Development of New Genome Editing Tools for the Treatment of Hyperlipidemia.

Hyperlipidemia is a medical condition characterized by high levels of lipids in the blood. It is often associated with an increased risk of cardiovascular diseases such as heart attacks and strokes. Traditional treatment approaches for hyperlipidemia involve lifestyle modifications, dietary changes, and the use of medications like statins. Recent advancements in genome editing technologies, including CRISPR-Cas9, have opened up new possibilities for the treatment of this condition. This review provides a general overview of the main target genes involved in lipid metabolism and highlights the progress made during recent years towards the development of new treatments for dyslipidemia.

Role of Lactone and Acid Forms in the Pleiotropic Effects of Statins.

Statins are a class of drugs used worldwide to lower low-density lipoprotein cholesterol [...].

Membrane Cholesterol Content and Lipid Organization Influence Melittin and Pneumolysin Pore-Forming Activity.

Melittin, the main toxic component in the venom of the European honeybee, interacts with natural and artificial membranes due to its amphiphilic properties. Rather than interacting with a specific receptor, melittin interacts with the lipid components, disrupting the lipid bilayer and inducing ion leakage and osmotic shock. This mechanism of action is shared with pneumolysin and other members of the cholesterol-dependent cytolysin family. In this manuscript, we investigated the inverse correlation for cholesterol dependency of these two toxins. While pneumolysin-induced damage is reduced by pretreatment with the cholesterol-depleting agent methyl-ß-cyclodextrin, the toxicity of melittin, after cholesterol depletion, increased. A similar response was also observed after a short incubation with lipophilic simvastatin, which alters membrane lipid organization and structure, clustering lipid rafts. Therefore, changes in toxin sensitivity can be achieved in cells by depleting cholesterol or changing the lipid bilayer organization.

New Insights Into Targeting Membrane Lipids for Cancer Therapy.

Modulation of membrane lipid composition and organization is currently developing as an effective therapeutic strategy against a wide range of diseases, including cancer. This field, known as membrane-lipid therapy, has risen from new discoveries on the complex organization of lipids and between lipids and proteins in the plasma membranes. Membrane microdomains present in the membrane of all eukaryotic cells, known as lipid rafts, have been recognized as an important concentrating platform for protein receptors involved in the regulation of intracellular signaling, apoptosis, redox balance and immune response. The difference in lipid composition between the cellular membranes of healthy cells and tumor cells allows for the development of novel therapies based on targeting membrane lipids in cancer cells to increase sensitivity to chemotherapeutic agents and consequently defeat multidrug resistance. In the current manuscript strategies based on influencing cholesterol/sphingolipids content will be presented together with innovative ones, more focused in changing biophysical properties of the membrane bilayer without affecting the composition of its constituents.

Pleiotropic effects of statins via interaction with the lipid bilayer: A combined approach.

Statins are effective inhibitors of cholesterol biosynthesis, largely used for prevention of cardiovascular diseases induced by hypercholesterolemia. However, their use in different clinical trials clearly indicate that the general benefits observed with statins are also related to effects beyond the cholesterol lowering. Increasing evidences suggest that some of these cholesterol-independent or "pleiotropic" effects of statins involve the interaction and modification of the membrane bilayers. In this manuscript, using a combined approach based on biophysical (electrochemical impedance spectroscopy on tethered bilayer lipid membranes) and biological methods (hemolysis on erythrocytes and immunocytochemistry on cancer cells), we demonstrate that lipophilic, but not hydrophilic statins are capable of reducing the damage caused by cholesterol-dependent cytolysins. This protection correlates with statins lipophilicity and capacity to interact with the lipid bilayer. Our data suggests lipophilic statins associate with membranes and interfere with the ability of cholesterol dependent cytolysins, to bind to membrane cholesterol. Evaluation of the capacity of statins to modulate cell membrane properties is essential for developing a correct therapeutic approach for cardiovascular diseases as well as for understanding the potential of this class of drugs as adjuvants for drug delivery.

Biological applications of tethered bilayer lipid membranes.

Lipid membranes have an essential role in most physiological processes including cell protection, cell-to-cell communication and regulation of intracellular signaling. These multiple roles of biological membranes prompted the study and the development of artificial lipid membranes with the primary aim to reconstitute the natural functions in vitro and understand the interaction between all membrane components at the molecular level. Tethered bilayer lipid membranes (tBLMs) are emerging as the ideal experimental platform for functional and structural studies on membrane-associated proteins due to their peculiar properties such as their stability and fluidity of both leaflets in the phospholipid bilayer. tBLMs have been used in many studies, ranging from the analysis of membrane structure and function, studies of the membrane-protein and membrane-peptide interactions, as well as applications as biosensors and energy generating devices. This review describes all the uses of tBLMs as a tool to investigate biological functions, pointing out limitations of this methodology and future applications once optimization of the technique will be achieved.

Lipids in the cell: organisation regulates function.

Lipids are fundamental building blocks of all cells and play important roles in the pathogenesis of different diseases, including inflammation, autoimmune disease, cancer, and neurodegeneration. The lipid composition of different organelles can vary substantially from cell to cell, but increasing evidence demonstrates that lipids become organised specifically in each compartment, and this organisation is essential for regulating cell function. For example, lipid microdomains in the plasma membrane, known as lipid rafts, are platforms for concentrating protein receptors and can influence intra-cellular signalling. Lipid organisation is tightly regulated and can be observed across different model organisms, including bacteria, yeast, Drosophila, and Caenorhabditis elegans, suggesting that lipid organisation is evolutionarily conserved. In this review, we summarise the importance and function of specific lipid domains in main cellular organelles and discuss recent advances that investigate how these specific and highly regulated structures contribute to diverse biological processes.

Inhibiting mevalonate pathway enzymes increases stromal cell resilience to a cholesterol-dependent cytolysin.

Animal health depends on the ability of immune cells to kill invading pathogens, and on the resilience of tissues to tolerate the presence of pathogens. Trueperella pyogenes causes tissue pathology in many mammals by secreting a cholesterol-dependent cytolysin, pyolysin (PLO), which targets stromal cells. Cellular cholesterol is derived from squalene, which is synthesized via the mevalonate pathway enzymes, including HMGCR, FDPS and FDFT1. The present study tested the hypothesis that inhibiting enzymes in the mevalonate pathway to reduce cellular cholesterol increases the resilience of stromal cells to PLO. We first verified that depleting cellular cholesterol with methyl-ß-cyclodextrin increased the resilience of stromal cells to PLO. We then used siRNA to deplete mevalonate pathway enzyme gene expression, and used pharmaceutical inhibitors, atorvastatin, alendronate or zaragozic acid to inhibit the activity of HMGCR, FDPS and FDFT1, respectively. These approaches successfully reduced cellular cholesterol abundance, but mevalonate pathway enzymes did not affect cellular resilience equally. Inhibiting FDFT1 was most effective, with zaragozic acid reducing the impact of PLO on cell viability. The present study provides evidence that inhibiting FDFT1 increases stromal cell resilience to a cholesterol-dependent cytolysin.

Understanding the Dr. Jekyll and Mr. Hyde nature of apoptosis-inducing factor: future perspectives.

Apoptosis-inducing factor (AIF) is emerging as a key protein in regulation of basic physiological processes including phagocytosis, mitophagy and regulation of the redox state. Recent evidences suggest that the enzymatic activity of AIF may play an active role in tumor progression controlling energy metabolism and redox balance. The present manuscript briefly describes the story of this protein from its initial discovery as caspase-independent apoptotic protein, throughout its role in oxidative phosphorylation and lately involvement in tumor progression. Understanding the dualistic nature of AIF is a critical starting point to clarify its contribution in tumor metabolic balance and to develop new AIF-specific therapeutic strategies.

Tethered bilayer membranes as a complementary tool for functional and structural studies: The pyolysin case.

We demonstrate the use of tethered bilayer lipid membranes (tBLMs) as an experimental platform for functional and structural studies of membrane associated proteins by electrochemical techniques. The reconstitution of the cholesterol-dependent cytolysin (CDC) pyolysin (PLO) from Trueperella pyogenes into tBLMs was followed in real-time by electrochemical impedance spectroscopy (EIS). Changes of the EIS parameters of the tBLMs upon exposure to PLO solutions were consistent with the dielectric barrier damage occurring through the formation of water-filled pores in membranes. Parallel experiments involving a mutant version of PLO, which is able to bind to the membranes but does not form oligomer pores, strengthen the reliability of this methodology, since no change in the electrochemical impedance was observed. Complementary atomic force microscopy (AFM) and neutron reflectometry (NR) measurements revealed structural details of the membrane bound PLO, consistent with the structural transformations of the membrane bound toxins found for other cholesterol dependent cytolysins. In this work, using the tBLMs platform we also observed a protective effect of the dynamin inhibitor Dynasore against pyolysin as well as pneumolysin. An effect of Dynasore in tBLMs, which was earlier observed in experiments with live cells, confirms the biological relevance of the tBLMs models, as well as demonstrates the potential of the electrochemical impedance spectroscopy to quantify membrane damage by the pore forming toxins. In conclusion, tBLMs are a reliable and complementary method to explore the activity of CDCs in eukaryotic cells and to develop strategies to limit the toxic effects of CDCs.

Dynasore - not just a dynamin inhibitor.

Dynamin is a GTPase protein that is essential for membrane fission during clathrin-mediated endocytosis in eukaryotic cells. Dynasore is a GTPase inhibitor that rapidly and reversibly inhibits dynamin activity, which prevents endocytosis. However, comparison between cells treated with dynasore and RNA interference of genes encoding dynamin, reveals evidence that dynasore reduces labile cholesterol in the plasma membrane, and disrupts lipid raft organization, in a dynamin-independent manner. To explore the role of dynamin it is important to use multiple dynamin inhibitors, alongside the use of dynamin mutants and RNA interference targeting genes encoding dynamin. On the other hand, dynasore provides an interesting tool to explore the regulation of cholesterol in plasma membranes.

Protective role of the dynamin inhibitor Dynasore against the cholesterol-dependent cytolysin of Trueperella pyogenes.

The virulence of many Gram-positive bacteria depends on cholesterol-dependent cytolysins (CDCs), which form pores in eukaryotic cell plasma membranes. Pyolysin (PLO) from Trueperella pyogenes provided a unique opportunity to explore cellular responses to CDCs because it does not require thiol activation. Sublytic concentrations of PLO stimulated phosphorylation of MAPK ERK and p38 in primary stromal cells, and induced autophagy as determined by protein light-chain 3B cleavage. Although, inhibitors of MAPK or autophagy did not affect PLO-induced cytolysis. However, 10 µM 3-hydroxynaphthalene-2-carboxylic acid-(3,4-dihydroxybenzylidene)-hydrazide (Dynasore), a dynamin guanosine 5'-triphosphatase inhibitor, protected stromal cells against PLO-induced cytolysis as determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (85 ± 17% versus 50 ± 9% cell viability), measuring extracellular ATP, and kinetic assays. This was a generalized mechanism because Dynasore also protected HeLa cells against streptolysin O. Furthermore, the effect was reversible, with stromal cell sensitivity to PLO restored within 30 minutes of Dynasore removal. The protective effect of Dynasore was not conferred by dynamin inhibition, induction of ERK phosphorylation, or Dynasore binding to PLO. Rather, Dynasore reduced cellular cholesterol and disrupted plasma membrane lipid rafts, similar to positive control methyl-ß-cyclodextrin. Dynasore is a tractable tool to explore the complexity of cholesterol homeostasis in eukaryotic cells and to develop strategies to counter CDCs.

Requirement of apoptotic protease-activating factor-1 for bortezomib-induced apoptosis but not for Fas-mediated apoptosis in human leukemic cells.

Bortezomib is a highly selective inhibitor of the 26S proteasome and has been approved for clinical use in the treatment of relapsing and refractory multiple myeloma and mantle cell lymphoma. Clinical trials are also underway to assess the role of bortezomib in several other human malignancies, including leukemia. However, the mechanism(s) by which bortezomib acts remain to be fully understood. Here, we studied the molecular requirements of bortezomib-induced apoptosis using the human T-cell leukemic Jurkat cells stably transfected with or without shRNA against apoptotic protease-activating factor-1 (Apaf-1). The Apaf-1-deficient Jurkat T cells were resistant to bortezomib-induced apoptosis, as assessed by caspase-3 activity, poly(ADP-ribose) polymerase cleavage, phosphatidylserine externalization, and hypodiploid DNA content. In contrast, Apaf-1-deficient cells were sensitive to Fas-induced apoptosis. Bortezomib induced an upregulation of the pro-apoptotic protein Noxa, loss of mitochondrial transmembrane potential, and release of cytochrome c in cells expressing or not expressing Apaf-1. Transient silencing of Apaf-1 expression in RPMI 8402 T-cell leukemic cells also diminished bortezomib-induced apoptosis. Fas-associated death domain (FADD)-deficient Jurkat cells were resistant to Fas-mediated apoptosis yet remained sensitive to bortezomib. Our results show that bortezomib induces apoptosis by regulating pathways that are mechanistically different from those activated upon death receptor ligation. Furthermore, in silico analyses of public transcriptomics databases indicated elevated Apaf-1 expression in several hematologic malignancies, including acute lymphoblastic and myeloid leukemia. We also noted variable Apaf-1 expression in a panel of samples from patients with acute lymphoblastic leukemia. Our results suggest that the expression of Apaf-1 may be predictive of the response to proteasome inhibition.

MAML1 acts cooperatively with EGR1 to activate EGR1-regulated promoters: implications for nephrogenesis and the development of renal cancer.

Mastermind-like 1 (MAML1) is a transcriptional coregulator of activators in various signaling pathways, such as Notch, p53, myocyte enhancer factor 2C (MEF2C) and beta-catenin. In earlier studies, we demonstrated that MAML1 enhanced p300 acetyltransferase activity, which increased the acetylation of Notch by p300. In this study, we show that MAML1 strongly induced acetylation of the transcription factor early growth response-1 (EGR1) by p300, and increased EGR1 protein expression in embryonic kidney cells. EGR1 mRNA transcripts were also upregulated in the presence of MAML1. We show that MAML1 physically interacted with, and acted cooperatively with EGR1 to increase transcriptional activity of the EGR1 and p300 promoters, which both contain EGR1 binding sites. Bioinformatics assessment revealed a correlation between p300, EGR1 and MAML1 copy number and mRNA alterations in renal clear cell carcinoma and p300, EGR1 and MAML1 gene alterations were associated with increased overall survival. Our findings suggest MAML1 may be a component of the transcriptional networks which regulate EGR1 target genes during nephrogenesis and could also have implications for the development of renal cell carcinoma.

AIF and Scythe (Bat3) regulate phosphatidylserine exposure and macrophage clearance of cells undergoing Fas (APO-1)-mediated apoptosis.

Phosphatidylserine (PS) exposure on the cell surface has been considered a characteristic feature of apoptosis and serves as a molecular cue for engulfment of dying cells by phagocytes. However, the mechanism of PS exposure is still not fully elucidated. Here we show that the cytosolic release from mitochondria of apoptosis-inducing factor (AIF) is required for PS exposure during death receptor-induced apoptosis and for efficient clearance of cell corpses by primary human macrophages. Fas-triggered PS exposure was significantly reduced upon siRNA-mediated silencing of AIF expression and by inhibition of the cytosolic translocation of AIF. In addition, AIF localizes to the plasma membrane upon Fas ligation and promotes activation of phospholipid scrambling activity. Finally, cytosolic stabilization of AIF through interaction with Scythe is shown to be involved in apoptotic PS exposure. Taken together, our results suggest an essential role for AIF and its binding partner Scythe in the pathway leading to apoptotic corpse clearance.

Scythe cleavage during Fas (APO-1)-and staurosporine-mediated apoptosis.

Scythe is a nuclear protein that has been implicated in the apoptotic process in Drosophila melanogaster; however, its role in apoptosis of mammalian cells is not fully elucidated. Here we show that cleavage of Scythe by caspase-3 occurs after activation of both the extrinsic (i.e. Fas/APO-1-mediated) and the intrinsic (i.e. staurosporine-induced) apoptosis pathway. Moreover, this caspase-dependent cleavage correlates with Scythe translocation from the nucleus to the cytosol. We also show that cytosolic re-localization of Scythe is required for Fas/APO-1-triggered phosphatidylserine (PS) exposure, a signal for macrophage clearance of apoptotic cells. Our data suggest that Scythe cleavage may represent a marker for caspase-3 activation and implicate cytosolic re-localization of Scythe in the pathway of PS exposure.

The Enigma of Tripeptidyl-Peptidase II: Dual Roles in Housekeeping and Stress.

The tripeptidyl-peptidase II complex consists of repeated 138 kDa subunits, assembled into two twisted strands that form a high molecular weight complex (>5 MDa). TPPII, like many other cytosolic peptidases, plays a role in the ubiquitin-proteasome pathway downstream of the proteasome as well as in the production and destruction of MHC class I antigens and degradation of neuropeptides. Tripeptidyl-peptidase II activity is increased in cells with an increased demand for protein degradation, but whether degradation of cytosolic peptides is the only cell biological role for TPPII has remained unclear. Recent data indicated that TPPII translocates into the nucleus to control DNA damage responses in malignant cells, supporting that cytosolic "housekeeping peptidases" may have additional roles in cell biology, besides their contribution to protein turnover. Overall, TPPII has an emerging importance in several cancer-related fields, such as metabolism, cell death control, and control of genome integrity; roles that are not understood in detail. The present paper reviews the cell biology of TPPII and discusses distinct roles for TPPII in the nucleus and cytosol.

MAP kinase-signaling controls nuclear translocation of tripeptidyl-peptidase II in response to DNA damage and oxidative stress.

Reactive oxygen species (ROS) are a continuous hazard in eukaroytic cells by their ability to cause damage to biomolecules, in particular to DNA. Previous data indicated that the cytosolic serine peptidase tripeptidyl-peptidase II (TPPII) translocates into the nucleus of most tumor cell lines in response to gamma-irradiation and ROS production; an event that promoted p53 expression as well as caspase-activation. We here observed that nuclear translocation of TPPII was dependent on signaling by MAP kinases, including p38MAPK. Further, this was caused by several types of DNA-damaging drugs, a DNA cross-linker (cisplatinum), an inhibitor of topoisomerase II (etoposide), and to some extent also by nucleoside-analogues (5-fluorouracil, hydroxyurea). In the minority of tumor cell lines where TPPII was not translocated into the nucleus in response to DNA damage we observed reduced intracellular ROS levels, and the expression levels of redox defense systems were increased. Further, treatment with the ROS-inducer gamma-hexa-chloro-cyclohexane (gamma-HCH, lindane), an inhibitor of GAP junctions, restored nuclear translocation of TPPII in these cell lines upon gamma-irradiation. Moreover, blocking nuclear translocation of TPPII in etoposide-treated cells, by using a peptide-derived inhibitor (Z-Gly-Leu-Ala-OH), attenuated expression of gamma-H2AX in gamma-irradiated melanoma cells. Our results indicated a role for TPPII in MAPK-dependent DNA damage signaling.

A role for nuclear translocation of tripeptidyl-peptidase II in reactive oxygen species-dependent DNA damage responses.

Responses to DNA damage are influenced by cellular metabolism through the continuous production of reactive oxygen species (ROS), of which most are by-products of mitochondrial respiration. ROS have a strong influence on signaling pathways during responses to DNA damage, by relatively unclear mechanisms. Previous reports have shown conflicting data on a possible role for tripeptidyl-peptidase II (TPPII), a large cytosolic peptidase, within the DNA damage response. Here we show that TPPII translocated into the nucleus in a p160-ROCK-dependent fashion in response to gamma-irradiation, and that nuclear expression of TPPII was present in most gamma-irradiated transformed cell lines. We used a panel of nine cell lines of diverse tissue origin, including four lymphoma cell lines (T, B and Hodgkins lymphoma), a melanoma, a sarcoma, a colon and two breast carcinomas, where seven out of nine cell lines showed nuclear TPPII expression after gamma-irradiation. Further, this required cellular production of ROS; treatment with either N-acetyl-Cysteine (anti-oxidant) or Rotenone (inhibitor of mitochondrial respiration) inhibited nuclear accumulation of TPPII. The local density of cells was important for nuclear accumulation of TPPII at early time-points following gamma-irradiation (at 1-4h), indicating a bystander effect. Further, we showed that the peptide-based inhibitor Z-Gly-Leu-Ala-OH, but not its analogue Z-Gly-(D)-Leu-Ala-OH, excluded TPPII from the nucleus. This correlated with reduced nuclear expression of p53 as well as caspase-3 and -9 activation in gamma-irradiated lymphoma cells. Our data suggest a role for TPPII in ROS-dependent DNA damage responses, through alteration of its localization from the cytosol into the nucleus.