Type I signal peptidase (SPase I) catalyzes the cleavage of the amino-terminal signal sequences from preproteins destined for cell export. Preproteins contain a signal sequence with a positively charged n-region, a hydrophobic h-region, and a neutral but polar c-region. Despite having no distinct consensus sequence other than a commonly found c-region "Ala-X-Ala" motif preceding the cleavage site, signal sequences are recognized by SPase I with high fidelity. Remarkably, other potential Ala-X-Ala sites are not cleaved within the preprotein. One hypothesis is that the source of this fidelity is due to the anchoring of both the SPase I enzyme (by way of its transmembrane segment) and the preprotein substrate (by the h-region in the signal sequence) in the membrane. This limits the enzyme-substrate interactions such that cleavage occurs at only one site. In this work we have, for the first time, successfully isolated Bacillus subtilis type I signal peptidase (SipS) and a truncated version lacking the transmembrane domain (SipS-P2). With purified full-length as well as truncated constructs of both B. subtilis and Escherichia coli (Lep) SPase I, in vitro specificity studies indicate that the transmembrane domains of either enzyme are not important determinants of in vitro cleavage fidelity, since enzyme constructs lacking them reveal no alternate site processing of pro-OmpA nuclease A substrate. In addition, experiments with mutant pro-OmpA nuclease A substrate constructs indicate that the h-region of the signal peptide is also not critical for substrate specificity. In contrast, certain mutants in the c-region of the signal peptide result in alternate site cleavage by both Lep and SipS enzymes.

A Soat1 deletion present in AKR mice, and resultant N-terminal ACAT1 truncation, was confirmed to be a significant modifier of macrophage cholesterol metabolism. Other Mcmm loci candidate genes were prioritized via bioinformatics.

High levels of high density lipoprotein-cholesterol (HDL-C) are associated with lower risk for cardiovascular disease in epidemiological studies ( 1 ). Although several mechanisms may play a role in HDL's protective effect, HDL and its major protein constituent, apoA1, are critical components of the reverse cholesterol transport (RCT) pathway, in which cholesterol is removed from peripheral tissues and transferred to the liver for excretion. In the fi rst step of the RCT pathway, lipid-poor apoA1 acts as an acceptor for cell cholesterol and phospholipids via the cell membrane protein ABCA1, generating nascent HDL.Recently, "HDL is the good cholesterol" hypothesis has suffered several setbacks. For example, several trials of HDL-C-raising drugs, including cholesteryl ester transfer protein inhibitors and niacin, failed to demonstrate clinical benefi t ( 2-5 ). Furthermore, a Mendelian randomization study did not associate gene variants that solely alter HDL-C with coronary artery disease (CAD) incidence ( 6 ). That said, human and mouse models with defective RCT, via mutations in ABCA1, apoA1, or scavenger receptor class B type I (SR-BI), are more susceptible to atherosclerosis independent Abstract HDL functions are impaired by myeloperoxidase (MPO), which selectively targets and oxidizes human apoA1. We previously found that the 4WF isoform of human apoA1, in which the four tryptophan residues are substituted with phenylalanine, is resistant to MPO-mediated loss of function. The purpose of this study was to generate 4WF apoA1 transgenic mice and compare functional properties of the 4WF and wild-type human apoA1 isoforms in vivo . Male mice had signifi cantly higher plasma apoA1 levels than females for both isoforms of human apoA1, attributed to different production rates. With matched plasma apoA1 levels, 4WF transgenics had a trend for slightly less HDL-cholesterol versus human apoA1 transgenics. While 4WF transgenics had 31% less reverse cholesterol transport (RCT) to the plasma compartment, equivalent RCT to the liver and feces was observed. Plasma from both strains had similar ability to accept cholesterol and facilitate ex vivo cholesterol effl ux from macrophages. Furthermore, we observed that 4WF transgenic HDL was partially ( ‫ف‬ 50%) protected from MPOmediated loss of function while human apoA1 transgenic HDL lost all ABCA1-dependent cholesterol acceptor activity. In conclusion, the structure and function of HDL from 4WF transgenic mice was not different than HDL derived from human apoA1 transgenic mice. -Berisha, S.

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The SRCBOCC proposes to lease approximately 328 acres of Air Force property for the purpose of constructing and operating a reclaimed water RIB system. The proposed RIB system would receive and distribute highly treated, reclaimed wastewater from three wastewater utilities operating on the Fairpoint Peninsula and Santa Rosa Island, Florida. These utilities are the Navarre Beach Utilities (NBU), the Holley Navarre Water System (HNWS) and the South Santa Rosa Utilities (SSRU). The reclaimed wastewater would be pumped via pipeline from these three locations to the RIB System, where it would filter through the soil to recharge the surficial aquifer. A portion of this pipeline, known as the South Holley Segment, would transverse a section of Eglin AFB (EA Section 1.0, page 1-1 and Figure 1-1). By implementing the proposed action, SRCBOCC would address their current and future regional needs for disposing of reclaimed water generated within the southern region of Santa Rosa County. The proposed site is located on Eglin AFB west of State Road (SR) 87 and south of Eglin AFB Range Road 726. A buffer distance of no less than 500 feet would be maintained from SR 87 and a huffer distance of no less than 10,000 feet would be maintained from the north-south runway of Choctaw Air Field. A series of RIB systems would be constructed in three phases over a 20-year period. Ofthe 328 acres, 200 acres would be a phased development as demands for wastewater disposal increase. Initially, Phase I (40 acres) would be constructed: then Phase II (90 acres); and then fmally Phase III (70 acres). The remaining 128 acres would be set aside as a contingency area that may be required in the event a regulatory review by the Florida Department of Environmental Protection determined the need for an additional area. •A 2-acre operations compound consisting of a combined office and equipment storage and maintenance shed would be constructed. Access to the facilities and infiltration basins would be provided by 15-foot wide gravel based roads. Several monitoring wells would be installed up gradient and down gradient from the RIBS system to monitor groundwater drinking water parameters. The number of installed wells would be determined by the Florida Department of Environmental Protection. (EA Section 2.4, pages 2-3 thru 2-10) Apart from the actual RIBs site, a small comer-section of Eglin land would be used to install a buried pipeline. This pipeline, referred to in the EA as the South Holley Segment, is part of a much larger pipeline system that would transport effluent to the RIBs. This comer-section of Eglin land, as well as the surrounding developed rural residential parcels, is located in the floodplain (Section 1.0, pg 1-1, Fig 1-1 and Fig 1-4, pg 1-3,-15). The South Holley Segment is approximately 500 feet long and 50 feet wide and is located immediately adjacent to the State Road 87 right-of-way. The only activity occurring here would be installation ofthe buried pipeline. No-Action Alternative: Under the no-action alternative, the RIB system...

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Introduction: apoAI-ABCA1 mediated nascent HDL assembly promotes cholesterol efflux and reverse cholesterol transport. Cell surface phosphatidylinositol 4,5- bisphosphate (PIP2) mediates this process, but whether PIP2 is effluxed via apoA1-ABCA1 pathway and is carried in plasma is unknown. Objective: To determine if PIP2 is effluxed during ABCA1-dependent nHDL biogenesis, whether PIP2 is found on plasma HDL, and if PIP2 can be delivered to target cells via SRB1. Methods and Results: We previously reported at this meeting that: 1) apoAI binds to PIP2 with a K d of ~100 nM; 2) ABCA1 expression translocates PIP2 from the inner to outer leaflet of the plasma membrane; 3) cell surface PIP2 is required for ABCA1-dependent cellular binding of apoA1 and cholesterol efflux. Here, we report that PIP2 incubation with lipid-free apoA1 promoted monomeric vs. dimeric apoA1. We found that ABCA1 expression increased efflux of PIP2 by ~20-fold in RAW264.7 cells labeled with [3H]myo-inositol, and chased with apoA1 (mean ± SD; ***, p<0.001, two-tailed t-test, n=3). Plasma from apoA1 knockout (A1 KO), wild type (WT), and human apoA1 transgenic (A1-Tg) mice contained apoA1-gene dosage dependent levels of both cholesterol and PIP2, with 64-fold higher PIP2 levels in the A1-Tg vs. A1 KO mice (mean ± SD; **, p<0.01, two-tailed t-test, n=3). PIP2 was also reverse transported in vivo from [3H]myo-inositol-labeled macrophages implanted s.c into mice. Labeled PIP2 was recovered in the plasma, with a 4-fold higher of the injected radioactivity found in the WT vs. A1 KO host mice (mean ± SD; **, p<0.01, two-tailed t-test, n=3). FPLC separation of human plasma determined that almost all of the plasma PIP2 was found in the HDL fraction; and, human HDL contained two PIP2 species with either 18:0, 20:4 fatty acids or 16:0, 20:4 fatty acids, as detected by liquid chromatography tandem mass spectrometry. SR-BI-inducible BHK cells exhibited 2-fold higher uptake of [3H]PIP2-labeled HDL after SR-BI induction, indicating that HDL can deliver PIP2 to target cells (mean ± SD; **, p<0.01, two-tailed t-test, n=3). Conclusions: PIP2 is effluxed from cells via the apoA1-ABCA1 pathway and is carried on HDL. HDL can deliver PIP2 to target tissues via SR-B1.

Introduction HDL functions, such as its cholesterol acceptor and anti-inflammatory activities, are impaired by myeloperoxidase (MPO), which selectively targets and oxidizes HDL and apoAI. We previously found that the 4WF apoAI isoform, in which the 4 Trp residues are substituted with Phe, is equally competent as wild type (WT) human apoAI to interact with lipids and accept cholesterol in an ABCA1-dependent manner, but is resistant to MPO mediated loss of function. Hypothesis The oxidant resistant 4WF apoAI isoform will be superior in mediating reverse cholesterol transport than WT human apoAI in vivo under conditions of elevated oxidative stress. Methods and Results We generated transgenic mice expressing the 4WF human apoAI (h-apoAI) isoform. A genomic fragment containing the h-apoAI gene was subjected to site directed mutagenesis to convert the Trp to Phe residues, and microinjected into C57BL/6 fertilized oocytes. We selected the line with the highest h-apoAI expression to compare with existing WT h-apoAI transgenics. We assessed plasma apoAI and HDL-C levels in male and female mice. Male mice have significantly higher h-apoAI levels than females for both isoforms (p<0.05). In general, the WT apoAI transgenics have higher plasma h-apoAI levels than the 4WF transgenics; however there is enough overlap that we were able to select male 4WF and WT h-apoAI transgenic mice having matched plasma h-apoAI levels of ~550 mg/dl, which are described below. HDL-C levels were slightly lower in 4WF vs. WT transgenics (157±4 vs 184±8 mg/dl, respectively, N=5 per group, p=0.03). We performed in vivo reverse cholesterol transport (RCT) assay by s.c. injection of cholesterol labeled and loaded bone-marrow macrophages. RCT to the plasma reflected the HDL-C levels and was lower in the 4WF vs. WT transgenic mice (4.5% vs 6.6%, respectively p=0.007). However, RCT to the liver and feces was equivalent in the 4WF and WT transgenics (NS). This suggests that the flux of HDL-C in the 4WF mice may be higher than in the WT apoAI transgenics, leading to equal RCT to the feces and liver despite lower HDL-C levels. Conclusion 4WF transgenic mice are now breeding with MPO transgenic mice in order to test our hypothesis that this isoform retains more HDL function under oxidative stress conditions.