Siqin Zhaorigetu

Apolipoprotein L1, a Novel Bcl-2 Homology Domain 3-only Lipid-binding Protein, Induces Autophagic Cell Death

The Bcl-2 family proteins are important regulators of type I programmed cell death apoptosis; however, their role in autophagic cell death (AuCD) or type II programmed cell death is still largely unknown. Here we report the cloning and characterization of a novel Bcl-2 homology domain 3 (BH3)-only protein, apolipoprotein L1 (apoL1), that, when overexpressed and accumulated intracellularly, induces AuCD in cells as characterized by the increasing formation of autophagic vacuoles and activating the translocation of LC3-II from the cytosol to the autophagic vacuoles. Wortmannin and 3-methyladenine, inhibitors of class III phosphatidylinostol 3-kinase and, subsequently, autophagy, blocked apoL1-induced AuCD. In addition, apoL1 failed to induce AuCD in autophagy-deficient ATG5-/- and ATG7-/- mouse embryonic fibroblast cells, suggesting that apoL1-induced cell death is indeed autophagy-dependent. Furthermore, a BH3 domain deletion construct of apoL1 failed to induce AuCD, demonstrating that apoL1 is a bona fide BH3-only pro-death protein. Moreover, we showed that apoL1 is inducible by p53 in p53-induced cell death and is a lipid-binding protein with high affinity for phosphatidic acid (PA) and cardiolipin (CL). Previously, it has been shown that PA directly interacted with mammalian target of rapamycin and positively regulated the ability of mammalian target of rapamycin to activate downstream effectors. In addition, CL has been shown to activate mitochondria-mediated apoptosis. Sequestering of PA and CL with apoL1 may alter the homeostasis between survival and death leading to AuCD. To our knowledge, this is the first BH3-only protein with lipid binding activity that, when overproduced intracellularly, induces AuCD.

ApoL1, a BH3-only lipid-binding protein, induces autophagic cell death

We recently reported the identification and characterization of a novel BH3-only pro-death protein, apolipoprotein L1 (ApoL1), that, when overexpressed, induces autophagic cell death (ACD) in a variety of cells, including those originated from normal and cancerous tissues. ApoL1 failed to induce ACD in autophagy-deficient Atg5-/- and Atg7-/- MEF cells, suggesting that ApoL1-induced cell death is indeed autophagy-dependent. In addition, a BH3 domain deletion allele of ApoL1 was unable to induce ACD, demonstrating that ApoL1 is a bona fide BH3-only pro-death protein. To further investigate regulation of ApoL1 expression, we showed that ApoL1 is inducible by interferon-γ and tumor necrosis factor-α in human umbilical vein endothelial cells, suggesting that ApoL1 may play a role in cytokine-induced inflammatory response. Moreover, we observed that ApoL1 is a lipid-binding protein with high affinity for phosphatidic acid and cardiolipin and less affinity for various phosphoinositides. Functional genomics analysis identified 5 nonsynonymous single nucleotide polymorphisms (NSNPs) in the coding exons of the human ApoL1 structural gene– all the 5 NSNPs may cause deleterious alteration of ApoL1 activity. Finally, we discuss the link between ApoL1 and various human diseases. Addendum to: Wan G, Zhaorigetu S, Liu Z, Kaini R, Jiang Z, Hu CA. Apolipoprotein L1, a novel Bcl-2 homology domain 3-only lipid-binding protein, induces autophagic cell death. J Biol Chem 2008; 283:21540-49.

Human Δ1-pyrroline-5-carboxylate synthase: function and regulation

Mammalian Δ1-pyrroline-5-carboxylate synthase (P5CS) is a bifunctional ATP- and NAD(P)H-dependent mitochondrial enzyme that catalyzes the coupled phosphorylation and reduction-conversion of l-glutamate to P5C, a pivotal step in the biosynthesis of l-proline, l-ornithine and l-arginine. Previously, we reported cloning and characterization of two P5CS transcript variants generated by exon sliding that encode two protein isoforms differing only by a two amino acid-insert at the N-terminus of the γ-glutamyl kinase active site. The short form (P5CS.short) is highly expressed in the gut and is inhibited by ornithine. In contrast, the long form (P5CS.long) is expressed ubiquitously and is insensitive to ornithine. Interestingly, we found that all the established human cell lines we have studied expressed P5CS.long but not P5CS.short. In addition, expression of P5CS.long can be modulated by hormones: downregulation by hydrocortisone and dexamethasone and upregulation by estradiol, for example. Using a quantitative proteomic approach, we showed that P5CS.long is upregulated by p53 in p53-induced apoptosis in DLD-1 colorectal cancer cells. Functional genomic analysis confirmed that there are two p53-binding consensus sequences in the promoter region and in the intron 1 of the human P5CS gene. Interestingly, overexpression of P5CS by adenoviruses harboring P5CS.long or P5CS.short in various cell types has no effect on cell growth or survival. It would be of importance to further investigate the role of P5CS as a p53 downstream effector and how P5CS.short expression is regulated by hormones and factors of alternative splicing in cells isolated from model animals.

Functional genomics and SNP analysis of human genes encoding proline metabolic enzymes

Proline metabolism in mammals involves two other amino acids, glutamate and ornithine, and five enzymatic activities, Δ1-pyrroline-5-carboxylate (P5C) reductase (P5CR), proline oxidase, P5C dehydrogenase, P5C synthase and ornithine-δ-aminotransferase (OAT). With the exception of OAT, which catalyzes a reversible reaction, the other four enzymes are unidirectional, suggesting that proline metabolism is purpose-driven, tightly regulated, and compartmentalized. In addition, this tri-amino-acid system also links with three other pivotal metabolic systems, namely the TCA cycle, urea cycle, and pentose phosphate pathway. Abnormalities in proline metabolism are relevant in several diseases: six monogenic inborn errors involving metabolism and/or transport of proline and its immediate metabolites have been described. Recent advances in the Human Genome Project, in silico database mining techniques, and research in dissecting the molecular basis of proline metabolism prompted us to utilize functional genomic approaches to analyze human genes which encode proline metabolic enzymes in the context of gene structure, regulation of gene expression, mRNA variants, protein isoforms, and single nucleotide polymorphisms.

Apolipoprotein L6, induced in atherosclerotic lesions, promotes apoptosis and blocks Beclin 1-dependent autophagy in atherosclerotic cells.

Inflammatory cytokine-regulated apoptosis and autophagy play pivotal roles in plaque rupture and thrombosis of atherosclerotic lesions. However, the molecular interplay between apoptosis and autophagy in vascular cells has not been investigated. Our prior study showed that human apolipoprotein L6 (ApoL6), a pro-apoptotic BH3-only member of the Bcl-2 family, was one of the downstream targets of interferon-γ (INFγ), which sensitizes atherosclerotic lesion-derived cells (LDCs) to Fas-induced apoptosis. To investigate whether ApoL6 plays a causal role in atherosclerotic apoptosis and autophagy, in this study, we demonstrate that IFNγ treatment itself strongly induces ApoL6, and ApoL6 is highly expressed and partially co-localized with activated caspase 3 in activated smooth muscle cells in atherosclerotic lesions. In addition, overexpression of ApoL6 promotes reactive oxygen species (ROS) generation, caspase activation, and subsequent apoptosis, which can be blocked by pan caspase inhibitor and ROS scavenger. Knockdown of ApoL6 expression by siApoL6 suppresses INFγ- and Fas-mediated apoptosis. Further, ApoL6 binds Bcl-XL, one of the most abundant anti-death proteins in LDCs. Interestingly, forced ApoL6 expression in LDCs induces degradation of Beclin 1, accumulation of p62, and subsequent attenuation of LC3-II formation and translocation and thus autophagy, whereas siApoL6 treatment reverts the phenotype. Taken together, our results suggest that ApoL6 regulates both apoptosis and autophagy in SMCs. IFNγ-initiated, ApoL6-induced apoptosis in vascular cells may be an important factor causing plaque instability and a potential therapeutic target for treating atherosclerosis and cardiovascular disease.

Autophagy regulates lipolysis and cell survival through lipid droplet degradation in androgen-sensitive prostate cancer cells.

Androgen deprivation therapy, one of the standard treatments for prostate cancer (PCa) induces apoptosis, as well as autophagy in androgen‐responsive PCa cells. As autophagy can promote either cell survival or death, it is important to understand its role in PCa treatment. The objective of this study was to elucidate the function of autophagy in lipid droplet (LD) homeostasis and survival in androgen‐sensitive PCa cells.

Delivery of negatively charged liposomes into the atherosclerotic plaque of apolipoprotein E-deficient mouse aortic tissue.

Abstract Liposomes have been used to diagnose and treat cancer and, to a lesser extent, cardiovascular disease. We previously showed the uptake of anionic liposomes into the atheromas of Watanabe heritable hyperlipidemic rabbits within lipid pools. However, the cellular distribution of anionic liposomes in atherosclerotic plaque remains undescribed. In addition, how anionic liposomes are absorbed into atherosclerotic plaque is unclear. We investigated the uptake and distribution of anionic liposomes in atherosclerotic plaque in aortic tissues from apolipoprotein E-deficient (ApoE–/–) mice. To facilitate the tracking of liposomes, we used liposomes containing fluorescently labeled non-silencing small interfering RNA. Confocal microscopy analysis showed the uptake of anionic liposomes into atherosclerotic plaque and colocalization with macrophages. Transmission electron microscopy analysis revealed anionic liposomal accumulation in macrophages. To investigate how anionic liposomes cross the local endothelial barrier, we examined the role of clathrin-mediated endocytosis in human coronary artery endothelial cells (HCAECs) treated with or without the inflammatory cytokine tumor necrosis factor (TNF)-α. Pretreatment with amantadine, an inhibitor of clathrin-mediated endocytosis, significantly decreased liposomal uptake in HCAECs treated with or without TNF-α by 77% and 46%, respectively. Immunoblot analysis showed that endogenous clathrin expression was significantly increased in HCAECs stimulated with TNF-α but was inhibited by amantadine. These studies indicated that clathrin-mediated endocytosis is partly responsible for the uptake of liposomes by endothelial cells. Our results suggest that anionic liposomes target macrophage-rich areas of vulnerable plaque in ApoE–/– mice; this finding may lead to the development of novel diagnostic and therapeutic strategies for treating vulnerable plaque in humans.

Intimacy and a deadly feud: the interplay of autophagy and apoptosis mediated by amino acids

Autophagy (i.e., “self-eating”) and apoptosis (i.e., type I programmed cell death) are essential and intimately involved in molecular, cellular, and whole-body homeostasis in humans and animals. Autophagy has been categorized as a mechanism of intracellular degradation, recycling, defense, and survival. To date, three types of autophagy have been identified: macroautophagy, microautophagy, and chaperone-mediated autophagy. Recent discoveries strongly suggest that macroautophagy also modulates type II programmed cell death under specific circumstances. Autophagy and apoptosis are fundamentally distinct processes, but are interconnected by common stress initiators and intermediate regulators. During the past two decades, the role of amino acid metabolism and signaling in the regulation of apoptosis and autophagy has been intensively studied. In this review, we summarize recent advances in our understanding of the molecular mechanisms that regulate both autophagy and apoptosis in the context of amino acid signaling.

Multimodal optical coherence tomography and fluorescence lifetime imaging with interleaved excitation sources for simultaneous endogenous and exogenous fluorescence

Multimodal imaging probes a variety of tissue properties in a single image acquisition by merging complimentary imaging technologies. Exploiting synergies amongst the data, algorithms can be developed that lead to better tissue characterization than could be accomplished by the constituent imaging modalities taken alone. The combination of optical coherence tomography (OCT) with fluorescence lifetime imaging microscopy (FLIM) provides access to detailed tissue morphology and local biochemistry. The optical system described here merges 1310 nm swept-source OCT with time-domain FLIM having excitation at 355 and 532 nm. The pulses from 355 and 532 nm lasers have been interleaved to enable simultaneous acquisition of endogenous and exogenous fluorescence signals, respectively. The multimodal imaging system was validated using tissue phantoms. Nonspecific tagging with Alexa Flour 532 in a Watanbe rabbit aorta and active tagging of the LOX-1 receptor in human coronary artery, demonstrate the capacity of the system for simultaneous acquisition of OCT, endogenous FLIM, and exogenous FLIM in tissues.

DELIVERY OF LIPOSOMAL FATTY ACID BINDING PROTEIN 4 SIRNA FOR THE TREATMENT OF ATHEROSCLEROSIS IN APOLIPOPROTEIN E-DEFICIENT MICE

Fatty acid binding protein 4 (FABP4) deficiency protects apolipoprotein E-deficient (ApoE−/−) mice against developing atherosclerosis, suggesting that FABP4 may be a potential drug target for treating atherosclerosis. We developed a liposomal siRNA delivery system for silencing FABP4 as a