Distinct roles of apolipoprotein components within the trypanosome lytic factor complex revealed in a novel transgenic mouse model

Humans express a unique subset of high-density lipoproteins (HDLs) called trypanosome lytic factors (TLFs) that kill many Trypanosoma parasite species. The proteins apolipoprotein (apo) A-I, apoL-I, and haptoglobin-related protein, which are involved in TLF structure and function, were expressed through the introduction of transgenes in mice to explore their physiological roles in vivo. Transgenic expression of human apolipoprotein L-I alone conferred trypanolytic activity in vivo. Coexpression of human apolipoprotein A-I and haptoglobin-related protein (Hpr) had an effect on the integration of apolipoprotein L-I into HDL, and both proteins were required to increase the specific activity of TLF, which was measurable in vitro. Unexpectedly, truncated apolipoprotein L-I devoid of the serum resistance gene interacting domain, which was previously shown to kill human infective trypanosomes, was not trypanolytic in transgenic mice despite being coexpressed with human apolipoprotein A-I and Hpr and incorporated into HDLs. We conclude that all three human apolipoproteins act cooperatively to achieve maximal killing capacity and that truncated apolipoprotein L-I does not function in transgenic animals.

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Abstract 20: Apolipoprotein A-I Influences Regulatory T Cell Development and Proliferation in Homeostasis and Atherogenesis

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvb.35.suppl_1.20 ◽

2015 ◽

Vol 35 (suppl_1) ◽

Author(s):

Dalia E Gaddis ◽

Amy Wu ◽

Debbi Yoakum ◽

Mary Sorci-Thomas ◽

Catherine C Hedrick

Keyword(s):

T Cell ◽

Atherogenic Diet ◽

Cholesterol Homeostasis ◽

Major Protein ◽

Apolipoprotein A ◽

Brdu Labeling ◽

And Function ◽

Treg Development

Apolipoprotein A-I (ApoAI) is the major protein component of HDL. HDL ApoAI is involved in the efflux of cholesterol from cells to maintain cellular cholesterol homeostasis. ApoAI also has anti-inflammatory properties. We previously showed that injecting mice fed an atherogenic diet with ApoAI decreased the number of activated CD4 lymphocytes. Since T regulatory lymphocytes (Treg) play a major role in inhibiting the immune response during atherosclerosis development, we wanted to determine if ApoAI influences Treg development, hypothesizing that ApoAI enhances Treg development. To test this hypothesis, we compared the numbers of Treg in ApoAI-/- mice to B6 mice, and found a 50% decrease in the numbers of Treg in the periaortic LNs (PaLN) of ApoAI-/- mice. BrdU labeling studies showed that ApoAI-/- Treg had a significant 30% reduction in proliferation, suggesting that in the absence of ApoAI and normal cholesterol homeostasis, Tregs have defective proliferation. Functionally, we discovered that ApoAI-/- Treg were significantly less suppressive than B6 Treg in reducing CD4 effector T cell proliferation, suggesting that ApoAI plays a role in both the development and function of Tregs. To determine if the addition of exogenous lipid-free ApoAI could rescue and promote Treg differentiation in ApoAI-/- mice, ApoAI-/- naïve T cells were incubated in vitro with TGFβ and exogenous ApoAI. Addition of ApoAI significantly increased development of naïve ApoA1-/- lymphocytes into Treg. To verify these results in vivo, we fed a novel Treg lineage tracker mouse (LT), Foxp3-YFP-Cre-Rosa26-RFP-ApoE-/- mice a western diet for 15 weeks and administered subcutaneous injections of ApoAI for the last 9 weeks of diet. These mice allow us to identify current functional Tregs and any exTregs that have lost active Treg function in vivo. We found that LT mice treated with ApoAI had a 37% decrease in exTregs and a concomitant 33% increase in current functional Tregs in the aorta. This was accompanied by decreased IFNγ and IL-17 production in PaLN, further confirming our in vitro findings that ApoAI promotes Treg development and function. In conclusion, we have identified a novel role for ApoAI by enhancing Treg development, emphasizing the immune properties of ApoAI for atheroprotection.

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Conservation of targeting but divergence in function and quality control of peroxisomal ABC transporters: an analysis using cross-kingdom expression

Biochemical Journal ◽

10.1042/bj20110249 ◽

2011 ◽

Vol 436 (3) ◽

pp. 547-557 ◽

Cited By ~ 27

Author(s):

Xuebin Zhang ◽

Carine De Marcos Lousa ◽

Nellie Schutte-Lensink ◽

Rob Ofman ◽

Ronald J. Wanders ◽

...

Keyword(s):

Quality Control ◽

Arabidopsis Thaliana ◽

Abc Transporters ◽

Plant Cells ◽

Related Protein ◽

Peroxisomal Membrane ◽

And Function ◽

Native Host

ABC (ATP-binding cassette) subfamily D transporters are found in all eukaryotic kingdoms and are known to play essential roles in mammals and plants; however, their number, organization and physiological contexts differ. Via cross-kingdom expression experiments, we have explored the conservation of targeting, protein stability and function between mammalian and plant ABCD transporters. When expressed in tobacco epidermal cells, the mammalian ABCD proteins ALDP (adrenoleukodystrophy protein), ALDR (adrenoleukodystrophy-related protein) and PMP70 (70 kDa peroxisomal membrane protein) targeted faithfully to peroxisomes and P70R (PMP70-related protein) targeted to the ER (endoplasmic reticulum), as in the native host. The Arabidopsis thaliana peroxin AtPex19_1 interacted with human peroxisomal ABC transporters both in vivo and in vitro, providing an explanation for the fidelity of targeting. The fate of X-linked adrenoleukodystrophy disease-related mutants differed between fibroblasts and plant cells. In fibroblasts, levels of ALDP in some ‘protein-absent’ mutants were increased by low-temperature culture, in some cases restoring function. In contrast, all mutant ALDP proteins examined were stable and correctly targeted in plant cells, regardless of their fate in fibroblasts. ALDR complemented the seed germination defect of the Arabidopsis cts-1 mutant which lacks the peroxisomal ABCD transporter CTS (Comatose), but neither ALDR nor ALDP was able to rescue the defect in fatty acid β-oxidation in establishing seedlings. Taken together, our results indicate that the mechanism for trafficking of peroxisomal membrane proteins is shared between plants and mammals, but suggest differences in the sensing and turnover of mutant ABC transporter proteins and differences in substrate specificity and/or function.

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Abstract 156: Development of a New Bioactivatable Fluorescent Probe for the Study of the Proteolytic Activities Degrading Apolipoprotein A-I in vitro and in vivo

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvb.35.suppl_1.156 ◽

2015 ◽

Vol 35 (suppl_1) ◽

Author(s):

Foued Maafi ◽

Baoqiang Li ◽

Catherine Gebhard ◽

Mathieu Brodeur ◽

Louis Villeneuve ◽

...

Keyword(s):

Fluorescent Probe ◽

Protease Inhibitors ◽

Near Infrared ◽

Ex Vivo ◽

Fold Increase ◽

High Density Lipoproteins ◽

Apolipoprotein A ◽

Proteolytic Activities

Introduction and Objective: The possible benefits of high-density lipoproteins (HDL) against atherosclerosis have been largely attributed to its major protein component, apolipoprotein A-I (apoA-I). However, apoA-I can be degraded by diverse processes, including proteases localized in atherosclerotic plaques, which could reduce the effectiveness of HDL-based therapies. Here we describe the development of a new bioactivatable near-infrared apoA-I-Cy5.5 fluorescent probe and its initial use in the assessment of proteolytic activities that degrade apoA-I in vitro, in vivo and ex vivo. Methods and Results: Fluorescence emission of our probe is quenched by saturation of Cy5.5 fluorophore molecules on the full-length apoA-I protein. In vitro proteolysis of the apoA-I probe showed up to 11-fold increase of near-infrared fluorescence (n=5, P ≤ 0.05). Using this apoA-I-Cy5.5 probe, we were able to quantify proteolytic activities from a wide range of proteases targeting serine (chymase), cysteine (cathepsin S) and metalloproteases (MMP-12). Also, we detected activation of the apoA-I-Cy5.5 probe on aortic cryosections from Ldlr-/--Tg for human apoB atherosclerotic (ATX) mice using an in situ zymography assay and observed that broad-spectrum protease inhibitors protect the probe from protease activities, as shown by decreased fluorescence compared to conditions without protease inhibitors (-54%, n= 6 per group, P ≤ 0.001). In vivo, using a combined Fluorescence Molecular Tomography-Magnetic Resonance imaging system, the injected probe exhibited a trend for increased fluorescence in the aorta when infused in ATX mice compared to C57BL/6J wild-type mice. Ex vivo imaging of these aortas showed a 10-fold increase of fluorescence in ATX (n=5) mice compared to CTL (n=3) mice (P ≤ 0.05). Conclusion: Given the potential importance of HDL functionality in the assessment of cardiovascular risk, this novel protease-activatable apoA-I probe may help to improve HDL-based therapies through better characterization of the alterations of functionality of apoA-I or lipid-poor HDL particles in different pathophysiological settings.

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Digestive development of the early-weaned pig

British Journal Of Nutrition ◽

10.1079/bjn19910079 ◽

1991 ◽

Vol 65 (2) ◽

pp. 181-188 ◽

Cited By ~ 78

Author(s):

D. Kelly ◽

J. A. Smyth ◽

K. J. Mccracken

Keyword(s):

Specific Activity ◽

Nutrient Supply ◽

Spare Capacity ◽

Protein Nitrogen ◽

Weaned Pig ◽

Mucosal Protein ◽

Stomach Weight ◽

And Function

Gastric intubation was adopted as a means of comparing the effect of two feeding levels, continuous nutrient supply (C) and restricted nutrient supply (R), on the digestive development of pigs weaned at 14 d of age, during the first 5 d post-weaning. The absolute weights of the stomach and the pancreas were significantly greater (P < 0.001) in C compared with R pigs. The effect was not significant for pancreas weight when expressed per kg body-weight but was significant (P < 0.05) for stomach weight. The weights of the small intestine (SI), SI mucosa and total mucosal protein were significantly higher (P < 0.001) in C pigs but protein content per g mucosa was similar in the C and R groups. There was no significant effect of treatment on the activity of lactase (β-glucosidase; EC 3.2.1.23) or sucrase (sucrose-α-glucosidase; EC 3.2.1.48) irrespective of the basis of comparison used. The specific activity (μmol/min per g protein) of maltase (α-glucosidase; EC 3.2.1.20) and of glucoamylase (glucan-1,4-α-glucosidase; EC 3.2.1.3) were similar in C and R groups but activities of maltase (μmol/g mucosa) (P < 0.05), and maltase and glucoamylase (mol/d) (P < 0.01) were significantly higher in C pigs. Villous height and crypt depth were significantly greater in C pigs (P < 0.001 and P < 0.05 respectively). Enteroglucagon was significantly (P < 0.05) higher in C compared with R pigs. Xylose absorption and the digestibility of energy were not affected by treatment. Digestibility of dry matter, organic matter, crude protein (nitrogen x 6.25) and carbohydrate were significantly higher (P < 0.001, P < 0.01, P < 0.05 and P < 0.001 respectively) in R pigs compared with C pigs but the differences were small, ranging from 1.3 to 2.5 %. These results demonstrate that (1) nutrient intake in the weaned pig affects the anatomy, morphology and function of the gut, (2) there is considerable ‘spare capacity’ for digestion of cereal-based diets even in pigs weaned at 14 d of age, (3) measurements in vitro of digestive function are of limited value unless supported by information in vivo on absorption/digestibility.

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Effects of human apolipoprotein A-I on endotoxin-induced leukocyte adhesion on endothelial cells in vivo and on the growth of Escherichia coli in vitro

Journal of Endotoxin Research ◽

10.1177/0968051907078611 ◽

2007 ◽

Vol 13 (1) ◽

pp. 58-64 ◽

Cited By ~ 13

Author(s):

Premtip Thaveeratitham ◽

Wanee Plengpanich ◽

Worakamol Naen-Udorn ◽

Suthiluk Patumraj ◽

Weerapan Khovidhunkit

Keyword(s):

Escherichia Coli ◽

Endothelial Cells ◽

Leukocyte Adhesion ◽

Human Apolipoprotein ◽

Apolipoprotein A

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Human apolipoprotein(a) kringle V inhibits angiogenesis in vitro and in vivo by interfering with the activation of focal adhesion kinases

Biochemical and Biophysical Research Communications ◽

10.1016/j.bbrc.2003.11.148 ◽

2004 ◽

Vol 313 (3) ◽

pp. 534-540 ◽

Cited By ~ 33

Author(s):

Jang-Seong Kim ◽

Hyun-Kyung Yu ◽

Jin-Hyung Ahn ◽

Ho-Jeong Lee ◽

Soon-Won Hong ◽

...

Keyword(s):

Focal Adhesion ◽

Human Apolipoprotein ◽

Apolipoprotein A

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Studies of intestinal morphology and cathepsin B expression in a transgenic mouse aiming at intestine-specific expression of Cath B-EGFP

Biological Chemistry ◽

10.1515/bc.2011.096 ◽

2011 ◽

Vol 392 (11) ◽

Cited By ~ 1

Author(s):

Maria Arampatzidou ◽

Kristina Mayer ◽

Maria E. Iolyeva ◽

Seblewongel Gebre Asrat ◽

Mirunalini Ravichandran ◽

...

Keyword(s):

Transgenic Mouse ◽

Cathepsin B ◽

Fluorescent Protein ◽

Transgenic Animals ◽

Intestinal Morphology ◽

Specific Expression ◽

Transgenic Expression ◽

Morphological Studies

AbstractCathepsin B has been shown to not only reside within endo-lysosomes of intestinal epithelial cells, but it was also secreted into the extracellular space of intestinal mucosa in physiological and pathological conditions. In an effort to further investigate the function of this protease in the intestine, we generated a transgenic mouse model that would enable us to visualize the localization of cathepsin Bin vivo. Previously we showed that the A33-antigen promoter could be successfully usedin vitroin order to express cathepsin B-green fluorescent protein chimeras in cells that co-expressed the intestine-specific transcription factor Cdx1. In this study an analog approach was used to express chi-meric cathepsin B specifically in the intestine of transgenic animals. No overt phenotype was observed for the transgenic mice that reproduced normally. Biochemical and morphological studies confirmed that the overall intestinal phenotype including the structure and polarity of this tissue as well as cell numbers and differentiation states were not altered in the A33-CathB-EGFP mice when compared to wild type animals. However, transgenic expression of chimeric cathepsin B could not be visualized because it was not translatedin situalthough the transgene was maintained over several generations.

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Fission yeast Msp1 is a mitochondrial dynamin-related protein

Journal of Cell Science ◽

10.1242/jcs.112.22.4151 ◽

1999 ◽

Vol 112 (22) ◽

pp. 4151-4161

Author(s):

L. Pelloquin ◽

P. Belenguer ◽

Y. Menon ◽

N. Gas ◽

B. Ducommun

Keyword(s):

Fission Yeast ◽

Mitochondrial Protein ◽

Related Protein ◽

Mitochondrial Inner Membrane ◽

Mitochondrial Processing Peptidase ◽

The Matrix ◽

Functional Peptide ◽

And Function

We recently identified Msp1p, a fission yeast Schizosaccharomyces pombe dynamin-related protein, which is essential for the maintenance of mitochondrial DNA. The Msp1p sequence displays typical features of a mitochondrial protein. Here we report in vitro and in vivo data that validate that prediction. We demonstrate that the targeting sequence of Msp1p is processed by recombinant mitochondrial processing peptidase and that Msp1p is imported into S. pombe mitochondria in vitro in the presence of cellular extracts. We show that the first 109 residues of Msp1p encompass a functional peptide signal that is sufficient to direct chimera to mitochondria. Immunofluorescence studies indicate that Msp1p staining colocalises with a mitochondrial marker and electron microscopy shows that the protein is located inside the mitochondria. Mitochondrial enrichment and fractionation further confirm that localisation and show that Msp1p is anchored to the matrix side of the mitochondrial inner membrane. Finally, we report that overexpression of the Msp1 protein results in gross alteration of the mitochondrial structure and function. All together our results suggest that Msp1p is an essential component for mitochondrial maintenance.

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