The ancestral type of the R-RAS protein has oncogenic potential.

BACKGROUND: The R-RAS2 is a small GTPase highly similar to classical RAS proteins at the regulatory and signaling levels. The high evolutionary conservation of R-RAS2, its links to basic cellular processes and its role in cancer, make R-RAS2 an interesting research topic. To elucidate the evolutionary history of R-RAS proteins, we investigated and compared structural and functional properties of ancestral type R-RAS protein with human R-RAS2. METHODS: Bioinformatics analysis were used to elucidate the evolution of R-RAS proteins. Intrinsic GTPase activity of purified human and sponge proteins was analyzed with GTPase-GloTM Assay kit. The cell model consisted of human breast cancer cell lines MCF-7 and MDA-MB-231 transiently transfected with EsuRRAS2-like or HsaRRAS2. Biological characterization of R-RAS2 proteins was performed by Western blot on whole cell lysates or cell adhesion protein isolates, immunofluorescence and confocal microscopy, MTT test, colony formation assay, wound healing and Boyden chamber migration assays. RESULTS: We found that the single sponge R-RAS2-like gene/protein probably reflects the properties of the ancestral R-RAS protein that existed prior to duplications during the transition to Bilateria, and to Vertebrata. Biochemical characterization of sponge and human R-RAS2 showed that they have the same intrinsic GTPase activity and RNA binding properties. By testing cell proliferation, migration and colony forming efficiency in MDA-MB-231 human breast cancer cells, we showed that the ancestral type of the R-RAS protein, sponge R-RAS2-like, enhances their oncogenic potential, similar to human R-RAS2. In addition, sponge and human R-RAS2 were not found in focal adhesions, but both homologs play a role in their regulation by increasing talin1 and vinculin. CONCLUSIONS: This study suggests that the ancestor of all animals possessed an R-RAS2-like protein with oncogenic properties similar to evolutionarily more recent versions of the protein, even before the appearance of true tissue and the origin of tumors. Therefore, we have unraveled the evolutionary history of R-RAS2 in metazoans and improved our knowledge of R-RAS2 properties, including its structure, regulation and function.

Transfection of Sponge Cells and Intracellular Localization of Cancer-Related MYC, RRAS2, and DRG1 Proteins.

The determination of the protein's intracellular localization is essential for understanding its biological function. Protein localization studies are mainly performed on primary and secondary vertebrate cell lines for which most protocols have been optimized. In spite of experimental difficulties, studies on invertebrate cells, including basal Metazoa, have greatly advanced. In recent years, the interest in studying human diseases from an evolutionary perspective has significantly increased. Sponges, placed at the base of the animal tree, are simple animals without true tissues and organs but with a complex genome containing many genes whose human homologs have been implicated in human diseases, including cancer. Therefore, sponges are an innovative model for elucidating the fundamental role of the proteins involved in cancer. In this study, we overexpressed human cancer-related proteins and their sponge homologs in human cancer cells, human fibroblasts, and sponge cells. We demonstrated that human and sponge MYC proteins localize in the nucleus, the RRAS2 in the plasma membrane, the membranes of the endolysosomal vesicles, and the DRG1 in the cell's cytosol. Despite the very low transfection efficiency of sponge cells, we observed an identical localization of human proteins and their sponge homologs, indicating their similar cellular functions.

Amyloid-ß plaque formation and BACE1 accumulation in the brains of a 5xFAD Alzheimer's disease mouse model is associated with altered distribution and not proteolysis of BACE1 substrates Sez6 and Sez6L.

The formation of amyloid-ß peptides (Aß), that accumulate in Alzheimer's disease (AD) brains, involves proteolytic processing of the amyloid precursor protein (APP) firstly by ß-secretase (BACE1). Since BACE1 cleaves a plethora of other substrates, in this work we investigated whether the proteolysis and/or distribution of other BACE1 substrates, such as seizure protein 6 (Sez6) and seizure 6-like protein (Sez6L), is altered in AD. To test this we used 5xFAD mouse model brains that show an early accumulation of Aß plaques already at 2-months of age. Here we show for the first time that accumulation of BACE1 in peri-plaque regions and its enhanced levels in AD brains does not affect proteolysis of BACE1 substrates other than APP, such as Sez6 and Sez6L. We observed altered distribution of Sez6 and Sez6L in the area of Aß plaques in 5xFAD brains which is distinct to that of APP, BACE1 and/or LAMP1, suggesting different localization and/or function of these BACE1 substrates. While it is necessary to further elucidate the potential role that this may play in the course of AD, it is likely that Aß-targeted therapies may have beneficial effects against accumulation and/or altered distribution of BACE1 and its substrates, in addition to APP.

Structure and function of cancer-related developmentally regulated GTP-binding protein 1 (DRG1) is conserved between sponges and humans.

Cancer is a disease caused by errors within the multicellular system and it represents a major health issue in multicellular organisms. Although cancer research has advanced substantially, new approaches focusing on fundamental aspects of cancer origin and mechanisms of spreading are necessary. Comparative genomic studies have shown that most genes linked to human cancer emerged during the early evolution of Metazoa. Thus, basal animals without true tissues and organs, such as sponges (Porifera), might be an innovative model system for understanding the molecular mechanisms of proteins involved in cancer biology. One of these proteins is developmentally regulated GTP-binding protein 1 (DRG1), a GTPase stabilized by interaction with DRG family regulatory protein 1 (DFRP1). This study reveals a high evolutionary conservation of DRG1 gene/protein in metazoans. Our biochemical analysis and structural predictions show that both recombinant sponge and human DRG1 are predominantly monomers that form complexes with DFRP1 and bind non-specifically to RNA and DNA. We demonstrate the conservation of sponge and human DRG1 biological features, including intracellular localization and DRG1:DFRP1 binding, function of DRG1 in α-tubulin dynamics, and its role in cancer biology demonstrated by increased proliferation, migration and colonization in human cancer cells. These results suggest that the ancestor of all Metazoa already possessed DRG1 that is structurally and functionally similar to the human DRG1, even before the development of real tissues or tumors, indicating an important function of DRG1 in fundamental cellular pathways.

Impaired Retromer Function in Niemann-Pick Type C Disease Is Dependent on Intracellular Cholesterol Accumulation.

Niemann-Pick type C disease (NPC) is a rare inherited neurodegenerative disorder characterized by an accumulation of intracellular cholesterol within late endosomes and lysosomes due to NPC1 or NPC2 dysfunction. In this work, we tested the hypothesis that retromer impairment may be involved in the pathogenesis of NPC and may contribute to increased amyloidogenic processing of APP and enhanced BACE1-mediated proteolysis observed in NPC disease. Using NPC1-null cells, primary mouse NPC1-deficient neurons and NPC1-deficient mice (BALB/cNctr-Npc1m1N), we show that retromer function is impaired in NPC. This is manifested by altered transport of the retromer core components Vps26, Vps35 and/or retromer receptor sorLA and by retromer accumulation in neuronal processes, such as within axonal swellings. Changes in retromer distribution in NPC1 mouse brains were observed already at the presymptomatic stage (at 4-weeks of age), indicating that the retromer defect occurs early in the course of NPC disease and may contribute to downstream pathological processes. Furthermore, we show that cholesterol depletion in NPC1-null cells and in NPC1 mouse brains reverts retromer dysfunction, suggesting that retromer impairment in NPC is mechanistically dependent on cholesterol accumulation. Thus, we characterized retromer dysfunction in NPC and propose that the rescue of retromer impairment may represent a novel therapeutic approach against NPC.

Involvement of the Choroid Plexus in the Pathogenesis of Niemann-Pick Disease Type C.

Niemann-Pick type C (NPC) disease, sometimes called childhood Alzheimer's, is a rare neurovisceral lipid storage disease with progressive neurodegeneration leading to premature death. The disease is caused by loss-of-function mutations in the Npc1 or Npc2 gene which both result into lipid accumulation in the late endosomes and lysosomes. Since the disease presents with a broad heterogenous clinical spectrum, the involved disease mechanisms are still incompletely understood and this hampers finding an effective treatment. As NPC patients, who carry NPC1 mutations, have shown to share several pathological features with Alzheimer's disease (AD) and we and others have previously shown that AD is associated with a dysfunctionality of the blood-cerebrospinal fluid (CSF) barrier located at choroid plexus, we investigated the functionality of this latter barrier in NPC1 pathology. Using NPC1-/- mice, we show that despite an increase in inflammatory gene expression in choroid plexus epithelial (CPE) cells, the blood-CSF barrier integrity is not dramatically affected. Interestingly, we did observe a massive increase in autophagosomes in CPE cells and enlarged extracellular vesicles (EVs) in CSF upon NPC1 pathology. Additionally, we revealed that these EVs exert toxic effects on brain tissue, in vitro as well as in vivo. Moreover, we observed that EVs derived from the supernatant of NPC1-/- choroid plexus explants are able to induce typical brain pathology characteristics of NPC1-/-, more specifically microgliosis and astrogliosis. Taken together, our data reveal for the first time that the choroid plexus and CSF EVs might play a role in the brain-related pathogenesis of NPC1.

GSK-3-TSC axis governs lysosomal acidification through autophagy and endocytic pathways.

Impaired lysosomal activity, which results in defective protein processing, waste accumulation, and protein aggregation, is implicated in a number of disease pathologies. Acidification of lysosomes is a crucial process required for lysosome function. Previously we showed that inhibition of glycogen synthase kinase-3 (GSK-3) enhanced lysosomal acidification in both normal and pathological conditions. However, how GSK-3 integrates into the lysosome networking is unknown. Here we show that inhibition of mTORC1 and increased autophagic activity are downstream to GSK-3 inhibition and contribute to lysosomal acidification. Strikingly, lysosomal acidification is also restored by GSK-3 inhibition in the absence of functional autophagy, and, independently of mTORC1. This is facilitated by increased endocytic traffic: We show that GSK-3 inhibition enhanced material internalization, increased recruitment of active Rab5 into endosomes, and increased Rab7/RILP clustering into lysosomes, all processes required for late endosome maturation. Consistently, in cells defective in endocytic traffic caused by either constitutively active Rab5, or, deletion of the Niemann-Pick C1 protein, GSK-3 inhibition could not restore lysosomal acidification. Finally we found that the tuberous sclerosis complex, TSC, is required for lysosomal acidification and is activated by GSK-3 inhibition. Thus, the GSK-3/TSC axis regulates lysosomal acidification via both the autophagic and endocytic pathways. Our study provides new insights into the therapeutic potential of GSK-3 inhibitors in treating pathological conditions associated with impaired cellular clearance.

Enhanced activity of superoxide dismutase is a common response to dietary and genetically induced increased cholesterol levels.

Objectives: Hypercholesterolaemia has been implicated in the pathogenesis of neurodegenerative diseases. In this work, we tested whether cholesterol-mediated neurodegeneration induced either by cholesterol-rich diet or genetic mutation may share a common mechanism involving increased oxidative stress and mitochondria oxidant status. Additionally, we analysed whether upon cholesterol-rich diet, different brain regions (prefrontal cortex, cortex, hippocampus, and cerebellum) show distinct vulnerability to an oxidative stress response.Methods: Oxidative stress parameters were measured both in vivo (in the liver and in different brain regions) in cholesterol-fed mice and in vitro in genetically induced cholesterol accumulation in NPC1-null cells.Results: Increased superoxide dismutase (SOD) activity was a common feature of cholesterol-mediated antioxidant response in both models. Moreover, upon high-cholesterol diet, all four brain regions analysed responded via somewhat different capacity of antioxidant defence, hippocampus showing the highest basal activity of SOD. Increased activity of SOD upon cholesterol accumulation in vitro involves mitochondrial SOD2. We found that SOD/SOD2 activities are modulated by cholesterol levels.Discussion: Hypercholesterolaemia could potentiate brain dysfunction and neurodegenerative processes via oxidative stress, and activity of mitochondrial SOD2 may play a key role in this process. Our findings suggest that preventing/reducing mitochondrial oxidative stress may represent a common approach against neurodegenerative diseases.

BACE1-cleavage of Sez6 and Sez6L is elevated in Niemann-Pick type C disease mouse brains.

It is intriguing that a rare, inherited lysosomal storage disorder Niemann-Pick type C (NPC) shares similarities with Alzheimer's disease (AD). We have previously reported an enhanced processing of ß-amyloid precursor protein (APP) by ß-secretase (BACE1), a key enzyme in the pathogenesis of AD, in NPC1-null cells. In this work, we characterized regional and temporal expression and processing of the recently identified BACE1 substrates seizure protein 6 (Sez6) and seizure 6-like protein (Sez6L), and APP, in NPC1-/- (NPC1) and NPC1+/+ (wt) mouse brains. We analysed 4-weeks old brains to detect the earliest changes associated with NPC, and 10-weeks of age to identify changes at terminal disease stage. Sez6 and Sez6L were selected due to their predominant cleavage by BACE1, and their potential role in synaptic function that may contribute to presentation of seizures and/or motor impairments in NPC patients. While an enhanced BACE1-cleavage of all three substrates was detected in NPC1 vs. wt-mouse brains at 4-weeks of age, at 10-weeks increased proteolysis by BACE1 was observed for Sez6L in the cortex, hippocampus and cerebellum of NPC1-mice. Interestingly, both APP and Sez6L were found to be expressed in Purkinje neurons and their immunostaining was lost upon Purkinje cell neurodegeneration in 10-weeks old NPC1 mice. Furthermore, in NPC1- vs. wt-mouse primary cortical neurons, both Sez6 and Sez6L showed increased punctuate staining within the endolysosomal pathway as well as increased Sez6L and BACE1-positive puncta. This indicates that a trafficking defect within the endolysosomal pathway may play a key role in enhanced BACE1-proteolysis in NPC disease. Overall, our findings suggest that enhanced proteolysis by BACE1 could be a part of NPC disease pathogenesis. Understanding the basic biology of BACE1 and the functional impact of cleavage of its substrates is important to better evaluate the therapeutic potential of BACE1 against AD and, possibly, NPC disease.

Glutathionylation: a regulatory role of glutathione in physiological processes.

Glutathione (γ-glutamyl-cysteinyl-glycine) is an intracellular thiol molecule and a potent antioxidant that participates in the toxic metabolism phase II biotransformation of xenobiotics. It can bind to a variety of proteins in a process known as glutathionylation. Protein glutathionylation is now recognised as one of important posttranslational regulatory mechanisms in cell and tissue physiology. Direct and indirect regulatory roles in physiological processes include glutathionylation of major transcriptional factors, eicosanoids, cytokines, and nitric oxide (NO). This review looks into these regulatory mechanisms through examples of glutathione regulation in apoptosis, vascularisation, metabolic processes, mitochondrial integrity, immune system, and neural physiology. The focus is on the physiological roles of glutathione beyond biotransformational metabolism.