128T was found to be associated with reduced plasma levels of LDL-cholesterol and apoB (all p < 0.001). Functional studies indicated that MTTP I128T is neither a classic loss nor gain of function allele. CONCLUSIONS: MTTP I128T is associated with reduced hepatic steatosis as well as reduced plasma lipids and apoB. This paradoxical profile is not consistent with a simple gain or loss of function in MTP activity and suggests a more complex effect on MTP function. Further investigation of MTTP I128T will provide insight into the structure-function of MTP and potentially new approaches to modulate MTP activity that could both reduce hepatic and circulating lipids.

miR-125b and miR-223 Contribute to Inflammation by Targeting the Key Molecules of NFκB Pathway.

The contribution of miRNA in the pathogenesis of ulcerative colitis (UC) has emerged in the past few decades. Differential miRNA expression has been demonstrated in UC patients, and their ability to target the genes involved in inflammatory pathway has also been explored in recent years. miR-125b and miR-223 have been demonstrated to get upregulated within the colonic mucosa of UC patients. Here, we explored the biological relevance of miR-125b and miR-223 altered expression during UC by identifying the potential gene targets for miR-125b and miR-223. TRAF6 and A20, the signaling molecules involved in the NFκB pathway, were identified as target genes for miR-125b while IKKα was identified as a gene target for miR-223. The colonic mucosal samples from UC patients exhibited a significant rise in miR-125b and miR-223 expression while a subsequent downregulation was observed in the expression of TRAF6, A20, and IKKα. This negative correlation between miRNAs and their respective target genes was validated by co-transfecting miR-125b and miR-223 in HT29 cells. Co-transfection with miR-125b resulted in a marked decline in the expression of TRAF6 and A20, while the miR-223 co-transfected cells exhibited lower IKKα expression levels. Additionally, co-transfection with miR-125b or miR-223 in HT29 cells caused higher p65 and pro-inflammatory cytokines (IL-8 and IL-1ß) expression upon LPS stimulation. From our findings, we highlight the possible contribution of miR-125b and miR-223 in regulating the inflammatory response during UC by negatively regulating the expression of TRAF6, A20, and IKKα. Therefore, we conclude that these two miRNAs could be considered as potential candidates for developing promising biomarkers for screening and diagnosis of UC.

MicroRNA exhibit altered expression in the inflamed colonic mucosa of ulcerative colitis patients.

AIM: To investigate the miRNA expression in colonic mucosal biopsies from endoscopically inflamed and non inflamed regions of ulcerative colitis (UC) patients. METHODS: Colonic mucosal pinch biopsies were analyzed from the inflamed and non inflamed regions of same UC patient. Total RNA was isolated and differential miRNA profiling was done using microarray platform. Quantitative Real Time PCR was performed in colonic biopsies from inflamed (n = 8) and non-inflamed (n = 8) regions of UC and controls (n = 8) to validate the differential expression of miRNA. Potential targets of dysregulated miRNA were identified by using in silico prediction tools and probable role of these miRNA in inflammatory pathways were predicted. RESULTS: The miRNA profile of inflamed colonic mucosa differs significantly from the non-inflamed. Real time PCR analysis showed that some of the miRNA were differentially expressed in the inflamed mucosa as compared to non inflamed mucosa and controls (miR-125b, miR-223, miR-138, and miR-155), while (miR-200a) did not show any significant changes. In contrast to microarray, where miR-378d showed downregulation in the inflamed mucosa, qRT-PCR showed a significant upregulation in the inflamed mucosa as compared to the non inflamed. The in silico prediction analysis revealed that the genes targeted by these miRNAs play role in the major signaling pathways like MAPK pathway, NF-κB signaling pathway, cell adhesion molecules which are all assciated with UC. CONCLUSION: The present study reports disease specific alteration in the expression of miR-125b, miR-155, miR-223 and miR-138 in UC patients and also predict their biological significance.