Zhenghui G. Jiang

Missense Mutation in APOC3 within the C-terminal Lipid Binding Domain of Human ApoC-III Results in Impaired Assembly and Secretion of Triacylglycerol-rich Very Low Density Lipoproteins EVIDENCE THAT ApoC-III PLAYS A MAJOR ROLE IN THE FORMATION OF LIPID PRECURSORS WITHIN THE MICROSOMAL LUMEN

Hepatic assembly of triacylglycerol (TAG)-rich very low density lipoproteins (VLDL) is achieved through recruitment of bulk TAG (presumably in the form of lipid droplets within the microsomal lumen) into VLDL precursor containing apolipoprotein (apo) B-100. We determined protein/lipid components of lumenal lipid droplets (LLD) in cells expressing recombinant human apoC-III (C3wt) or a mutant form (K58E, C3KE) initially identified in humans that displayed hypotriglyceridemia. Although expression of C3wt markedly stimulated secretion of TAG and apoB-100 as VLDL1, the K58E mutation (located at the C-terminal lipid binding domain) abolished the effect in transfected McA-RH7777 cells and in apoc3-null mice. Metabolic labeling studies revealed that accumulation of TAG in LLD was decreased (by 50%) in cells expressing C3KE. A Fat Western lipid protein overlay assay showed drastically reduced lipid binding of the mutant protein. Substituting Lys58 with Arg demonstrated that the positive charge at position 58 is crucial for apoC-III binding to lipid and for promoting TAG secretion. On the other hand, substituting both Lys58 and Lys60 with Glu resulted in almost entire elimination of lipid binding and loss of function in promoting TAG secretion. Thus, the lipid binding domain of apoC-III plays a key role in the formation of LLD for hepatic VLDL assembly and secretion.

Missense Mutations in APOB within the βα1 Domain of Human APOB-100 Result in Impaired Secretion of ApoB and ApoB-containing Lipoproteins in Familial Hypobetalipoproteinemia

Familial hypobetalipoproteinemia (FHBL) is associated with mutations in the APOB gene. We reported the first missense APOB mutation, R463W, in an FHBL kindred (Burnett, J. R., Shan, J., Miskie, B. A., Whitfield, A. J., Yuan, J., Tran, K., Mc-Knight, C. J., Hegele, R. A., and Yao, Z. (2003) J. Biol. Chem. 278, 13442-13452). Here we identified a second nonsynonymous APOB mutation, L343V, in another FHBL kindred. Heterozygotes for L343V (n = 10) had a mean plasma apoB at 0.31 g/liter as compared with 0.80 g/liter in unaffected family members (n = 22). The L343V mutation impaired secretion of apoB-100 and very low density lipoproteins. The secretion efficiency was 20% for B100wt and 10% for B100LV and B100RW. Decreased secretion of mutant apoB-100 was associated with increased endoplasmic reticulum retention and increased binding to microsomal triglyceride transfer protein and BiP. Reduced secretion efficiency was also observed with B48LV and B17LV. Biochemical and biophysical analyses of apoB domain constructs showed that L343V and R463W altered folding of the α-helical domain within the N terminus of apoB. Thus, proper folding of the α-helical domain of apoB-100 is essential for efficient secretion.

Functional analysis of the missense APOC3 mutation Ala23Thr associated with human hypotriglyceridemia

We have shown that expression of apolipoprotein (apo) C-III promotes VLDL secretion from transfected McA-RH7777 cells under lipid-rich conditions. To determine structural elements within apoC-III that confer to this function, we contrasted wild-type apoC-III with a mutant Ala23Thr originally identified in hypotriglyceridemia subjects. Although synthesis of [3H]glycerol-labeled TAG was comparable between cells expressing wild-type apoC-III (C3wt cells) or Ala23Thr mutant (C3AT cells), secretion of [3H]TAG from C3AT cells was markedly decreased. The lowered [3H]TAG secretion was associated with an inability of C3AT cells to assemble VLDL1. Moreover, [3H]TAG within the microsomal lumen in C3AT cells was 60% higher than that in C3wt cells, yet the activity of microsomal triglyceride-transfer protein in C3AT cells was not elevated. The accumulated [3H]TAG in C3AT microsomal lumen was mainly associated with lumenal IDL/LDL-like lipoproteins. Phenotypically, this [3H]TAG fractionation profiling resembled what was observed in cells treated with brefeldin A, which at low dose specifically blocked the second-step VLDL1 maturation. Furthermore, lumenal [35S]Ala23Thr protein accumulated in IDL/LDL fractions and was absent in VLDL fractions in C3AT cells. These results suggest that the presence of Ala23Thr protein in lumenal IDL/LDL particles might prevent effective fusion between lipid droplets and VLDL precursors. Thus, the current study reveals an important structural element residing within the N-terminal region of apoC-III that governs the second step VLDL1 maturation.

Lipoprotein metabolism in nonalcoholic fatty liver disease

Nonalcoholic fatty liver disease (NAFLD), an escalating health problem worldwide, covers a spectrum of pathologies characterized by fatty accumulation in hepatocytes in early stages, with potential progression to liver inflammation, fibrosis, and failure. A close, yet poorly understood link exists between NAFLD and dyslipidemia, a constellation of abnormalities in plasma lipoproteins including triglyceride-rich very low density lipoproteins. Apolipoproteins are a group of primarily liver-derived proteins found in serum lipoproteins; they not only play an extracellular role in lipid transport between vital organs through circulation, but also play an important intracellular role in hepatic lipoprotein assembly and secretion. The liver functions as the central hub for lipoprotein metabolism, as it dictates lipoprotein production and to a significant extent modulates lipoprotein clearance. Lipoprotein metabolism is an integral component of hepatocellular lipid homeostasis and is implicated in the pathogenesis, potential diagnosis, and treatment of NAFLD.

Dematin exhibits a natively unfolded core domain and an independently folded headpiece domain

Dematin is an actin‐binding protein originally identified in the junctional complex of the erythrocyte plasma membrane, and is present in many nonerythroid cells. Dematin headpiece knockout mice display a spherical red cell phenotype and develop a compensated anemia. Dematin has two domains: a 315‐residue, proline‐rich “core” domain and a 68‐residue carboxyl‐terminal villin‐type “headpiece” domain. Expression of full‐length dematin in E. coli as a GST recombinant protein results in truncation within a proline, glutamic acid, serine, threonine rich region (PEST). Therefore, we designed a mutant construct that replaces the PEST sequence. The modified dematin has high actin binding activity as determined by actin sedimentation assays. Negative stain electron microscopy demonstrates that the modified dematin also exhibits actin bundling activity like that of native dematin. Circular dichroism (CD) and NMR spectral analysis, however, show little secondary structure in the modified dematin. The lack of secondary structure is also observed in native dematin purified from human red blood cells. 15N‐HSQC NMR spectra of modified dematin indicate that the headpiece domain is fully folded whereas the core region is primarily unfolded. Our finding suggests that the core is natively unfolded and may serve as a scaffold to organize the components of the junctional complex.

Low LDL-C and High HDL-C Levels Are Associated with Elevated Serum Transaminases amongst Adults in the United States: A Cross-sectional Study

Background Dyslipidemia, typically recognized as high serum triglyceride, high low-density lipoprotein cholesterol (LDL-C) or low high-density lipoprotein cholesterol (HDL-C) levels, are associated with nonalcoholic fatty liver disease (NAFLD). However, low LDL-C levels could result from defects in lipoprotein metabolism or impaired liver synthetic function, and may serve as ab initio markers for unrecognized liver diseases. Whether such relationships exist in the general population has not been investigated. We hypothesized that despite common conception that low LDL-C is desirable, it might be associated with elevated liver enzymes due to metabolic liver diseases. Methods and Findings We examined the associations between alanine aminotransferase (ALT), aspartate aminotransferase (AST) and major components of serum lipid profiles in a nationally representative sample of 23,073 individuals, who had no chronic viral hepatitis and were not taking lipid-lowering medications, from the National Health and Nutrition Examination Survey (NHANES) from 1999 to 2010. ALT and AST exhibited non-linear U-shaped associations with LDL-C and HDL-C, but not with triglyceride. After adjusting for potential confounders, individuals with LDL-C less than 40 and 41–70 mg/dL were associated with 4.2 (95% CI 1.5–11.7, p = 0.007) and 1.6 (95% CI 1.1–2.5, p = 0.03) times higher odds of abnormal liver enzymes respectively, when compared with those with LDL-C values 71–100 mg/dL (reference group). Surprisingly, those with HDL-C levels above 100 mg/dL was associated with 3.2 (95% CI 2.1–5.0, p<0.001) times higher odds of abnormal liver enzymes, compared with HDL-C values of 61–80 mg/dL. Conclusions Both low LDL-C and high HDL-C, often viewed as desirable, were associated with significantly higher odds of elevated transaminases in the general U.S. adult population. Our findings underscore an underestimated biological link between lipoprotein metabolism and liver diseases, and raise a potential need for liver evaluation among over 10 million people with particularly low LDL-C or high HDL-C in the United States.

Purinergic signaling during intestinal inflammation

Inflammatory bowel disease (IBD) is a devastating disease that is associated with excessive inflammation in the intestinal tract in genetically susceptible individuals and potentially triggered by microbial dysbiosis. This illness markedly predisposes patients to thrombophilia and chronic debility as well as bowel, lymphatic, and liver cancers. Development of new therapies is needed to re-establish long-term immune tolerance in IBD patients without increasing the risk of opportunistic infections and cancer. Aberrant purinergic signaling pathways have been implicated in disordered thromboregulation and immune dysregulation, as noted in the pathogenesis of IBD and other gastrointestinal/hepatic autoimmune diseases. Expression of CD39 on endothelial or immune cells allows for homeostatic integration of hemostasis and immunity, which are disrupted in IBD. Our focus in this review is on novel aspects of the functions of CD39 and related NTPDases in IBD. Regulated CD39 activity allows for scavenging of extracellular nucleotides, the maintenance of P2-receptor integrity and coordination of adenosinergic signaling responses. CD39 together with CD73, serves as an integral component of the immunosuppressive machinery of dendritic cells, myeloid cells, T and B cells. Genetic inheritance and environental factors closely regulate the levels of expression and phosphohydrolytic activity of CD39, both on immune cells and released microparticles. Purinergic mechanisms associated with T regulatory and supressor T helper type 17 cells modulate disease activity in IBD, as can be modeled in experimental colitis. As a recent example, upregulation of CD39 is dependent upon ligation of the aryl hydrocarbon receptor (AHR), as with natural ligands such as bilirubin and 2-(1′ H-indole-3′-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE). Decreased expression of CD39 and/or dysfunctional AHR signaling, however, abrogates the protective effects of immunosuppressive AHR ligands. These factors could also serve as biomarkers of disease activity in IBD. Heightened thrombosis, inflammation, and immune disturbances as seen in IBD appear to be associated with aberrant purinergic signaling. Ongoing development of therapeutic strategies augmenting CD39 ectonucleotidase bioactivity via cytokines or AHR ligands offers promise for management of thrombophilia, disordered inflammation, and aberrant immune reactivity in IBD.

Interfacial properties of apolipoprotein B292-593 (B6.4-13) and B611-782 (B13-17). Insights into the structure of the lipovitellin homology region in apolipoprotein B.

The N-terminal sequence of apolipoprotein B (apoB) is critical in triacylglycerol-rich lipoprotein assembly. The first 17% of apoB (B17) is thought to consist of three domains: B5.9, a beta-barrel, B6.4-13, a series of 17 alpha-helices, and B13-17, a putative beta-sheet. B5.9 does not bind to lipid, while B6.4-13 and B13-17 contain hydrophobic interfaces that can interact with lipids. To understand how B6.4-13 and B13-17 might interact with triacylglycerol during lipoprotein assembly, the interfacial properties of both peptides were studied at the triolein/water interface. Both B6.4-13 and B13-17 are surface active. Once bound, the peptides can be neither exchanged nor pushed off the interface. Some residues of the peptides can be ejected from the interface upon compression but readsorb on expansion. B13-17 binds to the interface more strongly. The maximum pressure the peptide can withstand without being partially ejected (Pi(max)) is 19.2 mN/m for B13-17 compared to 16.7 mN/m for B6.4-13. B13-17 is purely elastic at the interface, while B6.4-13 forms a viscous-elastic film. When they are spread at an air/water interface, the limiting area and the collapse pressures are 16.6 A(2)/amino acid and 31 mN/m for B6.4-13 and 17.8 A(2)/amino acid and 35 mN/m for B13-17, respectively. The alpha-helical B6.4-13 contains some hydrophobic helices that stay bound and prevent the peptide from leaving the surface. The beta-sheets of B13-17 bind irreversibly to the surface. We suggest that during lipoprotein assembly, the N-terminal apoB starts recruiting lipid as early as B6.4, but additional sequences are essential for formation of a lipid pocket that can stabilize lipoprotein emulsion particles for secretion.

Steatohepatitis and liver fibrosis are predicted by the characteristics of very low density lipoprotein in nonalcoholic fatty liver disease

A major challenge in the management of nonalcoholic fatty liver disease (NAFLD) is to identify patients with nonalcoholic steatohepatitis (NASH) and early liver fibrosis. The progression of NAFLD is accompanied by distinctive changes in very low density lipoprotein (VLDL), a lipoprotein particle produced exclusively in the liver. Herein, we sought to determine the characteristics of VLDL profiles associated with NASH and liver fibrosis.

Surgery for Ulcerative Colitis Is Associated with a High Rate of Readmissions at 30 Days

Background:Currently, the predictors of readmission after colectomy specifically for ulcerative colitis (UC) are poorly investigated. We sought to determine the rates and predictors of 30-day readmissions after colectomy for UC. Methods:Patients undergoing total proctocolectomy and end ileostomy, abdominal colectomy with end ileostomy, proctocolectomy with ileoanal pouch anastomosis (IPAA) formation and diverting ileostomy, one stage IPAA, or abdominal colectomy with ileorectal anastomosis at a tertiary care center between January 2002 and January 2012 for UC were included. Patients were identified using ICD-9 code 556.x. Each record was manually reviewed. The electronic record system was reviewed for demographic information, medical histories, UC history, medications, and data regarding the admission and discharge. Charts were reviewed for readmissions within 30 days of surgery. Univariate and multivariate analyses were performed using Stata v.13. Results:Two hundred nine patients with UC underwent a colectomy. Forty-three percent had a proctocolectomy with IPAA and diverting ileostomy and 32% had abdominal colectomy with end ileostomy. Seventy-six percent of surgeries were due to failure of medical therapy and 68% of patients were electively admitted for surgery. Thirty-two percent (n = 67/209) of the cohort was unexpectedly readmitted within 30 days. In multivariate model, proctocolectomy with IPAA and diverting ileostomy (odds ratio [OR] = 2.11; 95% CI, 1.06–4.19; P = 0.033) was the only significant predictor of readmission. Hospital length of stay >7 days (OR = 1.82; 95% CI, 0.98–3.41; P = 0.060), presence of limited UC (OR = 2.10; 95% CI, 0.93–4.74; P = 0.074), and steroid before admission (OR = 1.69; 95% CI, 0.90–3.2; P = 0.100) trended toward significance. Conclusions:Surgery for UC is associated with a high rate of readmission. Further prospective studies are necessary to determine the means to reduce these readmissions.