Cholesteryl hemiazelate causes lysosome dysfunction impacting vascular smooth muscle cell homeostasis

In atherosclerotic lesions, vascular smooth muscle cells (VSMCs) represent half of the foam cell population, which is characterized by an aberrant accumulation of undigested lipids within lysosomes. Loss of lysosome function impacts VSMC homeostasis and disease progression. Understanding the molecular mechanisms underlying lysosome dysfunction in these cells is, therefore, crucial. We identify cholesteryl hemiazelate (ChA), a stable oxidation end-product of cholesteryl-polyunsaturated fatty acid esters, as an inducer of lysosome malfunction in VSMCs. ChA-treated VSMCs acquire a foam-cell-like phenotype, characterized by enlarged lysosomes full of ChA and neutral lipids. The lysosomes are perinuclear and exhibit degradative capacity and cargo exit defects. Lysosome luminal pH is also altered. Even though the transcriptional response machinery and autophagy are not activated by ChA, the addition of recombinant lysosomal acid lipase (LAL) is able to rescue lysosome dysfunction. ChA significantly affects VSMC proliferation and migration, impacting atherosclerosis. In summary, this work shows that ChA is sufficient to induce lysosomal dysfunction in VSMCs, that, in ChA-treated VSMCs, neither lysosome biogenesis nor autophagy are triggered, and, finally, that recombinant LAL can be a therapeutic approach for lysosomal dysfunction

Molecular Etiology of Atherogenesis – In Vitro Induction of Lipidosis in Macrophages with a New LDL Model

BACKGROUND: Atherosclerosis starts by lipid accumulation in the arterial intima and progresses into a chronic vascular inflammatory disease. A major atherogenic process is the formation of lipid-loaded macrophages in which a breakdown of the endolysomal pathway results in irreversible accumulation of cargo in the late endocytic compartments with a phenotype similar to several forms of lipidosis. Macrophages exposed to oxidized LDL exihibit this phenomenon in vitro and manifest an impaired degradation of internalized lipids and enhanced inflammatory stimulation. Identification of the specific chemical component(s) causing this phenotype has been elusive because of the chemical complexity of oxidized LDL. METHODOLOGY/PRINCIPAL FINDINGS: Lipid "core aldehydes" are formed in oxidized LDL and exist in atherosclerotic plaques. These aldehydes are slowly oxidized in situ and (much faster) by intracellular aldehyde oxidizing systems to cholesteryl hemiesters. We show that a single cholesteryl hemiester incorporated into native, non-oxidized LDL induces a lipidosis phenotype with subsequent cell death in macrophages. Internalization of the cholesteryl hemiester via the native LDL vehicle induced lipid accumulation in a time- and concentration-dependent manner in "frozen" endolysosomes. Quantitative shotgun lipidomics analysis showed that internalized lipid in cholesteryl hemiester-intoxicated cells remained largely unprocessed in those lipid-rich organelles. CONCLUSIONS/SIGNIFICANCE: The principle elucidated with the present cholesteryl hemiester-containing native-LDL model, extended to other molecular components of oxidized LDL, will help in defining the molecular etiology and etiological hierarchy of atherogenic agents

Interleukin-4 Alters Early Phagosome Phenotype by Modulating Class I PI3K Dependent Lipid Remodeling and Protein Recruitment

Phagocytosis is a complex process that involves membranelipid remodeling and the attraction and retention of key effector proteins. Phagosome phenotype depends on the type of receptor engaged and can be influenced by extracellular signals. Interleukin 4 (IL-4) is a cytokine that induces the alternative activation of macrophages (MΦs) upon prolonged exposure, triggering a different cell phenotype that has an altered phagocytic capacity. In contrast, the direct effects of IL-4 during phagocytosis remain unknown. Here, we investigate the impact of short-term IL-4 exposure (1 hour) during phagocytosis of IgG-opsonized yeast particles by MΦs. By time-lapse confocal microscopy of GFP-tagged lipid-sensing probes, we show that IL-4 increases the negative charge of the phagosomal membrane by prolonging the presence of the negatively charged second messenger PI(3,4,5)P3. Biochemical assays reveal an enhanced PI3K/Akt activity upon phagocytosis in the presence of IL-4. Blocking the specific class I PI3K after the onset of phagocytosis completely abrogates the IL-4-induced changes in lipid remodeling and concomitant membrane charge. Finally, we show that IL-4 direct signaling leads to a significantly prolonged retention profile of the signaling molecules Rac1 and Rab5 to the phagosomal membrane in a PI3K-dependent manner. This protracted early phagosome phenotype suggests an altered maturation, which is supported by the delayed phagosome acidification measured in the presence of IL-4. Our findings reveal that molecular differences in IL-4 levels, in the extracellular microenvironment, influence the coordination of lipid remodeling and protein recruitment, which determine phagosome phenotype and, eventually, fate. Endosomal and phagosomal membranes provide topological constraints to signaling molecules. Therefore, changes in the phagosome phenotype modulated by extracellular factors may represent an additional mechanism that regulates the outcome of phagocytosis and could have significant impact on the net biochemical output of a cell

In Vitro Surfactant Structure-Toxicity Relationships: Implications for Surfactant Use in Sexually Transmitted Infection Prophylaxis and Contraception

Background The need for woman-controlled, cheap, safe, effective, easy-to-use and easy-to-store topical applications for prophylaxis against sexually transmitted infections (STIs) makes surfactant-containing formulations an interesting option that requires a more fundamental knowledge concerning surfactant toxicology and structure-activity relationships. Methodology/Principal Findings We report in vitro effects of surfactant concentration, exposure time and structure on the viability of mammalian cell types typically encountered in the vagina, namely, fully polarized and confluent epithelial cells, confluent but non-polarized epithelial-like cells, dendritic cells, and human sperm. Representatives of the different families of commercially available surfactants – nonionic (Triton X-100 and monolaurin), zwitterionic (DDPS), anionic (SDS), and cationic (CnTAB (n = 10 to 16), C12PB, and C12BZK) – were examined. Triton X-100, monolaurin, DDPS and SDS were toxic to all cell types at concentrations around their critical micelle concentration (CMC) suggesting a non-selective mode of action involving cell membrane destabilization and/or destruction. All cationic surfactants were toxic at concentrations far below their CMC and showed significant differences in their toxicity toward polarized as compared with non-polarized cells. Their toxicity was also dependent on the chemical nature of the polar head group. Our results suggest an intracellular locus of action for cationic surfactants and show that their structure-activity relationships could be profitably exploited for STI prophylaxis in vaginal gel formulations. The therapeutic indices comparing polarized epithelial cell toxicity to sperm toxicity for all surfactants examined, except C12PB and C12BZK, does not justify their use as contraceptive agents. C12PB and C12BZK are shown to have a narrow therapeutic index recommending caution in their use in contraceptive formulations. Conclusions/Significance Our results contribute to understanding the mechanisms involved in surfactant toxicity, have a predictive value with regard to their safety, and may be used to design more effective and less harmful surfactants for use in topical applications for STI prophylaxis.Foundation for Science and Technology of the Portuguese Ministry of Science and Higher Educatio

Improved general regression network for protein domain boundary prediction

Background: Protein domains present some of the most useful information that can be used to understand protein structure and functions. Recent research on protein domain boundary prediction has been mainly based on widely known machine learning techniques, such as Artificial Neural Networks and Support Vector Machines. In this study, we propose a new machine learning model (IGRN) that can achieve accurate and reliable classification, with significantly reduced computations. The IGRN was trained using a PSSM (Position Specific Scoring Matrix), secondary structure, solvent accessibility information and inter-domain linker index to detect possible domain boundaries for a target sequence. Results: The proposed model achieved average prediction accuracy of 67% on the Benchmark_2 dataset for domain boundary identification in multi-domains proteins and showed superior predictive performance and generalisation ability among the most widely used neural network models. With the CASP7 benchmark dataset, it also demonstrated comparable performance to existing domain boundary predictors such as DOMpro, DomPred, DomSSEA, DomCut and DomainDiscovery with 70.10% prediction accuracy. Conclusion: The performance of proposed model has been compared favourably to the performance of other existing machine learning based methods as well as widely known domain boundary predictors on two benchmark datasets and excels in the identification of domain boundaries in terms of model bias, generalisation and computational requirements. © 2008 Yoo et al; licensee BioMed Central Ltd

Attenuated Leishmania induce pro-inflammatory mediators and influence leishmanicidal activity by p38 MAPK dependent phagosome maturation in Leishmania donovani co-infected macrophages

Promastigote form of Leishmania, an intracellular pathogen, delays phagosome maturation and resides inside macrophages. But till date limited study has been done to manipulate the phagosomal machinery of macrophages to restrict Leishmania growth. Attenuated Leishmania strain exposed RAW 264.7 cells showed a respiratory burst and enhanced production of pro-inflammatory mediators. The augmentation of pro-inflammatory activity is mostly attributed to p38 MAPK and p44/42 MAPK. In our study, these activated macrophages are found to induce phagosome maturation when infected with pathogenic Leishmania donovani. Increased co-localization of carboxyfluorescein succinimidyl ester labeled pathogenic L. donovani with Lysosome was found. Moreover, increased co-localization was observed between pathogenic L. donovani and late phagosomal markers viz. Rab7, Lysosomal Associated Membrane Protein 1, Cathepsin D, Rab9, and V-ATPase which indicate phagosome maturation. It was also observed that inhibition of V-type ATPase caused significant hindrance in attenuated Leishmania induced phagosome maturation. Finally, it was confirmed that p38 MAPK is the key player in acidification and maturation of phagosome in attenuated Leishmania strain preexposed macrophages. To our knowledge, this study for the first time reported an approach to induce phagosome maturation in L. donovani infected macrophages which could potentiate short-term prophylactic response in futur

Wheat TaRab7 GTPase Is Part of the Signaling Pathway in Responses to Stripe Rust and Abiotic Stimuli

Small GTP-binding proteins function as regulators of specific intercellular fundamental biological processes. In this study, a small GTP-binding protein Rab7 gene, designated as TaRab7, was identified and characterized from a cDNA library of wheat leaves infected with Puccinia striiformis f. sp. tritici (Pst) the wheat stripe rust pathogen. The gene was predicted to encode a protein of 206 amino acids, with a molecular mass of 23.13 KDa and an isoeletric point (pI) of 5.13. Further analysis revealed the presence of a conserved signature that is characteristic of Rab7, and phylogenetic analysis demonstrated that TaRab7 has the highest similarity to a small GTP binding protein gene (BdRab7-like) from Brachypodium distachyon. Quantitative real-time PCR assays revealed that the expression of TaRab7 was higher in the early stage of the incompatible interactions between wheat and Pst than in the compatible interaction, and the transcription level of TaRab7 was also highly induced by environmental stress stimuli. Furthermore, knocking down TaRab7 expression by virus induced gene silencing enhanced the susceptibility of wheat cv. Suwon 11 to an avirulent race CYR23. These results imply that TaRab7 plays an important role in the early stage of wheat-stripe rust fungus interaction and in stress tolerance

Topographical expression of class IA and class II phosphoinositide 3-kinase enzymes in normal human tissues is consistent with a role in differentiation

BACKGROUND: Growth factor, cytokine and chemokine-induced activation of PI3K enzymes constitutes the start of a complex signalling cascade, which ultimately mediates cellular activities such as proliferation, differentiation, chemotaxis, survival, trafficking, and glucose homeostasis. The PI3K enzyme family is divided into 3 classes; class I (subdivided into IA and IB), class II (PI3K-C2α, PI3K-C2β and PI3K-C2γ) and class III PI3K. Expression of these enzymes in human tissue has not been clearly defined. METHODS: In this study, we analysed the immunohistochemical topographical expression profile of class IA (anti-p85 adaptor) and class II PI3K (PI3K-C2α and PI3K-C2β) enzymes in 104 formalin-fixed, paraffin embedded normal adult human (age 33–71 years, median 44 years) tissue specimens including those from the gastrointestinal, genitourinary, hepatobiliary, endocrine, integument and lymphoid systems. Antibody specificity was verified by Western blotting of cell lysates and peptide blocking studies. Immunohistochemistry intensity was scored from undetectable to strong. RESULTS: PI3K enzymes were expressed in selected cell populations of epithelial or mesenchymal origin. Columnar epithelium and transitional epithelia were reactive but mucous secreting and stratified squamous epithelia were not. Mesenchymal elements (smooth muscle and endothelial cells) and glomerular epithelium were only expressed PI3K-C2α while ganglion cells expressed p85 and PI3K-C2β. All three enzymes were detected in macrophages, which served as an internal positive control. None of the three PI3K isozymes was detected in the stem cell/progenitor compartments or in B lymphocyte aggregates. CONCLUSIONS: Taken together, these data suggest that PI3K enzyme distribution is not ubiquitous but expressed selectively in fully differentiated, non-proliferating cells. Identification of the normal in vivo expression pattern of class IA and class II PI3K paves the way for further analyses which will clarify the role played by these enzymes in inflammatory, neoplastic and other human disease conditions

Involvement of the p62/NRF2 signal transduction pathway on erythrophagocytosis

This deposit is composed by the main article plus the supplementary materials of the publication.Erythrophagocytosis, the phagocytic removal of damaged red blood cells (RBC), and subsequent phagolysosome biogenesis are important processes in iron/heme metabolism and homeostasis. Phagolysosome biogenesis implies the interaction of nascent phagosomes with endocytic compartments and also autophagy effectors. Here, we report that besides recruitment of microtubule-associated protein-1-light chain 3 (LC3), additional autophagy machinery such as sequestosome 1 (p62) is also acquired by single-membrane phagosomes at very early stages of the phagocytic process and that its acquisition is very important to the outcome of the process. In bone marrow-derived macrophages (BMDM) silenced for p62, RBC degradation is inhibited. P62, is also required for nuclear translocation and activation of the transcription factor Nuclear factor E2-related Factor 2 (NRF2) during erythrophagocytosis. Deletion of the Nrf2 allele reduces p62 expression and compromises RBC degradation. In conclusion, we reveal that erythrophagocytosis relies on an interplay between p62 and NRF2, potentially acting as protective mechanism to maintain reactive oxygen species at basal levels and preserve macrophage homeostasis.Fundação para a Ciência e a Tecnologia grants: (HMSP-ICT/0024/2010, UID/Multi/04462/2013, SFRH/BD/62197/2009, SFRH/BD/90258/2012, SFRH /BD/51877/2012, SFRH/BD/52293/2013, PTDC/SAU-TOX/116627/2010, HMSP-ICT/0022/2010 ); European Union FEDER support: (COMPETE, QREN, PT2020 Partnership Agreement), ERC grant: (ERC-2011-AdG 294709-DAMAGECONTROL).info:eu-repo/semantics/publishedVersio

A multiplatform approach identifies miR-152-3p as a common epigenetically regulated onco-suppressor in prostate cancer targeting TMEM97

Prostate cancer (PCa) is a major cause of morbidity and mortality in men worldwide. MicroRNAs are globally downregulated in PCa, especially in poorly differentiated tumors. Nonetheless, the underlying mechanisms are still elusive. Herein, using combined analysis of microRNAs expression and genomewide DNA methylation, we aimed to identify epigenetically downregulated microRNAs in PCa.Research Center of Portuguese Oncology Institute of Porto (FB-GEBC-27 and 19-CI-IPOP-2016). JR-C and CSG are supported by FCT- Fundação para a Ciência e Tecnologia PhD fellowships (SFRH/BD/71293/2010 and SFRH/BD/92786/2013), SS is supported by a PhD fellowship IPO/ESTIMA-1 NORTE-01-0145-FEDER-000027, and IG is a research fellow from the strategic funding of FCT (PCT: PEst- UID/DTP/00776/2013 and COMPETE: POCI-01-0145-FEDER-006868). BMC is funded by FCT-Fundação para a Ciência e a Tecnologia (IF/00601/2012)info:eu-repo/semantics/publishedVersio