Haya Herscovitz

Boston University | BU · Department of Physiology and Biophysics

Population studies have found that a natural human apoA-I variant, apoA-I[K107del], is strongly associated with low HDL-C but normal plasma apoA-I levels. We aimed to reveal properties of this variant that contribute to its unusual phenotype associated with atherosclerosis. Our oil-drop tensiometry studies revealed that compared to WT, recombinant...

Lipid droplets (LDs) in all eukaryotic cells are coated with at least one of the perilipin (Plin) family of proteins. They all regulate key intracellular lipases but do so to significantly different extents. Where more than one Plin is expressed in a cell, they associate with LDs in a hierarchical manner. In vivo, this means that lipid flux control...

ApoA-I and the ATP-binding cassette transporter A1 (ABCA1) play important roles in nascent HDL (nHDL) biogenesis, the first step in the pathway of reverse cholesterol transport that protects against cardiovascular disease. On the basis of the crystal structure of a C-terminally truncated form of apoA-IΔ;[185-243]) determined in our laboratory, we h...

Apolipoprotein A-I (apoA-I) plays important structural and functional roles in reverse cholesterol transport. We have described the molecular structure of the N-terminal domain, Δ(185-243) by x-ray crystallography. To understand the role of the C-terminal domain, constructs with sequential elongation of Δ(185-243), by increments of 11-residue seque...

Fusion of modified LDL in the arterial wall promotes atherogenesis. Earlier we showed that thermal denaturation mimics LDL remodeling and fusion, and revealed kinetic origin of LDL stability. Here we report the first quantitative analysis of LDL thermal stability. Turbidity data show sigmoidal kinetics of LDL heat denaturation, which is unique amon...

Purified GST-tagged proteins (2 µg), fractionated and stained upon SDS-PAGE, used in tubulin polymerization assays, in vitro binding assays, and pull-downs from mouse brain lysates. The identity of the proteins was also confirmed by Western blot with Capzb2 and/or GST antibody (unpublished data). (0.69 MB TIF)

Controls for Capzb2Δ106–140 and Capzb2 bindings of F-actin. α-Actinin (10 µM) and BSA (51 µM) were incubated alone or in the presence of F-actin (23 µM). Upon centrifugation, the supernatant (S) and pellet (P) of each reaction were analyzed by SDS-PAGE followed by Coomassie Brilliant Blue staining. In the presence of F-actin, α-actinin cosediments...

The expression of EGFP-tagged RNAi-resistant Capzb2 full length and Capzb2Δ106–140 in CAD cells. Note that the levels of RNAi-resistant Capzb2-EGFP (protein used in experiments depicted in Figure 2A and 2F) and EGFP-Capzb2Δ106–140 (protein used in experiments depicted in Figure 6) are comparable. (0.09 MB TIF)

Capzb2 shRNA construct efficiently knocks down the expression of Capzb2 in CAD cells. CAD cells were transfected with either control shRNA or Capzb2 shRNA. Lysates were prepared 48 h posttransfection and analyzed by Western blot using Capzb2 antibody. (0.08 MB TIF)

Capzb2 is expressed in the developing and adult mouse brain. (A) Although the adult cortical Capzb2 expression is diminished in comparison to developmental levels (E16), the expression of Capzb2 in the hippocampus remains high in adulthood (representative Western blot). (B) Relative Capzb2 levels in the developing (E16) cortex, adult cortex, and ad...

Capping protein (CP) is a heterodimer that regulates actin assembly by binding to the barbed end of F-actin. In cultured nonneuronal cells, each CP subunit plays a critical role in the organization and dynamics of lamellipodia and filopodia. Mutations in either alpha or beta CP subunit result in retinal degeneration in Drosophila. However, the func...

Very-low-density lipoproteins (VLDL) are metabolic precursors of low-density lipoproteins (LDL) and a risk factor for atherosclerosis. Human VLDL are heterogeneous complexes containing a triacylglycerol-rich apolar lipid core and polar surface composed of phospholipids, a nonexchangeable apolipoprotein B, and exchangeable apolipoproteins E and Cs....

A simple and robust LC-MS-based methodology for the investigation of lipid mixtures is described, and its application to the analysis of human lipoprotein-associated lipids is demonstrated. After an optional initial fractionation on Silica 60, normal-phase HPLC-MS on a YMC PVA-Sil column is used first for class separation, followed by reversed-phas...

Epidemiological and clinical data demonstrate differences in atherosclerotic coronary heart disease prevalence between age-matched men and premenopausal women. Mechanisms underlying relative athero-susceptibility in men and athero-resistance in premenopausal women remain to be elucidated. Lack of informative animal models hinders research. We repor...

The antidiabetic drug metformin stimulates AMP-activated protein kinase (AMPK) activity in the liver and in skeletal muscle. To better understand the role of AMPK in the regulation of hepatic lipids, we studied the effect of metformin on AMPK and its downstream effector, acetyl-CoA carboxylase (ACC), as well as on lipid content in cultured human he...

We have previously demonstrated that endoplasmic reticulum (ER)-resident molecular chaperones interact with apolipoprotein B-100 (apoB) during its maturation. The initial stages of apoB folding occur while it is bound to the ER membrane, where it becomes partially lipidated to form a primordial intermediate. We determined whether this intermediate...

The mechanisms underlying the known interaction of two complex polygenic traits, hypertension and hyperlipidemia, resulting in exacerbation of coronary artery disease have not been elucidated. Identification of critical pathways underlying said exacerbation could identify mechanism-based targets for intervention and prevention. To investigate hyper...

The N-terminal 17% of apolipoprotein B (apoB-17) readily associates with dimyristoylphosphatidylcholine (DMPC) multilamellar vesicles (MLV) to form large (240-Å diameter) discoidal particles. Because apoB is normally secreted with triacylglycerol (TAG)-rich lipoproteins, we studied the binding of apoB-17 to triolein-rich emulsions modeling nascent...

The N-terminal 17% of apolipoprotein B (apoB-17) is secreted lipid-poor while apoB-41 particles are secreted with a triacylglycerol (TAG)-rich core. Thus, the sequence between apoB-17 and apoB-41 is necessary for the assembly of TAG-rich lipoproteins. To delineate this region, C127 cells were permanently transfected to secrete the N-terminal 29, 32...

Apolipoprotein (apo) B-100, an essential protein for the assembly and secretion of very low density lipoproteins depends on lipid binding (lipidation) for its secretion. Seven of its 8 disulfides are clustered within the N-terminal 21%. The role of these disulfides in the secretion of lipidated or unlipidated truncated forms of apoB was studied in...

The present study was undertaken to identify and characterize molecular chaperones that assist in the folding of apolipoprotein (apo) B, a secretory protein that requires assembly with lipids (lipidation) for its secretion. Both HepG2 cells, normally secreting full-length apoB (apoB-100), and C127 cells transfected to secrete truncated forms of apo...

The cDNA encoding the N-terminal 41% of human apolipoprotein B (apoB), apoB-41, was transfected into nonhepatic, nonintestinal, mammary-derived mouse cells (C127) to generate stably transfected cells expressing human apoB-41 (C127B-41). As determined by centrifugation, apoB-41 is secreted exclusively on lipoproteins (LPs) having a peak density of 1...

The interrelationships between activation of phospholipases and neutrophil stimulus-induced Ca2+ responses remain unclear. We report here that immune complexes activate a phosphatidylcholine-specific phospholipase A in a neutrophil only after the cytoplasmic Ca2+ transient has been initiated in the same cell, while chemotactic peptide activation do...

Transfected mouse mammary-derived cells (C127) expressing human apolipoprotein (apo) E (C127E) were used a) to determine whether the lipid-binding character of apoE is sufficient to promote its assembly with lipid to form lipoprotein-like particles when expressed by cells not normally expressing apolipoproteins; b) to characterize the secreted comp...

We have used a fluorescence-activated cytotoxicity protocol, 9-(1'-pyrene)nonanol (P9OH)/UV selection (Morand, O. H., Allen, L.-A. H., Zoeller, R. A., and Raetz, C. R. H. (1990) Biochim. Biophys. Acta 1034, 132-141), to isolate a series of plasmalogen-deficient mutants in a murine, macrophage-like cell line, RAW 264.7. Three of these mutants, RAW.7...

The N-terminal 17% of human apolipoprotein B (apoB-17) was expressed in murine C127 cells following transfection with a bovine papilloma virus-based expression vector. A permanent cell line overexpressing the expected 89-kDa protein was selected and characterized. Pulse-chase experiments showed that the depletion of intracellular apoB-17 follows an...

The N-terminal 17% of human apolipoprotein B (apoB-17) was expressed in murine C127 cells following transfection with a bovine papilloma virus-based expression vector. A permanent cell line overexpressing the expected 89-kDa protein was selected and characterized. Pulse-chase experiments showed that the depletion of intracellular apoB-17 follows an...

A comparison of the uptake and conversion of HDL cholesterol and cholesterol ester to bile acids by chick embryo hepatocytes showed the following. Considerable amounts of cholesterol but not cholesterol ester accumulated in the cells. Cholesterol ester was hydrolyzed to cholesterol, 60% of which was converted to bile acids. Approximately 3-5-times...

The availability of different sources of cholesterol for bile acid synthesis by cultured chick embryo hepatocytes was studied. (1) Mevalonolactone was taken up by the cells and converted to cholesterol, cholesterol ester and tauroconjugates of bile acids. The addition of mevalonolactone had little effect on the conversion of endogenous cholesterol...

The liver is the major organ for both synthesis and degradation of plasma cholesterol. These processes are carried out by the parenchymal cells. These cells have at least two distinct surfaces, sinusoidal and canalicular. The interaction with the plasma occurs on the sinusoidal surface; biliary lipids: cholesterol, bile acids and phosphatidylcholin...