Sphingosine kinases regulate ER contacts with late endocytic organelles and cholesterol trafficking.

Membrane contact sites (MCS), close membrane apposition between organelles, are platforms for interorganellar transfer of lipids including cholesterol, regulation of lipid homeostasis, and co-ordination of endocytic trafficking. Sphingosine kinases (SphKs), two isoenzymes that phosphorylate sphingosine to the bioactive sphingosine-1-phosphate (S1P), have been implicated in endocytic trafficking. However, the physiological functions of SphKs in regulation of membrane dynamics, lipid trafficking and MCS are not known. Here, we report that deletion of SphKs decreased S1P with concomitant increases in its precursors sphingosine and ceramide, and markedly reduced endoplasmic reticulum (ER) contacts with late endocytic organelles. Expression of enzymatically active SphK1, but not catalytically inactive, rescued the deficit of these MCS. Although free cholesterol accumulated in late endocytic organelles in SphK null cells, surprisingly however, cholesterol transport to the ER was not reduced. Importantly, deletion of SphKs promoted recruitment of the ER-resident cholesterol transfer protein Aster-B (also called GRAMD1B) to the plasma membrane (PM), consistent with higher accessible cholesterol and ceramide at the PM, to facilitate cholesterol transfer from the PM to the ER. In addition, ceramide enhanced in vitro binding of the Aster-B GRAM domain to phosphatidylserine and cholesterol liposomes. Our study revealed a previously unknown role for SphKs and sphingolipid metabolites in governing diverse MCS between the ER network and late endocytic organelles versus the PM to control the movement of cholesterol between distinct cell membranes.

Autophagy at the interface of endothelial cell homeostasis and vascular disease.

Autophagy is an essential intracellular process for cellular quality control. It enables cell homeostasis through the selective degradation of harmful protein aggregates and damaged organelles. Autophagy is essential for recycling nutrients, generating energy to maintain cell viability in most tissues and during adverse conditions such as hypoxia/ischaemia. The progressive understanding of the mechanisms modulating autophagy in the vasculature has recently led numerous studies to link intact autophagic responses with endothelial cell (EC) homeostasis and function. Preserved autophagic flux within the ECs has an essential role in maintaining their physiological characteristics, whereas defective autophagy can promote endothelial pro-inflammatory and atherogenic phenotype. However, we still lack a good knowledge of the complete molecular repertoire controlling various aspects of endothelial autophagy and how this is associated with vascular diseases. Here, we provide an overview of the current state of the art of autophagy in ECs. We review the discoveries that have so far defined autophagy as an essential mechanism in vascular biology and analyse how autophagy influences ECs behaviour in vascular disease. Finally, we emphasise opportunities for compounds to regulate autophagy in ECs and discuss the challenges of exploiting them to resolve vascular disease.

Thank ORP9 for FFAT: With endosomal ORP10, it's fission accomplished!

Heterogeneity in endosomal membrane phospholipid content is emerging as a regulator of endocytic trafficking pathways. Kawasaki et al. (2021. J. Cell. Biol.https://doi.org/10.1083/jcb.202103141) demonstrate exchange of endosomal PI4P for PS by ORP10 at ER-endosome contact sites, with the consequent recruitment of endosomal fission factors.

Cellular cholesterol and how to find it.

Cholesterol is an essential component of eukaryotic cellular membranes. Information about its subcellular localization and transport pathways inside cells are key for the understanding and treatment of cholesterol-related diseases. In this review we give an overview over the most commonly used methods that contributed to our current understanding of subcellular cholesterol localization and transport routes. First, we discuss methods that provide insights into cholesterol metabolism based on readouts of downstream effects such as esterification. Subsequently, we focus on the use of cholesterol-binding molecules as probes that facilitate visualization and quantification of sterols inside of cells. Finally, we explore different analogues of cholesterol which, when taken up by living cells, are integrated and transported in a similar fashion as endogenous sterols. Taken together, we highlight the challenges and advantages of each method such that researchers studying aspects of cholesterol transport may choose the most pertinent approach for their problem.

Exploiting Connections for Viral Replication.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of the COVID-19 (coronavirus disease 2019) pandemic, is a positive strand RNA (+RNA) virus. Like other +RNA viruses, SARS-CoV-2 is dependent on host cell metabolic machinery to survive and replicate, remodeling cellular membranes to generate sites of viral replication. Viral RNA-containing double-membrane vesicles (DMVs) are a striking feature of +RNA viral replication and are abundant in SARS-CoV-2-infected cells. Their generation involves rewiring of host lipid metabolism, including lipid biosynthetic pathways. Viruses can also redirect lipids from host cell organelles; lipid exchange at membrane contact sites, where the membranes of adjacent organelles are in close apposition, has been implicated in the replication of several +RNA viruses. Here we review current understanding of DMV biogenesis. With a focus on the exploitation of contact site machinery by +RNA viruses to generate replication organelles, we discuss evidence that similar mechanisms support SARS-CoV-2 replication, protecting its RNA from the host cell immune response.

Folic Acid-Peptide Conjugates Combine Selective Cancer Cell Internalization with Thymidylate Synthase Dimer Interface Targeting.

Drug-target interaction, cellular internalization, and target engagement should be addressed to design a lead with high chances of success in further optimization stages. Accordingly, we have designed conjugates of folic acid with anticancer peptides able to bind human thymidylate synthase (hTS) and enter cancer cells through folate receptor α (FRα) highly expressed by several cancer cells. Mechanistic analyses and molecular modeling simulations have shown that these conjugates bind the hTS monomer-monomer interface with affinities over 20 times larger than the enzyme active site. When tested on several cancer cell models, these conjugates exhibited FRα selectivity at nanomolar concentrations. A similar selectivity was observed when the conjugates were delivered in synergistic or additive combinations with anticancer agents. At variance with 5-fluorouracil and other anticancer drugs that target the hTS catalytic pocket, these conjugates do not induce overexpression of this protein and can thus help combating drug resistance associated with high hTS levels.

Depletion of Trichoplein (TpMs) Causes Chromosome Mis-Segregation, DNA Damage and Chromosome Instability in Cancer Cells.

Mitotic perturbations frequently lead to chromosome mis-segregation that generates genome instability, thereby triggering tumor onset and/or progression. Error-free mitosis depends on fidelity-monitoring systems that ensure the temporal and spatial coordination of chromosome segregation. Recent investigations are focused on mitotic DNA damage response (DDR) and chromosome mis-segregations with the aim of developing more efficient anti-cancer therapies. We previously demonstrated that trichoplein keratin filament binding protein (TpMs) exhibits hallmarks of a tumor suppressor gene in cancer-derived cells and human tumors. Here, we show that silencing of TpMs expression results in chromosome mis-segregation, DNA damage and chromosomal instability. TpMs interacts with Mad2, and TpMs depletion results in decreased levels of Mad2 and Cyclin B1 proteins. All the genetic alterations observed are consistent with both defective activation of the spindle assembly checkpoint and mitotic progression. Thus, low levels of TpMs found in certain human tumors may contribute to cellular transformation by promoting genomic instability.

Trichoplein binds PCM1 and controls endothelial cell function by regulating autophagy.

Autophagy is an essential cellular quality control process that has emerged as a critical one for vascular homeostasis. Here, we show that trichoplein (TCHP) links autophagy with endothelial cell (EC) function. TCHP localizes to centriolar satellites, where it binds and stabilizes PCM1. Loss of TCHP leads to delocalization and proteasome-dependent degradation of PCM1, further resulting in degradation of PCM1's binding partner GABARAP. Autophagic flux under basal conditions is impaired in THCP-depleted ECs, and SQSTM1/p62 (p62) accumulates. We further show that TCHP promotes autophagosome maturation and efficient clearance of p62 within lysosomes, without affecting their degradative capacity. Reduced TCHP and high p62 levels are detected in primary ECs from patients with coronary artery disease. This phenotype correlates with impaired EC function and can be ameliorated by NF-κB inhibition. Moreover, Tchp knock-out mice accumulate of p62 in the heart and cardiac vessels correlating with reduced cardiac vascularization. Taken together, our data reveal that TCHP regulates endothelial cell function via an autophagy-mediated mechanism.

Staying in touch with the endocytic network: The importance of contacts for cholesterol transport.

Cholesterol homeostasis is critical for cell function and human health. Cholesterol is heterogeneously distributed among cellular membranes, with the redistribution of endocytosed dietary cholesterol playing a pivotal role in the regulation of cholesterol homeostasis. While gaps remain in our understanding of intracellular dietary cholesterol transport, a highly complex network of pathways is starting to emerge, often involving inter-dependent vesicular and non-vesicular transport mechanisms. The last decade has seen a surge in interest in non-vesicular transport and inter-organellar communication at membrane contact sites. By providing platforms for protein interactions, signalling events, lipid exchange and calcium flux, membrane contact sites (MCS) are now appreciated as controlling the fate of large amounts of lipid and play central roles in the regulation and co-ordination of endocytic trafficking. Here, we review the role of MCS in multiple pathways for cholesterol export from the endocytic pathway and highlight the intriguing interplay between vesicular and non-vesicular transport mechanisms and relationship with neurodegenerative disease.

Promoter Methylation Leads to Decreased ZFP36 Expression and Deregulated NLRP3 Inflammasome Activation in Psoriatic Fibroblasts.

The mRNA-destabilizing protein tristetraprolin (TTP), encoded by the ZFP36 gene, is known to be able to end inflammatory responses by directly targeting and destabilizing mRNAs encoding pro-inflammatory cytokines. We analyzed its role in psoriasis, a disease characterized by chronic inflammation. We observed that TTP is downregulated in fibroblasts deriving from psoriasis patients compared to those deriving from healthy individuals and that psoriatic fibroblasts exhibit abnormal inflammasome activity compared to their physiological counterpart. This phenomenon depends on TTP downregulation. In fact, following restoration, TTP is capable of directly targeting for degradation NLRP3 mRNA, thereby drastically decreasing inflammasome activation. Moreover, we provide evidence that ZFP36 undergoes methylation in psoriasis, by virtue of the presence of long stretches of CpG dinucleotides both in the promoter and the coding region. Besides confirming that a perturbation of TTP expression might underlie the pathogenesis of psoriasis, we suggest that deregulated inflammasome activity might play a role in the disease alongside deregulated cytokine expression.

MicroRNA-532-5p Regulates Pericyte Function by Targeting the Transcription Regulator BACH1 and Angiopoietin-1.

MicroRNAs regulate endothelial function and angiogenesis, but their implication in pericyte biology remains undetermined. A PCR array, covering a panel of 379 human microRNAs, showed microRNA-532-5p to be one of the most differentially modulated by hypoxia, which was confirmed by qPCR in both skeletal muscle and adventitial pericytes. Furthermore, microRNA-532-5p was upregulated in murine muscular pericytes early after experimentally induced ischemia, decreasing below baseline after reperfusion. Transfection of human pericytes with anti-microRNA, microRNA-mimic, or controls indicates microRNA-532-5p modulates pro-angiogenic activity via transcriptional regulation of angiopoietin-1. Tie-2 blockade abrogated the ability of microRNA-532-5p-overexpressing pericytes to promote endothelial network formation in vitro. However, angiopoietin-1 is not a direct target of microRNA-532-5p. In silico analysis of microRNA-532-5p inhibitory targets associated with angiopoietin-1 transcription indicated three potential candidates, BACH1, HIF1AN, and EGLN1. Binding of microRNA-532-5p to the BACH1 3' UTR was confirmed by luciferase assay. MicroRNA-532-5p silencing increased BACH1, while a microRNA-532-5p mimic decreased expression. Silencing of BACH1 modulated angiopoietin-1 gene and protein expression. ChIP confirmed BACH1 transcriptional regulation of angiopoietin-1 promoter. Finally, microRNA-532-5p overexpression increased pericyte coverage in an in vivo Matrigel assay, suggesting its role in vascular maturation. This study provides a new mechanistic understanding of the transcriptional program orchestrating angiopoietin-1/Tie-2 signaling in human pericytes.

Phenotypic miRNA Screen Identifies miR-26b to Promote the Growth and Survival of Endothelial Cells.

Endothelial cell (EC) proliferation is a crucial event in physiological and pathological angiogenesis. MicroRNAs (miRNAs) have emerged as important modulators of the angiogenic switch. Here we conducted high-content screening of a human miRNA mimic library to identify novel regulators of EC growth systematically. Several miRNAs were nominated that enhanced or inhibited EC growth. Of these, we focused on miR-26b, which is a conserved candidate and expressed in multiple human EC types. miR-26b overexpression enhanced EC proliferation, migration, and tube formation, while inhibition of miR-26b suppressed the proliferative and angiogenic capacity of ECs. A combinatory functional small interfering RNA (siRNA) screening of 48 predicted gene targets revealed that miR-26b enhanced EC growth and survival through inhibiting PTEN expression. Local administration of miR-26b mimics promoted the growth of new microvessels in the Matrigel plug model. In the mouse model of hindlimb ischemia, miR-26b was found to be downregulated in endothelium in the first week following ischemia, and local overexpression of miR-26b improved the survival of capillaries and muscle fibers in ischemic muscles. Our findings suggest that miR-26b enhances EC proliferation, survival, and angiogenesis. miR-26b is a potential target for developing novel pro-angiogenic therapeutics in ischemic disease.

MicroRNA-148b Targets the TGF-ß Pathway to Regulate Angiogenesis and Endothelial-to-Mesenchymal Transition during Skin Wound Healing.

Transforming growth factor beta (TGF-ß) is crucial for regulation of the endothelial cell (EC) homeostasis. Perturbation of TGF-ß signaling leads to pathological conditions in the vasculature, causing cardiovascular disease and fibrotic disorders. The TGF-ß pathway is critical in endothelial-to-mesenchymal transition (EndMT), but a gap remains in our understanding of the regulation of TGF-ß and related signaling in the endothelium. This study applied a gain- and loss-of function approach and an in vivo model of skin wound healing to demonstrate that miR-148b regulates TGF-ß signaling and has a key role in EndMT, targeting TGFB2 and SMAD2. Overexpression of miR-148b increased EC migration, proliferation, and angiogenesis, whereas its inhibition promoted EndMT. Cytokine challenge decreased miR-148b levels in ECs while promoting EndMT through the regulation of SMAD2. Finally, in a mouse model of skin wound healing, delivery of miR-148b mimics promoted wound vascularization and accelerated closure. In contrast, inhibition of miR-148b enhanced EndMT in wounds, resulting in impaired wound closure that was reversed by SMAD2 silencing. Together, these results demonstrate for the first time that miR-148b is a key factor controlling EndMT and vascularization. This opens new avenues for therapeutic application of miR-148b in vascular and tissue repair.

O duplo especular nas psicoses não desencadeadas: um paradigma contemporâneo? / The specular double in the not triggered psychoses: a contemporary paradigm?

Para abordar a função do duplo especular nas psicoses, principalmente acerca de seu papel em relação ao desencadeamento e à estabilização, buscou-se investigar a regressão tópica ao estágio do espelho proposta por Lacan. Indaga-se se a saída encontrada para algumas psicoses pode indicar um movimento próprio da subjetividade contemporânea. Destaca-se um empuxo ao imaginário, cujas razões o artigo pretende esclarecer.

For an approach to the function of the specular double, mainly about its role in triggering and stabilization, this article investigates the topical regression to the Mirror Stage proposed by Lacan. It inquires if the solution found for some psychoses could indicate a proper movement of the contemporary subjectivity. It stands out an imaginary push towards, which reasons the article intends to elucidate.

O método analítico e as psicoses: do dispositivo freudiano à estrutura lacaniana / El método analítico y las psicosis: del dispositivo Freudiano a la estructura Lacaniana / The analytical method and the psychoses: from the Freudian device to the Lacanian structure

O artigo retoma as formulações teóricas de Freud acerca da diferença entre os campos da neurose e da psicose e dos limites do tratamento psicanalítico com psicóticos para, em seguida, apresentar as contribuições de Jacques Lacan à temática da psicose, no que alude à sua diferença estrutural, seu mecanismo específico e direção do tratamento. Demarca-se com o recorte teórico, uma revisão bibliográfica sobre o período a partir do qual Freud deixa de conceber a psicose pelo paradigma da neurose e em que o retorno efetivado por Lacan a Freud, com base em suas apropriações da linguística e da antropologia estrutural, deu centralidade à determinação do sujeito pela linguagem, à valorização do simbólico; permitindo com isso novas possibilidades para o tratamento da psicose

El artículo retoma las formulaciones teóricas de Freud acerca de la diferencia entre los campos de la neurosis y de la psicosis y de los límites del tratamiento psicoanalítico con psicóticos para en seguida, presentar las contribuciones de Jacques Lacan a la temática de la psicosis, lo que se refiere a la diferencia estructural, al mecanismo específico y a la dirección del tratamiento. Se demarca con el recorte teórico, una revisiónbibliográfica sobre el período a partir del cual Freud deja de concebir la psicosis por el paradigma de la neurosis y en que el retorno hecho efectivo por Lacan a Freud, con base en sus apropiaciones de la lingüística y de la antropología estructural, le da la centralidad a la determinación del sujeto por el lenguaje a la valorización de lo simbólico, permitiendo con ello, nuevas posibilidades para el tratamiento de la psicosis

This article takes up Freud's theoretical formulations about the difference between the neurosis and psychosis fields and the psychoanalytical limitations treatment with psychotic to, then, submit the Jacques Lacan's contributions to the psychosis theme, as referred to their structural differences, their specific mechanism and treatment direction. It is delimited with the theoretical framework, areview on the period from which Freud fails to conceive the psychosis by the neurosis paradigm and the return effected by Lacan to Freud, based on their appropriation of linguistics and anthropology structural, gave centrality to the subject determinationby the language, the symbolic appreciation; thereby providing new possibilities for the psychosis treatment

Pagamos com o nosso corpo? O gozo em jogo no discurso do mestre e no capitalismo / Nouspayons avec notre corps? La jouissance en jeu dans le discours du maîtreet dans le capitalisme / Do we pay with our body? The jouissance at stakein the master's discourse and in capitalism

O presente artigo faz uma reflexão sobre o corpo para a psicanálise,em relação ao que se paga ao entrar na linguagem. Para tanto o corpo será pensado a partir da metáfora extraída por Lacan (1963) em OMercador de Veneza, como uma libra de carne presa na máquina formal da linguagem. Esta metáfora nos permite estabelecer asrelações do corpo com a falta e a dívida, em relação à linguagem. Posteriormente, será articulado o corpo com o discurso capitalista, enquanto este é um fato da linguagem com uma incidência muito marcada na época atual. Abordaremos então, uma serie de consequências no funcionamento e no modo de usufruirmos de nosso corpo, como por exemplo a falta de limites ou o gozo sem medida(AU)

Cet article est une réflexion sur le corps de la psychanalyse en relation à ce qu’on paye pour entrer dans la langue. Le corps est pensé à partir de la métaphore extraite par Lacan (1963) de Le Marchand de Venise comme une livre de chair prise dans la machine formelle de la langue. Cette métaphore nous permet d'établir les relations de l'organisme avec le manque et la dette, en relation à la langue. Plus tard, nous articulons le corps avec le discours capitaliste, alors qu'il est un fait de la langue avec un impact très marqué sur l’époque actuelle. Nous discuterons ensuite une série de conséquences sur le fonctionnement de notre corps et dans le mode d’usufruit comme par exemple le manque de limites ou la jouissance sans mesure(AU)

The following article presents a reflection on the body for psychoanalysis in relation to what is paid to enter the language. In order to do so, the body is thought from the metaphor extracted by Lacan (1963) from The Merchant of Veniceas a pound of flesh caught in the formal machine of language. This metaphor allows us to establish the relations of the body with lack and debt, in relation to the language. Later we will articulate the body with the capitalist discourse, while this is a fact of language with a very incidence in the present time. We will then approach several consequences in the working of the body and the way we enjoy it, for example lack of limits or unmeasured enjoyment(AU)

Pagamos com o nosso corpo? O gozo em jogo no discurso do mestre e no capitalismo / Nouspayons avec notre corps? La jouissance en jeu dans le discours du maîtreet dans le capitalisme / Do we pay with our body? The jouissance at stakein the master's discourse and in capitalism

O presente artigo faz uma reflexão sobre o corpo para a psicanálise,em relação ao que se paga ao entrar na linguagem. Para tanto o corpo será pensado a partir da metáfora extraída por Lacan (1963) em OMercador de Veneza, como uma libra de carne presa na máquina formal da linguagem. Esta metáfora nos permite estabelecer asrelações do corpo com a falta e a dívida, em relação à linguagem. Posteriormente, será articulado o corpo com o discurso capitalista, enquanto este é um fato da linguagem com uma incidência muito marcada na época atual. Abordaremos então, uma serie de consequências no funcionamento e no modo de usufruirmos de nosso corpo, como por exemplo a falta de limites ou o gozo sem medida.

Cet article est une réflexion sur le corps de la psychanalyse en relation à ce qu’on paye pour entrer dans la langue. Le corps est pensé à partir de la métaphore extraite par Lacan (1963) de Le Marchand de Venise comme une livre de chair prise dans la machine formelle de la langue. Cette métaphore nous permet d'établir les relations de l'organisme avec le manque et la dette, en relation à la langue. Plus tard, nous articulons le corps avec le discours capitaliste, alors qu'il est un fait de la langue avec un impact très marqué sur l’époque actuelle. Nous discuterons ensuite une série de conséquences sur le fonctionnement de notre corps et dans le mode d’usufruit comme par exemple le manque de limites ou la jouissance sans mesure.

The following article presents a reflection on the body for psychoanalysis in relation to what is paid to enter the language. In order to do so, the body is thought from the metaphor extracted by Lacan (1963) from The Merchant of Veniceas a pound of flesh caught in the formal machine of language. This metaphor allows us to establish the relations of the body with lack and debt, in relation to the language. Later we will articulate the body with the capitalist discourse, while this is a fact of language with a very incidence in the present time. We will then approach several consequences in the working of the body and the way we enjoy it, for example lack of limits or unmeasured enjoyment.

Loss of ZFP36 expression in colorectal cancer correlates to wnt/ ß-catenin activity and enhances epithelial-to-mesenchymal transition through upregulation of ZEB1, SOX9 and MACC1.

The mRNA-destabilizing protein ZFP36 has been previously described as a tumor suppressor whose expression is lost during colorectal cancer development. In order to evaluate its role in this disease, we restored ZFP36 expression in different cell contexts, showing that the presence of this protein impairs the epithelial-to-mesenchymal transition (EMT) and induces a higher susceptibility to anoikis. Consistently, we found that ZFP36 inhibits the expression of three key transcription factors involved in EMT: ZEB1, MACC1 and SOX9. Finally, we observed for the first time that its expression negatively correlates with the activity of Wnt/ß-catenin pathway, which is constitutively activated in colorectal cancer. This evidence provides a clue on the mechanism leading to the loss of ZFP36 in CRC.

Intracellular quantitative detection of human thymidylate synthase engagement with an unconventional inhibitor using tetracysteine-diarsenical-probe technology.

Demonstrating a candidate drug's interaction with its target protein in live cells is of pivotal relevance to the successful outcome of the drug discovery process. Although thymidylate synthase (hTS) is an important anticancer target protein, the efficacy of the few anti-hTS drugs currently used in clinical practice is limited by the development of resistance. Hence, there is an intense search for new, unconventional anti-hTS drugs; there are approximately 1600 ongoing clinical trials involving hTS-targeting drugs, both alone and in combination protocols. We recently discovered new, unconventional peptidic inhibitors of hTS that are active against cancer cells and do not result in the overexpression of hTS, which is a known molecular source of resistance. Here, we propose an adaptation of the recently proposed tetracysteine-arsenic-binding-motif technology to detect and quantitatively characterize the engagement of hTS with one such peptidic inhibitor in cell lysates. This new model can be developed into a test for high-throughput screening studies of intracellular target-protein/small-molecule binding.